CA1308350C - Multiple antigen immunoassay - Google Patents
Multiple antigen immunoassayInfo
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- CA1308350C CA1308350C CA000566734A CA566734A CA1308350C CA 1308350 C CA1308350 C CA 1308350C CA 000566734 A CA000566734 A CA 000566734A CA 566734 A CA566734 A CA 566734A CA 1308350 C CA1308350 C CA 1308350C
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
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Abstract
ABSTRACT
The invention relates to an assay for one or more analytes which comprises contacting a sample suspected of containing one or more analytes with a solid phase containing one or more different antigen specific antibodies separately immobilized to defined areas on the solid phase, followed by indirectly detecting the presence of bound antigen by titrating the unbound immobilized antibodies with a titrating antibody which is specific for the first antibody. This assay allows the simultaneous detection of a multiplicity of antigens in a single assay.
A24.1.WP 051287
The invention relates to an assay for one or more analytes which comprises contacting a sample suspected of containing one or more analytes with a solid phase containing one or more different antigen specific antibodies separately immobilized to defined areas on the solid phase, followed by indirectly detecting the presence of bound antigen by titrating the unbound immobilized antibodies with a titrating antibody which is specific for the first antibody. This assay allows the simultaneous detection of a multiplicity of antigens in a single assay.
A24.1.WP 051287
Description
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TITL~ OF T~oe INYENTIONo MULTIPh~_~NTIGEN IMMUNOASSAY
Field of the In~ention The invention relates to a method for the assay of an analyte which may be present in a sample by r~acting a sample suspected o~ containing said analyte with antibodies immobi-lized to a solid support to form an antibody-antigen complex, followed by titrating the unoccupied antibody with a second labeled antibody which is specific to the first immobilized antibody, ~ollowed by detecting the label.
BAC~GROUND OF THE INVENTION
The detection and quantitation o~ antigenic substances in biological samples ~requently utilize immunoassay kechni-ques. These technigues are:based upon the formation of a ¢omplex between the antigenic substance being assayed and an antibody or antibodies in~which one or the other member of the ~omplex may be detectably labeled. ~ith competitiYe immunoassay ~echniques, ~he antigenic substance in a sample fluid being tested competes with a known quantity o~ labeled antigen ~or a limited quantity of antibody binding si~es.
The amount of labeled antigen bound to the antibody is inversely proportional to the amount of antigen in a sample.
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By contrast, most immunometric assays employ a labeled antibody. In such an assay, the amount of labeled antibody associated with the complex is directly proportional to the amount of antigenic substance in a fluid sample.
In sandwich immunome~ric assays, a ~uantity o~ unlabeled antibody is bound to a solid support which is insoluble in the fluid being tested. This immobilized antibody is first contacted with the sample being tested so that a binary antigen-antibody complex is ~ormed. After a suitable incubation period, the solid support is washed to remove unbound antigens, then contacted with a solution containing a known quantity of a second antibody. After a second incuba-tion period, the solid support is then washed a second time to remove the unreacted antibody. A labeled anti-antibody to the second antibody is then added, allowed ~o incubate for a sufficient amount of time, and the complex then ~ashed. The washed solid support is then tested to detect and quantify the presence of labeled antibody, for example by measuring the emitted radiation o~ a radioactive label. The amount of labeled antibody detected is compared to that for a negative control sample. This type of assay is frequently referred to as a two-site or sandwich assay, since the antigen has two antibodies bonded to its surface at different locations.
Despite their great utility, sandwich immunoassay has been recognized to be a slow procedure, in part because washing steps are required and lengthy incubation periods are required to reach equilibrium. David, et al., U.S. Patent No. 4,376,110~
To eliminate at least one of the washing steps associa-ted with this procedure, so-called simultaneous and reverse assays have been developed. A simultaneous assay inv41ves a single incubation step as the antibody bound to the solid support and the labeled antibody are both added to the sample A24.1.WP 051287 .
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being tested at the same time. Af~er incubation, the solid support is washed to remove unbound analyte and unbound antibody, and the bound antibody-analyte-labeled antibody "sandwich" is detected as with a conventional "forward~' sandwich assay. A reverse assay involves the stepwise addition ~irst of a solu~ion o labeled antibody to the fluid sample followed by the addition of unlabeled antibody bound to a solid support after a suitable incubation. After a second incubation, the solid phase is washed in conv ntional ~ashion and the amount of labeled complex is detected as before. United States Patent No. 4,098,876 to Piasio, et al.
However, all of these methods suffer from the requirement for two antigen-specific antibodies which are able to recognize separate and distinct epitopes on an antigen. This is a critical limitation which makes the application of such immunoassays impractical for small antigens.
Immunoassays which require only one antigen-specific antibody are pre~erred. Such an immunoassay was described by Hirano, K. et al., Anal. Biochem. 154:624-631 (1986) who di~close an assay for tumor-specific alkaline phosphatase using a nitxocellulose filter coated with monoclonal anti-bodies specific for alkaline phosphatase. The presence of the bound analyte was determined by taking advantage of the enzymatic activity of the bound analyte. Thus, this type of immunoassay can only be used for detecting analytes with enzymatic activity which is stable to the conditions of the assay protocol. In addition, such assays are not applicable to detection of all enzymes. For example, enæymes such as thiol protease require the addition of reducing agents to stabilize them. Such reducing agents destroy antibodies by cleavinq disulfide bonds.
A dot-blot assay for the detection and quantitation of ~he Leishmania glycoconjugate was developed which involves A24.1.WP 051287 .
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do~-blotting the solubilized protein from parasites onto nitrocellulose, blocking the ~ree protein-binding si~es with BLOTT0 (5% w/v skim-powdered milk) and detecting the presence o~ the glycoconjugate with an iodinated monoclonal anti~ody.
