CA1303983C - Solid phase assay - Google Patents

Solid phase assay

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Publication number
CA1303983C
CA1303983C CA000557276A CA557276A CA1303983C CA 1303983 C CA1303983 C CA 1303983C CA 000557276 A CA000557276 A CA 000557276A CA 557276 A CA557276 A CA 557276A CA 1303983 C CA1303983 C CA 1303983C
Authority
CA
Canada
Prior art keywords
tracer
analyte
binder
solid support
assay
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
CA000557276A
Other languages
French (fr)
Inventor
Robert W. Rosenstein
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Becton Dickinson and Co
Original Assignee
Becton Dickinson and Co
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Filing date
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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/805—Test papers
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/81—Packaged device or kit
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/97—Test strip or test slide
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/807—Apparatus included in process claim, e.g. physical support structures
    • Y10S436/81—Tube, bottle, or dipstick
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/829—Liposomes, e.g. encapsulation

Abstract

Abstract of the Disclosure A strip is used in an assay for an analyte wherein first and second portions of the strip are in capillary flow communication with each other, with the first portion including a tracer and the second portion a binder for at least the analyte whereby when the first portion is wetted with sample suspected of containing analyte, the analyte and tracer flow by capillarity to the second portion.

Description

bS / 13~3~ 3 ~,D SOLID PHASE ASSAY
P/~ 7 This invention relates to an assay for an ~nalyte, and more particul~rly to a solid ph~se assay.
Assays for various analytes have been accomplished by so-called solid phase assay. In a solid phase assay, a binder specific for at least the ligand to be determined (analyte) is supported on ~ solid ~upport, whereby, in the assay it is noi necessary to employ an additional agent for separating the bound and free phases formed in the assay.
In general, such solid supports have been in the form of tubes, soli~ particles, and in some cases, the solid phase has been in the form of a "dip-stick".
In a dip-stick solid phase assay, a binder may be supported on the dip-stick with the dip-stick, containing the binder, being dipped into an ~ssay solution containing the analyte, and in general, such solution further contains a tracer. The presence and/or ~mount of tracer on the dip-stick is then employed d9 a measure of analy~ (either a qualitative or quantit~tive mea~ure of analyte).
The present invention is directed to providing an improved solid phase assay for determining anAlyte, end more particularly to a solid phase assay.
In accordance with one asp~ct of the present invention, there i~ provided a solid support having a ~irst portion and a second portion with the first an~ second portions being in capillary flow communi~ation with ea~h other whsreby material flows by capillarity. The first and second portions are positioned on the solid support in a m~nner such that the firs~
portion may be contacted with mat~rial, in~luding any analyte, with material in said ~irst portion being transported by ~3(?3~3 capi}l~rity from the first portion of the support to the second portion thereof.
T~e second portion of the solid support ineludes ~ binder which is a binder for ~t least the analyte7 with the binder also being a binder for a tracer used in the ~ssay, when the RSSay format is a so-cQlled competiti~e ~ssay format.
The solid support also includes a tr~cer, which is comprised of a ligand portion and a detectable label portion conjugated to the ligand portion of the tracer. In the case where the æssay format is a so-called competiti~e assay format~
the ligand portion of the trQcer is bound by the blnder contained in the second portion of the solid support. In the case where the assay format is a so-called sandwich assay format, the ligand portion of the tracer is bound by the analyte .
The tracer is supported on the solid support on a tracer portion of the solid support in a m~nner such that when wetted, the trecer is eapable o~ being tr~nsported by capillarity to the second portion of the solid support, and thereafter, depending on the presence ~nd/or absence of analyte and/or the amount Or analyte, ~s hereina~ter explained in more detail, to a third portion of the solid support.
The tr~eer portion of the solid support may be a separats portion of tbe solid support or may be the first portion of the solid support (the portion to which sample is added~.
The binder which is supported on the secon~ portion of the solid ph~se is supported in a manner such th~t the binder remains immobile Qnd is not transported by capillarity to the third portion of the solid support~

