US20030153019A1 - Method and device for identifying a mycobacterium species responsible for a mycobacterial infection - Google Patents

Method and device for identifying a mycobacterium species responsible for a mycobacterial infection Download PDF

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US20030153019A1
US20030153019A1 US10/174,494 US17449402A US2003153019A1 US 20030153019 A1 US20030153019 A1 US 20030153019A1 US 17449402 A US17449402 A US 17449402A US 2003153019 A1 US2003153019 A1 US 2003153019A1
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antibody
imcrac
complexes
bound
substrate
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Pranab Das
Remco Van Es
Hendrik Houthoff
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RAPID MEDICAL DIAGNOSTICS Inc
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Kreatech Biotechnology BV
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Priority claimed from US08/454,122 external-priority patent/US5817473A/en
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Priority to US10/174,494 priority Critical patent/US20030153019A1/en
Assigned to KREATECH BIOTECHNOLOGY B.V. reassignment KREATECH BIOTECHNOLOGY B.V. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: VAN ES, REMCO MARIA, HOUTHOFF, HENDRICK JAN, DAS, PRANAB KHUMAR
Publication of US20030153019A1 publication Critical patent/US20030153019A1/en
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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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • 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
    • G01N33/56911—Bacteria
    • G01N33/5695—Mycobacteria
    • 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
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/35—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Mycobacteriaceae (F)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

Definitions

  • the invention relates to a method for identifying a Mycobacterium species responsible for a mycobacterial infection in human or animal.
  • the invention further relates to diagnostic kits for use in the method.
  • the genus Mycobacterium which contains about 50 species, is responsible for a number of human and animal diseases which are known collectively as the mycobacterioses.
  • the best known of these in humans are leprosy, caused by M. leprae, which affects more than ten million people worldwide, and tuberculosis, usually caused by M. tuberculosis, at least ten million new cases of which occur each year.
  • Most other mycobacteria normally occur only as environmental saprophytes but can also cause opportunist diseases. This happens usually, but not only, in the case of people who have problems with their immune system, such as AIDS patients and people undergoing immunosuppression.
  • These opportunist types comprise the slow-growing species M.
  • M. intracellulare and M. scrofulaceum (often referred to together as MAIS complex), M. kansasi, M marinum and M. ulcerans, and the fast-growing species M. chelonae and M. fortuitum.
  • mycobacteria or antigens thereof play a direct or indirect part in the etiology of a plurality of other diseases such as sarcoidosis and Crohn's disease and different auto-immune diseases such as auto-immune dermatitis, rheumatoid arthritis and diabetes. This could be attributed to a structural mimicry between epitopes of mycobacteria and those of the host.
  • the cell walls of mycobacteria are very complex and contain many lipids, some with structures unique to the genus. These structures comprise mycolinic acids and esters, peptido-glycolipde, arabino-galactane and lipo-arabino-manane.
  • the lipid-rich mycobacterial cell walls are responsible for the characterizing coloring properties of the mycobacteria. They also enable mycobacteria to counter an attack by the immune system of the host. A number of species, once taken up into macrophages, are capable of surrounding themselves with a thick layer of secreted lipids.
  • mycobacteria begins an interaction with the immune system.
  • these components comprise protein and hydrocarbon antigens, which can either be actively secreted by the mycobacteria or can form part of the cell wall or cell membrane.
  • they may be present in the cytoplasm, for instance in the cytoplasmic matrix, ribosomes and enzymes.
  • Mycobacteria also possess innuno-modulating components such as inmmunosuppressing compounds and adjuvants. Consequently, a single mycobacterial species can induce a large variety of immune responses in different forms and with diverse specificities. It is therefore difficult to distinguish immune responses against species-specific components from cross reactions.
  • Im-CRAC immuno-dominant mycobacterial cross-reactive antigen components
  • Im-CRAC mycobacterial immuno-cross-reactive antigen components
  • This discovery forms the basis of the present invention, whereby a method is provided for identifying a Mycobacterium species responsible for a mycobacterial infection in human or animal, comprising the steps of:
  • the antigen preparation is separated by electrophoresis prior to step (c) and the carrier is a membrane to which the antigen is bound by means of electroblotting. This process is called Western blotting.
  • the present invention is also a method for detecting or identifying a Mycobacterium species in a biological sample.
  • the method includes:
  • the present invention is also a method for detecting or identifying a Mycobacterium species in a biological sample.
  • the method includes:
  • the antigen preparation is separated by electrophoresis prior to step (d) and the carrier or substrate can be a membrane to which the lmCRACS are bound by means of electroblotting.
  • the membrane is a nitrocellulose membrane.
  • the antigen preparation of the standard Mycobacterium can be a total protein preparation. Additionally, the antigen preparation can be, but not limited to, a KP-100 or SP-100 fraction of the total protein preparation.
  • the carrier and substrate can be, but not limited to, a microtiter plate.
  • the ImCRACS of the antigen preparation is bound to the wells of a microtiter plate so that the carrier-bound ImCRACS is brought into contact with the biological sample under conditions that permit the antibodies in the biological sample to bind with the carrier-bound InCRACs to provide a pattern of carrier-bound antibody-ImCRAC complexes.
  • the non-binding antibodies can then be removed.
  • the pattern of the carrier-bound antibody-ImCRAC complexes and the substrate-bound antibody-ImCRAC complexes can be a banding pattern consisting of, but not limited to, 29/33 KDa, 45/48 KDa, 64 KDa and combinations thereof.
  • the carrier-bound antibody-ImCRAC complexes and the substrate-bound antibody-ImCRAC complexes can be made visualizable by any method known in the art.
  • the carrier-bound antibody-ImCRAC complexes can be made visualizable by using at least one antibody-enzyme conjugate directed against at least one antibody of an isotype selected from the group consisting of, but not limited to, IgG, IgM, IgA and combinations thereof.
  • the enzyme of the antibody-enzyme conjugate can be any enzyme known in the art which can make the carrier-bound antibody-ImCRAC complexes visualizable, preferably peroxidase.
  • the carrier and the substrate can be, but not limited to, a dip-stick.
  • the mycobacterial in the antigen preparation is brought into reaction with antibodies from a biological sample by dipping the stick in the biological sample for testing.
  • the carrier-bound antibody-ImCRAC complexes are made visible by subsequently dipping the stick in a solution with an antibody-enzyme conjugate and a color substrate for the relevant enzyme.
  • the present invention is also a diagnostic kit which includes (1) a test substrate having bound thereto an antigen preparation from a culture of the standard Mycobacterium, wherein the antigen preparation comprises at least two immuno-cross-reactive antigen components (ImCRACs) that are separable according to molecular weight, and wherein when the ImCRACs of the test substrate are contacted with a biological sample under conditions that permit antibodies in the biological sample to bind with the substrate-bound ImCRACs, a pattern of substrate-bound antibody-ImCRAC complexes is provided; and (2) a means for visualizing the pattern substrate-bound antibody-ImCRAC complexes.
  • ImCRACs immuno-cross-reactive antigen components
  • test substrate of the kit can be, but not limited to, a dip-stick, a microtiter plate, and an immunoblot.
  • FIG. 1 shows an example of Western blotting patterns which are developed after incubation respectively with representative negative and positive sera (positive for bovine tuberculosis).
  • FIG. 2 a shows an example of different Western blotting patterns developed after incubation with representative variable sera of tuberculous patients.
  • FIG. 2 b shows an example of different Western blotting patterns developed after incubation with representative sera of patients with Lepromatous Leprosy (LL) and Tuberculous Leprosy (TT).
  • FIG. 2 c shows an example of different Western blotting patterns developed after incubation with representative sera of patients with Crohn's Disease.
  • the Im-CRAC comprise namely a number of antigens with specific molecular weights which, as has now been found, after immunoblotting, exhibit a binding pattern which correlates to the disease or infection.
  • the specific band pattern is characterized by the presence or absence of four individual components, for instance:
  • region a region comprising different pronounced bands and/or overlapping bands, which can be observed as a smear (“region”);
  • extra bands are other positive bands.
  • Each of the mycobacterioses is characterized by a specific banding pattern which is formed when a blot having thereon an antigen preparation of mycobacteria separated to size is incubated with serum of an infected individual.
  • Tables 2a-2f below show a survey of the banding patterns of a number of mycobacterial and immunological diseases. TABLE 2a Bovine tuberculosis Regions (MW in KDa) and/or and/or and/or and Pattern 10-16 20-28 31-40 45-48 1. 14 KDa band 22 KDa band 31 KDa band 45/48 KDa 20-28 KDa doublet region
  • the method of the invention can be used to answer two questions.
  • the invention further relates to a heterogeneous enzyme immunoassay.
  • the antigens for a heterogeneous enzyme immunoassay is preferably chosen from the group which consists of mycobacterial immuno-cross-reactive antigen components with a molecular weight of 29/33 KDa, 45/48 KDa, 64/65 KDa and a fraction designated with the term KP-100.
  • These ImCRAC can be used separately or in combination with each other for serological diagnosis of the correlating diseases in a heterogeneous enzyme immunoassay (EIA).
  • microtiter plates (“Solid Phase”). By means of standard published techniques the antigens are irreversibly immobilized on the surface of the wells in such a microtiter plate.
  • binding antibodies are detected using an anti-antibody antibody labeled with an enzyme.
  • Binding of the enzyme is only possible when specific antibodies have adhered to the immobilized antigens. Substrate conversion by the binding enzyme to a visually or photometrically observable signal is thereby directly related to the presence of specific antibodies in the tested serum.
  • the choice of specificity of the enzyme-bound anti-antibody antibody determines the type of reaction that takes place. For instance, it may be desirable in some cases to demonstrate the specifically binding immunoglobulins of the IgG type, while in other cases immunoglobulins of the IgA and/or IgM type are just demonstrated.
  • the said methods that is, the immunoblot and the EIA, can be used as mutual confirmation.
  • test stick for the serological diagnosis based on the said antigens, use can be made of a test stick as solid phase.
  • a particularly advantageous embodiment of the invention relates to a test stick, the so-called “dip-stick”, which is used as solid phase in the heterogeneous enzyme immunoassay.
  • the said mycobacterial antigens can be irreversibly bound to such a dip-stick.
  • the antigen is brought into reaction with antibody from serum for testing by dipping the dip-stick in a serum sample for testing.
  • the formed antigen-antibody complex can be made visible by subsequently dipping the dip-stick in an anti-antibody antibody-enzyme conjugate solution.
  • a substrate With the binding enzyme a substrate can then be converted to a visually or photometrically observable signal.
  • the invention is a diagnostic kit for:
  • an immunoblot assay comprising IMCRAC antigens separated by electrophoresis as described above, immobilized on a solid carrier, in addition to an associated suitable detection system.