Handmant E. ~ . Imm~nol. ~eth. 83:113-123 (1985). A
second two-site i~munoradiometric assay disclosed by Handman for glycoconjugate involved i~mobiliza~ion of monoclonal antibody on nitrocellulose, blocking remaining protein binding sites with BLOTT0, binding with antigen, followed by a second incubation wi~h ~he same monoclonal antibody, which was radioiodinated. This assay was ba~ed on the fact that Lli~ glycoconjugate possesses a large number of epitopes recognized by the monoclorlal antibody. This suggests ~hat IrL~h=~ai~ glycoconjugate contains a repetitive polymeric structure. Handman, su~ra. Thus, this immunoassay is limited to antigens capable of binding two or more identical antibodies. In addition, assays which rely on radiodinated monoclonal antibody are relatively expensive because of the high costs of monoclonal antibodies and the limited shelf lives of most labeled antibodies.
Anothe~ immunoassay which involves a single antihody comprises immobilization o~ a monoclonal antibody on nitro-cellulose, blocking the additional binding sites, and using a labeled antigen to detect khe desired antibodies. Suresh, .R. et al., Anal. Biochem. 151~19~-195 (1985~. This method is used to screen hybridoma supernatants and to detect monoclonal antibodies. ~owever, in many instances, the antigen i~ not available in 6ufficient quantities to allow labeling for use in such an assay. Fur~her, labeling the antigen can re~ult in deleterious al~eration of the immuno-~pecifici~y of the antigen.
~ nited States Patent No. ~,279,885 to Reese et al., describes a solid pha~e competitive pro~ein binding assay *Trade Mark A24.1.WP 051287 .~
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where an antigen or hapten can be assayed. The method involves competition between the analyte and a labelPd form thereof for a limited number of receptor or binding sites which are i~mobilized to a solid support. The assay may be conducted by mixing the componen~s simultaneously or sequentially. The sequential assay involves contacting a solu~ion of an analyte with a support containing immobilized receptors or antibodies, ~ollowed by contacting the mixturP
with a tracer. The tracer may be the analyte, or analog thereo~, which contains a label or tag. Competitive assays are generally recognized to be less preferable to non-competitive assays.
It is also pos~ible to have assays which do not utilize antibodies at all. For example, a sample containing protein to be assayed is mixed with a marker protein in contact with a polystyrene latex. A competition is created between the marker enzyme and the analyte protein for the limited surface binding sites. The inactivation of the enzyme upon binding to the hydrophobic latex surface allows m~asurement of the bound/free enzyme ratio, and thus, the competing protein concentration~ However, this method is not able to distin-guish betwean different proteins, and only gives a measure of the total protein content. Sandwick, et al., Aaal~ Biochem.
147:210-216 (1985).
Further improvements to immunoassay techniques involve the use of amplification strategies to increase the detection limit. These strategies include substrate cycling and enzyme channeling. Mosbach, K., Ann. N. Y. Acad._Sçi. 434:239-248 (1984). However, neither system has been widely adopted.
Thus, it would be desirable to have an immunoassay whioh is fast and reliable, and requires the preparation of only one specific antibody. Further, it is de~irable to have an A24.1.WP 051287 .~
3~0 immunoassay for a multiplicity of analytes utilizing one specific antibody for each a~alyte to effect detection.
Summary of the Invention The invention relates to a method for detecting and quantitating an analyte in a sample, which include ~ a) contac~ing a sample suspec~ed o~ containing the analyte with a solid p~ase suppor~ onto which an analyte-specific first antibody ha been immobilized;
(b) incubating said sample wi~h said support ~or a sufficient amount of time ~o allow the analyte present in the sample to bind to said first antibody;
(c) separating said solid suppor~ from the incubation mixture obtained in step (b);
(d) contacting said solid phase support with a detect-ably labeled titrating antibody which is specific for said first antibody;
(e) incubating the mixture formed in step (d) for a time sufficient to allow ~aid titrating antibody to bind to said first antibody;
(f) separating said solid phase support from the incubation mixture obtained in step (e); and ~ g) detecting the analyte in the sample by measuring the amount of bound labeled antibody.
The invention relates as well to an assay for a multi-plicity of analytes comprising contacting a ~ample suspected of containing a multiplicity of analytes with a solid support containing different analyte-specific antibodies separately immobilized onto defined areas o~ the solid phase support, followed by titration and detection with a common titrating antibody.
The invention also relates to a kit for the detection of an analyte in a sample comprising a carrier being compart A24.1.WP 051287 ~3083~
mentalized to receiYe in close confinement therein one or more containers wherein (a~ a first oontainer contains a solid support containing a first antibody immobilized to said solid support wherein said ~irst antibody is speci~ic ~o an analyte;
(b) a second container contains washing buf~ers: and (c) a third container contains a titrating antibody specific for the ~irst immobilized antibody.
The invention also relates to a kit for the detection of a multiplicity o~ analytes in a sample which includes a carrier means being compar~mentalized to receive in close confinement therein one or more containers wherein (a) a first container contains a solid support contain-ing a multiplicity of analyte-specific first antibodies separately immobilized to separate de~ined areas of said solid support:
(h) a second container contains washing buffers; and (c) a third container contains a second titrating antibody specific for each analyte-specific first antibody.
The invention offers a convenient, flexible and rapid method to detect and quantify one or more analytes in solution. In addition, the invention provides for r~cycling the solid phase support by elution with a chaotropic salt.
Thus, th~ solid phase support may be reused and the antigen recovered from the assay system.
DESCRIPTION O~l THE FIGURES
Fiqure 1. This ~igure show~ the general scheme of the assay, the titration of immobilized IgG not bound to antigen by iodinated IgG-specific antibody, and subsequent recycling of ~he nitrocellulose disk.
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Figure 2. This figure is a plot oP the number of disks per tube versus the amount of bound radioactivity.
Figure 3. This figure shows a comparison of the binding of iodinated sheep anti-rabbit (SAR) antibodies onto differ-ent types of immobilization matrices, with and without 20%
methanol.
Fi~ure 4. This figure ranks the relative ability of different papers to absorb iodinated SAR with or without 20%
methanol.
Fi~ure 5. This figure shows the ef~ect of recycling antibody coated disks on the amoun~ of residual radioactivity associated with the absorbed antibody.
