US20070128587A1 - Method of diagnosis of foot and mouth disease and the diagnostic kit - Google Patents
Method of diagnosis of foot and mouth disease and the diagnostic kit Download PDFInfo
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- US20070128587A1 US20070128587A1 US10/555,059 US55505903A US2007128587A1 US 20070128587 A1 US20070128587 A1 US 20070128587A1 US 55505903 A US55505903 A US 55505903A US 2007128587 A1 US2007128587 A1 US 2007128587A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
- G01N33/525—Multi-layer analytical elements
- G01N33/526—Multi-layer analytical elements the element being adapted for a specific analyte
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
- G01N33/54387—Immunochromatographic test strips
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
- G01N33/54387—Immunochromatographic test strips
- G01N33/54388—Immunochromatographic test strips based on lateral flow
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/085—Picornaviridae, e.g. coxsackie virus, echovirus, enterovirus
- G01N2333/09—Foot-and-mouth disease virus
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/811—Test for named disease, body condition or organ function
Definitions
- the present invention relates to a method and kit for diagnosing an infection of the subject with foot-and-mouth disease virus (FMDV), and more particularly, a method and kit for rapidly detecting infection of the subjects with FMDV by observing a change in appearance of a reactivity zone containing at least more than one immobilized phase selected from antigens, antibodies or haptens derived from FMDV, or obtainable from FMDV via an immunological reaction, which is immunologically reactive with the test sample from animals as a target.
- FMDV foot-and-mouth disease virus
- Foot and mouth disease is a devastating disease of livestock and an Office International des Epizooties list A disease. All species of cloven-hoofed animals (cattle, pigs, sheep and goats) are susceptible and the disease is extremely contagious. Financial losses as a result of FMD can be significant. There are direct losses due to deaths in young animals, loss of milk, loss of meat and a decrease in productive performance. The costs associated with eradication or control can be high and, in addition, there are indirect losses due to the imposition of trade restrictions.
- the causative agent is FMDV, anaphthovirus of the Picornaviridae family (Bittle et al., 1982 and Fross et al., 1984).
- the FMDV genome consists of a single RNA positive strand of approximately 8,000 nucleotide bases.
- the RNA is initially translated as a single polypeptide which is subsequently cleaved by viral-encoded proteases to produce four capsid proteins (VP1-VP4) and non-structural polypeptides (2C, 3A, 3ABC and 3D) in infected cells.
- VP1-VP4 capsid proteins
- 2C, 3A, 3ABC and 3D non-structural polypeptides
- FMD FMDV-specific antigens or antibodies
- the detection of antibody to FMD virus in serum has several usefulness.
- the antibody detection evidences previous infection in animals from which vesicular material is not available. Diagnosis of FMD by clinical signs may be difficult, especially for sheep and goats, in which clinical signs are often mild (Barnett, P. V et al., 1999 and Callens, M., K. et al., 1998).
- vesicular virus infections including those caused by swine vesicular disease (SVD) virus, vesicular stomatitis virus, and others, cannot be distinguished from FMDV infection by the clinical findings.
- FMDV can establish a persistent or carrier stage in ruminants and they show no signs of FMD.
- Such carrier animals can become the source of new outbreaks of the disease. Because of these problems, a rapid serological method is needed to identify infected and/or asymptomatic carrier animals and distinguish them from vaccinated animals. This antibody detection method also can be used in epidemiological surveys and to measure the effectiveness of vaccination.
- the capsid protein alone used in the diagnostic assays, it will detect both vaccinated and infected animal based on the detection of antibody to structural protein. For this reason, the antibody test to structural protein can be used only in vaccine-free region, such as the USA or the UK, but not in regions where vaccination practice is established. But even in areas where animals are vaccinated, the FMD occurs frequenctly. In such a region, diagnostic tests that can differentiate the infection from the vaccination are required.
- PCR polymerase chain reaction
- RT-PCR reverse transcription-polymerase chain reaction
- the present invention has been made in view of the above problems, and it is an object of the present invention to provide a method for diagnosing infection of the subject animal with foot-and-mouth disease virus, making it possible to simply and rapidly identify whether the animal is infected with that virus or not, upon using biological samples obtained from the animal.
- the diagnostic method according to the present invention includes a sandwich assay or a competition assay.
- the present invention provides a kit for diagnosing foot-and-mouth disease virus infection comprising:
- a strip 1 including a reactivity zone 13 containing at least more than one immobilized phase selected from antigen, antibody or hapten thereon, derived from FMDV or obtainable from FMDV through an immunological reaction, and a control zone 14 for confirming normal operation of the kit, provided on a predetermined region of a wicking membrane 9 ; and
- a housing 20 protecting the strip 1 from a variety of contaminants, and including at least a test sample application port 2 and an indicia window 4 for observing results of reaction in the reactivity zone 13 and the control zone 14 on the strip 1 .
- the test sample is preferably a body fluid which is secreted out of the body and includes blood, serum, plasma, urine, tears, saliva, milk, etc.
- the analyte of interest which is contained in the test sample to be analyzed may include any substances containing specific-binding members which may be naturally formed or artificially imparted, including antigen-presenting substances, antibodies (including monoclonal and polyclonal antibody), haptens, and combinations thereof, for example.
- an immobilized phase (or, capture reagent) is an unlabeled specific bonding member which specifically binds to the analyte, an indicator reagent, an auxiliary specific-binding member, or the like and then captures the analyte, and is immobilized directly or indirectly on the wicking membrane 9 of the strip 1 .
- the detection reagent may bind diffusibly or non-diffusibly to a pad and includes a labeled reagent, the auxiliary specific-binding member and/or a component of a signal generating system.
- the signal generating system includes at least a catalytic member and solute.
- the solute may be catalyzed by the catalytic member to induce a reaction, and generates a signal recognizable from membrane surface or inside.
- the catalytic member may be enzyme or non-enzyme.
- the solute may carry out a reaction which is catalyzed by the catalytic member. Such a reaction produces a large amount of signal-generating compound which may be directly or indirectly detectable.
- the signal detectable by these components includes spectrophotometric, visible signal, electrochemical signal, and other electrically detectable signals.
- FIGS. 1 a - b are, respectively, separate perspective views of exemplified diagnostic kits as a preferred embodiment used in the present invention.
- the diagnostic kit includes a strip 1 and a housing 20 .
- the housing is required for spotting a test sample from a test sample application port 2 and a developing reagent application port 3 on a filter pad (or a dye pad: it refers to a pad containing a detection reagent).
- the kit also includes a main body 7 having a cover 5 comprising an indicia widow 4 for showing test results, and a strip-mounting member 6 for placing and fixing the strip 1 in place therein.
- the cover 5 and main body 7 are interconnected via a fastening member 8 . They are required for fixing the strip 1 , and for preventing contact with a reactivity zone or contamination thereof, and are preferably made of a non-reactive material which does not react with any other reagents used in the test, such as plastics or the like.
- a separate developing reagent application port 3 is required.
- the developing reagent application port 3 is configured to have a curvature in a cup-like shape so as to receive a predetermined amount of the developing reagent. Further, the lower part thereof is preferably in close contact with a reservoir pad 10 so as to prevent the developing reagent from flowing out into other regions of the test device.
- the application port 2 ′ is configured to have a curvature in a cup-like shape so as to receive a predetermined amount of the test sample.
- the lower part thereof is preferably in close contact with a reservoir pad so as to prevent the test sample from flowing out into other regions of the test device.
- a pad 11 ′ is a dye pad containing the detection reagent, or a second filter pad, or may be eliminated.
- An indicia window 4 is designed for externally observing changes occurring in a reactivity zone 13 and a control zone 14 on the wicking membrane 9 constituting the strip 1 , and is provided on the housing cover 5 so as to be positioned immediately over the reactivity zone 13 and control zone 14 .
- the housing cover 5 may be provided with given discrimination symbols, for example ‘Date’ for test date, ‘It 0000’ for the subject, ‘C’ for the control zone, ‘T’ (Test) for the reactivity zone, ‘S’ (Sample) for the test sample application port, ‘D’ for the developing reagent, and the like, such that the test date, subject, test sample application port, developing reagent application port, indicia window for showing test result, etc. may be easily distinguished.
- Those symbols may be any letter, number, icon, or the like and any combinations thereof different from the foregoing.
- FIGS. 1 a - b and FIG. 2 a structure of a strip constituting the diagnostic device in accordance with the present invention will be described with reference to FIGS. 1 a - b and FIG. 2 .
- FIG. 1 a shows one embodiment in accordance with a first aspect of the present invention.
- the inventive strip 1 includes a wicking membrane 9 , a reservoir pad 10 , a filter pad 11 (also, serving as a dye pad), an absorbent pad 12 , and a reactivity zone 13 and a control zone 14 provided on the wicking membrane 9 .
- a base member 15 for fixing the strip 1 on a mounting member 6 of the main housing body 7 .
- the base member 15 is preferably made of plastic or a glass plate.
- FIG. 1 b shows another embodiment in accordance with the second aspect of the present invention.
- the inventive strip 1 ′ has the same configuration as in the strip shown in FIG. 1 a , except for a reservoir pad 10 ′, and a filter pad 11 ′ (which may also serve as a dye pad).
- a reservoir pad 10 ′ which may also serve as a dye pad.
- a filter pad 11 ′ which may also serve as a dye pad.
- the detailed description of respective configuration thereof will be based on the first aspect of the present invention shown in FIG. 1 a , but only the difference therebetween will be mentioned.
- a filter pad 11 is in contact with a back surface of a wicking membrane 9 for chromatography to form a connection passage for fluid flow into the wicking membrane 9 .
- the back surface of the filter pad 11 is in contact with the reservoir pad 10 to form one connection passage for fluid flow therebetween.
- the absorbent pad 12 is attached to the upper part of the wicking membrane 9 .
- On the predetermined region of the wicking membrane 9 are spaced apart a reactivity zone 13 containing at least more than one immobilized phase which specifically binds to an analyte to be detected, a labeled reagent, an auxiliary specific-binding member, or the like, and a control zone 14 for determining whether the kit is normally operating.
- the reservoir pad 10 absorbs the test sample, or a solution necessary for other tests, for example, a developing reagent, and the like and includes a capillary membrane to transfer analyte to the filter pad or wicking membrane.
- the reservoir pad 10 is required to have voids and volume sufficient to receive the test sample or developing reagent.
- Material suitable for the reservoir pad is preferably low molecular weight protein binding substances, including cellulose, polyester, polyurethane, glass fiber having a pore size of 0.45 to 60 ⁇ m, etc.
- the filter pad 11 filters unnecessary components in the test sample and may contain a detection reagent (in this case, the filter pad may also function as a dye pad). Where the detection reagent is contained in the filter pad, there is an advantage of eliminating the step of premixing the detection reagent with the test sample for the test.
- material suitable for the filter pad 11 there may be mentioned polyester, polyurethane, polyacetate, cellulose, glass fiber, nylon having a pore size of 0.45 to 60 ⁇ m, etc.
- the reservoir pad 10 and filter pad 11 may be made of the same material, and in this case, the detection reagent may be contained within the bottom of one long filter pad 11 .
- the detection reagent is provided with a labeled reagent, auxiliary specific-binding member, and/or a constitutional component of a signal generating system, which enable it to identify the presence of analyte of interest by naked eye or other instrumentation from the outside.
- Labeled detection reagents are well known to those skilled in the art.
- labels include catalysts, enzymes (for example, phosphatase, peroxydase, etc., and more specifically, alkaline phosphatase and horseradish peroxidase, or the like, which is used in combination with a substrate for an enzyme), substrate for enzyme (for example, nitrobluetetrazolium, 3,5′,5,5′ tetranitrobenzidine, 4-methoxy-1-naphthol, 4-chloro-1-naphthol, 5-bromo-4-chloro-3-indolylphosphate, chemoluminescent substrates for enzymes, for example, dioxethane, and derivatives and analogs thereof), fluorescent compounds (for example, fluorescein, phycobiliprotein, rhodamine, derivatives and analogs thereof), chemoluminescent compounds, radioactive elements, and the like.
- enzymes for example, phosphatase, peroxydase, etc., and more specifically, alkaline phosphatase and horserad
- the above-mentioned labeling reagents may form a conjugate with a given auxiliary specific-binding member having a property of easily binding to an analyte of interest.
- An auxiliary specific-binding member is not particularly limited and includes antigen, antibody, hapten, or the like, for example, protein G, protein A, protein G/A, known as material binding well to an antibody in case the analyte is antibody and various antibodies known as binding well to other antibodies IgG and IgM. These materials are presently commercially available as recombinants from Sigma, etc.