~3~3~33 The third portion of the solid support may be a portion for detecting trQcer which has been transpor~ed by capill~rity from the second portion to the third portion. The third portion may or may not include a substance supported ~hereon for detecting ~r~cer. Alternatively9 the third portion may function only to receive materials not bound in the second portion.
In ~ccordQnce with the present invention, the amount of tra~er which is immobilized in the second portion of the solid support by beinK bound either directly to the binder in the second portion (in a competitive assay form~t), or by being indirectly bound to the binder ~tra~er is bound to analyte which is bound to the binder in a sandwich assay format) i5 dependent upon the presence and/or amount of Qnalyte in the sample. In a so-called sandwich assay format, the amount of tracer which is passed from the second portion to the third portion of the solid support by capillarity is indirectly proportional to t~e ~mount of anQlyee in the s~mplet and in the so-called competitive a~s~y format9 the amount of tracer which passes from the second portion to the third portion of the solid ~upport, by capillarity, is directly proportional to t~e amount of ~nalyte in t~e sample.
In a preferred embodirnent of the present invention, the solid support and the various components are produced and employed in a manner for determining analyte by ~ competitive assay format, with the tr~cer bein~ supported on the first portion of the solid support.
In a particularly pre~erred embodiment, as hereinQfter explained in more detail, the detectable label portion of the ~3~3~13 tracer is comprised of a sac or lipid vesicle (often referred to as a liposome), which includes a detectable label.
In employing a preferred embodiment wherein the ass~y is a competitive ~ssay, the tracer is supported on the solid support on the first portion thereof, and the first portion of the solid support is wetted with the sample contRining analyte to be determined. Upon wetting of the solid support with the sample, both sample and tracer flow by capillarity into the second portion of the solid support which contains a binder specifie for both the ~n~lyte and tracer, with the binder being immobili~ed on the second portion of the solid support.
Depending upon the presence ~nd/or amount of Pnalyte in the sample portion, trQcer becomes bound to the binder on the second portion of the solid sùpport. The tr~cer which is not bound by the binder on the second portion, then flows by capill~rity into the third portion of the solid support for detection and/or determination therein. If the assay format is to be a simple "yes or no" ~ormQt (only determining whether or not ~nalyte i9 present in the sample), then the binder supported on the second portion of the solid support is supported in an ~mount such that in the absence of a detect~ble amount of analyte in the sample, there is no detectable presence of tracer in the third portion of the solid support.
As should be app~rent, as the amount of analyte in the sample increases, the amount of tracer which is not bound to the binder in the second portion of the solid support increases, thereby increasing the amount o~ tracer present in the third portion of the solid support. Aceordingly3 a quantitative y m~y be run by determining tracer which rem~ins in the second portion of the solid support and/or whieh flows by ~L3~3~
c~pillarity into the third portion of the solid support, and comp~ring such detected amount of tracer in the second and/or third portion with a "standard curve" to determine the amount of analyte in the sample. T~us, in an assay the determination of tracer and/or anQlyte may be either qu~l i tative or qu~ntitative.
In the sandwich assay format, tr~cer is preferably supported on a ~racer portion of the solid support which is different from the fir~t portion of the solid support. The ligsnd portion of the tracer is bound by the ~nRlyte, with the binder in the second portion o~ the solid support being specific for the analyte. T~e first portion of the solid support is contacted with the sample containing analy~e, and the tracer portion of the solid support is ~etted to cause both the tracer and ~nalyte to flow by capillarity to the binder supported by t~e second portion of the support. The amount of tracer whi~ becomes bound to analyte is directly proportional to the amount of anRlyte in the sample, and tracer bound ~o analyte, as well 85 any unbound tracer, flow by capillarity to the second portion of the solid support. In the second portion of the solid support, analy~e be~omes bound to immobilized binder specific for the analyte, with the unbollnd tracer (tracer not bound to analyte w~ich is bound to the i~mobilized binder) flows by ~apill~rity to the third portion of the solid support. The tracer on the third portion of the solid support may be detected as a measure of the presenee and/or arnount of analyte in the sample.
In Q '1yes or no" sandwich ~ssay type form~t, the amount of tr~eer which is employed on the first portion of the solid support as well ~s the amount of binder on the second portion ~..3~3~
of the solid support are such that in the presence of a detectable ~mount of analyte, essentially no detectRble tracer flows into the third portion of the solid support.
In a sandwich assay format, the Qmount o~ binder which is employed on the second portion of the solid support is an amount such that essentially all of the an~ly~e which is suspected of being present in the sample is bound by the binder on the second portion.
The solid support which is employed in the assay is one which is capable of absorbing analyte from the sample, ~nd which, when wetted, provides for flow of analyte and tracer by capillary ~ttraction from the first portion, and through the second portion into the third portion of the solid support. In addition, the solid support is one which is capable of supporting tracer and the binder. As repreSentQtiVe examples of suitable solid supports there may be mentioned: glass fiber, eellulose, nylon, crosslinked dextran, various chrom~tographic papers, nitrocellulose, etc. A pArticul~rly preferred material is nitrocellulose.