Figure 6. This figure shows a standard curve for the titration of nitrocellulose-immobilized affinity purified SAR
IgG immobilized onto nitrocellulose.
Figure 7. This figure shows a determination of the amount of IgG bound to nitrocellulose disks for a range of different SAR dilutions.
Fiqure 8. ~his figure depicts a comparison of a 10-minute incubation with an overniqht incubation for a series of SAR dilutions.
Fiqure 9. This figure shows a standard curve for various concentrations of NIRS using SAR-coated nitrocel-lulose disks, obtained by ti~ration of unbound free antibody sites with iodinated titrating antibody.
Fi~ure 10. This ~iqure compares the amount of bound titrating antibody for two different incubation times and varying concentrations of NIRS.
FiGurç 11. This figure shows the effect of amplifica-tion of titratinq antibody f~r dif~erent concentrations of SAR.
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:, ' 3~)83S~) _g_ DESCRIPTION~ OF_THE PREFERRED EMBODIMENTS
This invention is directed towards methods of assay by immobilizing a first antibody specific to an analyte on a solid phase support, contacting the sample suspected of containing the analyte with the immobilized ankibody, and titrating the unbound antibody with a second labeled antibody which is specific for the first immobilized antibody.
By '~solid phase support" is intended any support capable of binding antibodies. Such supports include but are not limited to nitrocellulose, diazocellulose, microtiter plates, glass, polystyrene, polyvinylchloride, polypropylene, polyethylene, dextran, affini~y support gels such as Sepharose or agar, starch, and nylon. Preferred supports are nitrocellulose and diazocellulose. Those skilled in the art will note that many other suitable carriers for binding monoclonal antibody exist, or will be able to ascertain th~
same by use of routine experimentation.
The term "antibody" refers both to monoclonal antibodies which have a substantially homogeneous population and to polyclonal antibodies which have heterogeneous populations.
Both the first and second antibodies may be monoclonal or polyclonal. Polyclonal antibodies are derived from the antisera of animals immunized with the analyte. Monoclonal antibodies to specific antigens may be obtained by methods known to those skilled in the art~ See, for example Kohler and Milstein, Nature 256:495-497 (1975). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.
The term "antibody'~ i6 meant as wall to include both intact molecules as well as fragments thereof, such as, for example, Fab and F(ab')2, which are capable of binding antigen.
A24.1.WP 051287 ~L3~33S~
By the term "analyte" is intended any molecule with an antigenic site capable of binding to an antibody. Such analytes may include but are not limited to proteins, drugs, viruses, cells, haptens, ~ubcellular particles, carbo-hydrates, hormones, vitamins, metabolites and their binding materials.
In one e~bodiment, the first analyte-specific antibody is a polyclonal antibody derived from an animal immunized with the analyte. The second titrating antibody may be heterologous polyclonal antibodies which are specific against the immunoglobulins comprising the firs~ an~ibody. The second antibody may be obtained by isolating the antibodies from a second animal species which has been immunized with antisera from the same species of animal uséd to prepare the first antibody.
In another embodiment, a first IgG monoclonal antibody which is specific to the analyte to be assayed is used. The second titrating antibody comprises a detectably labeled anti-IgG polyclonal antibody specific to the first antibody.
In still another embodiment, a first IgG polyclonal antibody, which is specific to the analyte to be assayed, is used. The second titrating antibody comprises a detectably labeled anti-IgG monoclonal antibody specific to the first antibody.
In a preferred embodiment, a number of different analyte-specific antibodies deriYed from the same animal species may be separately immobilized on defined areas of a solid support which is attached to a dip stick. Thus, the dip stick may be incubated with a sample to assay for many different analytes simultaneously. The unoccupied antibody sites on each defined area can then be titrated with co~mon titrating antibodies which are specific for each antigen-specific antibody. For instance, each analyte-specific A24.1.WP 051287 ~. . ~ , . . - .
13~)~335~) antibody may be of the IgG class. The common titrating antibodies will then be anti-IgG antibodies. As used herein, the term "common titrating antibodiesl' is used in the plu.al, although it will be understood that only one class of antibodies is intended. This asp~ct of the invention provides for the simultaneous detection and quantitation of a multiplicity of antigens by a universal labeling method.
The amount of bound analyte is determined indirectly by measuring the amount of label associated with the second antibody which binds to the unoccupied Pirst antibody. The amount of analyte present in a sample is inversely propor-tional to the amount o~ label present. There are many different labels and methods of labeling known to those of ordinary skill in the art. Examples of the types of labels which can be used in the present invention include, but are not limited to, enzvmes, radioisotopes, dyes, fluorescent compounds, chemiluminescent compounds, bioluminescent compounds and metal chelates. Those of ordinary skill in the art will know of other suitable labels for binding to the antibody, or will be able to ascertain the same by the use of routine experimentation. ~urthermore, the binding of these labels to the antibodies can be accomplished using standard techniques commonly known to those of ordinary skill in the art.
One of the ways in which the titrating antibody of the present invention can be detectably labeled is by linking the same to an enzyme. This enzymeO in turn, when later exposed to its substrate, will react with the substrate in such a manner as to produce a chemical moiety which can be detected as, for example, by spectrophotometric, fluorometric or visual means. Examples of enzymes which can be used to detectably label the antibody of the present invention include malate dehydrogenase, staphylococcal nuclease, delta-A24.1.WP 051287 83~
~2-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphat2 dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholine esterase~ ~vidin-biotin binding may be used to facilitate the enzyme labeling.
The itrating antibody of the present invention can also be labeled with a radioactive i~otope which can then be determined by such means as ~he l~se of a gamma counter or a scintillation counter. Isotopes which are particularly use~ul for the purpose of the present invention are: 3H, 125I 131I 32p, 35S, 14c, 51Cr, 36cl, 57Co, 5~Co, 59Fe and 75Se.