- the detection reagent needs not necessarily be included in the filter pad 11 .
- the detection reagent may be provided at any point between the reactivity zone 13 on the wicking membrane showing a detection result and the test sample application port. This detection reagent may be applied to upper or inside of any point of the filter pad 11 or wicking membrane 9 , in the dried or freeze-dried state.
- the strip 1 may further include a given control reagent to determine whether the kit is normally operating or not. Similar to the detection reagent, the control reagent may also be provided at any point between the filter pad 11 or the reactivity zone 13 on the wicking membrane 9 and the test sample application port.
- the control reagent may be selected from labeled protein, antigen, antibody, and the like which specifically bind to an immobilized phase (for example, protein, antigen, antibody, or the like) forming a control zone (or a control band) on the wick membrane 9 .
- immobilized phase for example, protein, antigen, antibody, or the like
- control zone or a control band
- the labeling reagent which may be included in the control reagent those as described in the detection reagent may be applied.
- the auxiliary specific-binding member is not particularly limited and includes one species selected from avidin, biotin, FITC, anti-FITC mouse antibody, mouse immunoglobulin, or anti-mouse immunoglobulin antibody, for example.
- the wicking membrane 9 should have sufficient voids, and be able to absorb substantial portions of test sample which has passed through the filter pad 11 .
- material suitable for such a wicking membrane there may be mentioned at least more than one material selected from nylon, polyester, cellulose, polysulfone, polyvinylidene difluoride, cellulose acetate, polyurethane, glass fiber, nitrocellulose, or the like.
- an example of material suitable for the developing reagent may include phosphate buffer, saline, Tris-HCl, water, etc.
- the developing reagent is required where the test sample application port 2 is positioned over the immediately upper part of the filter pad 11 so as to spot the test sample. Therefore, as in the embodiment in accordance with the second aspect of the FIG. 1 b , the developing reagent is not particularly required where the test sample application port 2 ′ is positioned on the reservoir pad 10 ′ so as to load a predetermined amount of the test sample thereon.
- the complex labeled with the detection reagent contains the analyte to be detected, it binds to the immobilized phase located on the reactivity zone 13 of the wicking membrane 9 and then results in externally discernable change.
- Material which may be used as such an immobilized phase may include at least more than one selected from antigen, antibody or hapten, which constitutes foot-and-mouse disease virus, or may be derived therefrom through an immunological reaction.
- Material which may be used as the antigen includes non-structural and/or structural proteins.
- Structural proteins include inactivated FMDV disrupted material or constituents thereof, VP1-VP4 polypeptide.
- Non-structural proteins may include at least more than one polypeptide selected from leader peptide (Lb), 2B, 2C, 3A, 3D, 3AB and 3ABC.
- FIG. 3 shows a map of a polyprotein precursor comprising the structural and non-structural proteins.
- the structural proteins with the same name may also exhibit some difference in constitutional amino acids among them, depending on serological classification of FMDV.
- the structural proteins which may be used in the present invention are not particularly limited, so long as they may specifically react with antibodies formed against all the sero-types.
- An example of these structural proteins includes, but is not limited to, VP1 represented by SEQ ID NO: 118.
- an example of antigen employed in the vaccine production includes, in addition to the structural protein, a non-structural protein 3D.
- a non-structural protein 3D is shown in SEQ ID NO: 121.
- the non-structural proteins are proteins that have not been used in conventional vaccine production and an antibody to those proteins is observable only in a virus-infected animal. Thus, when this protein is applied as an immobilized phase, it will be possible to make an exact diagnosis for the infected animal.
- a non-structural protein also has some difference in constituent amino acids thereof, depending on respective sero-type of FMDV.
- Usable non-structural proteins are not particularly limited, as long as preferably, they may specifically react with antibodies produced against all sero-types. Examples of these structural proteins include 2C represented by SEQ ID NO: 119 and 3ABC represented by SEQ ID NO: 120.
- FIG. 4 shows an example to which the non-structural protein was applied as a single immobilized phase.
- T represents a reactivity zone in which the non-structural protein (2C or 3ABC) was immobilized, which has never been used in vaccine production up to now.
- C represents a control zone.
- the kit having both the discolored T and C ( FIG. 4 a ) represents the infected animal, whereas the kit having only the discolored C ( FIG. 4 b ) represents a negative animal prior to vaccination or a vaccinated animal. If C was not discolored in any case, it means that the kit of interest was not normally operated with the result thus obtained being unreliable.
- the most preferred embodiment of the present invention provides a diagnostic method and kit which make it possible to distinguish an FMDV-infected animal as well as a vaccinated animal.
- a first reactivity zone in which as an immobilized phase, an antigen usable in vaccine production known until now was immobilized at a particular site on a wicking membrane
- a second reactivity zone in which as an immobilized phase, a non-structural protein that was known as never used before in vaccine production was spaced and immobilized at a particular site different from the first reactivity zone on the wicking membrane. Therefore, it is possible to diagnose whether animal was vaccinated, virus-infected or negative prior to vaccination, through change in appearance produced from a binding reaction between these first and second reactivity zones and a labeled complex.
- the immobilized phase bonding to a control reagent forms a control zone at a different site spaced from the reactivity zones.
- various reagents and immobilized phases which are applied in other commercially available diagnostic kits may be used. Details on that will be described in examples as follows.
- a test sample such as animal serum (or plasma, whole blood) is spotted on the test sample application port 2 formed on the housing cover.
- the filter pad 10 constituting the strip is positioned at the lower end of the application port 2 .
- the filter pad 10 also contains a protein G-gold conjugate as a detection reagent.
- the protein G-gold conjugate can form complexes with all the antibodies present in the test sample.
- a predetermined amount of a developing reagent is loaded on the developing reagent application port 3 in which the reservoir pad 10 constituting the strip is positioned on the lower end thereof.
- a complex between the labeled conjugate and an antibody in the test sample. Then, this complex is chromatographed along the longitudinal axis of the wicking membrane 9 (preferably, nitrocellulose membrane).
- FMDV recombinant antigen (construction thereof will be described in detail with reference to the following examples) was previously applied and immobilized on the reactivity zone 13 of the wicking membrane 9 , and thus if the complex contains a specific antibody to the recombinant antigen, it will undergo reaction and then show discoloration in the form of a red line.
- the recombinant antigen includes both the structural and non-structural proteins, there is described for example, a method to diagnose whether the animal was vaccinated, virus-infected or negative prior to vaccination.
- FIG. 5 shows this example.
- T1 represents a line on which an antigen (structural protein or non-structural protein 3D) which had been introduced in vaccine production up to now was immobilized.
- T2 represents a line on which a non-structural protein (2C or 3ABC) which has never been used before in vaccine production was immobilized.
- C represents a control line.
- the kit ( FIG. 5 a ) in which all the T1, T2 and C were discolored represents a virus-infected animal.
- a kit ( FIG. 5 b ) in which only the T1 and C were discolored represents a vaccinated animal.
- a kit ( FIG. 5 c ) means a negative animal prior to vaccination, as only the C was discolored. If C was not discolored in any case, it means that the kit was not normally operated with the result thus obtained being unreliable.
- the diagnostic device usable in the present invention can be made of various configurations and modifications as disclosed in U.S. Pat. No. 5,728,587, and the particular disclosure of the strip construction (i.e., arrangement of the pads) included in this separate device does not constitute the essence of the present invention.
- the arrangement of the pad is not limited to those described above, and other arrangement such as a reservoir pad/a first filter pad/a second filter pad/a wicking membrane/an absorbent pad may be considered.
- the first filter pad or the second filter pad serves to filter and separate blood cells from blood, or filter and remove foreign materials unnecessary for sample test.
- the present invention was conveniently explained by way of example of structural and non-structural proteins constituting foot-and-mouth disease virus as an immobilized phase contained in the reactivity zone, the scope and sprit of the present invention should be construed to encompass any material which had been already supplied as antigen for vaccine production at the time of filing the present invention or would be supplied as antigen in the near future and a certain material capable of inducing an antibody in vivo (including hapten), or various antibodies obtainable from FMDV through an immunological reaction.
- FIG. 1 a shows a separate perspective view of a device (a rapid kit) for diagnosing infection of the subject animal with foot-and-mouth disease virus according to a first aspect of the present invention.
- FIG. 1 b shows a separate perspective view of a device (a rapid kit) for diagnosing infection of the subject animal with foot-and-mouth disease virus according to a second aspect of the present invention
- FIG. 2 shows a configuration of a strip constituting a diagnostic device according to the present invention
- FIG. 3 shows a structure of a polyprotein expressed from foot-and-mouth disease virus
- FIG. 4 shows an exemplified test result for the one-line test kit
- FIG. 5 shows an exemplified test result for the two-line test kit
- FIG. 6 shows a map of plasmid pBM-VPITw97F
- FIG. 7 shows a map of plasmid pBM-2CTw97F
- FIG. 8 shows a map of plasmid pBM-3ABCTw97F.
- FIG. 9 shows a map of plasmid pBM-3DTw97F.
- Oligonucleotides for gene construction and sequencing were synthesized at ResGen (Huntsville, Ala.). Unless otherwise indicated, DNA sequencing was also performed at ResGen.
- PCR polymerase chain reaction
- New England Biolabs, Inc. (Beverly, Mass.) and a mixture of dNTPs was purchased from Amersham-Pharmacia (Piscataway, N.J.) and used according to the manufacturer's specifications unless otherwise indicated.
- PCR amplifications were performed on a GeneAmp 2400 thermal cycler from Perkin-Elmer Corporation (Foster City, Calif.).
- the PCR product was purified using Qiagen PCR spin column (Qiagen Inc., Chatsworth, Calif.) as recommended by the manufacturer.
- restriction enzymes were purchased from New England BioLabs, and DNA fragments were isolated on agarose (Sigma-Aldrich) gels, treated with restriction enzymes and then used for cloning.
- Desired fragment was excised and the DNA was extracted with a QIAEX II gel extraction kit as recommended by the manufacturer.
- DNA was resuspended in H 2 O or TE (1 mM ethylenediaminetetraacetic acid (EDTA; pH 8.0; Sigma-Aldrich), 10 mM tris(hydroxymethyl)aminomethane-hydrochloride (Tris-HCl; pH 8.0; Sigma-Aldrich)).
- Ligations were performed using DNA ligase (Boehringer Mannheim Corporation, Indianapolis, Ind.) as recommended by the manufacturer. Ligation reaction was incubated at 16° C. overnight. Bacterial transformations were performed using E. coli XL1-Blue competent cells.
- transformations and bacterial restreaks were plated on LB agar (Lennox) plates supplemented with 100 ug/ml ampicillin. All bacterial incubations (plates and liquid cultures) were conducted overnight (16 hours) at 37° C.
- Miniprep DNA was prepared according to Molecular Cloning: A Laboratory Manual, unless otherwise indicated. Colonies containing desired clones were propagated from the transfer plate or stocked in glycerol at ⁇ 70° C.
- Taiwan Type O 97 sequence was retrieved from NCBI GenBank data and oligonucleotides for syntheictc gene were synthesized at ResGen (Huntsville, Ala.). In the synthetic oligonucleotides, the native FMDV codons were altered to conform to E. coli codon bias in an effort to increase expression levels of the recombinant protein in E. coli . See, for example, M. Gouy and C. Gautier, Nucleic Acids Research 10:7055 (1982); H. Grosjean and W. Fiers, Gene 18:199 (1982); J. Watson et al.
- the recursive PCR method was used to assemble the oligonucleotides into full VP1 gene.
- the gene construction strategy involved synthesis of a series of overlapping oligonucleotides with complementary ends. When annealed, the ends served as primers for the extension of the complementary strand. The fragments then were amplified by excessive outside primers.
- Oligonucleotide was designed to contain a BamHI restriction site for cloning into the expression vector pGEX-4T-1.
- Reverse oligonucleotide contains a translation stop codon (TAA) and EcoRI restriction site.
- TAA translation stop codon
- EcoRI EcoRI restriction site
- Vent DNA polymerase (1U) and 1 ⁇ buffer, along with 25 uM of each dNTP (dATP, dCTP, dGTP, and dTTP), 50 pmol each of oligonucleotides TW97-1 (SEQ ID NO: 1) and TW97-16 (SEQ ID NO: 1), and 0.25 pmol each of oligonucleotides TW97-2 (SEQ ID NO: 2) through TW97-15 (SEQ ID NO: 15).