  • a heterogeneous enzyme immunological assay comprising a microtiter plate, the wells of which are coated with above mentioned antigens or antigen preparations, in addition to an associated suitable detection system.
  • a dip-stick assay comprising test sticks coated with antigen or antigen preparations, in addition to an associated suitable detection system.
  • the probe may be an antibody, preferably a monoclonal antibody.
  • the antibodies may be prepared as described above.
  • Assays for detecting the presence of proteins with antibodies have been previously described, and follow known formats, such as standard blot and ELISA formats. These formats are normally based on incubating an antibody with a sample suspected of containing the protein and detecting the presence of a complex between the antibody and the protein. The antibody is labeled either before, during, or after the incubation step.
  • the protein is preferably immobilized prior to detection. Immobilization may be accomplished by directly binding the protein to a solid surface, such as a microtiter well, or by binding the protein to immobilized antibodies. This and other immunoassays are described by David et al., U.S. Pat. No. 4,376,110 which entirety is incorporated herein by reference.
  • Immunoassays may involve one step or two steps.
  • the target molecule if it is present, is immobilized and incubated with a labeled antibody.
  • the labeled antibody binds to the immobilized target molecule. After washing to remove unbound molecules, the sample is assayed for the presence of the label.
  • immobilized target molecule is incubated with an unlabeled first antibody.
  • the target molecule-antibody complex if present, is then bound to a second, labeled antibody that is specific for the unlabeled antibody.
  • the sample is washed and assayed for the presence of the label, as described above.
  • the immunometric assays described above include simultaneous sandwich, forward sandwich, and reverse sandwich immunoassays. These terms are well known to those skilled in the art.
  • a sample is first incubated with a solid phase immunosorbent containing antibody against the protein. Incubation is continued for a period of time sufficient to allow the protein in the sample to bind to the immobilized antibody in the solid phase. After the first incubation, the solid phase immunoabsorbent is separated from the incubation mixture and washed to remove excess protein and other interfering substances which also may be present in the sample. Solid phase immunoabsorbent-containing protein bound to the immobilized antibodies is subsequently incubated for a second time with soluble labeled antibody cross-reactive with a different domain on the protein.
  • a reverse sandwich assay the sample is initially incubated with labeled antibody.
  • the solid phase immunoabsorbent containing immobilized antibody cross-reactive with a different domain on the protein is added to the labeled antibody, and a second incubation is carried out.
  • the initial washing step required by a forward sandwich assay is not required, although a wash is performed after the second incubation.
  • Reverse sandwich assays have been described, for example, in U.S. Pat. Nos. 4,098,876 and 4,376,110.
  • a simultaneous sandwich assay In a simultaneous sandwich assay, the sample, the immunoabsorbent with immobilized antibody, and labeled soluble antibody specific to a different domain are incubated simultaneously in one incubation step.
  • the simultaneous assay requires only a single incubation and does not require any washing steps.
  • the use of a simultaneous assay is a very useful technique, providing ease of handling, homogeneity, reproducibility, linearity of the assays, and high precision. See U.S. Pat. No.4,376,110 to David et al.
  • the sample containing antigen, solid phase immmunoabsorbent with immobilized antibody and labeled soluble antibody are incubated under conditions and for a period of time sufficient to allow antigen to bind to the immobilized antibodies and to the soluble antibodies.
  • the specific concentrations of labeled and immobilized antibodies, the temperature and time of incubation, as well as other such assay conditions can be varied, depending upon various factors including the concentration of antigen in the sample, the nature of the sample and the like. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.
  • solid phase immunoabsorbents there are many solid phase immunoabsorbents which have been employed and which can be used in the present invention.
  • Well known immunoabsorbents include beads formed from glass, polystyrene, polypropylene, dextran, nylon, and other material; and tubes formed from or coated with such materials, and the like.
  • the immobilized antibodies may be covalently or physically bound to the solid phase immunoabsorbent, by techniques such as covalent bonding via an amide or ester linkage or by absorption.
  • the proteins may be labeled and used as probes in standard immunoassays to detect antibodies against the proteins in samples, such as in the sera or other bodily fluids of patients being tested for.
  • a protein in accordance with the invention is incubated with the sample suspected of containing antibodies to the protein.
  • the protein is labeled either before, during, or after incubation.
  • the detection of labeled protein bound to an antibody in the sample indicates the presence of the antibody.
  • the antibody is preferably immobilized.
  • Suitable assays are known in the art, such as the standard ELISA protocol described by R H Kenneth, “Enzyme-linked antibody assay with cells attached to polyvinyl chloride plates” in Kenneth et al., Monoclonal Antibodies, Plenum Press, New York, page 376 (1981).
  • plates are coated with antigenic protein at a concentration sufficient to bind detectable amounts of the antibody.
  • a suitable blocking agent such as, for example, 10% normal goat serum.
  • the sample such as patient sera, is added and titered to determine the endpoint. Positive and negative controls are added simultaneously to quantitate the amount of relevant antibody present in the unknown samples.
  • the samples are probed with goat anti-human Ig conjugated to a suitable label, such as an enzyme. The presence of anti-protein antibodies in the sample is indicated by the presence of the label.
  • the probe may be the entire protein or may be functional analogs thereof.
  • Functional analogs of these proteins include fragments and substitution, addition and deletion mutations that do not destroy the ability of the proteins to bind to their antibodies. As long as the proteins are able to detect antibodies specific for the protein, they are useful in the present invention.
  • the probes described above can be detectably labeled in accordance with methods known in the art.
  • the probe can be modified by attachment of a detectable label moiety to the probe, or a detectable probe can be manufactured with a detectable label moiety incorporated therein.
  • the detectable label moiety can be any detectable moiety, many of which are known in the art, including radioactive atoms, electron dense atoms, enzymes, chromogens and colored compounds, fluorogens and fluorescent compounds, members of specific binding pairs, and the like.
  • the label moiety may be radioactive.
  • Some examples of useful radioactive labels include 32 P, 125 I, 131 I, and 3 H. Use of radioactive labels have been described in U.K. patent document No. 2,034,323, U.S. Pat. Nos. 4,358,535, and 4,302,204, each incorporated herein by reference.
  • non-radioactive labels include enzymes, chromogens, atoms and molecules detectable by electron microscopy, and metal ions detectable by their magnetic properties.
  • Some useful enzymatic labels include enzymes that cause a detectable change in a substrate.
  • Some useful enzymes include, for example, horseradish peroxidase (pyrogallol and o-phenylenediamine), beta-galactosidase (fluorescein beta-D-galactopyranoside), and alkaline phosphatase (5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium).
  • horseradish peroxidase pyrogallol and o-phenylenediamine
  • beta-galactosidase fluorescein beta-D-galactopyranoside
  • alkaline phosphatase 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium.
  • Useful reporter moieties include, for example, fluorescent, phosphorescent, chemiluminescent, and bioluminescent molecules, as well as dyes.
  • Some specific colored or fluorescent compounds useful in the present invention include, for example, fluoresceins, coumarins, rhodamines, Texas red, phycoerythrins, umbelliferones, LUMINOL®, and the like.
  • Chromogens or fluorogens i.e., molecules that can be modified (e.g., oxidized) to become colored or fluorescent or to change their color or emission spectra, are also capable of being incorporated into probes to act as reporter moieties under particular conditions.
  • the label moieties may be conjugated to the probe by methods that are well known in the art.
  • the label moieties may be directly attached through a functional group on the probe.
  • the probe either contains or can be caused to contain such a functional group.
  • suitable functional groups include, for example, amino, carboxyl, sulfhydryl, maleimide, isocyanate, isothiocyanate.
  • label moieties such as enzymes and chromogens may be conjugated to antibodies or nucleotides by means of coupling agents, such as dialdehydes, carbodiimides, dimaleimides, and the like.
  • the label moiety may also be conjugated to the probe by means of a ligand attached to the probe by a method described above and a receptor for that ligand attached to the label moiety.
  • a ligand attached to the probe by a method described above and a receptor for that ligand attached to the label moiety.
  • Any of the known ligand-receptor binding pair combinations is suitable.
  • Some suitable ligand-receptor pairs include, for example, biotin-avidin or -streptavidin, and antibody-antigen.
  • the biotin-avidin combination may be preferred.
  • the mycobacteria were cultured in commercially available Sauton medium supplemented with 2 g MgSO 4 , 8 g citric acid, 2 g K 2 HPO 4 , 16 g asparagine, 2 g (Fe + ) ammonium citrate, and 240 mL glycerol.
  • the bacteria were cultured under standard conditions.
  • the cells were harvested by filtration of the culture medium with a 12 ⁇ m filter.
  • the cells were subsequently resuspended in 20 mL PBS (phosphate-buffered salt solution) (pH 7.4) and the harvested cells were autoclaved under a pressure of 15 psi for 20 minutes in order to deactivate and sterilize the bacteria.
  • the thus obtained bacterial mass can be stored at ⁇ 80° C.
  • the bacterial mass was subsequently broken open using an automatic French-X-press or RIBI press (American Instruments Company, Trevenollab. Inc. Maryland).
  • the buckets were pre-cooled overnight at ⁇ 20° C. Before use the buckets were held in a mixture of ethanol and dry ice ( ⁇ 20° C.). After the buckets were filled with 1 g bacterial mass per bucket of 5 mL and cooled at ⁇ 80° C. for 20 minutes, the buckets were placed in the French-X-press and twelve tons of pressure was applied by pushing the plunger of the press. The buckets were then removed and cooled again at ⁇ 80° C. for 20 minutes.
  • the buckets were inverted and treated for the second time under the same conditions as the first time with the exception of pressure being ten tons.
  • the sequence of cooling and breaking was then repeated five times.
  • the disrupted cells were eluted with a suitable volume of PBS and subsequently centrifuged at 4° C. at 300 ⁇ g for 10 minutes in order to remove the unbroken bacteria with the sediment.
  • the collected supernatant was then centrifuged at 4° C. and 145,000 ⁇ g for 2 hours.
  • the pellet was suspended in 0.1 M Tris-HCl (pH 7.2), 0.01 M EDTA which contained 20 mM MgSO 4 ⁇ 7H 2 O in a concentration of about 1 g per 10 mL. 1 mg RNase and 1 mg DNase were added per 10 mL volume.
  • starting material This material is referred to hereinafter as “starting material”.
  • a 12% polyacrylamide analytical gel of 1.5 mm thickness was casted according to normal standard procedures. No comb was used in the stacking gel. Five milligrams (5 mg) of the starting material, KP-100, or SP-100 (see Example 2) were used for each gel. Forty microlitres (40 ⁇ L) of this material was diluted with 1.2 ⁇ L PBS. 300 ⁇ L 5 ⁇ loading mixture (0.3 g 250 mM Tris-HCl, 1.0 mL 10% SDS, 1.0 mL 10% dithioerythritol, 5 mg 0.05% bromophenol blue) was then added.