The solid support is preferably shaped in the form of a strip, with the first, second and third portions being Hrranged on the strip in the same plane in a manner such that material can flow by capillary attr~ction from the first zone and through the second zone to the third zone. Although the preferred shape i~ in the form of a strip, any other of a wide variety of shapes or forms may be employed as long as the shape and form permits separate portions for performing the various functions; as hereinabove described.
The tracer employed in the assay, as hereinabove indicated, is comprised of ~ ligand portion and A deteCtQble - ~3~3~3 la~el portion conjugated to the ligand portion. The detectable label of the detectable label portion m~y be any one of a wide variety of detect~ble labels; however, in accordance with a preferred em~odiment, the detect~ble label is one which provides a color change in the second and/or third portion of the solid support, which is either a visible color change, or one which requires an instrwnent to detect the change in color.
In accordance with a preferred embodiment, the l~bel which is employed provides a change in color in the second and/or third portion of the solid support which is visible without the use of an instrument. For ex~rnple, such a change in color may be provided by employing an en~yme as the detectable label, and ~y providing a sùbstrate for the enzyme in the third portion of the solid support, which substrate, when contacted with the enzyme, provides Q visible detectable change in color.
Alt0rnatively, the detectable label may be the substrate, and the third portion of the solid support may be provided with the en2yme, whereby ther2 is a detectable ~hange in color in the third portion by contacting of the enzyme with the substrate label. As represent~tiv~ examples of other detectable labels, which may or m~y not require an instrument for detecting a color change9 there m~y be menSioned various chromogens, such as fluorescent materials, absorbing dyes, and the lik~. As hereinafter indicated in a compe~itive assay~ a preferred label portion is a vesicle, which includes a det~table marker, with the detectable marker being one which is visible.
The ligand portion of the tr~cer is dependent upon the assay format. If the assay is a competitive assay, then the ligand portion of the tracer is ~ither the analyte or an appropriate analogue thereo~. An appropriate analogue means 3~3~3~l~3 that the analogue of the ligand is also speci f iCQI ly bound by the birlder for the an~lyte. If the assay format is ~ sandwich type of assay, then the lig~nd portion of the tracer is ~
ligand which is specifically bound by the an~lyte or by an antibody which is specifi~ally bound by the analyte.
The binder w~ich is employed in the assay is one which ~t least binds the ~nalyte. As hereinabove indicated, if the ass~y format is 6 competitive type of assay format, then the binder also binds the lig~nd portion of the tracer.
As generally known in the art, i~ the analyte is an antigen or a hapten, then the binder may be either a naturally occuring binder or an ~ntibody which is specific for the ~nalyte (either a polyclonal and/or monoclonal antibody). If the analyte is an antibody, the binder may be either an antigen specific for the antibody or an antibody which specifically binds the antibody analyte.
The binder may be supported on the solid support in manner whic~ immobilizes the binder; e.g., adsorption, eovalent coupling, etc. The procedures ~or immobilizing binders on a solid support ~re generally known in the ~rt.
The tracer9 when supported on the first portion of the solid support, is supported in ~ manner such th~t when the first portion is wetted the tracer 10ws by capillary action.
Thus9 for example1 the tra~er may be absorbed on the first portion of t~e support.
In accordance w;th a particularly preferred embodiment of the present invention, in a competitive assay, the tracer is comprised of a ligand conjugated to ~ vesicle, which vesicle contains ~ detectable marker, with the tr~cer being supported on the solid support. Applicant has foun~ that i~ is possible ~3~3~
to support such ~ tr~er on a solid support of the type hereinabove described, and that such tr~cer will flow by capillarity when the solid support is wetted with ~ s~mple containing or suspected of eontaining an anQlyte.
The lipid vesicles (liposomes) which ~re employe~ may be prep~red from a wide variety of lipids, including phospholipids, glycol lipids, and as representative examples there may be mentioned lecithin, spingomyelin, dipalmitoyl le~ithin, distearoylphosphatidylcholine, etc. The ~mphiphilic lipids employed for producing liposomes generally have a hydrophilic group, such as a phosphato, carboxyli~, sulfato, or amino group, and a hydrophobic group, such as saturated and unsaturated aliphatic hydrocarbons, and aliphatic hydrocarbon groups substituted by one or more aromHtic or cycloaliphatic groups. The wall forming compounds for producing the liposomes may further include a steroid component such as cholesterol, cholest~nol, and the like. The compounds for producing liposomes are generally known in the art, and no further detai 15 in this respect are deemed necessary for ~ complete understsnding of the present invention.
The }iposomes m~y be produced by procedures generally available in the art. For example, liposomes mfly be produced by ~ revers~ phase ev~por~tion ~echnique wherein the compound or compounds used in producing liposomes are initially dissolved in an organic ph~se9 followed by ~ddition of an aqueous phase and forming of a homogeneous emulsion. After forming the emulsion, the organic solvent is evaporated to form Q gel like materi~l, and such gel may be converted to a liposome by agit~tion or dispersion in an agueous media.