- dNTP dATP, dCTP, dGTP, and dTTP
- reaction was incubated at 95° C. for 5 minutes, and then amplified with 30 cycles of 95° C. for 15 seconds, 58 CC for 15 seconds and 72° C. for 60 seconds, followed by incubation at 72° C. for 5 minutes.
- PCR-derived product was purified using Qiagen PCR spin column.
- the PCR product amplified as described hereinabove was digested with the restriction endonucleases Bam HI+Eco RI and ligated into the vector pGEX-4T-1 that had been digested with Bam HI+Eco RI and gel-isolated.
- the ligation product was used to transform XL-1 Blue competent cells.
- the transformed cells were plated on LB plates supplemented with 100 ug/ml ampicillin.
- Miniprep DNAs were prepared from overnight cultures of colonies and digested with Bam HI+Eco RI to screen the desired clones.
- the clone with right insert was designated as pBM-VPITw97F ( FIG. 6 ).
- the pBM-VPITw97F clone was sequenced with the oligonucleotide primers pGEX5 (SEQ ID NO: 116) and pGEX3 (SEQ ID NO: 117).
- Frozen cells obtained from Example B were resuspended in PBS with 1 mM PMSF.
- the cells were disrupted by ultrasonication (Branson). Inclusion bodies were separated from soluble proteins by centrifugation. Pelletized inclusion bodies were washed sequentially in (1) PBS; and (2) water. The washed inclusion bodies were resuspended in a solution of PBS and 5 M urea with brief sonication. Once again, the centrifugally pelleted inclusion bodies were fully solubilized in 7M guanidine-HCl. The solubilized recombinant antigens were clarified by centrifugation, and passed through a 0.2 um filter.
- Guanidine-HCl solubilized fusion protein was denatured by diluting in water and the denatured protein was precipitated by centrifugation. The pellet was washed with water and suspended in water. 2M NaOH solution was added to solubilize the denatured protein completely and then was added to neutralize the pH of protein solution.
- FMDV 2C protein was retrieved from NCBI GenBank data (GI: 5921457, O strain Chu-Pei) and oligonucleotides for the synthesis of whole 2C gene and sequencing were synthesized at ResGen (Huntsville, Ala.).
- the coding DNA sequence is 954 nucleotides long, which encodes 318 amino acids (SEQ ID NO: 119).
- the gene construction strategy involved synthesis of a series of overlapping oligonucleotides with complementary ends. When annealed, the ends served as primers for the extension of the complementary strand. The fragments then were amplified by excessive outside primers. Because of the large size of 2C gene to be synthesized, the oligonucleotides were divided into three groups and respective recursive PCRs were performed. The produced DNAs were designated as A, B and C fragment. B and C fragment were joined with PCR and then the B-C fragment was joined with A fragment to produce full 2C gene.
- One of the oligonucleotides was designed to contain a BamHI restriction site for cloning into the expression vector pGEX-4T-1.
- the reaction was incubated at 95° C. for 5 minutes, and then amplified with 35 cycles of 95° C. for 30 seconds, 53° C. for 30 seconds and 73° C. for 100 seconds, followed by incubation at 73° C. for 5 minutes. Aliquot of the reaction mixture was analyzed by electrophoresis on agarose mini-gel.
- the PCR product amplified as described herein above was digested with the restriction endonucleases Bam HI+Hind III and ligated into the vector pGEX-4T-1 that had been digested with Bam HI+Hind III previously.
- the ligation product was used to transform E. coli XL-1 Blue competent cells.
- the transformed cells were plated on LB plates supplemented with 100 ug/ml ampicillin.
- Miniprep DNAs were prepared from overnight cultures of transformed colonies using QIAprep plasmid DNA mini-preparation kit and digested with Bam HI+Hind III to screen the desired clones.
- the clone with right insert was designated as pGEX-2CTw97F ( FIG. 7 ).
- the pGEX-2CTw97F clone was sequenced with the oligonucleotide primers pGEX5 (SEQ ID NO: 116), pGEX3 (SEQ ID NO: 117), 2C-25 (SEQ ID NO: 41) and 2C-26 (SEQ ID NO: 42).
- Overnight seed cultures of pGEX-2CTw97F were prepared in 500 ml sterile LB supplemented with 100 ug/ml ampicillin, and placed in a shaking orbital incubator at 37° C. 50 ml inoculum from seed cultures was transferred to flask containing 0.5 liter sterile LB supplemented with 100 ug/ml ampicillin. Cultures were incubated at 37° C. until it reached mid-logarithmic growth and then induced with 1 mM ITPG (isopropylthiogalactoside) for 3 hours at 37° C. After the induction period, cells were pelleted by centrifugation and harvested following standard procedures. Pelleted cells were stored at ⁇ 70° C. until further process.
- ITPG isopropylthiogalactoside
- Frozen cells obtained from Example 2B were resuspended in PBS with 1 mM PMSF and Triton X-100 detergent, and then disrupted by ultrasonication (Branson). Inclusion bodies were separated from soluble proteins by centrifugation. Protein fraction enriched with 2C was obtained through 3-4 rounds of washing off the contaminants and solubilization of cell lysate pellet in urea or Guanidin-HCl.
- Recombinant 2C was purified through size exclusion chromatography (FPLC, Sephacryl S 200 HR) under denaturing condition (5N GuHCl, PBS (pH7.4)) and eluted fraction containing 2C was identified by SDS-PAGE and dialyzed against 20 mM phosphate buffer (pH 9.0). Protein solution was stored refrigerated after adding sodium azide to 0.05%. For longer storage (over 1 month), protein solution was aliquoted and frozen at ⁇ 70° C.
- FPLC size exclusion chromatography
- FMDV 3ABC protein was retrieved from NCBI GenBank data (GI: 5921457, O strain Chu-Pei) and oligonucleotides for the synthesis of whole 3ABC gene and sequencing were synthesized at ResGen (Huntsville, Ala.).
- the coding DNA sequence is 1281 nucleotides long, which encodes 427 amino acids (SEQ ID NO: 120).
- the gene construction strategy involved synthesis of a series of overlapping oligonucleotides with complementary ends. When annealed, the ends served as primers for the extension of the complementary strand. The fragments then were amplified by excessive outside primers.
- the oligonucleotides were divided into four groups and respective recursive PCRs were performed.
- the produced DNAs were designated as A, B, C and D fragment.
- a and B fragment were joined and C and D fragment were joined through PCR.
- A-B fragment was joined with C-D fragment to produce full 3ABC gene.
- One of the oligonucleotide was designed to contain a BamHI restriction site for cloning into the expression vector pGEX-4T-1.
- the anti-sense oligonucleotide contains a translational termination codon (TAA) and an EcoRI restriction site.
- TAA translational termination codon
- EcoRI EcoRI restriction site
- Vent DNA polymerase (1U) and 1 ⁇ buffer, along with 25 uM of each dNTP (dATP, dCTP, dGTP, and dTTP), 4 ul 100 mM MgSO 4 and 100 pmol of each oligonucleotide.
- the template was mixture of A-B fragment and C-D fragment described above.
- the reaction was incubated at 95° C. for 5 minutes, and then amplified with 35 cycles of 95° C. for 30 seconds, 60° C. for 30 seconds and 73° C. for 120 seconds, followed by incubation at 73° C. for 5 minutes.
- PCR-derived product was run on the agarose gel and the DNA band was excised and eluted from the gel using Quigen gel extraction kit.
- the PCR product amplified as described herein above was digested with the restriction endonucleases Bam HI+Hind III and ligated into the vector pGEX-4T-1 that had been digested with Bam HI+Hind III previously.
- the ligation product was used to transform E. coli XL-1 Blue competent cells.
- the transformed cells were plated on LB plates supplemented with 100 ug/ml ampicillin.
- Miniprep DNAs were prepared from overnight cultures of transformed colonies using QIAprep plasmid DNA mini-preparation kit and digested with Bam HI+Hind III to screen the desired clones.
- the clone with right insert was designated as pBM-3ABCTw97F ( FIG. 8 ).
- the pBM-3ABCTw97F clone was sequenced with the oligonucleotide primers pGEX5 (SEQ ID NO: 116), pGEX3 (SEQ ID NO: 117), 3ABC-36 (SEQ ID NO: 78) and 3ABC-37 (SEQ ID NO: 79).
- Overnight seed cultures of pGEX-3ABCTw97F were prepared in 500 ml sterile LB supplemented with 100 ug/ml ampicillin, and placed in a shaking orbital incubator at 37° C. 50 ml inoculum from seed cultures was transferred to flask containing 0.5 liter sterile LB supplemented with 100 ug/ml ampicillin. Cultures were incubated at 37° C. until it reached mid-logarithmic growth and then induced with 1 mM ITPG (isopropylthiogalactoside) for 3 hours at 37° C. After the induction period, cells were pelleted by centrifugation and harvested following standard procedures. Pelleted cells were stored at ⁇ 70° C. until further process.
- ITPG isopropylthiogalactoside
- Frozen cells obtained from Example 3B were resuspended in PBS with 1 mM PMSF and Triton X-100 detergent and disrupted by ultrasonication (Branson). Inclusion bodies were separated from soluble proteins by centrifugation. Protein fraction enriched with 3ABC was obtained through 3-4 rounds of washing off the contaminants and solubilization of cell lysate pellet in urea. Recombinant 3ABC was run through ion-exchange chromatography (FPLC, Q-Sepharose FF) under denaturing condition (8M urea, 10 mM DTT, 20 mM potassium phosphate, pH 7.0) and eluted by NaCl gradient.
- FPLC ion-exchange chromatography
- the eluted fraction was dialyzed against 20 mM phosphate buffer (pH 9.0). After measuring the protein concentration by Bradford method and adding sodium azide to 0.05%, protein solution was stored refrigerated. For longer storage (over 1 month), protein solution was aliquoted and frozen at ⁇ 70° C.
- oligonucleotides were synthesized, each with complementary ends, at Resgen.
- the gene construction strategy involved synthesis of a series of overlapping oligonucleotides with complementary ends. When annealed, the ends served as primers for the extension of the complementary strand. The fragments then were amplified by excessive outside primers.
- the oligonucleotides were divided into three groups and recursive PCRs were performed. The produced DNAs were designated as A, B and C fragment. B and C fragments were joined with PCR and then the B-C fragment was joined with A fragment to produce full 3D gene.
- Oligonucleotide was designed to contain a BamHI restriction site for cloning into the expression vector pGEX-4T-1.
- the anti-sense oligonucleotide contains a translational termination codons (TAA) and an EcoRI restriction site.
- TAA translational termination codons
- 3d-1A SEQ ID NO: 80
- 3d-36A SEQ ID NO: 115
- Vent DNA polymerase (1U) and 1 ⁇ buffer, along with 25 uM of each dNTP (dATP, dCTP, dGTP, and dTTP), 4 ul 100 mM MgSO 4 , 100 pmol each of oligonucleotides 3d-1A (SEQ ID NO: 80) and 3d-14 (SEQ ID NO: 93).
- the template was mixture of 0.83 pmol of each oligonucleotides 3d-1A to 3d-14.
- the reaction was incubated at 95° C. for 5 minutes, and then amplified with 35 cycles of 95° C. for 30 seconds, 53° C. for 30 seconds and 73° C. for 100 seconds, followed by incubation at 73° C. for 5 minutes. Aliquot of the reaction mixture was analyzed by electrophoresis on agarose mini-gel.
- Vent DNA polymerase (1U) and 1 ⁇ buffer, along with 25 uM of each dNTP (dATP, dCTP, dGTP, and dTTP), 4 ul 100 mM MgSO 4 , 100 pmol each of oligonucleotides 3d-13 (SEQ ID NO: 92) and 3d-24 (SEQ ID NO: 103).
- the template was mixture of 0.83 pmol of each oligonucleotides 3d-13 to 3d-24.
- the reaction was incubated at 95° C. for 5 minutes, and then amplified with 35 cycles of 95° C. for 30 seconds, 55° C. for 30 seconds and 72° C. for 90 seconds, followed by incubation at 72° C. for 5 minutes. Aliquot of the reaction mixture was analyzed by electrophoresis on agarose mini-gel.
- Vent DNA polymerase (1U) and 1 ⁇ buffer, along with 25 uM of each dNTP (dATP, dCTP, dGTP, and dTTP), 4 ul 100 mM MgSO 4 , 100 pmol each of oligonucleotides 3d-25 (SEQ ID NO: 104) and 3d-36A (SEQ ID NO: 115).
- the template was mixture of 0.83 pmol of each oligonucleotides 3d-25 to 3d-36A.