  • Incubation was carried out for 20 minutes at 65° C. 1.5 mL of total sample were subsequently applied to the gel and electrophoresis performed under the following conditions: 150 V for the run through the stacking gel for 30 minutes and 100 V through the running gel for 6 hours.
  • the strips were incubated with human serum diluted 1:200 with PBS containing 3% BSA for 1 hour at room temperature. The strips were subsequently washed three times (for 3 minutes at a time) in PBS. The strips were then incubated with a goat anti-human immunoglobulin-alkaline phosphatase conjugate in a dilution of 1 to 1000 in PBS with 3% BSA and 0.05% Tween 80. The strips were then washed again three times in PBS, as before. The color was developed with an NBT/BCIP (nitroblue tetrazolium/Bromo, Chloro Indolyl phosphate) color solution (1 mg per 10 mL) to which 10 ⁇ L H 2 O 2 were added.
  • NBT/BCIP nitrogen blue tetrazolium/Bromo, Chloro Indolyl phosphate
  • the strips were incubated for a maximum of 2 hours in 1 mL of this solution per strip.
  • the color reaction was stopped by transferring the strips to 0.1 M Tris-HCl (pH 8.3), 0:01 M EDTA.
  • the obtained patterns are interpreted by comparison with a reference pattern.
  • FIG. 1 shown in the blots A and B an example of Western blotting patterns which are developed after incubation respectively with representative negative and positive sera (positive for bovine tuberculosis).
  • Blots C and D are exemplary Western blotting patterns which are developed after incubation with a representative negative serum sample (Blot C) or positive serum sample (Blot D) (positive for Cattle Jones Disease).
  • Blots A and B Lane 1: BCG (Bacillus Calmette-Guérin) crude extract, Lane 2: crude extract of an M. tuberculosis strain, Lane 3: M. bovis crude extract.
  • Blots C and D Lane 1: BCG derived KP-100, Lane 2: RIVM 7114 derived KP-100.
  • RIVH is the abbreviation from the Netherlands National Institute of Public Health and Environmental Protection (Rijksinstituut voor Herbstgezondheid en Milieuhgiene, Bilthoren, the Netherlands).
  • Blot A only the background bands can be observed in blots incubated with negative serunm.
  • Blot B region in the 10-16 KDa region in lane 3, 22 KDa band in lane 2, 31 KDa bands in lane 1 and 2, 14 KDa band in lane 2.
  • Blot C only background bands can be observed in blots incubated with negative serum.
  • Blot D 45/48 KDa doublet in lane 1 and 2, 22 and 25 KDa band in lane 1 and 2, 66 KDa band in lane 1 and 2, 27 KDa band in lane 1.
  • FIG. 2 a is an example of different Western blotting patterns developed after incubation with representative variable sera of tuberculosis patients. Noticeable is the combination of different patterns demonstrating the presence of different dominant bands, as shown in table 1. These band patterns function as “hallmarks” for TB patients as diagnosed serologically.
  • Lane 1 BCG crude extract
  • Lane 2 crude extract of an M. tuberculosis strain.
  • Blot A Mycobacterium avium infected patient.
  • Blot B-F Tuberculosis patients.
  • Blot G non-endemic negative serum.
  • Blot H endemic negative serum (known recent contact, blot developed 2 weeks after patient returned to Netherlands from endemic range). Only “hallmarks” are mentioned.
  • Blot A Mycobacterium avium infected patients, sera, band at 68 KDa in lane 1 and 2, range in 10-16 KDa in lane 1, band in the 58-60 KDa region in lane 2. Patient shows low IgA titer in P-90 ELISA).
  • Blot B 38-40 KDa band in lane 1 and 2, 10-16 KDa band in lane 1 and 2, band in 58-60 KDa region in lane 2, smear in 22-28 KDa region in lane 1.
  • Blot C 16 KDa band in lane 1 and 2, bands in 58-60* KDa region in lane 1 and 2, bands in 38-40 KDa region in lane 1 and 2, smear in 22-28 KDa region in lane 1, 33 KDa band in lane 1 and 2.
  • Blot D 10 KDa band in 10-16 KDa region in lane 1, 16 KDa band in 10-16 KDa region in lane 2, 68 KDa band in lane 1 and 2, bands in 58-60* KDa region in lane 1 and 2.
  • Blot E smear in 33-38 KDa region in lane 1 and 2, 16 KDa bands in lane 1 and 2, bands in 58-60* KDa region in lane 2.
  • Blot F bands in 10-16, 22-28, 38-40, 58-60 regions and 68 KDa band in both lanes 1 and 2.
  • Blot G non-endemic negative serum.
  • Blot H endemic negative serum (known contact).
  • FIG. 2 b is an example of different Western blotting patterns developed after incubation with representative sera of patients with Lepromatous Leprosy (LL), Blot A and C, and Tuberculous Leprosy (TT), Blot B and D.
  • LL Lepromatous Leprosy
  • TT Tuberculous Leprosy
  • the “hallmark” patterns are shown in table 1 and are for LL: distinctive 29/33 KDa doublet, and for TT: distinctive 64/65 KDa doublet (often observed as single band) or a very pronounced smear in the 30-64 KDa region.
  • Lane 1 BCG crude extract
  • Lane 2 crude extract of a M. tuberculosis strain
  • Blot C Lane 1: Molecular marker
  • Lane 2 not relevant
  • Lane 3 BCG crude extract
  • Lane 4 crude extract of an M. tuberculosis strain
  • Blot D Lane 1: BCG crude extract
  • Lane 2 crude extract of an M. tuberculosis strain
  • Lane 3 Molecular marker.
  • Blot A/C 29/33 KDa doublet in lane 1 and 2.
  • Blot B/D 64/65 KDa doublet in lane 1 and 2.
  • FIG. 2 c is an example of different western blotting patterns developed after incubation with representative sera of patients with Crohn's Disease.
  • the “hallmark” patterns are shown in table 3 and are for Crohn's Disease a pronounced 45/48 KDa doublet.
  • blot A BCG crude extract
  • blot B crude extract of an M. tuberculosis stain
  • blot C Mycobacterium aviurn crude extract
  • blot D molecular marker
  • the starting material prepared according to Example 1 was, depending on the chosen M. tuberculosis strain, centrifuged at 70,000 ⁇ g to 120,000 ⁇ g at 4° C. for 2 hours. The pellet was washed three times with PBS. Between the washing steps centrifuging took place at 70,000 ⁇ g to 120,000 ⁇ g at 4° C. for 2 hours. The pellet was collected and resuspended in 10 mL PBS. In addition to the supernatant in Example 1, SP-100 can also be used for immunoblots (Example 1) and enzyme immunoassays (this example). The suspension was subsequently sonicated for 2 minutes at 80 watts at 4° C. After the protein concentration was determined, quantities of 100 ⁇ L were frozen at a concentration of 1 mg/mL and stored at ⁇ 80° C. until time of use (this preparation is designated with the term KP-100).
  • Microtiter plates are coated (via standard techniques) with either KP-100, SP-100, starting material, whole bacteria, 29/33 KDa, 64/65 KDa or 45/48 KDa antigens.
  • the plates are blocked with a 3% BSA solution in order to prevent a nonspecific binding of serum components. Plates are then dried and stored at 4° C.
  • Test sera are pipetted in a 1:100 dilution into the coated wells of a microtiter plate. The reaction takes place for 1 hour at 37° C. Nonspecific serum components and non-binding serum components are washed away with a washing buffer. A second incubation with a suitable dilution of an anti-human IgA peroxidase conjugate is carried out again for 1 hour at 37° C., and excess conjugate is then washed away.
  • Detection of human antibodies of the sub-type IgA binding specifically to KP-100 takes place by adding TMB (tetramethylbenzidine) to the wells.
  • Binding enzyme results in the occurrence of a blue color which, after addition of a coloring stop solution, changes to yellow. This yellow color has an absorption maximum of 450 nm.
  • the intensity of the resulting color is proportional to the amount of bound KP-100specific IgA.
  • A Endemic area (Africa, Ghana)
  • B Non-endemic area (Europe, the Netherlands).
  • Each population is sub-divided into 4 sub-groups, namely:
  • Group 1 culture confirmed TB patients
  • Group 2 negative control group (normal healthy individuals)
  • Group 3 suspected positives (TB contacts)
  • Group 4 suspected negatives (no known data, but certainly no TB, possibly leprosy or other nonspecific mycobacteriosis)
  • the test is performed with two kits having different lot numbers and production dates.
  • test serum can be considered positive when the result found in the test scores higher than 2.1 units.
  • test serum can be considered negative when the result found scores lower than 1.2 units.

Abstract

The invention relates to a method for identifying a Mycobacterium species responsible for a mycobacterial infection in human or animal, comprising selecting a suitable mycobacterial species and strain; preparing at least one mycobacterial antigen, respectively antigen preparation; binding the antigen, respectively the antigen preparation to a suitable carrier; causing the binding antigen to react with antibodies from serum of an individual infected with a Mycobacterium species; making visible antigen-antibody reactions for a suitable antibody (sub-)class; and identifying the responsible Mycobacterium species on the basis of the reactions which are made visible. The invention further provides a diagnostic kit which takes the form of a dip-stick on which is arranged a carrier strip with mycobacterial antigens binding thereto, and means for visualizing antigen-antibody reactions occurring on the carrier after contact with the serum for testing. In another embodiment the diagnostic kit comprises a microtiter plate, in the wells of which a specified antibody is arranged, and means for making visible antigen-antibody reactions occurring in the wells after contact with the serum for testing. The third embodiment is an immunoblot with mycobacterial antigens separated by electrophoresis binding thereto, and means for visualizing antigen-antibody reactions occurring on the immunoblot after contact with the serum for testing.