It is also po~sible to label the titrating antibody with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can then be detected due to the fluorescence of the dye. Among the most commonly used fluorescent labelling compounds are fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
The titrating antibody of the invention can also be detectably labeled using fluorescent emitting metals such a~
52Eu, or others of the lanthanide series. These metals can be attached to the antibody molecule using such metal chelating groups as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
The titrating antibody o~ the present inven~ion also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged titrating antibody is then determined by detecting the pre~ence of luminescence that arises during the course o~ a A24.1.WP 051287 ~.3~
chemical reaction. Examples o~ particularly useful chemi-luminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
Likewise, a bioluminescent compound may be used to label the titratiny antibody of the present in~ention. Biolumi-nescence is a type of chemiluminescence ~ound in biological systems in which a catalytic protein increases the Pfficiency of the chemiluminescent reaction. The presence of a biolumi-nescent antibody is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.
Another techni~ue which may also result in greater sensitivity when used in conjunction with the present invention consists of coupling the titrating antibody of the present invention to low molecular weight haptens. The haptens can then be specifically detected by means of a second reaction. For example, it is common to use such haptens as biotin (reacting with avidin) or dinitrophenyl, pyridoxal and fluorescamine (reacting with specific anti-hapten antibodies) in this manner.
In addition, the sensitivity of the assay may be increased by use of amplification strategies including substrate cycling and enzyme channeling as taught by Mosbach, supra, incorporated by reference herein.
For the purposes o~ the present invention, the analyte which is detected by this assay may be present in a sample solution. Normally, the sample is a biological sample such as, for example, saliva, cerebrospinal fluid, blood, serum, urine, water, food and the like. However, the invention is not limited to assays using only these samples, it being possible for one of ordinary skill in the art to determine suitable conditions which allow the use of other samples.
A2401.WP 051287 , ~ ~
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For instance, various chemicals and drugs are also capable of being antigens, and thus may ~e suitable assayed in water, food, or othPr samples by the methods of this invention.
In carrying out the titrating immunoassay of the presPnt invention on a sample containing a multiplicity of analytes, the process comprises:
a~ contacting a sample suspected of containing a ~ultiplicity of analytes with a solid support on which dif~erent analyte-specific first antibodie have been separately immobilized to separately defined areas of said solid support:
b) incubating said sample with said support for a sufficient amount of time to allow the analytes present in the sample to bind to said first antibodies;
c) separating the solid phase support from the incubation mixture obtained in step b~:
d) contacting said solid support with a detectably labeled titrating antibodies which are specific for said first antibodies;
e) incubating the mixture formed in step d) for a time sufficient to allow said titrating antibodies to bind to said first antibodies:
f) separating said solid phase support from the incubation mixture obtained in step e); and g) detecting the analyte in the sample by measuring the amount of bound labeled titrating antibodies to each separately defined area.
Of course, the specific concentration~ of label and analyte, the temperature and timP of incubation, as well as other assay conditions may be varied, depending on various factors including the concentration of antigen in the sample, the n~ture of the sample, and the like. Those skilled in the art will be able to determine operative and optlmal assay A24.1.WP 051287 .
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conditions for each determina~ion hy employing routine experimentation. In addition, the eluted titrating antibody may be separated from the eluted analyte accoding to means ~nown in the art and also recycled for reuse in the assay.
The eluted analyte may further be recovered.
Detection of the labeled antibody may be accompli~hed by a scintillation count~r, Por example, if the label is a radioactive gamma emitter, or by a fluorometer, for example, if the label is a fluorescent ma~erial. In ~he case of an enzyme label, the detection can be accomplished by colori-metric methods which employ a substra~e for the enzyme.
Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with standards.
Other such stsps as washing, stirring, shaking, filter-ing and the like may of course be added to the assays as is customary or necessary for the particular situation.
The solid phase i~munosorbent may be recycled by elution of the antigen and the titrating antibody with a chaotropic salt such as MgC12. However, the invention is not limited to the use of MgC12, it being possible for one of ordinary skill in the art to determine other chaotropic salts which may be used to recycle the solid phase immunosorbent, without undue experimentation. In addition, the eluted titrating antibody may be separated from the eluted analyte according to means known in the art and may also then be recovered for re-use.
The eluted analyte may further be recovered.
The assay of the present invention is ideally suited for the preparation of a kit. Such a kit may comprise a carrier means being compartmentalized tc receive in close confinement therewith one or more container mean~ such as vials, tubes and the like, each of ~aid container means comprising the separate elements of the immunoassay. ~or example, there may A24.1.WP 051287 ~L301~35~
be a container means containing the first antibody immobil-ized on a solid phase support, and further container means containing detectably labeled titrating antibodiPs in solution. Further container means may contain standard solutions comprising serial dilutions o~ analytes to be detected. The standard solutions of these an~lytes may be used to prepare a standard curve with the concentration of the analyte plotted on the abscissa and the detection signal on the ordinate. The results obtained from a sample ¢ontain-ing an analyte may be interpolated from such a plot to give the concentration of the analyte.
In another e~bodiment of the ki~, there may be a container means containing a dipstick which comprises a multiplicity of different analyte-specific antibodies separately immobilized to separate defined areas of the solid phase support. Further container means may contain a common titrating antibody which is specific for each analyte-specific antibody. Further container means may contain standard solutions of analytes to be detected. The standard solutions of these analytes may be used to provide a standard reference dipstick for comparison with the sample dipstick.
The various aspects of the invention are further described in the following examples. These examples are not intended to limit the invention in any manner.
EXAMPLES
EXAMPL~ 1 General Procedure For the ImmunoassaY
In the examples that follow, a simple antibody-antigen system illustrates the protocol used for the assay for one antigen by isotopic detection.
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Sheep anti-rabbit serum (SAR) was induced by sheep by standard procedures and heat inactivated at 56C fsr 30 ~inutes. Rabbit IgG was purchased from Sigma (St. Louis, M0). Iodination was achieved using iodo-beads (Pierce Chemical Company, Rockford, IL) using carrier-free ~odium iodid~ 25 (Dupont N~w England Nuclear, 8Os~on, MA). All other chemicals wer~ reagent grade and were purchased from either 5igma Chemical Corporation (S~. Louis, M0~ or Fisher (Medford, ~A).