- the reaction was incubated at 95° C. for 5 minutes, and then amplified with 35 cycles of 95° C. for 30 seconds, 53° C. for 30 seconds and 73° C. for 100 seconds, followed by incubation at 73° C. for 5 minutes. Aliquot of the reaction mixture was analyzed by electrophoresis on agarose mini-gel.
- Vent DNA polymerase (1U) and 1 ⁇ buffer, along with 25 uM of each dNTP (dATP, dCTP, dGTP, and dTTP), 4 ul 100 mM MgSO 4 , 100 pmol each of oligonucleotides 3d-13 (SEQ ID NO: 92) and 3d-36A (SEQ ID NO: 115).
- the template was mixture of B and C fragments described above.
- the reaction was incubated at 95° C. for 5 minutes, and then amplified with 35 cycles of 95° C. for 30 seconds, 55° C. for 30 seconds and 73° C. for 90 seconds, followed by incubation at 73° C. for 5 minutes. Aliquot of the reaction mixture was analyzed by electrophoresis on agarose mini-gel.
- Vent DNA polymerase (1U) and 1 ⁇ buffer, along with 25 uM of each dNTP (dATP, dCTP, dGTP, and dTTP), 4 ul 100 mM MgSO 4 , 100 pmol each of oligonucleotides 3d-1A (SEQ ID NO: 80) and 3d-36A (SEQ ID NO: 115).
- the template was mixture of A, B and C fragments described above.
- the reaction was incubated at 95° C. for 5 minutes, and then amplified with 35 cycles of 95° C. for 30 seconds, 60° C. for 30 seconds and 73° C. for 120 seconds, followed by incubation at 73° C. for 5 minutes.
- PCR-derived product was run on the agarose gel and the DNA band was cut from the gel and then the DNA was eluted using Quigen gel extraction kit.
- the PCR product amplified as described hereinabove was digested with the restriction endonucleases Bam HI+Eco RI and ligated into the vector pGEX-4T-1 that had been digested with Bam HI+Eco RI and gel-isolated.
- the ligation product was used to transform XL-1 Blue competent cells.
- the transformed cells were plated on LB plates supplemented with 100 ug/ml ampicillin.
- Miniprep DNAs were prepared from overnight cultures of colonies and digested with Bam HI+Eco RI to screen the desired clones.
- the clone with right insert was designated as pGEX-3Df ( FIG. 9 ).
- pGEX-3Df plasmid was transformed into E. coli BL21(DE3) and transformants were spreaded on LB-agar plate supplemented with 100 ug/ml ampicillin.
- Overnight seed cultures of pGEX-3Df clone were prepared in 500 ml sterile LB supplemented with 100 ug/ml ampicillin, and placed in a shaking orbital incubator at 37° C. 50 ml inoculums from seed cultures were transferred to flasks containing 0.5 liter sterile LB supplemented with 100 ug/ml ampicillin. Cultures were incubated at 37° C. until the cultures reached mid-logarithmic growth and then induced with 1 mM ITPG (isopropylthiogalactoside) for 3 hours at 37° C. After the induction period, cells were pelleted by centrifugation and harvested following standard procedures. Pelleted cells were stored at ⁇ 70° C. until further processed.
- ITPG isopropylthiogalactoside
- Frozen cells obtained from Example were resuspended in PBS with 1 mM PMSF.
- the cells were lysed by sonication (Branson, model S-125). Soluble crude lysate was prepared by centrifugation of the cell-lysate (10,000 rpm, 30 min) and filtered with 0.45 um syringe filter (Sartorius).
- Glutathione affinity chromatography was carried out to purify rGST-3D protein, Soluble cell lysate was loaded onto glutathione sepharose 4B (Pharmacia) column equilibrated with PBS. After washing the column with three bed volume of PBS, GST-3D was eluted with 10 mM reduced glutathione, 50 mM Tris-HCl, pH 8.0 buffer solution. The elution fractions were analyzed on the 8% SDS-PAGE. The fractions which contained the fusion protein were dialyzed in PBS overnight.
- Recombinant Protein G engineered to eliminate non-specific-binding with serum albumin was purchased from Sigma and was made to a concentration of 1 mg/ml. Protein G was added dropwise to gold solution while stirring to make a final concentration of 10 ⁇ g/ml and the solution was kept stirring for 15 min. Then 15% BSA solution was added to gold particle suspension used. After stirring for another 15 min, coupled gold solution was centrifuged and supernatant was discarded in order to remove unbound Protein G. To the coupled gold solution, 2% BSA was added and sonicated in sonic bath (Branson model #2200 or equivalent) in order to resuspend the pellet. The suspension was centrifuged again and the final pellet was suspended in 2% BSA and stored in refrigerator.
- Biotinylated BSA purchased from Pierce was used for gold coupling. The conjugation procedures were basically the same as described above as for Protein G. 10 ⁇ g of biotinylated BSA per every ml of gold particle suspension was added to gold solution with vigorous stirring. At the end of the coupling reaction, 15% BSA solution was added per ml of gold particle suspension. After stirring for another 15 min, Biotin-BSA coupled gold conjugate suspension was centrifuged to discard supernatant to remove unbound Biotin-BSA. To the pellet of coupled gold solution, 2% BSA (10 mM Sodium phosphate, pH 7.5) was added and suspension was centrifuged a gain to wash. The pellet was resuspended in 2% BSA and stored in refrigerator.
- Protein G coupled gold solution was diluted using dye dilution buffer (1% casein, 100 mM sodium phosphate, pH 7.0). Biotin-BSA coupled gold solution was added for generation of the control line which binds to avidin on the membrane. The diluted gold solution was spread onto the Lydall pad strip (microglass paper) and dried in lyophilizer. The Lydall pad was stored in low humidity room until use
- Cellulose filter paper was presoaked in pretreatment buffer (100 mM sodium phosphate, pH 7.0) and dried on a fan after blotting off excessive liquid.
- pretreatment buffer 100 mM sodium phosphate, pH 7.0
- the prepared reservoir pad was stored in a low humidity room.
- Absorbent pad was attached along the long axis of the plate after protective sheet from the tape at the top was peeled off.
- Filter pad was attached beneath test membrane area along the long axis of the plate after protective sheet from the tape at the bottom of the plate was peeled off.
- the dye pad should overlap the bottom of the test membrane.
- reservoir pad was attached to the plate to cover the bottom of filter pad.
- the dressed membrane plate was cut into a strip having a width so as to fit into housing.
- a total of 1540 identified cattle, swine, goat and sheep sera were used.
- a test serum consists of the negative animal prior to vaccination, the uninfected and vaccinated animal and the infected animal.
- 3ABC ELISA (Italy and USDA, USA) was used as a reference test, for each test cattle. Overall, relative sensitivity, specificity and overall accuracy were 98.6% ( 69/70), 98.6% ( 1449/1470) and 98.6% ( 1518/1540), respectively.
- FMDV foot-and-mouth disease virus
- the present invention is applicable to a variety of disease markers in organisms, such as cancer diagnostic markers, hormones, enzymes, drugs and various antigens in the test sample.
Abstract
Description
- The present invention relates to a method and kit for diagnosing an infection of the subject with foot-and-mouth disease virus (FMDV), and more particularly, a method and kit for rapidly detecting infection of the subjects with FMDV by observing a change in appearance of a reactivity zone containing at least more than one immobilized phase selected from antigens, antibodies or haptens derived from FMDV, or obtainable from FMDV via an immunological reaction, which is immunologically reactive with the test sample from animals as a target.
- Foot and mouth disease (FMD) is a devastating disease of livestock and an Office International des Epizooties list A disease. All species of cloven-hoofed animals (cattle, pigs, sheep and goats) are susceptible and the disease is extremely contagious. Financial losses as a result of FMD can be significant. There are direct losses due to deaths in young animals, loss of milk, loss of meat and a decrease in productive performance. The costs associated with eradication or control can be high and, in addition, there are indirect losses due to the imposition of trade restrictions.
- The causative agent is FMDV, anaphthovirus of the Picornaviridae family (Bittle et al., 1982 and Fross et al., 1984). The FMDV genome consists of a single RNA positive strand of approximately 8,000 nucleotide bases. The RNA is initially translated as a single polypeptide which is subsequently cleaved by viral-encoded proteases to produce four capsid proteins (VP1-VP4) and non-structural polypeptides (2C, 3A, 3ABC and 3D) in infected cells. The coding region for structural and nonstructural proteins is shown schematically in
FIG. 3 . - FMD virus is antigenically heterogeneous. Seven distinct serotypes have been recognized, O, A, C, ASIA1, SAT1, SAT2 and SAT3 (SAT=Southern African Territories). Each serotype of FMDV is antigenically distinct from the other six serotypes. Serotype A viruses are the most variable, having more than 30 subtypes. Furthermore, each serotype can be subdivided into antigenically distinct multiple subtypes. The serotypes of FMD virus were originally identified by cross-immunity experiments in animals. An animal recovered from infection with one serotype being resistant to challenge by the same serotype but remaining susceptible to infection by any other serotype. The different serotypes of FMDV have different geological distributions. In Asia, serotypes A, O, and Asia are most common. In Europe and South America, serotypes A, O, and C are found. In Africa, serotypes A, O, and SAT are prevalent. Some countries in Africa, Asia and South America are endemic area.
- Primary diagnosis of FMD commonly involves recognition of typical clinical signs in affected animals. Clinical signs of FMD are essentially similar in all species although the severity may vary considerably. The principal signs are pyrexia followed by vesicle formation in the mouth and feet resulting in salivation and lameness. Serological diagnosis is determined by the presence of FMDV-specific antigens or antibodies in the suspected animals and can be usually performed by ELISA and Virus neutralization test.
- After animals have been infected with FMDV, specific antibodies against structural proteins (SPs) and non-structural proteins (NSPs) begin to appear and titers increase and remain long. Thus, the presence of specific FMD virus antibody in a serum indicates that the animal from which the sample was collected has had contact with FMD virus or antigen.
- The detection of antibody to FMD virus in serum has several usefulness. The antibody detection evidences previous infection in animals from which vesicular material is not available. Diagnosis of FMD by clinical signs may be difficult, especially for sheep and goats, in which clinical signs are often mild (Barnett, P. V et al., 1999 and Callens, M., K. et al., 1998). Furthermore, several other vesicular virus infections, including those caused by swine vesicular disease (SVD) virus, vesicular stomatitis virus, and others, cannot be distinguished from FMDV infection by the clinical findings. FMDV can establish a persistent or carrier stage in ruminants and they show no signs of FMD. Such carrier animals can become the source of new outbreaks of the disease. Because of these problems, a rapid serological method is needed to identify infected and/or asymptomatic carrier animals and distinguish them from vaccinated animals. This antibody detection method also can be used in epidemiological surveys and to measure the effectiveness of vaccination.
- Both vaccination and infection induce antibodies to the structural capsid proteins. Therefore, if the capsid protein alone used in the diagnostic assays, it will detect both vaccinated and infected animal based on the detection of antibody to structural protein. For this reason, the antibody test to structural protein can be used only in vaccine-free region, such as the USA or the UK, but not in regions where vaccination practice is established. But even in areas where animals are vaccinated, the FMD occurs frequenctly. In such a region, diagnostic tests that can differentiate the infection from the vaccination are required. Several studies reported that using nonstructural proteins of FMDV such as 2C and 3ABC, animals which have been infected with FMDV could be differentiated from the vaccinated animals on the basis of the detection of antibody to one or more non-structural proteins of the virus (Rodriguez A et al, Mackey D k et al., Sorensen K J et al.).
- Another method to detect FMDV is PCR (polymerase chain reaction). To detect specific RNA sequence from FMDV, RT-PCR (reverse transcription-polymerase chain reaction) assay also has been developed (Munez et al). This technique can provide specific and highly sensitive, and so it can detect FMD viral RNA in poorly preserved samples when insufficient virus is present to initiate infection in tissue culture. But this method requires equipments for PCR and electricity, which make it impractical in field assay, and if inhibitory substances to PCR reaction are present in some samples, some samples that contain virus or viral genome will not give a positive result by PCR.
- Therefore, the present invention has been made in view of the above problems, and it is an object of the present invention to provide a method for diagnosing infection of the subject animal with foot-and-mouth disease virus, making it possible to simply and rapidly identify whether the animal is infected with that virus or not, upon using biological samples obtained from the animal.
- It is another object of the present invention to provide a diagnostic kit for realizing the above-mentioned method.