Description

    CROSS REFERENCE TO RELATED APPLICATIONS
  • This application is a continuation-in-part of copending U.S. application Ser. No. 08/454,122 filed on Jun. 7, 1995, the disclosure of which is incorpored herein by reference.[0001]
    • BACKGROUND OF THE INVENTION
  • 1. Field of the Invention [0002]
  • The invention relates to a method for identifying a Mycobacterium species responsible for a mycobacterial infection in human or animal. The invention further relates to diagnostic kits for use in the method. [0003]
  • 2. Description of the Related Art [0004]
  • The genus Mycobacterium, which contains about 50 species, is responsible for a number of human and animal diseases which are known collectively as the mycobacterioses. The best known of these in humans are leprosy, caused by [0005] M. leprae, which affects more than ten million people worldwide, and tuberculosis, usually caused by M. tuberculosis, at least ten million new cases of which occur each year. Most other mycobacteria normally occur only as environmental saprophytes but can also cause opportunist diseases. This happens usually, but not only, in the case of people who have problems with their immune system, such as AIDS patients and people undergoing immunosuppression. These opportunist types comprise the slow-growing species M. avium, and the closely related M. intracellulare and M. scrofulaceum (often referred to together as MAIS complex), M. kansasi, M marinum and M. ulcerans, and the fast-growing species M. chelonae and M. fortuitum. Although once rare, the incidence of opportunist mycobacterial diseases and tuberculosis shows a parallel increase in the western world with the incidence of AIDS. In addition there is limited but increasing evidence that mycobacteria or antigens thereof play a direct or indirect part in the etiology of a plurality of other diseases such as sarcoidosis and Crohn's disease and different auto-immune diseases such as auto-immune dermatitis, rheumatoid arthritis and diabetes. This could be attributed to a structural mimicry between epitopes of mycobacteria and those of the host.
  • The cell walls of mycobacteria are very complex and contain many lipids, some with structures unique to the genus. These structures comprise mycolinic acids and esters, peptido-glycolipde, arabino-galactane and lipo-arabino-manane. The lipid-rich mycobacterial cell walls are responsible for the characterizing coloring properties of the mycobacteria. They also enable mycobacteria to counter an attack by the immune system of the host. A number of species, once taken up into macrophages, are capable of surrounding themselves with a thick layer of secreted lipids. [0006]
  • Many different components of the mycobacteria begin an interaction with the immune system. These components comprise protein and hydrocarbon antigens, which can either be actively secreted by the mycobacteria or can form part of the cell wall or cell membrane. In addition they may be present in the cytoplasm, for instance in the cytoplasmic matrix, ribosomes and enzymes. Mycobacteria also possess innuno-modulating components such as inmmunosuppressing compounds and adjuvants. Consequently, a single mycobacterial species can induce a large variety of immune responses in different forms and with diverse specificities. It is therefore difficult to distinguish immune responses against species-specific components from cross reactions. For this reason it has therefore been found difficult to derive protein antigens suitable for the detection of species-specific humoral responses as a basis for a very sensitive and specific sero-diagnostic test for tuberculosis. Because the mycobacteria occur a great deal in the environment, human serum nearly always contains anti-mycobacterial antibodies. [0007]
  • In view of the problems with the specificity of protein antigens, a number of researchers, including the present inventors, have focused their attention on species-specific glycolipid antigens for the detection of specific humoral immune responses. This approach is for example illustrated in Vega-Lopez et al., [0008] J. Clin. Microbiol. 26(12), 2474-2479 (1988) and Roche et al., Int. J. Mycobact. Dis. 60(2), 201-207 (1992) who both stated that in serodiagnosis species-specific antigens and antibodies are required. Although the immune reactivity against mycobacteria is of the cell-mediated type and the humoral immune responses probably play a minor part in the total effector mechanism of mycobacterial immunity and immunopathology, studies in the antibody response to immuno-dominant mycobacterial cross-reactive antigen components (referred to hereinafter as “Im-CRAC”) could shed light on the varying capability of the host to recognize different mycobacterial antigens. They could therefore provide indirect information relating to the nature of the immune recognition of, and response to, a specific mycobacterial pathogen.
    • SUMMARY OF THE INVENTION
  • It has now been found that the clinical manifestation of mycobacterial diseases appears to be related to the varying capability of an individual host to produce a humoral response to different mycobacterial immuno-cross-reactive antigen components (Im-CRAC). Each mycobacterial infection generates its own specific antibody response to a number of specified antigens. Analysis of the antibody-response by means of immunoblotting has demonstrated that the immuno-dominant Im-CRAC vary in accordance with the immunopathological manifestation of the mycobacterial diseases. It has been found that the sera of individuals which are infected with different Mycobacterium species cause different and distinguishing band patterns on immunoblots of mycobacterial antigens. [0009]
  • This discovery forms the basis of the present invention, whereby a method is provided for identifying a Mycobacterium species responsible for a mycobacterial infection in human or animal, comprising the steps of: [0010]
  • (a) selecting a suitable mycobacterial species and strain; [0011]
  • (b) preparing an antigen preparation comprising at least one mycobacterial antigen; [0012]
  • (c) binding the antigen, respectively the antigen preparation to a suitable carrier; [0013]
  • (d) causing the binding antigen to react with antibodies from serum of an individual infected with a Mycobacterium species; [0014]
  • (e) making visible antigen-antibody reactions for a suitable antibody (sub-)class; and [0015]
  • (f) identifying the responsible Mycobacterium species on the basis of the reactions which are made visible. [0016]
  • In preference, the antigen preparation is separated by electrophoresis prior to step (c) and the carrier is a membrane to which the antigen is bound by means of electroblotting. This process is called Western blotting. [0017]
  • The present invention is also a method for detecting or identifying a Mycobacterium species in a biological sample. The method includes: [0018]
  • (a) selecting a standard Mycobacterium; [0019]
  • (b) preparing an antigen preparation from a culture of the standard Mycobacterium, wherein the antigen preparation comprises at least two immuno-cross-reactive antigen components (ImCRACs); [0020]
  • (c) separating the ImCRACs of the antigen preparation according to molecular weight; [0021]
  • (d) binding the separated InCRACS of the antigen preparation to a solid carrier; [0022]
  • (e) contacting the bound ImCRACs with the biological sample under conditions that permit antibodies in the biological sample to bind with the carrier-bound InCRAds to provide a pattern of carrier-bound antibody-InCRAC complexes; [0023]
  • (f) detecting the pattern of carrier-bound antibody-ImCRAC complexes; and [0024]
  • (g) comparing the pattern of carrier-bound antibody-ImCRAC complexes from the sample to a library of standard patterns of carrier-bound antibody-ImCRAC complexes characteristic of particular Mycobacterium species, [0025]
  • wherein matching of the pattern of carrier-bound antibody-ImCRAC complexes from the sample to one of the standard patterns of carrier-bound antibody-ImCRAC complexes from the library permits detection or identification of the Mycobacterium species in the sample. [0026]
  • The present invention is also a method for detecting or identifying a Mycobacterium species in a biological sample. The method includes: [0027]
  • (a) providing a test substrate having bound thereto an antigen preparation from a culture of the standard Mycobacterium, wherein the antigen preparation comprises at least two immuno-cross-reactive antigen components (ImCRACs) such that the ImCRACs of the antigen preparation separate according to molecular weight; [0028]
  • (b) contacting the bound ImCRACs of the test substrate with the biological sample under conditions that permit antibodies in the biological sample to bind with the substrate-bound ImCRACs to provide a pattern of substrate-bound antibody-ImCRAC complexes; [0029]
  • (c) detecting the pattern of substrate-bound antibody-ImCRAC complexes; and [0030]
  • (d) comparing the pattern of substrate-bound antibody-ImCRAC complexes from the sample to a library of standard patterns of substrate-bound antibody-ImCRAC complexes characteristic of particular Mycobacterium species, [0031]
  • wherein matching of the pattern of substrate-bound antibody-ImCRAC complexes from the sample to one of the standard patterns of substrate-bound antibody-ImCRAC complexes from the library permits detection or identification of the Mycobacterium species in the sample. [0032]
  • In one preferred embodiment of the present invention, the antigen preparation is separated by electrophoresis prior to step (d) and the carrier or substrate can be a membrane to which the lmCRACS are bound by means of electroblotting. In addition, the membrane is a nitrocellulose membrane. [0033]
  • In another preferred embodiment of the present invention, the antigen preparation of the standard Mycobacterium can be a total protein preparation. Additionally, the antigen preparation can be, but not limited to, a KP-100 or SP-100 fraction of the total protein preparation. [0034]
  • In another preferred embodiment of the present invention, the carrier and substrate can be, but not limited to, a microtiter plate. The ImCRACS of the antigen preparation is bound to the wells of a microtiter plate so that the carrier-bound ImCRACS is brought into contact with the biological sample under conditions that permit the antibodies in the biological sample to bind with the carrier-bound InCRACs to provide a pattern of carrier-bound antibody-ImCRAC complexes. The non-binding antibodies can then be removed. [0035]
  • The pattern of the carrier-bound antibody-ImCRAC complexes and the substrate-bound antibody-ImCRAC complexes can be a banding pattern consisting of, but not limited to, 29/33 KDa, 45/48 KDa, 64 KDa and combinations thereof. [0036]
  • The carrier-bound antibody-ImCRAC complexes and the substrate-bound antibody-ImCRAC complexes can be made visualizable by any method known in the art. Preferably, the carrier-bound antibody-ImCRAC complexes can be made visualizable by using at least one antibody-enzyme conjugate directed against at least one antibody of an isotype selected from the group consisting of, but not limited to, IgG, IgM, IgA and combinations thereof. The enzyme of the antibody-enzyme conjugate can be any enzyme known in the art which can make the carrier-bound antibody-ImCRAC complexes visualizable, preferably peroxidase. [0037]
  • In another preferred embodiment of the present invention, the carrier and the substrate can be, but not limited to, a dip-stick. The mycobacterial in the antigen preparation is brought into reaction with antibodies from a biological sample by dipping the stick in the biological sample for testing. The carrier-bound antibody-ImCRAC complexes are made visible by subsequently dipping the stick in a solution with an antibody-enzyme conjugate and a color substrate for the relevant enzyme. [0038]
  • The present invention is also a diagnostic kit which includes (1) a test substrate having bound thereto an antigen preparation from a culture of the standard Mycobacterium, wherein the antigen preparation comprises at least two immuno-cross-reactive antigen components (ImCRACs) that are separable according to molecular weight, and wherein when the ImCRACs of the test substrate are contacted with a biological sample under conditions that permit antibodies in the biological sample to bind with the substrate-bound ImCRACs, a pattern of substrate-bound antibody-ImCRAC complexes is provided; and (2) a means for visualizing the pattern substrate-bound antibody-ImCRAC complexes. [0039]
  • The test substrate of the kit can be, but not limited to, a dip-stick, a microtiter plate, and an immunoblot.[0040]
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • Preferred embodiments of the invention have been chosen for purposes of illustration and description, but are not intended in any way to restrict the scope of the invention. The preferred embodiments of certain aspects of the invention are shown in the accompanying drawing, wherein: [0041]
  • FIG. 1 shows an example of Western blotting patterns which are developed after incubation respectively with representative negative and positive sera (positive for bovine tuberculosis). [0042]
  • FIG. 2[0043] a shows an example of different Western blotting patterns developed after incubation with representative variable sera of tuberculous patients.
  • FIG. 2[0044] b shows an example of different Western blotting patterns developed after incubation with representative sera of patients with Lepromatous Leprosy (LL) and Tuberculous Leprosy (TT).