Nitrocellulo~e paper (~illipore filter type HAHY 000-lO, O.45 uM pore size, Millipore Corporation, 9edford, MA) was used unless stated otherwise. The other papers te~ted include: Zeta Probe blotting membrane (cationized nylon, BioRad, Ric~mond, C~), mixed cellulose acetate and nitrate paper (GSWP 013-00, O.22 uM pore size3 and Millipore MF
filters (HAWG04750, 0.45 uM pore size, ~illipore Corporation, Bedford, MA), high binding capacity S&S Nytran (positively charged hydrophilic nylon-66, pore siæes 0.2 uM and 0.45 uM), S&S pure 100% hydrophilic nitrocellulose in different pore sizes (no cellulose acetate added, PH70, 0.025 uM pore size and BA83, pore size 0.2 uM, and ~A85, pore size 0.45 uM, Schleicher and Schuell, Xeene, NH), Hydrophilic Hybond-C 87 mm nitrocellulose and Hybond-N 132 mm nylon membranes (Amersham Corporation, Arlington Heights, ~L).
Sev~n millimeter diameter disks of nitrocellulose (Millipore ~A 0.45 uM pore, unless stated otherwise) were made using a standard o~fice hole puncher and transferred by needle to a 15 x 75 millimeter polystyrene tube. Volumes between 50-300 ul o~ diluted antisera (SAR) were incubated with the disXs at room temperature for 10 minu~es. After washing in Tris-EDTA-azide, the samples were inubated in 1-2 ~l of blocker. All ~itration and wash bufPers utilized a base buffer o~ 50 mM Tris, 5 mM EDTA, O. 01% sodium azide at *Trade Marks A24.1.WP 051287 :~ ~
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pH 7.4. Blocking buffers included 10% (v/v) bovine serum, 3 BSA, or 5% Carnation non~at milk. The antigen (xabbit IgG) was t~en added (50~300 ul) and ~ncuba~ed for 30-60 minutes, as indicated in the following Examples. After washing three ti~est iodinated non-immune rabbit serum (NIRS-I125) was ~dded (50-300 ul) for the ~ime ~tated (30 minutes minimum).
After a second wash, the ~a~ples were counted for gamma radiation on a 40% efficient Packard Auto-Gamma Scintillation Spectrophotometer Model 5220.
EXAMPL~_2 The Effic,iency of ~qG Immobilization The efficiency of IqG immobilization on nitrocellulose disks was determined as follows~ Serial dilutions of SAR
were incubated with a ni~rocellulose disk. The nitrocellu-lose ~ilter were then washed and titrated with iodinated antisera. A plot o~ the amount of bound IgG, as represented by the amount o~ radioactivity, appears sc~ematically in the inset to Figure 2. The competition between IgG and the larger proportion o~ other serum proteins for protein binding sites on nitrocellulo6e results in the affinity displacement (A) of IgG at high serum concentrations. As the antiserum concentration diminishes, the amount of immobilized IgG
increases until the inflection point (X) is reached, where all the sample protein i8 absorb~d equally. Diminishing concentrations of IgG beyond the in~lection point are ~hen reflected by diminishing counts of ti~rating antibody (B).
This ln~lection point can be displaced to higher serum concentration~ i~ desired ~imply by providing more nitrocel-lulose binding 8ite5. Figure 2 shows the relationship between the number of nitrocellulose disks and the amount o~
bound radioactivityO 500 uL of ~heep anti-rabbit serum was *Trade Marks A24~1.WP 051287 ~,, ~3~183S~) incubated ~or 10 minutes with 1-10 disks as shown in Figure 2, washed with Tris buffer, blocked with 1 ml of 10% bovine serum blocker for 20 minutes, wàshed, ~itrated with 1 ml of NIRS-I125 for 60 minutes, washed, and the radioactivity bound to the disk determined. A~ a tenfold increase in nitrocel-lulose surface area, the number oP binding sites was no longer limitiny for that concentrat~on of sample. This illustrates that it is advantageous to use dilute sera for coating the matrix. Alternatively, puri~ied IgG may be used to achieve high coating densities.
Stability of the Immobilized IaG
The stability of immobilized IgG on different immobili-zation matrices, with and without 20% methanol, was deter-mined as follows. Disks of cellulose or nylon papers were incubated with iodinated SAR in Tris-EDTA for 20 minutes, with or without 20% methanol. The disks were then washed with Tris-EDTA and the radioactivity bound to the disks measured. The following papers were tested: 1) BioRad Zeta Probe, 2) Millipore nitrocellulose HA 0.~5 uM pore, 3) Millipore mixed esters 0.22 uM pore, 4) Millipore MF 0.45 uM
pore, 5) S&S Nytran 0.2 uM pore, 6) S&S 100% nitrocellulose 0.025 uM pore, 7) S~S Nytran 0.45 uM pore, 8) S&S 100%
nitrocellulose, 0.2 uM pore, 9) S~S 100% nitrocellulose, 0.45 uM pore, 10) Amersham Hybond Nitrocellose, 11) Amersham Hybond nylon. The amount of bound radioactivity is depicted in Figure 3. The relative abilities of different papers to absorb iodinated SA~ under the best conditions (with (+) or without (-) 20% methanol) is depicted in Figure 4. Millipore 0.45 uM pore nitrocellulose gave the best results and was chosen for all subsequent experiments. Mixed acetate paper A24.1.WP 051287 ~ 3 [)~
also performed well despite reports in the literature that such papers poorly absorbed proteins (Gershoni, J.M., et al., Anal. Biochem. 131:1-15 (1983)~. For S&S pure nitrocellulose paper, smaller pore sizes performed better (0.02 v~rsus 0.45 13M ) . ~
The e~fsct on stability o~ the immobilized IgG was then examined during recycling. Nitrocellulose disks in this experiment, were recycled four times. Nitrocellulose disks containing immobilized sheep anti-rabbit IgG and titrating anti~ody NIRS-I125 were e~uted with 2.5 M MgCl2 and the loss of antibody during the elution cycle determined. Four elutions with MgCl2 resulted in a 20% loss of absorbed antibody (as determined by iodinated sheep anti-rabbit serum3 as shown in Figure 5. To test the ~unctional integrity of the SAR remaining after each cycle of the elution, the disks were inc~bated with 150 uL o~ iodinated NIRS-antigen for 60 minutes, followed by washing in milk bu~fer, and counting the radioactivity remaining on the disk. Background counts were determined using the counts adhering after elution of the iodinated antigen. All data as shown in Figure 5 is normali-zed as a percentage of the starting value. During regenera-tion cycles, nonspecific binding increased gradually, which indicates the absorption of label onto sites vacated by the blocker protein or the sheep anti-rabbit serum.