- In accordance with the present invention, the above and other objects can be accomplished by the provision of a method for diagnosing foot-and-mouth disease virus infection, comprising the steps of:
- (1) applying a predetermined amount of a test sample to a loading region of a strip; (2) coupling a detection reagent including a given labeling reagent to an analyte of interest in the test sample to form a complex therebetween; (3) developing the complex onto a wicking membrane; and (4) observing changes in appearance of a reactivity zone having at least more than one immobilized phase selected from antigen, antibody or hapten on the predetermined region of the wicking membrane, derived from FMDV or obtainable from FMDV through an immunological reaction to determine the presence or absence of foot-and-mouth disease virus infection.
- The diagnostic method according to the present invention includes a sandwich assay or a competition assay.
- As a diagnostic kit in order to realize the diagnostic method as described above, the present invention provides a kit for diagnosing foot-and-mouth disease virus infection comprising:
- a strip 1 including a
reactivity zone 13 containing at least more than one immobilized phase selected from antigen, antibody or hapten thereon, derived from FMDV or obtainable from FMDV through an immunological reaction, and acontrol zone 14 for confirming normal operation of the kit, provided on a predetermined region of awicking membrane 9; and - a
housing 20 protecting the strip 1 from a variety of contaminants, and including at least a testsample application port 2 and anindicia window 4 for observing results of reaction in thereactivity zone 13 and thecontrol zone 14 on the strip 1. - The test sample is preferably a body fluid which is secreted out of the body and includes blood, serum, plasma, urine, tears, saliva, milk, etc.
- Further, the analyte of interest which is contained in the test sample to be analyzed may include any substances containing specific-binding members which may be naturally formed or artificially imparted, including antigen-presenting substances, antibodies (including monoclonal and polyclonal antibody), haptens, and combinations thereof, for example.
- In addition, an immobilized phase (or, capture reagent) is an unlabeled specific bonding member which specifically binds to the analyte, an indicator reagent, an auxiliary specific-binding member, or the like and then captures the analyte, and is immobilized directly or indirectly on the
wicking membrane 9 of the strip 1. - The detection reagent may bind diffusibly or non-diffusibly to a pad and includes a labeled reagent, the auxiliary specific-binding member and/or a component of a signal generating system. The signal generating system includes at least a catalytic member and solute. The solute may be catalyzed by the catalytic member to induce a reaction, and generates a signal recognizable from membrane surface or inside. The catalytic member may be enzyme or non-enzyme. The solute may carry out a reaction which is catalyzed by the catalytic member. Such a reaction produces a large amount of signal-generating compound which may be directly or indirectly detectable. The signal detectable by these components includes spectrophotometric, visible signal, electrochemical signal, and other electrically detectable signals.
- Now, the present invention will be described in detail with reference to the accompanying drawings.
-
FIGS. 1 a-b are, respectively, separate perspective views of exemplified diagnostic kits as a preferred embodiment used in the present invention. - The diagnostic kit includes a strip 1 and a
housing 20. The housing is required for spotting a test sample from a testsample application port 2 and a developingreagent application port 3 on a filter pad (or a dye pad: it refers to a pad containing a detection reagent). The kit also includes amain body 7 having acover 5 comprising anindicia widow 4 for showing test results, and a strip-mountingmember 6 for placing and fixing the strip 1 in place therein. - The
cover 5 andmain body 7 are interconnected via afastening member 8. They are required for fixing the strip 1, and for preventing contact with a reactivity zone or contamination thereof, and are preferably made of a non-reactive material which does not react with any other reagents used in the test, such as plastics or the like. - As shown in a configuration of
FIG. 1 a in accordance with the first aspect of the present invention, where the test sample is spotted through theapplication port 2 on the filter pad 11 (also serving as a dye pad), a separate developingreagent application port 3 is required. The developingreagent application port 3 is configured to have a curvature in a cup-like shape so as to receive a predetermined amount of the developing reagent. Further, the lower part thereof is preferably in close contact with areservoir pad 10 so as to prevent the developing reagent from flowing out into other regions of the test device. - Differently from the configuration in
FIG. 1 a, where a testsample application port 2′ is positioned over an immediately upper part of areservoir pad 10′ constituting the strip so as to immediately apply the test sample to thereservoir pad 10′ (in this case, the reservoir pad also has a function of the filter pad as in following example below, thereby no separate developing reagent application port is required), as shown in a configuration ofFIG. 1 b in accordance with the second aspect of the present invention, theapplication port 2′ is configured to have a curvature in a cup-like shape so as to receive a predetermined amount of the test sample. Further, the lower part thereof is preferably in close contact with a reservoir pad so as to prevent the test sample from flowing out into other regions of the test device. In this case, apad 11′ is a dye pad containing the detection reagent, or a second filter pad, or may be eliminated. - An
indicia window 4 is designed for externally observing changes occurring in areactivity zone 13 and acontrol zone 14 on thewicking membrane 9 constituting the strip 1, and is provided on thehousing cover 5 so as to be positioned immediately over thereactivity zone 13 andcontrol zone 14. - Further, the
housing cover 5 may be provided with given discrimination symbols, for example ‘Date’ for test date, ‘It 0000’ for the subject, ‘C’ for the control zone, ‘T’ (Test) for the reactivity zone, ‘S’ (Sample) for the test sample application port, ‘D’ for the developing reagent, and the like, such that the test date, subject, test sample application port, developing reagent application port, indicia window for showing test result, etc. may be easily distinguished. Those symbols may be any letter, number, icon, or the like and any combinations thereof different from the foregoing. - Now, as the preferred embodiment, a structure of a strip constituting the diagnostic device in accordance with the present invention will be described with reference to
FIGS. 1 a-b andFIG. 2 . -
FIG. 1 a shows one embodiment in accordance with a first aspect of the present invention. The inventive strip 1 includes awicking membrane 9, areservoir pad 10, a filter pad 11 (also, serving as a dye pad), anabsorbent pad 12, and areactivity zone 13 and acontrol zone 14 provided on thewicking membrane 9. To the back surface of the strip 1 is attached a base member 15 for fixing the strip 1 on a mountingmember 6 of themain housing body 7. The base member 15 is preferably made of plastic or a glass plate. -
FIG. 1 b shows another embodiment in accordance with the second aspect of the present invention. The inventive strip 1′ has the same configuration as in the strip shown inFIG. 1 a, except for areservoir pad 10′, and afilter pad 11′ (which may also serve as a dye pad). The detailed description of respective configuration thereof will be based on the first aspect of the present invention shown inFIG. 1 a, but only the difference therebetween will be mentioned. - A
filter pad 11 is in contact with a back surface of awicking membrane 9 for chromatography to form a connection passage for fluid flow into thewicking membrane 9. The back surface of thefilter pad 11 is in contact with thereservoir pad 10 to form one connection passage for fluid flow therebetween. Theabsorbent pad 12 is attached to the upper part of thewicking membrane 9. On the predetermined region of thewicking membrane 9 are spaced apart areactivity zone 13 containing at least more than one immobilized phase which specifically binds to an analyte to be detected, a labeled reagent, an auxiliary specific-binding member, or the like, and acontrol zone 14 for determining whether the kit is normally operating. - The
reservoir pad 10 absorbs the test sample, or a solution necessary for other tests, for example, a developing reagent, and the like and includes a capillary membrane to transfer analyte to the filter pad or wicking membrane. Thereservoir pad 10 is required to have voids and volume sufficient to receive the test sample or developing reagent. Material suitable for the reservoir pad is preferably low molecular weight protein binding substances, including cellulose, polyester, polyurethane, glass fiber having a pore size of 0.45 to 60 μm, etc. - The
filter pad 11 filters unnecessary components in the test sample and may contain a detection reagent (in this case, the filter pad may also function as a dye pad). Where the detection reagent is contained in the filter pad, there is an advantage of eliminating the step of premixing the detection reagent with the test sample for the test. As material suitable for thefilter pad 11, there may be mentioned polyester, polyurethane, polyacetate, cellulose, glass fiber, nylon having a pore size of 0.45 to 60 μm, etc. Where appropriate, thereservoir pad 10 andfilter pad 11 may be made of the same material, and in this case, the detection reagent may be contained within the bottom of onelong filter pad 11. - The detection reagent is provided with a labeled reagent, auxiliary specific-binding member, and/or a constitutional component of a signal generating system, which enable it to identify the presence of analyte of interest by naked eye or other instrumentation from the outside. Labeled detection reagents are well known to those skilled in the art. Examples of such labels include catalysts, enzymes (for example, phosphatase, peroxydase, etc., and more specifically, alkaline phosphatase and horseradish peroxidase, or the like, which is used in combination with a substrate for an enzyme), substrate for enzyme (for example, nitrobluetetrazolium, 3,5′,5,5′ tetranitrobenzidine, 4-methoxy-1-naphthol, 4-chloro-1-naphthol, 5-bromo-4-chloro-3-indolylphosphate, chemoluminescent substrates for enzymes, for example, dioxethane, and derivatives and analogs thereof), fluorescent compounds (for example, fluorescein, phycobiliprotein, rhodamine, derivatives and analogs thereof), chemoluminescent compounds, radioactive elements, and the like. In addition to those, metal sol, dye sol, particulate latex, color indicator, color matter contained in liposome, carbon sol and non-metal sol such as selenium may be mentioned as disclosed in U.S. Pat. No. 5,728,587 as well. Further, this patent, from columns 8-10, discloses a large number of immunochemical labels as labels usable in the diagnostic method of the present invention.
- The above-mentioned labeling reagents may form a conjugate with a given auxiliary specific-binding member having a property of easily binding to an analyte of interest. An auxiliary specific-binding member is not particularly limited and includes antigen, antibody, hapten, or the like, for example, protein G, protein A, protein G/A, known as material binding well to an antibody in case the analyte is antibody and various antibodies known as binding well to other antibodies IgG and IgM. These materials are presently commercially available as recombinants from Sigma, etc.
- From the foregoing, the detection reagent needs not necessarily be included in the
filter pad 11. The detection reagent may be provided at any point between thereactivity zone 13 on the wicking membrane showing a detection result and the test sample application port. This detection reagent may be applied to upper or inside of any point of thefilter pad 11 orwicking membrane 9, in the dried or freeze-dried state. - Then, if desired, in order to enhance sensitivity of the test, reactivity, etc., a variety of auxiliary agents may be added, such as buffer, detergent, anti-coagulating stock solution, or the like, for example. In addition, the strip 1 may further include a given control reagent to determine whether the kit is normally operating or not. Similar to the detection reagent, the control reagent may also be provided at any point between the
filter pad 11 or thereactivity zone 13 on thewicking membrane 9 and the test sample application port. The control reagent may be selected from labeled protein, antigen, antibody, and the like which specifically bind to an immobilized phase (for example, protein, antigen, antibody, or the like) forming a control zone (or a control band) on thewick membrane 9. These immobilized phase and control reagent are well known to those skilled in the art. As the labeling reagent which may be included in the control reagent, those as described in the detection reagent may be applied. The auxiliary specific-binding member is not particularly limited and includes one species selected from avidin, biotin, FITC, anti-FITC mouse antibody, mouse immunoglobulin, or anti-mouse immunoglobulin antibody, for example. - The wicking
membrane 9 should have sufficient voids, and be able to absorb substantial portions of test sample which has passed through thefilter pad 11. As an example of material suitable for such a wicking membrane, there may be mentioned at least more than one material selected from nylon, polyester, cellulose, polysulfone, polyvinylidene difluoride, cellulose acetate, polyurethane, glass fiber, nitrocellulose, or the like. - Where a developing reagent is employed, an example of material suitable for the developing reagent may include phosphate buffer, saline, Tris-HCl, water, etc. The developing reagent is required where the test
sample application port 2 is positioned over the immediately upper part of thefilter pad 11 so as to spot the test sample. Therefore, as in the embodiment in accordance with the second aspect of theFIG. 1 b, the developing reagent is not particularly required where the testsample application port 2′ is positioned on thereservoir pad 10′ so as to load a predetermined amount of the test sample thereon. - Where the complex labeled with the detection reagent contains the analyte to be detected, it binds to the immobilized phase located on the
reactivity zone 13 of thewicking membrane 9 and then results in externally discernable change. Material which may be used as such an immobilized phase may include at least more than one selected from antigen, antibody or hapten, which constitutes foot-and-mouse disease virus, or may be derived therefrom through an immunological reaction. - Material which may be used as the antigen includes non-structural and/or structural proteins. Structural proteins include inactivated FMDV disrupted material or constituents thereof, VP1-VP4 polypeptide. Non-structural proteins may include at least more than one polypeptide selected from leader peptide (Lb), 2B, 2C, 3A, 3D, 3AB and 3ABC.