  • FIG. 2[0045] c shows an example of different Western blotting patterns developed after incubation with representative sera of patients with Crohn's Disease.
    • DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
  • The Im-CRAC comprise namely a number of antigens with specific molecular weights which, as has now been found, after immunoblotting, exhibit a binding pattern which correlates to the disease or infection. The specific band pattern is characterized by the presence or absence of four individual components, for instance: [0046]
  • a region comprising different pronounced bands and/or overlapping bands, which can be observed as a smear (“region”); [0047]
  • sharp single bands which are strongly positive (“band”); [0048]
  • sharp double bands which are strongly positive (“doublet”); and [0049]
  • other positive bands (“extra bands”). [0050]
  • For a survey of the different antigens, their molecular weights and binding characteristics, see Table 1. [0051]
    TABLE 1
    Survey of characteristic bind patterns of mycobacterial
    Immuno-Cross-reactive Antigen Components.
    MW range Binding
    Antigen Diagnostic for (in KDa) characteristic
    A J <8 band
    B T, B, J, 10-16 band
    C B, J, T, 20-28 band or region
    D L
    29/33 doublet
    E B, J, T, 31-40 band or region
    F T 38-40 band or region
    G C, B, J, 45/48 doublet
    H T 58-60 band or region
    I L
    64/65 doublet
    J J
    66 band
    K T
    68 band
    L L 30-64 region
  • Each of the mycobacterioses is characterized by a specific banding pattern which is formed when a blot having thereon an antigen preparation of mycobacteria separated to size is incubated with serum of an infected individual. [0052]
  • Tables 2a-2f below show a survey of the banding patterns of a number of mycobacterial and immunological diseases. [0053]
    TABLE 2a
    Bovine tuberculosis
    Regions (MW in KDa)
    and/or and/or and/or and
    Pattern 10-16 20-28 31-40 45-48
    1. 14 KDa band 22 KDa band 31 KDa band 45/48 KDa
    20-28 KDa doublet
    region
  • [0054]
    TABLE 2b
    Johne's Disease
    Regions (MW in KDa)
    and/or and and/or and/or and and
    Pattern >8 10-16 20-28 31-40 45-48 66
    1. region 14 KDa and 22 31 KDa 45/48 66 KDa
    band (25) band KDa band
    - KDa doublet
    band,
    and/or
    27 KDa
    band
  • [0055]
    TABLE 2c
    Human Tuberculosis
    Regions (MW in KDa)
    Pattern 10-16 20-28 31-38 38-40 58-60 68 other*
    1. 10 and / 33 KDa region band / +/−
    16 KDa band
    bands
    2. 16 KDa region 33 KDa bands region / +/−
    single band
    band
    3. 10 or / / bands bands / +/−
    16 KDa
    band
    4. 16 KDa / 33 KDa region bands / +/−
    band band
    5. 10 region 33 KDa bands bands 68 +/−
    and/or band and/or KDa
    16 KDa region band
    band
  • [0056]
    TABLE 2d
    Leprosy
    Regions (MW in KDa)
    Pattern 29-33 30-65 64-65 other*
    LL pattern 1 29/33 KDa / / +/−
    doublet
    TT pattern 1 / regions and/or / +/−
    bands
    TT pattern 2 / regions and/or 64/65 KDa +/−
    bands doublet
    TT pattern 3 / / 64/65 KDa +/−
    doublet
  • [0057]
    TABLE 2e
    Crohn's Disease
    Regions (MW in KDa)
    Pattern 45-48 other*
    1. 45/48 KDa doublet +/−
  • [0058]
    TABLE 2f
    Rheumatoid Arthritis
    Regions (MW in KDa)
    and/or and/or and/or
    Pattern and 42 KDa 80-90 KDa 58-60 KDa 14-18 KDa
    1. band region region region
  • The method of the invention, such as an immunoblot, can be used to answer two questions. First, the presence of any positive band pattern will answer the question of whether a mycobacterial infection is present. Second, the presence of specific banding patterns indicates which mycobacterial species has caused the infection, and therefore what the nature and etiology of the disease will be. From the patterns in the immunoblotting it follows which mycobacterial antigen preparations are suitable for diagnosis of any particular disease. [0059]
  • The invention further relates to a heterogeneous enzyme immunoassay. The antigens for a heterogeneous enzyme immunoassay is preferably chosen from the group which consists of mycobacterial immuno-cross-reactive antigen components with a molecular weight of 29/33 KDa, 45/48 KDa, 64/65 KDa and a fraction designated with the term KP-100. These ImCRAC can be used separately or in combination with each other for serological diagnosis of the correlating diseases in a heterogeneous enzyme immunoassay (EIA). [0060]
  • In this form of assay, antibody-conjugates labeled with a standard enzyme are used. An important detail is that the enzyme activity does not change during the immunological reaction. [0061]
  • To test the immune response in patients to the selected antigens, use is made for instance of microtiter plates (“Solid Phase”). By means of standard published techniques the antigens are irreversibly immobilized on the surface of the wells in such a microtiter plate. [0062]
  • This binding takes place while retaining specific antigen determinants on the used antigens. After incubation with serum, in the wells of the microtiter plate, antibodies present therein can specifically form a complex with the irreversibly bound antigens. [0063]
  • After removal of non-binding serum components, binding antibodies are detected using an anti-antibody antibody labeled with an enzyme. [0064]
  • Binding of the enzyme is only possible when specific antibodies have adhered to the immobilized antigens. Substrate conversion by the binding enzyme to a visually or photometrically observable signal is thereby directly related to the presence of specific antibodies in the tested serum. [0065]
  • The choice of specificity of the enzyme-bound anti-antibody antibody determines the type of reaction that takes place. For instance, it may be desirable in some cases to demonstrate the specifically binding immunoglobulins of the IgG type, while in other cases immunoglobulins of the IgA and/or IgM type are just demonstrated. [0066]
  • The combination of antigen and immunoglobulin type defines the specificity of the test. [0067]
  • The said methods, that is, the immunoblot and the EIA, can be used as mutual confirmation. [0068]
  • In addition, for the serological diagnosis based on the said antigens, use can be made of a test stick as solid phase. [0069]
  • A particularly advantageous embodiment of the invention relates to a test stick, the so-called “dip-stick”, which is used as solid phase in the heterogeneous enzyme immunoassay. [0070]
  • The said mycobacterial antigens can be irreversibly bound to such a dip-stick. [0071]
  • The antigen is brought into reaction with antibody from serum for testing by dipping the dip-stick in a serum sample for testing. The formed antigen-antibody complex can be made visible by subsequently dipping the dip-stick in an anti-antibody antibody-enzyme conjugate solution. [0072]
  • With the binding enzyme a substrate can then be converted to a visually or photometrically observable signal. [0073]
  • In another embodiment, the invention is a diagnostic kit for: [0074]
  • an immunoblot assay; comprising IMCRAC antigens separated by electrophoresis as described above, immobilized on a solid carrier, in addition to an associated suitable detection system. [0075]
  • a heterogeneous enzyme immunological assay; comprising a microtiter plate, the wells of which are coated with above mentioned antigens or antigen preparations, in addition to an associated suitable detection system. [0076]
  • a dip-stick assay; comprising test sticks coated with antigen or antigen preparations, in addition to an associated suitable detection system. [0077]
  • Detecting Protein with Antibodies [0078]
  • The probe may be an antibody, preferably a monoclonal antibody. The antibodies may be prepared as described above. [0079]
  • Assays for detecting the presence of proteins with antibodies have been previously described, and follow known formats, such as standard blot and ELISA formats. These formats are normally based on incubating an antibody with a sample suspected of containing the protein and detecting the presence of a complex between the antibody and the protein. The antibody is labeled either before, during, or after the incubation step. The protein is preferably immobilized prior to detection. Immobilization may be accomplished by directly binding the protein to a solid surface, such as a microtiter well, or by binding the protein to immobilized antibodies. This and other immunoassays are described by David et al., U.S. Pat. No. 4,376,110 which entirety is incorporated herein by reference. [0080]
  • Immunoassays may involve one step or two steps. In a one-step assay, the target molecule, if it is present, is immobilized and incubated with a labeled antibody. The labeled antibody binds to the immobilized target molecule. After washing to remove unbound molecules, the sample is assayed for the presence of the label. [0081]
  • In a two-step assay, immobilized target molecule is incubated with an unlabeled first antibody. The target molecule-antibody complex, if present, is then bound to a second, labeled antibody that is specific for the unlabeled antibody. The sample is washed and assayed for the presence of the label, as described above. [0082]
  • The immunometric assays described above include simultaneous sandwich, forward sandwich, and reverse sandwich immunoassays. These terms are well known to those skilled in the art. [0083]
  • In a forward sandwich immunoassay, a sample is first incubated with a solid phase immunosorbent containing antibody against the protein. Incubation is continued for a period of time sufficient to allow the protein in the sample to bind to the immobilized antibody in the solid phase. After the first incubation, the solid phase immunoabsorbent is separated from the incubation mixture and washed to remove excess protein and other interfering substances which also may be present in the sample. Solid phase immunoabsorbent-containing protein bound to the immobilized antibodies is subsequently incubated for a second time with soluble labeled antibody cross-reactive with a different domain on the protein. After the second incubation, another wash is performed to remove the unbound labeled antibody from the solid immunoabsorbent and to remove non-specifically bound labeled antibody. Labeled antibody bound to the solid phase immunoabsorbent is then detected and the amount of labeled antibody detected serves as a direct measure of the amount of antigen present in the original sample. Alternatively, labeled antibody that is not associated with the immunoabsorbent complex can also be detected, in which case the measure is in inverse proportion to the amount of antigen present in the sample. Forward sandwich assays are described, for example, in U.S. Pat. Nos. 3,867,517, 4,012,294, and 4,376,110. [0084]
  • In a reverse sandwich assay, the sample is initially incubated with labeled antibody. The solid phase immunoabsorbent containing immobilized antibody cross-reactive with a different domain on the protein is added to the labeled antibody, and a second incubation is carried out. The initial washing step required by a forward sandwich assay is not required, although a wash is performed after the second incubation. Reverse sandwich assays have been described, for example, in U.S. Pat. Nos. 4,098,876 and 4,376,110. [0085]
  • In a simultaneous sandwich assay, the sample, the immunoabsorbent with immobilized antibody, and labeled soluble antibody specific to a different domain are incubated simultaneously in one incubation step. The simultaneous assay requires only a single incubation and does not require any washing steps. The use of a simultaneous assay is a very useful technique, providing ease of handling, homogeneity, reproducibility, linearity of the assays, and high precision. See U.S. Pat. No.4,376,110 to David et al. [0086]
  • In each of the above assays, the sample containing antigen, solid phase immmunoabsorbent with immobilized antibody and labeled soluble antibody are incubated under conditions and for a period of time sufficient to allow antigen to bind to the immobilized antibodies and to the soluble antibodies. In general, it is desirable to provide incubation conditions sufficient to bind as much antigen as possible, since this maximizes the binding of labeled antibody to the solid phase, thereby increasing the signal. The specific concentrations of labeled and immobilized antibodies, the temperature and time of incubation, as well as other such assay conditions, can be varied, depending upon various factors including the concentration of antigen in the sample, the nature of the sample and the like. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation. [0087]
  • There are many solid phase immunoabsorbents which have been employed and which can be used in the present invention. Well known immunoabsorbents include beads formed from glass, polystyrene, polypropylene, dextran, nylon, and other material; and tubes formed from or coated with such materials, and the like. The immobilized antibodies may be covalently or physically bound to the solid phase immunoabsorbent, by techniques such as covalent bonding via an amide or ester linkage or by absorption. [0088]
  • Detecting Antibodies with Protein [0089]
  • The proteins may be labeled and used as probes in standard immunoassays to detect antibodies against the proteins in samples, such as in the sera or other bodily fluids of patients being tested for. In general, a protein in accordance with the invention is incubated with the sample suspected of containing antibodies to the protein. The protein is labeled either before, during, or after incubation. The detection of labeled protein bound to an antibody in the sample indicates the presence of the antibody. The antibody is preferably immobilized. [0090]
  • Suitable assays are known in the art, such as the standard ELISA protocol described by R H Kenneth, “Enzyme-linked antibody assay with cells attached to polyvinyl chloride plates” in Kenneth et al., [0091] Monoclonal Antibodies, Plenum Press, New York, page 376 (1981).