In thi~ example, the amount of IgG necessary to give a readable ~ignal was determined. Five ul aliquots of serial dilutions of SAR were spotted onto disks o~ nitrocellulose and dried. They were blocked for 30 minutes in 2 ml of 5%
non~at milk, washed in Tris-EDTA, and incubated with 150 ul A24.1.WP 051287 , ~L3~3~q~
of iodinated NIRS in 3~ BSA for 60 minutes. Following thorough washing, the radioactivity bound to the disks was determined. As can be seen in Figure 6, the lowest detect-able amount of bound IgG under these conditions was approxi mately 25 ng. A parallel series of experiment~ were con-ducted by amplifying the detection signal with iodinated S~R.
Again, the lowest detection limit was about 25 ny.
The amount of Ig~ bound to the dis~s after brief incubation with a range of serum dilutions is depicted in Figure 7. 100% efficient absorption was achieved at a titre of approximately 1:500. At lower dilutions, decreasing amounts of sample IgG were absorbed onto the matrix. The capacity of the nitrocellulose is approximately 80 ug/cm2.
The area of the disk used was about 1 cm2. Thus, the amount of specific IgG is far short of saturating the disk. About 50% absorption of total sample IgG was achieved by using 1:128 dilution of serum.
O~timizatiQn_of Incubation Time for Disk Coatinq To achieve maximal immobilization of antisera, serial dilutions of SAR were incubated with nitrocellulose disks for minutes or overnight. A comparison of a 10 minute incubation with an overnight incubation for a wide range of SAR dilutions is shown in Figure 8. A lO-minute incubation bound more IgG than an overnight incubation. In addition, an overnight incubation showed an inflection point. The development of the inflection point only in the longer incubation time is due to the displacement of IgG by other serum proteins, due to either mass action or af~inity differences. At all dilutions o~ SAR, the binding of Ig~ was diminished with the longer incubation time~ After incuba A24.1.WP 051287 ~3~)~335(~
tion, the samples were washed, blocked with 1 ml of 5% nonfat milk for 20 minutes, washed again and incubated with 450 ml of iodinated NI~S for 60 minutes in non~at milk buffer.
After thorough washing, the radioactivity bound to the disks was measured and is presented in Figure 8. ~he relationship of the time of incubation ~or shorter time periods and the radioactivity bound to the disks appears in Table 1.
Radioactivity bound to disk SAR concentration Time of incubation (minutes) Undiluted 1:4 0 (blank) -1 171 + ~0 158 + 12523 ~ 2 155 + 9 199 + 16984 + 14 151 + 33 171 + 26738 + 13 162 ~ 7 318 + 159 1036 + 136 126 ~ 4 172 + 0718 + 147 130 146 + 33 215 + 571190 + 8 .
Undiluted, 1:4, and samples with no SAR were spiked with~
iodinated SA~, and 100 ul incubated with each disk for th~
time shown. After washing with 2 ml volumes of 5% nonfat milk buf~er, the counts remaining on the disks were deter-mined. The total counts added initially were 3610 + 596.
Thus, the percentage of the total that became bound ranged ~rom 14% at one minute to 33% at 130 minutes. The counts are expressed as mPan and SD o~ duplicates. Maximal binding of IgG in a 100 ul sample occurred at 20 minutes at a 1:4 A24.1.WP 051287 . ........... -.
, ` :
~L308350 dilution~ However, this data was no~ significantly dif~erent from the 10 minute incubation. Table 2 depicts the percen-tage of total counts bound to the blank disk for each length of time. The binding plateaus between 10-20 minutes.
Adequate coating oP disks was achieved using 100 ul of SAR at a dilution of 1:200 for 10 minutes.
TABLE Z
Time (minutes) ~ of Total Counts Bound to Blank Disk _ 1 14.5 + 0.3 27.2 + 2 20.4 + 2 28.7 ~ 13 19.9 + 2 134 32O9 + 0.6 LRgend: Timecourse of SAR adsorption onto nitrocellulose disks. The data is derived from Table 1.
a~
Optimization of Incubation Time With Titratin~ Antibody ~ he relationship between the time of incubation of the titrating labeled antibody (60 or 140 minutes) and the amount of bound label for various serial dilutions of NIRS appears Figure 10. The disks were coated with SAR at a 1:~ dilution (100 ul for 20 minutes), blocked for 20 minutes in 1 ml of 5%
nonat milk buffer, and incubated with 200 ul of serial ~24.1.WP 051287 ' .
TITL~ OF T~oe INYENTIONo MULTIPh~_~NTIGEN IMMUNOASSAY
Field of the In~ention The invention relates to a method for the assay of an analyte which may be present in a sample by r~acting a sample suspected o~ containing said analyte with antibodies immobi-lized to a solid support to form an antibody-antigen complex, followed by titrating the unoccupied antibody with a second labeled antibody which is specific to the first immobilized antibody, ~ollowed by detecting the label.
BAC~GROUND OF THE INVENTION
The detection and quantitation o~ antigenic substances in biological samples ~requently utilize immunoassay kechni-ques. These technigues are:based upon the formation of a ¢omplex between the antigenic substance being assayed and an antibody or antibodies in~which one or the other member of the ~omplex may be detectably labeled. ~ith competitiYe immunoassay ~echniques, ~he antigenic substance in a sample fluid being tested competes with a known quantity o~ labeled antigen ~or a limited quantity of antibody binding si~es.
The amount of labeled antigen bound to the antibody is inversely proportional to the amount of antigen in a sample.