FIG. 3 shows a map of a polyprotein precursor comprising the structural and non-structural proteins. - The structural proteins with the same name may also exhibit some difference in constitutional amino acids among them, depending on serological classification of FMDV. Preferably, the structural proteins which may be used in the present invention are not particularly limited, so long as they may specifically react with antibodies formed against all the sero-types. An example of these structural proteins includes, but is not limited to, VP1 represented by SEQ ID NO: 118.
- However, even when the above-described structural protein alone is used as an immobilized phase for constructing the diagnostic kit, there is a case in which it is possible to precisely diagnose whether or not cattle are infected with foot-and-mouth disease virus, for non-vaccinated cattle, but it is difficult to distinguish infected cattle from vaccinated cattle. An example of antigen employed in the vaccine production includes, in addition to the structural protein, a
non-structural protein 3D. Thus, even when thenon-structural protein 3D is used as an immobilized phase, the same limitations as described above exist. An example of thenon-structural protein 3D is shown in SEQ ID NO: 121. - The non-structural proteins (except for 3D) are proteins that have not been used in conventional vaccine production and an antibody to those proteins is observable only in a virus-infected animal. Thus, when this protein is applied as an immobilized phase, it will be possible to make an exact diagnosis for the infected animal. Of course, to such a non-structural protein also has some difference in constituent amino acids thereof, depending on respective sero-type of FMDV. Usable non-structural proteins are not particularly limited, as long as preferably, they may specifically react with antibodies produced against all sero-types. Examples of these structural proteins include 2C represented by SEQ ID NO: 119 and 3ABC represented by SEQ ID NO: 120.
-
FIG. 4 shows an example to which the non-structural protein was applied as a single immobilized phase. T represents a reactivity zone in which the non-structural protein (2C or 3ABC) was immobilized, which has never been used in vaccine production up to now. C represents a control zone. The kit having both the discolored T and C (FIG. 4 a) represents the infected animal, whereas the kit having only the discolored C (FIG. 4 b) represents a negative animal prior to vaccination or a vaccinated animal. If C was not discolored in any case, it means that the kit of interest was not normally operated with the result thus obtained being unreliable. - Where only the non-structural protein as described above is employed as an immobilized phase, it is difficult to distinguish between the negative animal prior to vaccination and the vaccinated animal, in any case. Therefore, the most preferred embodiment of the present invention provides a diagnostic method and kit which make it possible to distinguish an FMDV-infected animal as well as a vaccinated animal. For this purpose, it is preferable to have a first reactivity zone in which as an immobilized phase, an antigen usable in vaccine production known until now was immobilized at a particular site on a wicking membrane, and a second reactivity zone in which as an immobilized phase, a non-structural protein that was known as never used before in vaccine production was spaced and immobilized at a particular site different from the first reactivity zone on the wicking membrane. Therefore, it is possible to diagnose whether animal was vaccinated, virus-infected or negative prior to vaccination, through change in appearance produced from a binding reaction between these first and second reactivity zones and a labeled complex.
- The immobilized phase bonding to a control reagent forms a control zone at a different site spaced from the reactivity zones. As such an immobilized phase, various reagents and immobilized phases which are applied in other commercially available diagnostic kits may be used. Details on that will be described in examples as follows.
- Now, a method for diagnosing FMDV infection using the kit having a configuration as described above will be described with reference to preferred embodiments. A test sample such as animal serum (or plasma, whole blood) is spotted on the test
sample application port 2 formed on the housing cover. Thefilter pad 10 constituting the strip is positioned at the lower end of theapplication port 2. Thefilter pad 10 also contains a protein G-gold conjugate as a detection reagent. The protein G-gold conjugate can form complexes with all the antibodies present in the test sample. A predetermined amount of a developing reagent is loaded on the developingreagent application port 3 in which thereservoir pad 10 constituting the strip is positioned on the lower end thereof. Application of the developing reagent results in formation of a complex between the labeled conjugate and an antibody in the test sample. Then, this complex is chromatographed along the longitudinal axis of the wicking membrane 9 (preferably, nitrocellulose membrane). FMDV recombinant antigen (construction thereof will be described in detail with reference to the following examples) was previously applied and immobilized on thereactivity zone 13 of thewicking membrane 9, and thus if the complex contains a specific antibody to the recombinant antigen, it will undergo reaction and then show discoloration in the form of a red line. - If the recombinant antigen includes both the structural and non-structural proteins, there is described for example, a method to diagnose whether the animal was vaccinated, virus-infected or negative prior to vaccination.
-
FIG. 5 shows this example. T1 represents a line on which an antigen (structural protein ornon-structural protein 3D) which had been introduced in vaccine production up to now was immobilized. T2 represents a line on which a non-structural protein (2C or 3ABC) which has never been used before in vaccine production was immobilized. C represents a control line. First, the kit (FIG. 5 a) in which all the T1, T2 and C were discolored represents a virus-infected animal. Secondly, a kit (FIG. 5 b) in which only the T1 and C were discolored represents a vaccinated animal. Finally, a kit (FIG. 5 c) means a negative animal prior to vaccination, as only the C was discolored. If C was not discolored in any case, it means that the kit was not normally operated with the result thus obtained being unreliable. - As described above, the diagnostic device usable in the present invention can be made of various configurations and modifications as disclosed in U.S. Pat. No. 5,728,587, and the particular disclosure of the strip construction (i.e., arrangement of the pads) included in this separate device does not constitute the essence of the present invention. Thus, the arrangement of the pad is not limited to those described above, and other arrangement such as a reservoir pad/a first filter pad/a second filter pad/a wicking membrane/an absorbent pad may be considered. In this case, the first filter pad or the second filter pad serves to filter and separate blood cells from blood, or filter and remove foreign materials unnecessary for sample test.
- Although the present invention was conveniently explained by way of example of structural and non-structural proteins constituting foot-and-mouth disease virus as an immobilized phase contained in the reactivity zone, the scope and sprit of the present invention should be construed to encompass any material which had been already supplied as antigen for vaccine production at the time of filing the present invention or would be supplied as antigen in the near future and a certain material capable of inducing an antibody in vivo (including hapten), or various antibodies obtainable from FMDV through an immunological reaction.
-
FIG. 1 a shows a separate perspective view of a device (a rapid kit) for diagnosing infection of the subject animal with foot-and-mouth disease virus according to a first aspect of the present invention. -
FIG. 1 b shows a separate perspective view of a device (a rapid kit) for diagnosing infection of the subject animal with foot-and-mouth disease virus according to a second aspect of the present invention; -
FIG. 2 shows a configuration of a strip constituting a diagnostic device according to the present invention; -
FIG. 3 shows a structure of a polyprotein expressed from foot-and-mouth disease virus; -
FIG. 4 shows an exemplified test result for the one-line test kit; -
FIG. 5 shows an exemplified test result for the two-line test kit; -
FIG. 6 shows a map of plasmid pBM-VPITw97F; -
FIG. 7 shows a map of plasmid pBM-2CTw97F; -
FIG. 8 shows a map of plasmid pBM-3ABCTw97F; and -
FIG. 9 shows a map of plasmid pBM-3DTw97F. - Now, the present invention will be described in more detail with reference to following examples. These examples are provided only for illustrating the present invention and should not be construed as limiting the scope and spirit of the present invention.
- Materials
- Oligonucleotides for gene construction and sequencing were synthesized at ResGen (Huntsville, Ala.). Unless otherwise indicated, DNA sequencing was also performed at ResGen.
- For polymerase chain reaction (PCR), Vent DNA polymerase and buffer were purchased from New England Biolabs, Inc. (Beverly, Mass.) and a mixture of dNTPs was purchased from Amersham-Pharmacia (Piscataway, N.J.) and used according to the manufacturer's specifications unless otherwise indicated. PCR amplifications were performed on a GeneAmp 2400 thermal cycler from Perkin-Elmer Corporation (Foster City, Calif.). The PCR product was purified using Qiagen PCR spin column (Qiagen Inc., Chatsworth, Calif.) as recommended by the manufacturer. Unless indicated otherwise, restriction enzymes were purchased from New England BioLabs, and DNA fragments were isolated on agarose (Sigma-Aldrich) gels, treated with restriction enzymes and then used for cloning.
- Desired fragment was excised and the DNA was extracted with a QIAEX II gel extraction kit as recommended by the manufacturer. DNA was resuspended in H2O or TE (1 mM ethylenediaminetetraacetic acid (EDTA; pH 8.0; Sigma-Aldrich), 10 mM tris(hydroxymethyl)aminomethane-hydrochloride (Tris-HCl; pH 8.0; Sigma-Aldrich)). Ligations were performed using DNA ligase (Boehringer Mannheim Corporation, Indianapolis, Ind.) as recommended by the manufacturer. Ligation reaction was incubated at 16° C. overnight. Bacterial transformations were performed using E. coli XL1-Blue competent cells. Unless indicated otherwise, transformations and bacterial restreaks were plated on LB agar (Lennox) plates supplemented with 100 ug/ml ampicillin. All bacterial incubations (plates and liquid cultures) were conducted overnight (16 hours) at 37° C.
- Screening of transformants to identify desired clones was accomplished by restriction enzyme digestion of miniprep DNA and/or by colony PCR. Miniprep DNA was prepared according to Molecular Cloning: A Laboratory Manual, unless otherwise indicated. Colonies containing desired clones were propagated from the transfer plate or stocked in glycerol at −70° C.
- Antigen Production
- Preparation of Recombinant FMDV VP1 Antigen
- A. Construction of FMDV VP1 Expression Vectors
- (i) Construction of Synthetic VP1 Gene
- VP1 protein of Foot and Mouth Disease virus Taiwan Type O 97 sequence was retrieved from NCBI GenBank data and oligonucleotides for syntheictc gene were synthesized at ResGen (Huntsville, Ala.). In the synthetic oligonucleotides, the native FMDV codons were altered to conform to E. coli codon bias in an effort to increase expression levels of the recombinant protein in E. coli. See, for example, M. Gouy and C. Gautier, Nucleic Acids Research 10:7055 (1982); H. Grosjean and W. Fiers, Gene 18:199 (1982); J. Watson et al. (eds.), Molecular Biology of the Gene, 4th Ed., Benjamin Kumming Publishing Co., pp. 440 (1987). The recursive PCR method was used to assemble the oligonucleotides into full VP1 gene. The gene construction strategy involved synthesis of a series of overlapping oligonucleotides with complementary ends. When annealed, the ends served as primers for the extension of the complementary strand. The fragments then were amplified by excessive outside primers.
- Oligonucleotide was designed to contain a BamHI restriction site for cloning into the expression vector pGEX-4T-1.
- Reverse oligonucleotide contains a translation stop codon (TAA) and EcoRI restriction site. When external primer TW97-1 (SEQ ID NO: 1) and TW97-16 (SEQ ID NO: 16) were used, whole VP1 (213 amino acids) gene was synthesized (SEQ ID NO: 118).
- These steps for recursive PCR are detailed hereinbelow.
- PCR reaction (100 ul volume) was set up as follows:
- Vent DNA polymerase (1U) and 1× buffer, along with 25 uM of each dNTP (dATP, dCTP, dGTP, and dTTP), 50 pmol each of oligonucleotides TW97-1 (SEQ ID NO: 1) and TW97-16 (SEQ ID NO: 1), and 0.25 pmol each of oligonucleotides TW97-2 (SEQ ID NO: 2) through TW97-15 (SEQ ID NO: 15).
- The reaction was incubated at 95° C. for 5 minutes, and then amplified with 30 cycles of 95° C. for 15 seconds, 58 CC for 15 seconds and 72° C. for 60 seconds, followed by incubation at 72° C. for 5 minutes. PCR-derived product was purified using Qiagen PCR spin column.
- (ii) Cloning of the PCR Product.
- The PCR product amplified as described hereinabove was digested with the restriction endonucleases Bam HI+Eco RI and ligated into the vector pGEX-4T-1 that had been digested with Bam HI+Eco RI and gel-isolated. The ligation product was used to transform XL-1 Blue competent cells. The transformed cells were plated on LB plates supplemented with 100 ug/ml ampicillin. Miniprep DNAs were prepared from overnight cultures of colonies and digested with Bam HI+Eco RI to screen the desired clones. The clone with right insert was designated as pBM-VPITw97F (
FIG. 6 ). - The pBM-VPITw97F clone was sequenced with the oligonucleotide primers pGEX5 (SEQ ID NO: 116) and pGEX3 (SEQ ID NO: 117).