_g _ ~3~?3~
Procedures for producing liposomes are described~ for ex~mple, in U.S. Patent No. 4,241,~46; U.S Patent No.
4,342,826 and PCT Intern~tional Publica~ion No. WO 80-01515.
If a m~terial is to be encapsulated in the liposome, such m~terial may be encapsul~ted in the liposome by including the material in the aqueous solution in which the- liposome is formed. Alternatively, the material may be encapsulated into a previously formed e~ty liposome (without materiAl to be encapsulated) The liposomes may also be produced by the procedures disclosed in U.S. P~tent No. 4,522,803.
The material which is entrapped or encapsul~ted wit~in the liposome (the material is within the aqueous compartment or within the membrane bilayer of the liposome) is a detectable marker, such as dyesg radiolabels, fluorescent materials, chemilumines~ent materials, electton spin reSonQnCe materials, and the like; substrates for detectable m~rkers; and the like.
AlternQtively, thé liposome may be derivatized with Q
detectable marker, rather than entrapping a marker in the liposome.
The liposome is derivatized with a ligand for producing a tracer. The liposome may be derivatized with a ligand by procedures known in the art, such as covQle~t coupling, derivatization or activation, etc. In derivatizing the liposomes with a ligand, a compound or compounds used in forming the liposome may be derivRtized with the ligand, prior to forming the liposome~ or alternatively, the liposome may be derivatized with the ligand, subsequent to forming of the liposome. Procedures for derivatizin~ liposomes with ligQnds, ~3~3~3 and suitable coupling agents, and the like for preparing derivatized liposomes are known in the ~rt, and no further detsils in this respect are deemed necessary for a complete understanding of the present invention.
In employing a preferred tracer in which the detectable m~rker portion thereof is comprised of a liposome including a detectable marker for use in a competitive assay, the assay may be accomplished as herein~bove described with gener~l reference to a variety of traeers, except that the tracer includes a liposome 8S the detectable marker portion of the tracer.
In a particularly pre~erred embodiment, at tracer used in the ~ssay is a ligand conjugated to a particulate l~bel which is visible. The particulate label may be a metal or alloy (e.g. colloidal gold) or a sac in particul~r a liposome containing a visible dye. The m~rker pre~erably included in the SQC iS a dye or some other material which is visible, without lysing of the sacs.
The tracer comprised of ligand and particulate label may ~lso be produced by labeling the ligand with an aqueous disper~ion of a hydrophobic dye or pigment, or of polymer nuclei coated with such a dye or pigment. Such labels are described in more detail in U~S. Patent No. 4,373,932, which issued on February 15, 1983. The tracers produced in accordance with such p~tent may also be employed as tracers in the present invention.
As indicPted in the aforesaid patent, the colored organic compounds which are used as labels are in the form of a hydrophobic sol, which hydrophobic organic dyes or pigments are insoluble in water or soluble only to a very limited extent.