.
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By contrast, most immunometric assays employ a labeled antibody. In such an assay, the amount of labeled antibody associated with the complex is directly proportional to the amount of antigenic substance in a fluid sample.
In sandwich immunome~ric assays, a ~uantity o~ unlabeled antibody is bound to a solid support which is insoluble in the fluid being tested. This immobilized antibody is first contacted with the sample being tested so that a binary antigen-antibody complex is ~ormed. After a suitable incubation period, the solid support is washed to remove unbound antigens, then contacted with a solution containing a known quantity of a second antibody. After a second incuba-tion period, the solid support is then washed a second time to remove the unreacted antibody. A labeled anti-antibody to the second antibody is then added, allowed ~o incubate for a sufficient amount of time, and the complex then ~ashed. The washed solid support is then tested to detect and quantify the presence of labeled antibody, for example by measuring the emitted radiation o~ a radioactive label. The amount of labeled antibody detected is compared to that for a negative control sample. This type of assay is frequently referred to as a two-site or sandwich assay, since the antigen has two antibodies bonded to its surface at different locations.
Despite their great utility, sandwich immunoassay has been recognized to be a slow procedure, in part because washing steps are required and lengthy incubation periods are required to reach equilibrium. David, et al., U.S. Patent No. 4,376,110~
To eliminate at least one of the washing steps associa-ted with this procedure, so-called simultaneous and reverse assays have been developed. A simultaneous assay inv41ves a single incubation step as the antibody bound to the solid support and the labeled antibody are both added to the sample A24.1.WP 051287 .
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- - ~3~3S~
being tested at the same time. Af~er incubation, the solid support is washed to remove unbound analyte and unbound antibody, and the bound antibody-analyte-labeled antibody "sandwich" is detected as with a conventional "forward~' sandwich assay. A reverse assay involves the stepwise addition ~irst of a solu~ion o labeled antibody to the fluid sample followed by the addition of unlabeled antibody bound to a solid support after a suitable incubation. After a second incubation, the solid phase is washed in conv ntional ~ashion and the amount of labeled complex is detected as before. United States Patent No. 4,098,876 to Piasio, et al.
However, all of these methods suffer from the requirement for two antigen-specific antibodies which are able to recognize separate and distinct epitopes on an antigen. This is a critical limitation which makes the application of such immunoassays impractical for small antigens.
Immunoassays which require only one antigen-specific antibody are pre~erred. Such an immunoassay was described by Hirano, K. et al., Anal. Biochem. 154:624-631 (1986) who di~close an assay for tumor-specific alkaline phosphatase using a nitxocellulose filter coated with monoclonal anti-bodies specific for alkaline phosphatase. The presence of the bound analyte was determined by taking advantage of the enzymatic activity of the bound analyte. Thus, this type of immunoassay can only be used for detecting analytes with enzymatic activity which is stable to the conditions of the assay protocol. In addition, such assays are not applicable to detection of all enzymes. For example, enæymes such as thiol protease require the addition of reducing agents to stabilize them. Such reducing agents destroy antibodies by cleavinq disulfide bonds.
A dot-blot assay for the detection and quantitation of ~he Leishmania glycoconjugate was developed which involves A24.1.WP 051287 .
.
~30~
do~-blotting the solubilized protein from parasites onto nitrocellulose, blocking the ~ree protein-binding si~es with BLOTT0 (5% w/v skim-powdered milk) and detecting the presence o~ the glycoconjugate with an iodinated monoclonal anti~ody.
Handmant E. ~ . Imm~nol. ~eth. 83:113-123 (1985). A
second two-site i~munoradiometric assay disclosed by Handman for glycoconjugate involved i~mobiliza~ion of monoclonal antibody on nitrocellulose, blocking remaining protein binding sites with BLOTT0, binding with antigen, followed by a second incubation wi~h ~he same monoclonal antibody, which was radioiodinated. This assay was ba~ed on the fact that Lli~ glycoconjugate possesses a large number of epitopes recognized by the monoclorlal antibody. This suggests ~hat IrL~h=~ai~ glycoconjugate contains a repetitive polymeric structure. Handman, su~ra. Thus, this immunoassay is limited to antigens capable of binding two or more identical antibodies. In addition, assays which rely on radiodinated monoclonal antibody are relatively expensive because of the high costs of monoclonal antibodies and the limited shelf lives of most labeled antibodies.
Anothe~ immunoassay which involves a single antihody comprises immobilization o~ a monoclonal antibody on nitro-cellulose, blocking the additional binding sites, and using a labeled antigen to detect khe desired antibodies. Suresh, .R. et al., Anal. Biochem. 151~19~-195 (1985~. This method is used to screen hybridoma supernatants and to detect monoclonal antibodies. ~owever, in many instances, the antigen i~ not available in 6ufficient quantities to allow labeling for use in such an assay. Fur~her, labeling the antigen can re~ult in deleterious al~eration of the immuno-~pecifici~y of the antigen.
~ nited States Patent No. ~,279,885 to Reese et al., describes a solid pha~e competitive pro~ein binding assay *Trade Mark A24.1.WP 051287 .~
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where an antigen or hapten can be assayed. The method involves competition between the analyte and a labelPd form thereof for a limited number of receptor or binding sites which are i~mobilized to a solid support. The assay may be conducted by mixing the componen~s simultaneously or sequentially. The sequential assay involves contacting a solu~ion of an analyte with a support containing immobilized receptors or antibodies, ~ollowed by contacting the mixturP
with a tracer. The tracer may be the analyte, or analog thereo~, which contains a label or tag. Competitive assays are generally recognized to be less preferable to non-competitive assays.
It is also pos~ible to have assays which do not utilize antibodies at all. For example, a sample containing protein to be assayed is mixed with a marker protein in contact with a polystyrene latex. A competition is created between the marker enzyme and the analyte protein for the limited surface binding sites. The inactivation of the enzyme upon binding to the hydrophobic latex surface allows m~asurement of the bound/free enzyme ratio, and thus, the competing protein concentration~ However, this method is not able to distin-guish betwean different proteins, and only gives a measure of the total protein content. Sandwick, et al., Aaal~ Biochem.