  • Briefly, plates are coated with antigenic protein at a concentration sufficient to bind detectable amounts of the antibody. After incubating the plates with the protein, the plates are blocked with a suitable blocking agent, such as, for example, 10% normal goat serum. The sample, such as patient sera, is added and titered to determine the endpoint. Positive and negative controls are added simultaneously to quantitate the amount of relevant antibody present in the unknown samples. Following incubation, the samples are probed with goat anti-human Ig conjugated to a suitable label, such as an enzyme. The presence of anti-protein antibodies in the sample is indicated by the presence of the label. [0092]
  • For use in immunoassays, the probe may be the entire protein or may be functional analogs thereof. Functional analogs of these proteins include fragments and substitution, addition and deletion mutations that do not destroy the ability of the proteins to bind to their antibodies. As long as the proteins are able to detect antibodies specific for the protein, they are useful in the present invention. [0093]
  • The probes described above can be detectably labeled in accordance with methods known in the art. In general, the probe can be modified by attachment of a detectable label moiety to the probe, or a detectable probe can be manufactured with a detectable label moiety incorporated therein. The detectable label moiety can be any detectable moiety, many of which are known in the art, including radioactive atoms, electron dense atoms, enzymes, chromogens and colored compounds, fluorogens and fluorescent compounds, members of specific binding pairs, and the like. [0094]
  • Methods for labeling antibodies have been described, for example, by Hunter et al. (1962) and by David et al., [0095] Biochemistry 13:1014-1021 (1974). Additional methods for labeling antibodies have been described in U.S. Pat. Nos. 3,940,475 and 3,645,090, each of which is incorporated herein by reference.
  • Methods for labeling oligonucleotide probes have been described, for example, by Leary et al., [0096] Proc Natl Acad Sci USA (1983) 80:4045; Renz and Kurz, Nucl Acids Res 12:3435 (1984); Richardson and Gumport, Nucl Acids Res 11:6167 (1983); Smith et al., Nucl Acids Res 13:2399 (1985); Meinkoth and Wahl, Anal Biochem 138:267 (1984). Other methods for labeling nucleic acids are described, for example, in U.S. Pat. Nos. 4,711,955, 4,687,732, 5,241,060, 5,244,787, 5,328,824, 5,580,990, and 5,714,327, each of which is incorporated herein by reference.
  • The label moiety may be radioactive. Some examples of useful radioactive labels include [0097] 32P, 125I, 131I, and 3H. Use of radioactive labels have been described in U.K. patent document No. 2,034,323, U.S. Pat. Nos. 4,358,535, and 4,302,204, each incorporated herein by reference.
  • Some examples of non-radioactive labels include enzymes, chromogens, atoms and molecules detectable by electron microscopy, and metal ions detectable by their magnetic properties. [0098]
  • Some useful enzymatic labels include enzymes that cause a detectable change in a substrate. Some useful enzymes (and their substrates) include, for example, horseradish peroxidase (pyrogallol and o-phenylenediamine), beta-galactosidase (fluorescein beta-D-galactopyranoside), and alkaline phosphatase (5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium). The use of enzymatic labels has been described, for example, in U.K. 2,019,404, EP 63,879, and by Rotman, [0099] Proc Natl Acad Sci USA 47:1981-91 (1961).
  • Useful reporter moieties include, for example, fluorescent, phosphorescent, chemiluminescent, and bioluminescent molecules, as well as dyes. Some specific colored or fluorescent compounds useful in the present invention include, for example, fluoresceins, coumarins, rhodamines, Texas red, phycoerythrins, umbelliferones, LUMINOL®, and the like. Chromogens or fluorogens, i.e., molecules that can be modified (e.g., oxidized) to become colored or fluorescent or to change their color or emission spectra, are also capable of being incorporated into probes to act as reporter moieties under particular conditions. [0100]
  • The label moieties may be conjugated to the probe by methods that are well known in the art. The label moieties may be directly attached through a functional group on the probe. The probe either contains or can be caused to contain such a functional group. Some examples of suitable functional groups include, for example, amino, carboxyl, sulfhydryl, maleimide, isocyanate, isothiocyanate. [0101]
  • Alternatively, label moieties such as enzymes and chromogens may be conjugated to antibodies or nucleotides by means of coupling agents, such as dialdehydes, carbodiimides, dimaleimides, and the like. [0102]
  • The label moiety may also be conjugated to the probe by means of a ligand attached to the probe by a method described above and a receptor for that ligand attached to the label moiety. Any of the known ligand-receptor binding pair combinations is suitable. Some suitable ligand-receptor pairs include, for example, biotin-avidin or -streptavidin, and antibody-antigen. The biotin-avidin combination may be preferred. [0103]
  • The present invention will be further elucidated with reference to a number of examples which are given herein by way of illustration and are not intended to limit the invention. [0104]
    • EXAMPLE 1 Immunoblot
  • 1. Preparation of Crude Mycobacterial Mass (“Starting Material”) [0105]
  • The mycobacteria were cultured in commercially available Sauton medium supplemented with 2 g MgSO[0106] 4, 8 g citric acid, 2 g K2HPO4, 16 g asparagine, 2 g (Fe+) ammonium citrate, and 240 mL glycerol. The bacteria were cultured under standard conditions. The cells were harvested by filtration of the culture medium with a 12 μm filter. The cells were subsequently resuspended in 20 mL PBS (phosphate-buffered salt solution) (pH 7.4) and the harvested cells were autoclaved under a pressure of 15 psi for 20 minutes in order to deactivate and sterilize the bacteria. The thus obtained bacterial mass can be stored at −80° C.
  • To determine the quantity of starting material a 1/100 dilution of the harvested autoclaved suspension in PBS was made. The optical density thereof, measured at 420 nm (O.D.[0107] 420) must be 0.1. If necessary the concentrated bacterial mass is supplemented with PBS (pH 7.4) until the correct O.D. is obtained. An O.D.420 of 0.1 indicates the presence of 7×1011 bacteria per 30 mL, which is equivalent to 12 g wet weight of the bacterial mass.
  • For preparation of a crude mycobacterial extract, 5 g wet weight of the bacterial mass was washed three times with PBS (pH 7.4). Centrifuging was then carried out at 3000×g until the mass precipitated. The pellet was suspended in 50 mL PBS and stirred carefully to reduce formation of lumps to a minimum. To prevent lump formation, 0.05% Tween 80 was added. To avoid bacterial contamination 3 mg penicillin/streptomycin was added to this solution. This material was then diluted with PBS to a final concentration of 2 g wet weight/mL. [0108]
  • The bacterial mass was subsequently broken open using an automatic French-X-press or RIBI press (American Instruments Company, Trevenollab. Inc. Maryland). The buckets were pre-cooled overnight at −20° C. Before use the buckets were held in a mixture of ethanol and dry ice (−20° C.). After the buckets were filled with 1 g bacterial mass per bucket of 5 mL and cooled at −80° C. for 20 minutes, the buckets were placed in the French-X-press and twelve tons of pressure was applied by pushing the plunger of the press. The buckets were then removed and cooled again at −80° C. for 20 minutes. The buckets were inverted and treated for the second time under the same conditions as the first time with the exception of pressure being ten tons. The sequence of cooling and breaking was then repeated five times. The disrupted cells were eluted with a suitable volume of PBS and subsequently centrifuged at 4° C. at 300×g for 10 minutes in order to remove the unbroken bacteria with the sediment. The collected supernatant was then centrifuged at 4° C. and 145,000×g for 2 hours. The pellet was suspended in 0.1 M Tris-HCl (pH 7.2), 0.01 M EDTA which contained 20 mM MgSO[0109] 4·7H2O in a concentration of about 1 g per 10 mL. 1 mg RNase and 1 mg DNase were added per 10 mL volume. Samples were then incubated overnight at 4° C. with careful stirring, followed by incubation for 1 hour at 37° C. The lysate was centrifuged at 300×g and 4° C. for 10 minutes in order to remove the last-remaining unbroken bacteria (this material is referred to hereinafter as “starting material”).
  • 2. Manufacture of Membrane for Assays [0110]
  • A 12% polyacrylamide analytical gel of 1.5 mm thickness was casted according to normal standard procedures. No comb was used in the stacking gel. Five milligrams (5 mg) of the starting material, KP-100, or SP-100 (see Example 2) were used for each gel. Forty microlitres (40 μL) of this material was diluted with 1.2 μL PBS. 300 μL 5×loading mixture (0.3 g 250 mM Tris-HCl, 1.0 [0111] mL 10% SDS, 1.0 mL 10% dithioerythritol, 5 mg 0.05% bromophenol blue) was then added.