- B. Growth and Induction of E. coli Strains with VP1 Plasmids
- Overnight seed cultures of each E. coli clones were prepared in 500 ml sterile LB supplemented with 100 ug/ml ampicillin, and placed in a shaking orbital incubator at 37° C.
- 50 ml inoculums from seed cultures were transferred
- to flasks containing 0.5 liter sterile LB supplemented with 100 ug/ml ampicillin. Cultures were incubated at 37° C. until the cultures reached mid-logarithmic growth and then induced with 1 mM ITPG (isopropylthiogalactoside) for 3 hours at 37° C. After the induction period, cells were pelleted by centrifugation and harvested following standard procedures. Pelleted cells were stored at −70° C. until further processed.
- C. Preparation of VP1 Antigen
- Frozen cells obtained from Example B were resuspended in PBS with 1 mM PMSF.
- The cells were disrupted by ultrasonication (Branson). Inclusion bodies were separated from soluble proteins by centrifugation. Pelletized inclusion bodies were washed sequentially in (1) PBS; and (2) water. The washed inclusion bodies were resuspended in a solution of PBS and 5 M urea with brief sonication. Once again, the centrifugally pelleted inclusion bodies were fully solubilized in 7M guanidine-HCl. The solubilized recombinant antigens were clarified by centrifugation, and passed through a 0.2 um filter.
- Guanidine-HCl solubilized fusion protein was denatured by diluting in water and the denatured protein was precipitated by centrifugation. The pellet was washed with water and suspended in water. 2M NaOH solution was added to solubilize the denatured protein completely and then was added to neutralize the pH of protein solution.
- Preparation of
Recombinant FMDV 2C Antigen - A. Construction of
FMDV 2C Expression Vector - The genome sequence of
FMDV 2C protein was retrieved from NCBI GenBank data (GI: 5921457, O strain Chu-Pei) and oligonucleotides for the synthesis of whole 2C gene and sequencing were synthesized at ResGen (Huntsville, Ala.). The coding DNA sequence is 954 nucleotides long, which encodes 318 amino acids (SEQ ID NO: 119). - (i) Construction of Synthetic Full-
Length 2C Gene - To obtain the 2C gene of FMD virus, 24 oligonucleotide primers were synthesized, each with complementary ends, at Resgen.
- We used the recursive PCR method to assemble the oligonucleotides into full 2C gene. The gene construction strategy involved synthesis of a series of overlapping oligonucleotides with complementary ends. When annealed, the ends served as primers for the extension of the complementary strand. The fragments then were amplified by excessive outside primers. Because of the large size of 2C gene to be synthesized, the oligonucleotides were divided into three groups and respective recursive PCRs were performed. The produced DNAs were designated as A, B and C fragment. B and C fragment were joined with PCR and then the B-C fragment was joined with A fragment to produce full 2C gene. One of the oligonucleotides was designed to contain a BamHI restriction site for cloning into the expression vector pGEX-4T-1.
- The reaction was incubated at 95° C. for 5 minutes, and then amplified with 35 cycles of 95° C. for 30 seconds, 53° C. for 30 seconds and 73° C. for 100 seconds, followed by incubation at 73° C. for 5 minutes. Aliquot of the reaction mixture was analyzed by electrophoresis on agarose mini-gel.
- (ii) Cloning of the PCR Product.
- The PCR product amplified as described herein above was digested with the restriction endonucleases Bam HI+Hind III and ligated into the vector pGEX-4T-1 that had been digested with Bam HI+Hind III previously. The ligation product was used to transform E. coli XL-1 Blue competent cells. The transformed cells were plated on LB plates supplemented with 100 ug/ml ampicillin. Miniprep DNAs were prepared from overnight cultures of transformed colonies using QIAprep plasmid DNA mini-preparation kit and digested with Bam HI+Hind III to screen the desired clones. The clone with right insert was designated as pGEX-2CTw97F (
FIG. 7 ). - The pGEX-2CTw97F clone was sequenced with the oligonucleotide primers pGEX5 (SEQ ID NO: 116), pGEX3 (SEQ ID NO: 117), 2C-25 (SEQ ID NO: 41) and 2C-26 (SEQ ID NO: 42).
- B. Growth and Induction of E. coli Strains with 2C Plasmid
- Overnight seed cultures of pGEX-2CTw97F were prepared in 500 ml sterile LB supplemented with 100 ug/ml ampicillin, and placed in a shaking orbital incubator at 37° C. 50 ml inoculum from seed cultures was transferred to flask containing 0.5 liter sterile LB supplemented with 100 ug/ml ampicillin. Cultures were incubated at 37° C. until it reached mid-logarithmic growth and then induced with 1 mM ITPG (isopropylthiogalactoside) for 3 hours at 37° C. After the induction period, cells were pelleted by centrifugation and harvested following standard procedures. Pelleted cells were stored at −70° C. until further process.
- C. Preparation of
FMDV 2C Antigen - Frozen cells obtained from Example 2B were resuspended in PBS with 1 mM PMSF and Triton X-100 detergent, and then disrupted by ultrasonication (Branson). Inclusion bodies were separated from soluble proteins by centrifugation. Protein fraction enriched with 2C was obtained through 3-4 rounds of washing off the contaminants and solubilization of cell lysate pellet in urea or Guanidin-HCl. Recombinant 2C was purified through size exclusion chromatography (FPLC, Sephacryl S 200 HR) under denaturing condition (5N GuHCl, PBS (pH7.4)) and eluted fraction containing 2C was identified by SDS-PAGE and dialyzed against 20 mM phosphate buffer (pH 9.0). Protein solution was stored refrigerated after adding sodium azide to 0.05%. For longer storage (over 1 month), protein solution was aliquoted and frozen at −70° C.
- Preparation of Recombinant FMDV 3ABC Antigen
- A. Construction of FMDV 3ABC Expression Vector
- The genome sequence of FMDV 3ABC protein was retrieved from NCBI GenBank data (GI: 5921457, O strain Chu-Pei) and oligonucleotides for the synthesis of whole 3ABC gene and sequencing were synthesized at ResGen (Huntsville, Ala.). The coding DNA sequence is 1281 nucleotides long, which encodes 427 amino acids (SEQ ID NO: 120).
- (i) Construction of Synthetic Full-Length 3ABC Genes
- To obtain the 3ABC gene of FMD virus, 33 oligonucleotide primers were synthesized, each with complementary ends, at Resgen.
- We used the recursive PCR method to assemble the oligonucleotides into full 3ABC gene. The gene construction strategy involved synthesis of a series of overlapping oligonucleotides with complementary ends. When annealed, the ends served as primers for the extension of the complementary strand. The fragments then were amplified by excessive outside primers.
- Because of the large size of 3ABC gene to be synthesized, the oligonucleotides were divided into four groups and respective recursive PCRs were performed. The produced DNAs were designated as A, B, C and D fragment. A and B fragment were joined and C and D fragment were joined through PCR. Then A-B fragment was joined with C-D fragment to produce full 3ABC gene.
- One of the oligonucleotide was designed to contain a BamHI restriction site for cloning into the expression vector pGEX-4T-1. The anti-sense oligonucleotide contains a translational termination codon (TAA) and an EcoRI restriction site. When N- and C-terminal primers, 3ABC-1 (SEQ ID NO: 43) and 3ABC-33 (SEQ ID NO: 75), were used, a full-length 3ABC (427 amino acids) gene was synthesized.
- PCR reaction (100 ul volume) was set up as follows:
- Vent DNA polymerase (1U) and 1× buffer, along with 25 uM of each dNTP (dATP, dCTP, dGTP, and dTTP), 4 ul 100 mM MgSO4 and 100 pmol of each oligonucleotide. The template was mixture of A-B fragment and C-D fragment described above.
- The reaction was incubated at 95° C. for 5 minutes, and then amplified with 35 cycles of 95° C. for 30 seconds, 60° C. for 30 seconds and 73° C. for 120 seconds, followed by incubation at 73° C. for 5 minutes. PCR-derived product was run on the agarose gel and the DNA band was excised and eluted from the gel using Quigen gel extraction kit.
- (ii) Cloning of the PCR Product.
- The PCR product amplified as described herein above was digested with the restriction endonucleases Bam HI+Hind III and ligated into the vector pGEX-4T-1 that had been digested with Bam HI+Hind III previously. The ligation product was used to transform E. coli XL-1 Blue competent cells. The transformed cells were plated on LB plates supplemented with 100 ug/ml ampicillin. Miniprep DNAs were prepared from overnight cultures of transformed colonies using QIAprep plasmid DNA mini-preparation kit and digested with Bam HI+Hind III to screen the desired clones. The clone with right insert was designated as pBM-3ABCTw97F (
FIG. 8 ). - The pBM-3ABCTw97F clone was sequenced with the oligonucleotide primers pGEX5 (SEQ ID NO: 116), pGEX3 (SEQ ID NO: 117), 3ABC-36 (SEQ ID NO: 78) and 3ABC-37 (SEQ ID NO: 79).
- B. Growth and Induction of E. coli Strains with E3ABC Plasmid.
- Overnight seed cultures of pGEX-3ABCTw97F were prepared in 500 ml sterile LB supplemented with 100 ug/ml ampicillin, and placed in a shaking orbital incubator at 37° C. 50 ml inoculum from seed cultures was transferred to flask containing 0.5 liter sterile LB supplemented with 100 ug/ml ampicillin. Cultures were incubated at 37° C. until it reached mid-logarithmic growth and then induced with 1 mM ITPG (isopropylthiogalactoside) for 3 hours at 37° C. After the induction period, cells were pelleted by centrifugation and harvested following standard procedures. Pelleted cells were stored at −70° C. until further process.
- C. Preparation of FMDV 3ABC Antigen
- Frozen cells obtained from Example 3B were resuspended in PBS with 1 mM PMSF and Triton X-100 detergent and disrupted by ultrasonication (Branson). Inclusion bodies were separated from soluble proteins by centrifugation. Protein fraction enriched with 3ABC was obtained through 3-4 rounds of washing off the contaminants and solubilization of cell lysate pellet in urea. Recombinant 3ABC was run through ion-exchange chromatography (FPLC, Q-Sepharose FF) under denaturing condition (8M urea, 10 mM DTT, 20 mM potassium phosphate, pH 7.0) and eluted by NaCl gradient. The eluted fraction was dialyzed against 20 mM phosphate buffer (pH 9.0). After measuring the protein concentration by Bradford method and adding sodium azide to 0.05%, protein solution was stored refrigerated. For longer storage (over 1 month), protein solution was aliquoted and frozen at −70° C.
- Preparation of
Recombinant FMDV 3D Antigen - A. Construction of
FMDV 3D Expression Vector - (i) Construction of Synthetic Full-
Length 3D Genes - To obtain the 3D gene of FMD virus, 36 oligonucleotides were synthesized, each with complementary ends, at Resgen. We used the recursive PCR method to assemble the oligonucleotides into full 3D gene (SEQ ID NO: 121). The gene construction strategy involved synthesis of a series of overlapping oligonucleotides with complementary ends. When annealed, the ends served as primers for the extension of the complementary strand. The fragments then were amplified by excessive outside primers.
- Because of the large size of 3D gene to be synthesized, the oligonucleotides were divided into three groups and recursive PCRs were performed. The produced DNAs were designated as A, B and C fragment. B and C fragments were joined with PCR and then the B-C fragment was joined with A fragment to produce full 3D gene.
- Oligonucleotide was designed to contain a BamHI restriction site for cloning into the expression vector pGEX-4T-1.
- The anti-sense oligonucleotide contains a translational termination codons (TAA) and an EcoRI restriction site. When N- and C-terminal primers, 3d-1A (SEQ ID NO: 80) and 3d-36A (SEQ ID NO: 115), were used, a full-
length 3D (470 amino acids) gene was synthesized. - These steps are detailed herein below.
- 1. 3DA Fragment PCR
- PCR reaction (100 ul volume) was set up as follows:
- Vent DNA polymerase (1U) and 1× buffer, along with 25 uM of each dNTP (dATP, dCTP, dGTP, and dTTP), 4 ul 100 mM MgSO4, 100 pmol each of oligonucleotides 3d-1A (SEQ ID NO: 80) and 3d-14 (SEQ ID NO: 93). The template was mixture of 0.83 pmol of each oligonucleotides 3d-1A to 3d-14.