~3~3~
The visible particulate label may be visible polymer p~rticles, such as colored polystyrene particles, prefer~bly of spherical shape.
As representative examples of oth~r p~rtieulate lsbels which may be employed in producing a tracer for use in the assay of the present invention; in which the tracer would be visible, there may be mentioned; ferritin, phycoerythrins o~
other phycobili-proteins; precipitated or insoluble metals or ~lloys; fungal, ~lgal, or bacterial pigments or derivatives such as bacterial chlorophylls; plant mater i81s or derivative metal sols and the like. In such an embodiment, at least the portion of the product which includes the binder is formed of a material having fl ~urface area cap~ble of supporting the binder thereon in an amount such that tracer bound in such portion is visible. In general, the surface ~rea is caphble o~ supporting the binder in a concentration of at least 1 ug/em2, and most generally in a concentration of ~t le~st 10 ug/cm2. .
particularly preferred m~terial is nitro-~ellulose.

The invention will be further described with reference to the ~ccompQnying drawing, wherein:
The drawing is a schematic drawin~ of a dip-stick in accordance with the present invention.
Referrin~ to the drawing, there is shown a strip including a first portion A on which a tracer is supported; a second portion B on whi~h ~ binder is supp4rted and a third portion D
in which tracer may be determined. As p~rticularly shown, a portion C is between portions B and D to provide spacing ! ,~ r ` 9~3~3~