  • Incubation was carried out for 20 minutes at 65° C. 1.5 mL of total sample were subsequently applied to the gel and electrophoresis performed under the following conditions: 150 V for the run through the stacking gel for 30 minutes and 100 V through the running gel for 6 hours. [0112]
  • To prepare a Western blot the proteins present in the gel were transferred at 50 V for 3 hours to a nitrocellulose membrane. After completion of the transfer the membrane was colored with 0.2% amido black for 2 minutes to check the membrane for irregularities and air bubbles. The membrane was the decolorized in 0.05% Tween 80 in PBS with 1% BSA (bovine serum albumin). The membrane was then cut into strips and was ready for use. [0113]
  • 3. Immunodetection [0114]
  • The strips were incubated with human serum diluted 1:200 with PBS containing 3% BSA for 1 hour at room temperature. The strips were subsequently washed three times (for 3 minutes at a time) in PBS. The strips were then incubated with a goat anti-human immunoglobulin-alkaline phosphatase conjugate in a dilution of 1 to 1000 in PBS with 3% BSA and 0.05% Tween 80. The strips were then washed again three times in PBS, as before. The color was developed with an NBT/BCIP (nitroblue tetrazolium/Bromo, Chloro Indolyl phosphate) color solution (1 mg per 10 mL) to which 10 μL H[0115] 2O2 were added. The strips were incubated for a maximum of 2 hours in 1 mL of this solution per strip. The color reaction was stopped by transferring the strips to 0.1 M Tris-HCl (pH 8.3), 0:01 M EDTA. The obtained patterns are interpreted by comparison with a reference pattern.
  • The results are shown in FIGS. 1 and 2[0116] a-2 c.
  • FIG. 1 shown in the blots A and B an example of Western blotting patterns which are developed after incubation respectively with representative negative and positive sera (positive for bovine tuberculosis). [0117]
  • Blots C and D are exemplary Western blotting patterns which are developed after incubation with a representative negative serum sample (Blot C) or positive serum sample (Blot D) (positive for Cattle Jones Disease). [0118]
  • Blots A and B: Lane 1: BCG (Bacillus Calmette-Guérin) crude extract, Lane 2: crude extract of an [0119] M. tuberculosis strain, Lane 3: M. bovis crude extract.
  • Blots C and D: Lane 1: BCG derived KP-100, Lane 2: RIVM 7114 derived KP-100. RIVH is the abbreviation from the Netherlands National Institute of Public Health and Environmental Protection (Rijksinstituut voor Volksgezondheid en Milieuhgiene, Bilthoren, the Netherlands). [0120]
  • Interpretation of Banding Patterns from Left to Right is as Follows [0121]
  • Only the specific characteristics are stated herein. [0122]
  • Blot A: only the background bands can be observed in blots incubated with negative serunm. [0123]
  • Blot B: region in the 10-16 KDa region in [0124] lane 3, 22 KDa band in lane 2, 31 KDa bands in lane 1 and 2, 14 KDa band in lane 2.
  • Blot C: only background bands can be observed in blots incubated with negative serum. [0125]
  • [0126] Blot D: 45/48 KDa doublet in lane 1 and 2, 22 and 25 KDa band in lane 1 and 2, 66 KDa band in lane 1 and 2, 27 KDa band in lane 1.
  • FIG. 2[0127] a is an example of different Western blotting patterns developed after incubation with representative variable sera of tuberculosis patients. Noticeable is the combination of different patterns demonstrating the presence of different dominant bands, as shown in table 1. These band patterns function as “hallmarks” for TB patients as diagnosed serologically.
  • Applicable to all blots (from left to right): Lane 1=BCG crude extract, Lane 2=crude extract of an M. tuberculosis strain. [0128]
  • Interpretation of Banding Patterns is as Follows. Different Blots (Originating from Different PAGE Gels) are Herein Compared with Each Other [0129]
  • Blot A: Mycobacterium avium infected patient. [0130]
  • Blot B-F: Tuberculosis patients. [0131]
  • Blot G: non-endemic negative serum. [0132]
  • Blot H: endemic negative serum (known recent contact, blot developed 2 weeks after patient returned to Netherlands from endemic range). Only “hallmarks” are mentioned. [0133]
  • Blot A: Mycobacterium avium infected patients, sera, band at 68 KDa in lane 1 and 2, range in 10-16 KDa in lane 1, band in the 58-60 KDa region in lane 2. Patient shows low IgA titer in P-90 ELISA). [0134]
  • Blot B: 38-40 KDa band in lane 1 and 2, 10-16 KDa band in lane 1 and 2, band in 58-60 KDa region in lane 2, smear in 22-28 KDa region in lane 1. [0135]
  • Blot C: 16 KDa band in lane 1 and 2, bands in 58-60* KDa region in lane 1 and 2, bands in 38-40 KDa region in lane 1 and 2, smear in 22-28 KDa region in [0136] lane 1, 33 KDa band in lane 1 and 2.
  • [0137] Blot D: 10 KDa band in 10-16 KDa region in lane 1, 16 KDa band in 10-16 KDa region in lane 2, 68 KDa band in lane 1 and 2, bands in 58-60* KDa region in lane 1 and 2.
  • Blot E: smear in 33-38 KDa region in [0138] lane 1 and 2, 16 KDa bands in lane 1 and 2, bands in 58-60* KDa region in lane 2.
  • Blot F: bands in 10-16, 22-28, 38-40, 58-60 regions and 68 KDa band in both lanes 1 and 2. [0139]
  • Blot G: non-endemic negative serum. [0140]
  • Blot H: endemic negative serum (known contact). [0141]
  • FIG. 2[0142] b is an example of different Western blotting patterns developed after incubation with representative sera of patients with Lepromatous Leprosy (LL), Blot A and C, and Tuberculous Leprosy (TT), Blot B and D.
  • The “hallmark” patterns are shown in table 1 and are for LL: distinctive 29/33 KDa doublet, and for TT: distinctive 64/65 KDa doublet (often observed as single band) or a very pronounced smear in the 30-64 KDa region. [0143]
  • To Blot A and B are applied: Lane 1: BCG crude extract, Lane 2: crude extract of a M. tuberculosis strain, Blot C: Lane 1: Molecular marker, Lane 2: not relevant, Lane 3: BCG crude extract, Lane 4: crude extract of an M. tuberculosis strain, Blot D: Lane 1: BCG crude extract, Lane 2: crude extract of an M. tuberculosis strain, Lane 3: Molecular marker. [0144]
  • Interpretation of Banding Patterns, Wherein Only the “Hallmarks” are Mentioned, is as Follows [0145]
  • Blot A/C: 29/33 KDa doublet in lane 1 and 2. [0146]
  • Blot B/D: 64/65 KDa doublet in lane 1 and 2. [0147]
  • The very intensive smear in the 30-64 KDa range on blot D is distinct. [0148]
  • Finally, FIG. 2[0149] c is an example of different western blotting patterns developed after incubation with representative sera of patients with Crohn's Disease. The “hallmark” patterns are shown in table 3 and are for Crohn's Disease a pronounced 45/48 KDa doublet.
  • Applied to blot A is: BCG crude extract, blot B: crude extract of an M. tuberculosis stain, blot C: Mycobacterium aviurn crude extract, blot D: molecular marker. [0150]
  • Interpretation of Banding Patterns is as Follows [0151]
  • All lanes show a distinctive coloring of the 45/48 KDa doublet positively, which indicates Crohn's Disease. The 45/48 KDa doublet reacts positively in 65% of all Crohn patients. [0152]
    • EXAMPLE 2 Enzyme Immunoassay
  • 1. Preparation of Antigens [0153]
  • The starting material prepared according to Example 1 was, depending on the chosen [0154] M. tuberculosis strain, centrifuged at 70,000×g to 120,000×g at 4° C. for 2 hours. The pellet was washed three times with PBS. Between the washing steps centrifuging took place at 70,000×g to 120,000×g at 4° C. for 2 hours. The pellet was collected and resuspended in 10 mL PBS. In addition to the supernatant in Example 1, SP-100 can also be used for immunoblots (Example 1) and enzyme immunoassays (this example). The suspension was subsequently sonicated for 2 minutes at 80 watts at 4° C. After the protein concentration was determined, quantities of 100 μL were frozen at a concentration of 1 mg/mL and stored at −80° C. until time of use (this preparation is designated with the term KP-100).
  • Thirty milligrams (30 mg) of the starting material was then applied in the presence of loading buffer onto a preparative 12% polyacrylamide gel of 0.5 cm thickness after 20 minutes of incubation at 65° C. Electrophoresis was carried out for 30 minutes at 150 V (stacking gel) and for 6 hours at 100 V (running or separating gel). The electrophoresis was stopped after the blue colorant band (“dry front”) had ran off the gel. The gel was then cut into horizontal strips of 2 mm thickness which in turn were divided into pieces of 1 cm length. The gel pieces were each eluted overnight at 4° C. in a tube with 5 mL sterile distilled water. Thorough mixing thereafter took place and the remaining gel pieces were centrifuged to the bottom. [0155]
  • The elution was checked using a 12% polyacrylamide analytical gel of 1.5 mm thickness. The gel was cast with a comb. After 20 minutes incubation at 65° C., 40 μL of each tube with gel pieces was placed in the slots in the presence of 10μL 5×loading mixture. The electrophoresis was carried out for 30 minutes at 150 V in the “stacking gel” and for 6 hours at 100 V in the “running or separating gel”. The electrophoresis was stopped and the gel made ready for preparation of a Western blot according to the procedure described in Example 1. Similar results were obtained using HPLC, FPLC, and other routine separating procedures. [0156]
  • To establish which fractions contain the relevant antigens, strips of the blot were incubated with sera of patients with lepromatous leprosy, tuberculous leprosy and Crohn's Disease. Shown herewith are respectively the 29/33 KDa antigens, the 64/65 KDa antigen and the 45/48 KDa antigens. The complex formation was visualized using anti-human IgG peroxidase conjugate and DAB. The desired fractions were collected, combined and used to cast a microtiter plate (see below). [0157]
  • 2. EIA [0158]
  • Microtiter plates are coated (via standard techniques) with either KP-100, SP-100, starting material, whole bacteria, 29/33 KDa, 64/65 KDa or 45/48 KDa antigens. [0159]
  • After coating, the plates are blocked with a 3% BSA solution in order to prevent a nonspecific binding of serum components. Plates are then dried and stored at 4° C. [0160]
  • 2.1. Tuberculosis EIA Test (Microtiter Plates Coated with KP-100) [0161]
  • Test sera are pipetted in a 1:100 dilution into the coated wells of a microtiter plate. The reaction takes place for 1 hour at 37° C. Nonspecific serum components and non-binding serum components are washed away with a washing buffer. A second incubation with a suitable dilution of an anti-human IgA peroxidase conjugate is carried out again for 1 hour at 37° C., and excess conjugate is then washed away. [0162]
  • Detection of human antibodies of the sub-type IgA binding specifically to KP-100 takes place by adding TMB (tetramethylbenzidine) to the wells. [0163]
  • Binding enzyme results in the occurrence of a blue color which, after addition of a coloring stop solution, changes to yellow. This yellow color has an absorption maximum of 450 nm. [0164]
  • The intensity of the resulting color is proportional to the amount of bound KP-100specific IgA. [0165]
  • The results are shown in the tables below. [0166]
  • In the described test patient and control sera are used from two different populations. [0167]
  • A=Endemic area (Africa, Ghana) [0168]
  • B=Non-endemic area (Europe, the Netherlands). [0169]
  • Each population is sub-divided into 4 sub-groups, namely: [0170]
  • Group 1=culture confirmed TB patients [0171]
  • Group 2=negative control group (normal healthy individuals) [0172]
  • Group 3=suspected positives (TB contacts) [0173]
  • Group 4=suspected negatives (no known data, but certainly no TB, possibly leprosy or other nonspecific mycobacteriosis) [0174]
  • The test is performed with two kits having different lot numbers and production dates. [0175]
  • Interpretation of the test results takes place on the basis of the so-called calibration line which is made up of control sera with a determined arbitrary unit definition which corresponds to a known OD value (1 unit, 4 units, and 8 units). [0176]
  • Each time a test is carried out the units are included in the assay. Found sample values can then be related to the unit definition. [0177]
  • A test serum can be considered positive when the result found in the test scores higher than 2.1 units. [0178]
  • A test serum can be considered negative when the result found scores lower than 1.2 units. [0179]
  • Test sera with unit values between 2.1 and 1.2 units fall into the set so-called reconfirmation zone. This means that in the first instance positivity or negativity for tuberculosis cannot be determined with this test. [0180]
  • Reconfirmation of these sera takes place using the described Western blot strips with which, after serum incubation on the basis of banding patterns and specific “hallmarks”, an answer can be given to the question of whether the test serum is positive (bands present) or negative (bands absent). [0181]
    TABLE 3
    Population A. Endemic range:
    Group 1:
    SERUM NO. # UNITS TEST SCORE REMARKS
    1 2.44 positive culture positive
    2 3.43 positive culture positive
    3 1.78 reconfirmation culture positive
    4 1.37 reconfirmation culture positive
    5 5.74 positive culture positive
    6 3.21 positive culture positive
    7 1.66 reconfirmation culture positive
    8 2.00 reconfirmation culture positive
    9 2.00 reconfirmation culture positive
  • [0182]
    TABLE 4
    Population A.