- The reaction was incubated at 95° C. for 5 minutes, and then amplified with 35 cycles of 95° C. for 30 seconds, 53° C. for 30 seconds and 73° C. for 100 seconds, followed by incubation at 73° C. for 5 minutes. Aliquot of the reaction mixture was analyzed by electrophoresis on agarose mini-gel.
- 2. 3 DB Fragment PCR
- PCR reaction (100 ul volume) was set up as follows:
- Vent DNA polymerase (1U) and 1× buffer, along with 25 uM of each dNTP (dATP, dCTP, dGTP, and dTTP), 4 ul 100 mM MgSO4, 100 pmol each of oligonucleotides 3d-13 (SEQ ID NO: 92) and 3d-24 (SEQ ID NO: 103). The template was mixture of 0.83 pmol of each oligonucleotides 3d-13 to 3d-24.
- The reaction was incubated at 95° C. for 5 minutes, and then amplified with 35 cycles of 95° C. for 30 seconds, 55° C. for 30 seconds and 72° C. for 90 seconds, followed by incubation at 72° C. for 5 minutes. Aliquot of the reaction mixture was analyzed by electrophoresis on agarose mini-gel.
- 3. 3DC Fragment PCR
- PCR reaction (100 ul volume) was set up as follows:
- Vent DNA polymerase (1U) and 1× buffer, along with 25 uM of each dNTP (dATP, dCTP, dGTP, and dTTP), 4 ul 100 mM MgSO4, 100 pmol each of oligonucleotides 3d-25 (SEQ ID NO: 104) and 3d-36A (SEQ ID NO: 115). The template was mixture of 0.83 pmol of each oligonucleotides 3d-25 to 3d-36A.
- The reaction was incubated at 95° C. for 5 minutes, and then amplified with 35 cycles of 95° C. for 30 seconds, 53° C. for 30 seconds and 73° C. for 100 seconds, followed by incubation at 73° C. for 5 minutes. Aliquot of the reaction mixture was analyzed by electrophoresis on agarose mini-gel.
- 4. 3DB-C Fragment PCR
- PCR reaction (100 ul volume) was set up as follows:
- Vent DNA polymerase (1U) and 1× buffer, along with 25 uM of each dNTP (dATP, dCTP, dGTP, and dTTP), 4 ul 100 mM MgSO4, 100 pmol each of oligonucleotides 3d-13 (SEQ ID NO: 92) and 3d-36A (SEQ ID NO: 115). The template was mixture of B and C fragments described above.
- The reaction was incubated at 95° C. for 5 minutes, and then amplified with 35 cycles of 95° C. for 30 seconds, 55° C. for 30 seconds and 73° C. for 90 seconds, followed by incubation at 73° C. for 5 minutes. Aliquot of the reaction mixture was analyzed by electrophoresis on agarose mini-gel.
- 5. Full-
Length 3D (ABC) PCR - PCR reaction (100 ul volume) was set up as follows:
- Vent DNA polymerase (1U) and 1× buffer, along with 25 uM of each dNTP (dATP, dCTP, dGTP, and dTTP), 4 ul 100 mM MgSO4, 100 pmol each of oligonucleotides 3d-1A (SEQ ID NO: 80) and 3d-36A (SEQ ID NO: 115). The template was mixture of A, B and C fragments described above.
- The reaction was incubated at 95° C. for 5 minutes, and then amplified with 35 cycles of 95° C. for 30 seconds, 60° C. for 30 seconds and 73° C. for 120 seconds, followed by incubation at 73° C. for 5 minutes. PCR-derived product was run on the agarose gel and the DNA band was cut from the gel and then the DNA was eluted using Quigen gel extraction kit.
- (ii) Cloning of the PCR Product.
- The PCR product amplified as described hereinabove was digested with the restriction endonucleases Bam HI+Eco RI and ligated into the vector pGEX-4T-1 that had been digested with Bam HI+Eco RI and gel-isolated. The ligation product was used to transform XL-1 Blue competent cells. The transformed cells were plated on LB plates supplemented with 100 ug/ml ampicillin. Miniprep DNAs were prepared from overnight cultures of colonies and digested with Bam HI+Eco RI to screen the desired clones. The clone with right insert was designated as pGEX-3Df (
FIG. 9 ). - B. Growth and Induction of E. coli Strains with pGEX-3Df
- To expressed recombinant GST-3D protein, pGEX-3Df plasmid was transformed into E. coli BL21(DE3) and transformants were spreaded on LB-agar plate supplemented with 100 ug/ml ampicillin.
- Overnight seed cultures of pGEX-3Df clone were prepared in 500 ml sterile LB supplemented with 100 ug/ml ampicillin, and placed in a shaking orbital incubator at 37° C. 50 ml inoculums from seed cultures were transferred to flasks containing 0.5 liter sterile LB supplemented with 100 ug/ml ampicillin. Cultures were incubated at 37° C. until the cultures reached mid-logarithmic growth and then induced with 1 mM ITPG (isopropylthiogalactoside) for 3 hours at 37° C. After the induction period, cells were pelleted by centrifugation and harvested following standard procedures. Pelleted cells were stored at −70° C. until further processed.
- C. Preparation of GST-3D Protein
- Frozen cells obtained from Example were resuspended in PBS with 1 mM PMSF.
- The cells were lysed by sonication (Branson, model S-125). Soluble crude lysate was prepared by centrifugation of the cell-lysate (10,000 rpm, 30 min) and filtered with 0.45 um syringe filter (Sartorius).
- Glutathione affinity chromatography was carried out to purify rGST-3D protein, Soluble cell lysate was loaded onto glutathione sepharose 4B (Pharmacia) column equilibrated with PBS. After washing the column with three bed volume of PBS, GST-3D was eluted with 10 mM reduced glutathione, 50 mM Tris-HCl, pH 8.0 buffer solution. The elution fractions were analyzed on the 8% SDS-PAGE. The fractions which contained the fusion protein were dialyzed in PBS overnight.
- Kit Assay
- FMDV Antibody Detection Kit Formulation
- A. Preparation of Antigen Printed Membrane
- From the stock solution, recombinant 2C and 3ABC were adjusted and mixed to 0.5 mg/ml and filtered through 0.22 μm filter unit Millex-GV (Millipore). Avidin solution in PBS (pH 7.4) was used as internal control after filtered. The antigen mixture and control solution were applied to nitrocellulose membrane using Bio-Dot equipment (Bio-Dot). After dried in the low humidity room overnight, the membrane was blocked with 3% BSA in PBS for 20 min and then dried on a fan at least for 2 hours. The processed membrane plates must be stored in an enclosed container with desiccant or low humidity room.
- B. Preparation of Protein G-Gold Conjugate
- Recombinant Protein G engineered to eliminate non-specific-binding with serum albumin was purchased from Sigma and was made to a concentration of 1 mg/ml. Protein G was added dropwise to gold solution while stirring to make a final concentration of 10 μg/ml and the solution was kept stirring for 15 min. Then 15% BSA solution was added to gold particle suspension used. After stirring for another 15 min, coupled gold solution was centrifuged and supernatant was discarded in order to remove unbound Protein G. To the coupled gold solution, 2% BSA was added and sonicated in sonic bath (Branson model #2200 or equivalent) in order to resuspend the pellet. The suspension was centrifuged again and the final pellet was suspended in 2% BSA and stored in refrigerator.
- C. Preparation of Biotin-BSA-Gold Conjugate: Control Indicator
- Biotinylated BSA purchased from Pierce was used for gold coupling. The conjugation procedures were basically the same as described above as for Protein G. 10 μg of biotinylated BSA per every ml of gold particle suspension was added to gold solution with vigorous stirring. At the end of the coupling reaction, 15% BSA solution was added per ml of gold particle suspension. After stirring for another 15 min, Biotin-BSA coupled gold conjugate suspension was centrifuged to discard supernatant to remove unbound Biotin-BSA. To the pellet of coupled gold solution, 2% BSA (10 mM Sodium phosphate, pH 7.5) was added and suspension was centrifuged a gain to wash. The pellet was resuspended in 2% BSA and stored in refrigerator.
- D. Preparation of Filter Pad (Also Serving as Dye Pad)
- Protein G coupled gold solution was diluted using dye dilution buffer (1% casein, 100 mM sodium phosphate, pH 7.0). Biotin-BSA coupled gold solution was added for generation of the control line which binds to avidin on the membrane. The diluted gold solution was spread onto the Lydall pad strip (microglass paper) and dried in lyophilizer. The Lydall pad was stored in low humidity room until use
- E. Preparation of Reservoir Pad
- Cellulose filter paper was presoaked in pretreatment buffer (100 mM sodium phosphate, pH 7.0) and dried on a fan after blotting off excessive liquid. The prepared reservoir pad was stored in a low humidity room.
- F. Device Assembly
- Absorbent pad was attached along the long axis of the plate after protective sheet from the tape at the top was peeled off. Filter pad was attached beneath test membrane area along the long axis of the plate after protective sheet from the tape at the bottom of the plate was peeled off. The dye pad should overlap the bottom of the test membrane. Then reservoir pad was attached to the plate to cover the bottom of filter pad. The dressed membrane plate was cut into a strip having a width so as to fit into housing.
- Result
- A total of 1540 identified cattle, swine, goat and sheep sera were used. A test serum consists of the negative animal prior to vaccination, the uninfected and vaccinated animal and the infected animal. 3ABC ELISA (Italy and USDA, USA) was used as a reference test, for each test cattle. Overall, relative sensitivity, specificity and overall accuracy were 98.6% ( 69/70), 98.6% ( 1449/1470) and 98.6% ( 1518/1540), respectively.
- The Inventive Test vs. Reference Test
BioSign ™ FMDV Positive Negative Total Reference Infected(+) 69 1 70 Test Naïve (−) 11 1236 1247 Vaccinated(−) Single 4 149 153 Multi 6 64 70 Total 90 1450 1540 - In accordance with the present invention, it is possible to rapidly and accurately diagnose whether an animal is infected with foot-and-mouth disease virus (FMDV), based on test samples obtained from the animal. Further, where appropriate, it is also possible to distinguish between FMV vaccinated animals and an infected animal with only a small volume of test sample. Thus, it allows easy and rapid determination of whether FMV-susceptible animal was infected with FMV regardless of FMV vaccination.
- Although the preferred embodiments of the preset invention have been disclosed for illustrative purpose, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and sprit of the invention as disclosed in the accompanying claims. Therefore, embodiments of the present invention were illustrated by way of FMDV antigen only, but the scope and sprit of the present invention, of course, can also be applied to detection of FMDV antibody, PRRSV (Porcine Respiratory and Reproductive Symptom Virus) antigen and antibody, FeLV (Feline Leukemia Virus) antigen, FIV (Feline Immunodeficiency Virus) antibody, diagnostic marker for hydrophobia, CSF (Classical Swine Fever) antigen and antibody, B. canis (Brucellosis canis) antigen and antibody, Johnes antibody, BVDV (Bovine Viral Diarrhea Virus) antigen and antibody.
- Further, the present invention is applicable to a variety of disease markers in organisms, such as cancer diagnostic markers, hormones, enzymes, drugs and various antigens in the test sample.
Claims (28)
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PCT/KR2003/000896 WO2004097418A1 (en) | 2003-04-28 | 2003-05-06 | Method of diagnosis of foot and mouth disease and the diagnostic kit |
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US16/030,331 Active 2026-10-01 US11598775B2 (en) | 2003-04-28 | 2018-07-09 | Method and devices for rapid diagnosis of foot-and-mouth disease |
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CN1798976A (en) | 2006-07-05 |
KR20040095824A (en) | 2004-11-16 |
EP1618381A4 (en) | 2007-02-21 |
AU2003230254A8 (en) | 2004-11-23 |
US9459251B2 (en) | 2016-10-04 |
CN1798976B (en) | 2011-03-23 |
ATE514945T1 (en) | 2011-07-15 |
AU2003230254A1 (en) | 2004-11-23 |
EP1618381A1 (en) | 2006-01-25 |
KR100531760B1 (en) | 2005-11-29 |
US20060127885A1 (en) | 2006-06-15 |
WO2004097418A1 (en) | 2004-11-11 |
US7732145B2 (en) | 2010-06-08 |
BRPI0318312B1 (en) | 2017-01-17 |
BR0318312A (en) | 2006-07-11 |
US20170115290A1 (en) | 2017-04-27 |
CA2523939A1 (en) | 2004-11-11 |
EP1618381B1 (en) | 2011-06-29 |
US20190324039A1 (en) | 2019-10-24 |
US20110065088A1 (en) | 2011-03-17 |
US11598775B2 (en) | 2023-03-07 |
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