between portions B and D, whereby the portion for determining tracer is separ~ted by a distance from the portion cont~ining binder.
In a competitive ~ssay format, employing an enzyme ~s a detect~ble label, portion A would contain lig~nd labeled with enzyme, with the ligand portion being the analyte or appropriate an~logue thereof; portion B would contain a binder specific for the analyte and the ligand portion of the tracer;
Hnd portion D would contain a substrate for the enzyme which interacts with the enzyme to provide a change in color.
In use, portion A of the strip 10 would be contacted with a sample containing analyte, whereby portion A would be wet with the sample. The tracer in portion A, as well as, sample would be transported by caplllarity to portion B, where tracer and anQlyee compete for binding sites on the binder. Unbound tracer and unbound analyte move by capillarity through portion C to portion D where any tracer interacts with the substrate in portion D to provide a change in color. As hereinabove indicated, the assay may be a "yes-no" assay or a quantitative assay and detection of tracer in portion D is dependent upon the Q~ say employed.
In ~he case where the tr~cer hss Q detect~ble l&bel which doss not r2gui~e an additional substance for determination thereof, the portion D would not require an additional substance, i.e., portion D would also be bl~nk. Thus, for example if the tracer included a liposome h~ving a dye as a detectable label, then tsacer may be determined without supporting an additional substance on portion D.
; Alternatively, if for example, it was required to release detectable lable from the liposome, portion D c~uld contain R

``` ~3~3~3 suitable lysing agent, such as an enzyme or detergent which lyses liposomes to release label from the liposome in portion D
for detection of tracer.
In ~ddition, it is also possible to determine tracer in portion C, with or without determining tracer in portion D.
For example, a substrate could be ~dded eO portion C in the case where the label is an enzyme.
The product may be used ~s 8 dip stick. Alternatively, Q
sample may be applied to portion A. Accordingly, the product may be used in either a horizontal or vertical orientation.
The invention is applicable ~o detecting ~nd/or measuring a wide varie~y of analytes, such as: drugs, including therapeutic drugs and drugs of sbuse; hormones, vitamins, proteins, including antibodies of all classes, peptides;
steroids; bacteria; fungi; viruses; parasites~ components or products of bacteria, fungi, viruses, or parasites; allergens of all types; products or components of norm~l or malignant cells; etc. As pnr~icular examples, there may be mentione~ T~;
T3; digoxin; h~G; insulin; theophylline; leutinizing hormone;
organisms causing or associated with v~rious dis~ase states, such as streptococcus pyrogenes (group A), Herpes Simplex I and Il, cytomegalovirus, c~lamydiae, rubella antibody, etc.
The invention will be further described with reference ~o the following example:

Dipstieks were constructed by iirst coating 005 x 8 cm strips of polystyrene with 5cotch(~) #96~ adhesive transfer tape (3M, St. Paul Minnesota 55144). Zone B, consisting of a 0.5 ~ 0.5 cm square of 5 um pore nitrocellulose (S~S7 Keene, New Hampshire) was spotted with 3 ~1 of affinity purified ~3V3~3~3 rabbit anti-Group A Streptococcus antigen and then blocked with 3~ bovine serum ~lbumin. After drying, it was applied to the taped side of the dipstick, approximately 1 cm from the bottom of the stick. A strip of filter paper 0.5 x 6.S cm. (Whatman 3 mm) was applied just above and touc~ing the nitrocellulose, at the positions indicated by zones C and D. Zone A, consisting of dry Sephadex G50 fine grade (PhQrmacia) WQS then applied.
Detector liposomes packed with sulfo-rhodamine dye were prepared by the method outlined in O'Connell et al. (Clin.
Chem. 31:1424 [1985]). They were covalently coupled to affinity purified rQbbit anti~Group A Streptococcus antigen.
The detector liposomes were spotted (2 ul~ onto Zone A, 0.5 cm from the bottom and air dried. The liposomes are in a 0.05 M Tris buffer, pH 6.8, containing 2% glycerol, 0.05%
dimethyl sulfoxide, 20 m M EDTA.
Group A Streptococcus org~nisms were harvested from culture plates, washed with saline (0O9% NQC1), and adjusted to 1 x 109 organi~ms/ml. An aliquot (0.1 ml) ~ontaining 1 x 108 org~nisms was subjected to the micro nitrous acid extraction method for exposing the Group A carbohydrate antigen. This method consists of mixing 0.3 ml o~ 0.1 M HCl with 4D ul of 4 NaN02, adding this to the ~ organism3 and, after 3 minutes, neturalizing with 40 ul of lM Tris base. To faciliate the extractisn and the dips~ick ~5say9 ~he HCl and the subsequent diluting ~luid contain 0.1% Tween-20 non-ionic detergent.
Using the extracted Qntigen, a dilution series was prepared ranging from 8 x 106 organisms/ml ~o 1025 X 105 organisms/ml. Aliquots of these dilutions (0.5 ml) were placed in 12 x 75 mm test tubes and a dipstick placed into the fluid * Trademarks 1~-~3~3~3 in each test tube. As the fluid containing extracted antigen wicks up the stick, it carries the liposome detector past the spot of capture antibody. In the presence of antigen, which binds to the capture antibody spot, some oP the liposomes also bind, resulting in the appearance of a red spot in zone B. The remainder of the liposomes and antigen solution pass into zone D.
The assay can be "reAd" by observing the lowest ~oncentration of organisms resulting in ~ red spot in zone B.
The results of this example are given in the following table and indicate an end point of 5 x 105 organisms/ml, close to the sensitivity required for a direct throat sw~b diagnostic for Group A Streptococcus pharyngitis.

Group A Strep_Ant~en (organisms/ml) x 10-5 40 20 10 5 2.5 1.25 0 _ .
+ + +
tl) = positive indication of antigen (red spot) (-) = negative i~dieation of antigen (red spot) The present invention is advantageous in that there is provided c product and process whi~h may be easily employed for acoomplishing an assay. The product and process do not require the addition of tracer in that traeer is included in the product. In addition, the product ~nd process are capable of providing for a rapid assay.
These and other advantages should be apparent to those skilled in the are from the teachings herein.
Numerous modific~tions and variations of the present invention are possible in light of the above teachings;

.

- " ' . , . ~ :

13~?39~

therefore, the invention may be practiced otherwise than ~s p~rticul~rly described.

! 17

Claims (10)

1. An article for use in an assay for an analyte in a liquid sample, comprising:
a solid absorbent support, including at least a first portion and a second portion, said first and second portions being spaced from each other along a surface of the support, said second portion containing a binder immobilized in said second portion, said binder being specific for the analyte; said first portion containing a tracer comprised of a ligand conjugated to a liposome having a detectable marker, said ligand being bound directly or indirectly to the binder in an assay, said tracer being movably supported in said first portion, said first and second portions being in capillary flow communication with each other whereby when the first portion is wetted and a liquid sample suspected of containing analyte is applied to the support, tracer from the first portion and any analyte flow by capillarity to the second portion and across said binder in said second portion.
2. The article of Claim 1 wherein the solid support is a strip and the first and second portions are in the same plane.
3. The article of Claim 1 wherein the tracer is comprised of a ligand conjugated to a sac having a detectable marker.
4. The article of Claim 2 wherein the second portion is comprised of a nitrocellulose.
5. The article of Claim 2 wherein the ligand portion of the tracer is bound by the binder immobilized on the second portion.
6. The article of Claim 3 wherein the sac includes a visible marker.
7. The article of Claim 6 wherein the sac is a liposome.
8. The article of Claim 1 wherein the solid support includes a third portion in capillary flow communication with the second portion whereby material flows by capillarity from the second portion to the third portion.
9. The article of Claim 2 wherein the tracer is comprised of a ligand conjugated to a sac having a detectable marker.
10. An assay for an analyte, comprising:
contacting a sample suspected of containing analyte to be assayed with the first portion of the article of any one of Claims 1-9; and determining at least one of the tracers which is bound or not bound in the second portion.
CA000557276A 1987-03-27 1988-01-25 Solid phase assay Expired - Lifetime CA1303983C (en)

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