    Group 2:
    SERUM NO. # UNITS TEST SCORE REMARKS
    1.16 negative healthy individual
    11 0.86 negative healthy individual
    12 0.77 negative healthy individual
    13 0.64 negative healthy individual
    14 0.74 negative healthy individual
    15 0.79 negative healthy individual
  • [0183]
    TABLE 5
    Population A.
    Group 3:
    SERUM NO. # UNITS TEST SCORE REMARKS
    16 3.13 positive sick individual
    17 1.57 reconfirmation sick individual
    18 1.59 reconfirmation sick individual
    19 5.39 positive sick individual
    20 1.97 reconfirmation sick individual
    21 2.29 positive sick individual
    22 0.48 negative sick individual
  • [0184]
    TABLE 6
    Population A.
    Group 4:
    SERUM NO. # UNITS TEST SCORE REMARKS
    23 1.54 reconfirmation normal control
    24 1.76 reconfirmation normal control
    25 0.58 negative culture negative
    26 1.03 negative culture negative
    27 0.79 negative culture negative
    28 0.89 negative culture negative
  • [0185]
    TABLE 7
    Population B. Non-Endemic range:
    Group 1:
    SERUM NO. # UNITS TEST SCORE REMARKS
    1 2.24 positive culture positive
    2 2.53 positive culture positive
    3 4.40 positive culture positive
    4 14.95 positive culture positive
    5 16.82 positive culture positive
    6 10.54 positive culture positive
    7 5.70 positive culture positive
    8 6.72 positive culture positive
    9 5.06 positive culture positive
  • [0186]
    TABLE 8
    Population B.
    Group 2:
    SERUM NO. # UNITS TEST SCORE REMARKS
    10 1.04 negative healthy individual
    11 0.92 negative healthy individual
    12 0.85 negative healthy individual
    13 0.20 negative healthy individual
    14 0.42 negative healthy individual
    15 0.90 negative healthy individual
    16 0.35 negative healthy individual
    17 0.73 negative healthy individual
  • [0187]
    TABLE 9
    Population B.
    Group 3:
    SERUM NO. # UNITS TEST SCORE REMARKS
    18 2.23 positive sick individual
    19 4.70 positive sick individual
    20 1.22 reconfirmation sick individual
    21 1.43 reconfirmation sick individual
    22 2.21 positive sick individual
    with TB history
    23 6.38 positive sick individual
    24 1.59 reconfirmation sick individual
  • [0188]
    TABLE 10
    Population B.
    Group 4:
    SERUM NO. # UNITS TEST SCORE REMARKS
    25 0.20 negative patient resistant to
    drug therapy
  • Thus, while there have been described what are presently believed to be the preferred embodiments of the present invention, those skilled in the art will realize that other and further embodiments can be made without departing from the spirit of the invention, and it is intended to include all such further modifications and changes as come within the true scope of the claims set forth herein. [0189]

Claims (22)

What is claimed is:
1. A method for detecting or identifying a Mycobacterium species in a biological sample, comprising:
(a) selecting a standard Mycobacterium;
(b) preparing an antigen preparation from a culture of the standard Mycobacterium, wherein the antigen preparation comprises at least two immuno-cross-reactive antigen components (ImCRACs);
(c) separating the ImCRACs of the antigen preparation according to molecular weight;
(d) binding the separated IMCRACS of the antigen preparation to a solid carrier;
(e) contacting the bound ImCRACs with the biological sample under conditions that permit antibodies in the biological sample to bind with the carrier-bound ImCRACs to provide a pattern of carrier-bound antibody-ImCRAC complexes;
(f) detecting the pattern of carrier-bound antibody-ImCRAC complexes; and
(g) comparing the pattern of carrier-bound antibody-ImCRAC complexes from the sample to a library of standard patterns of carrier-bound antibody-ImCRAC complexes characteristic of particular Mycobacterium species,
wherein matching of the pattern of carrier-bound antibody-ImCRAC complexes from the sample to one of the standard patterns of carrier-bound antibody-ImCRAC complexes from the library permits detection or identification of the Mycobacterium species in the sample.
2. The method of claim 1, wherein the antigen preparation is separated by electrophoresis prior to step (d) and the carrier is a membrane to which the ImCRACS are bound by means of electroblotting.
3. The method of claim 2, wherein the membrane is a nitrocellulose membrane.
4. The method of claim 1, wherein the antigen preparation of the standard Mycobacterium is a total protein preparation.
5. The method of claim 4, wherein the antigen preparation is a KP-100 or SP-100 fraction of the total protein preparation.
6. The method of claim 1, wherein the carrier is a microtiter plate.
7. The method of claim 1, wherein the pattern of the carrier-bound antibody-ImCRAC complexes is a banding pattern consisting of 29/33 KDa, 45/48 KDa, 64 KDa and combinations thereof.
8. The method of claim 1, wherein the carrier-bound antibody-ImCRAC complexes are made visualizable by using at least one antibody-enzyme conjugate directed against at least one antibody of an isotype selected from the group consisting of IgG, IgM, IgA and combinations thereof.
9. The method of claim 8, wherein the enzyme of the antibody-enzyme conjugate is peroxidase.
10. The method of claim 1, wherein the carrier is a dip-stick.
11. A method for detecting or identifying a Mycobacterium species in a biological sample, comprising:
(a) providing a test substrate having bound thereto an antigen preparation from a culture of the standard Mycobacterium, wherein the antigen preparation comprises at least two immuno-cross-reactive antigen components (ImCRACs) such that the ImCRACs of the antigen preparation separate according to molecular weight;
(b) contacting the bound ImCRACs of the test substrate with the biological sample under conditions that permit antibodies in the biological sample to bind with the substrate-bound ImCRACs to provide a pattern of substrate-bound antibody-ImCRAC complexes;
(c) detecting the pattern of substrate-bound antibody-ImCRAC complexes; and
(d) comparing the pattern of substrate-bound antibody-ImCRAC complexes from the sample to a library of standard patterns of substrate-bound antibody-ImCRAC complexes characteristic of particular Mycobacterium species,
wherein matching of the pattern of substrate-bound antibody-ImCRAC-complexes from the sample to one of the standard patterns of substrate-bound antibody-ImCRAC complexes from the library permits detection or identification of the Mycobacterium species in the sample.
12. The method of claim 11, wherein the antigen preparation is separated by electrophoresis and the substrate is a membrane to which the IMCRACS are bound by means of electroblotting.
13. The method of claim 12, wherein the membrane is a nitrocellulose membrane.
14. The method of claim 11, wherein the antigen preparation of the standard Mycobacterium is a total protein preparation.
15. The method of claim 14, wherein the antigen preparation is a KP-100 or SP-100 fraction of the total protein preparation.
16. The method of claim 11, wherein the substrate is a microtiter plate.
17. The method of claim 11, wherein the pattern of the substrate-bound antibody-ImCRAC complexes is a banding pattern consisting of 29/33 KDa, 45/48 KDa, 64 KDa and combinations thereof.
18. The method of claim 11, wherein the substrate-bound antibody-ImCRAC complexes are made visualizable by using at least one antibody-enzyme conjugate directed against at least one antibody of an isotype selected from the group consisting of IgG, IgM, IgA and combinations thereof.
19. The method of claim 18, wherein the enzyme of the antibody-enzyme conjugate is peroxidase.
20. The method of claim 11, wherein the substrate is a dip-stick.
21. Diagnostic kit comprising:
(1) a test substrate having bound thereto an antigen preparation from a culture of standard Mycobacterium,
wherein the antigen preparation comprises at least two immuno-cross-reactive antigen components (ImCRACs) that are separable according to molecular weight, and
wherein when the ImCRACs of the test substrate are contacted with a biological sample under conditions that permit antibodies in the biological sample to bind with the substrate-bound ImCRACs, a pattern of substrate-bound antibody-ImCRAC complexes is provided; and
(2) a means for visualizing the pattern substrate-bound antibody-ImCRAC complexes.
22. The diagnostic kit of claim 21, wherein the test substrate is selected from the group consisting of a dip-stick, a microtiter plate, and an immunoblot.
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WO2005045441A1 (en) * 2003-10-31 2005-05-19 Plasmacute As Assay
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RU2473095C2 (en) * 2007-12-28 2013-01-20 БиЭл КО., ЛТД Analysis of mycobacterium tuberculosis complex immunodetection
US9739773B1 (en) 2010-08-13 2017-08-22 David Gordon Bermudes Compositions and methods for determining successful immunization by one or more vaccines

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