147:210-216 (1985).
Further improvements to immunoassay techniques involve the use of amplification strategies to increase the detection limit. These strategies include substrate cycling and enzyme channeling. Mosbach, K., Ann. N. Y. Acad._Sçi. 434:239-248 (1984). However, neither system has been widely adopted.
Thus, it would be desirable to have an immunoassay whioh is fast and reliable, and requires the preparation of only one specific antibody. Further, it is de~irable to have an A24.1.WP 051287 .~
3~0 immunoassay for a multiplicity of analytes utilizing one specific antibody for each a~alyte to effect detection.
Summary of the Invention The invention relates to a method for detecting and quantitating an analyte in a sample, which include ~ a) contac~ing a sample suspec~ed o~ containing the analyte with a solid p~ase suppor~ onto which an analyte-specific first antibody ha been immobilized;
(b) incubating said sample wi~h said support ~or a sufficient amount of time ~o allow the analyte present in the sample to bind to said first antibody;
(c) separating said solid suppor~ from the incubation mixture obtained in step (b);
(d) contacting said solid phase support with a detect-ably labeled titrating antibody which is specific for said first antibody;
(e) incubating the mixture formed in step (d) for a time sufficient to allow ~aid titrating antibody to bind to said first antibody;
(f) separating said solid phase support from the incubation mixture obtained in step (e); and ~ g) detecting the analyte in the sample by measuring the amount of bound labeled antibody.
The invention relates as well to an assay for a multi-plicity of analytes comprising contacting a ~ample suspected of containing a multiplicity of analytes with a solid support containing different analyte-specific antibodies separately immobilized onto defined areas o~ the solid phase support, followed by titration and detection with a common titrating antibody.
The invention also relates to a kit for the detection of an analyte in a sample comprising a carrier being compart A24.1.WP 051287 ~3083~
mentalized to receiYe in close confinement therein one or more containers wherein (a~ a first oontainer contains a solid support containing a first antibody immobilized to said solid support wherein said ~irst antibody is speci~ic ~o an analyte;
(b) a second container contains washing buf~ers: and (c) a third container contains a titrating antibody specific for the ~irst immobilized antibody.
The invention also relates to a kit for the detection of a multiplicity o~ analytes in a sample which includes a carrier means being compar~mentalized to receive in close confinement therein one or more containers wherein (a) a first container contains a solid support contain-ing a multiplicity of analyte-specific first antibodies separately immobilized to separate de~ined areas of said solid support:
(h) a second container contains washing buffers; and (c) a third container contains a second titrating antibody specific for each analyte-specific first antibody.
The invention offers a convenient, flexible and rapid method to detect and quantify one or more analytes in solution. In addition, the invention provides for r~cycling the solid phase support by elution with a chaotropic salt.
Thus, th~ solid phase support may be reused and the antigen recovered from the assay system.
DESCRIPTION O~l THE FIGURES
Fiqure 1. This ~igure show~ the general scheme of the assay, the titration of immobilized IgG not bound to antigen by iodinated IgG-specific antibody, and subsequent recycling of ~he nitrocellulose disk.
A24.1.WP 05128 - ~3~)~33~q~
Figure 2. This figure is a plot oP the number of disks per tube versus the amount of bound radioactivity.
Figure 3. This figure shows a comparison of the binding of iodinated sheep anti-rabbit (SAR) antibodies onto differ-ent types of immobilization matrices, with and without 20%
methanol.
Fi~ure 4. This figure ranks the relative ability of different papers to absorb iodinated SAR with or without 20%
methanol.
Fi~ure 5. This figure shows the ef~ect of recycling antibody coated disks on the amoun~ of residual radioactivity associated with the absorbed antibody.
Figure 6. This figure shows a standard curve for the titration of nitrocellulose-immobilized affinity purified SAR
IgG immobilized onto nitrocellulose.
Figure 7. This figure shows a determination of the amount of IgG bound to nitrocellulose disks for a range of different SAR dilutions.
Fiqure 8. ~his figure depicts a comparison of a 10-minute incubation with an overniqht incubation for a series of SAR dilutions.
Fiqure 9. This figure shows a standard curve for various concentrations of NIRS using SAR-coated nitrocel-lulose disks, obtained by ti~ration of unbound free antibody sites with iodinated titrating antibody.
Fi~ure 10. This ~iqure compares the amount of bound titrating antibody for two different incubation times and varying concentrations of NIRS.
FiGurç 11. This figure shows the effect of amplifica-tion of titratinq antibody f~r dif~erent concentrations of SAR.
A24.1.WP 051287 , , ~
:, ' 3~)83S~) _g_ DESCRIPTION~ OF_THE PREFERRED EMBODIMENTS
This invention is directed towards methods of assay by immobilizing a first antibody specific to an analyte on a solid phase support, contacting the sample suspected of containing the analyte with the immobilized ankibody, and titrating the unbound antibody with a second labeled antibody which is specific for the first immobilized antibody.
By '~solid phase support" is intended any support capable of binding antibodies. Such supports include but are not limited to nitrocellulose, diazocellulose, microtiter plates, glass, polystyrene, polyvinylchloride, polypropylene, polyethylene, dextran, affini~y support gels such as Sepharose or agar, starch, and nylon. Preferred supports are nitrocellulose and diazocellulose. Those skilled in the art will note that many other suitable carriers for binding monoclonal antibody exist, or will be able to ascertain th~
same by use of routine experimentation.
The term "antibody" refers both to monoclonal antibodies which have a substantially homogeneous population and to polyclonal antibodies which have heterogeneous populations.
Both the first and second antibodies may be monoclonal or polyclonal. Polyclonal antibodies are derived from the antisera of animals immunized with the analyte. Monoclonal antibodies to specific antigens may be obtained by methods known to those skilled in the art~ See, for example Kohler and Milstein, Nature 256:495-497 (1975). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.
The term "antibody'~ i6 meant as wall to include both intact molecules as well as fragments thereof, such as, for example, Fab and F(ab')2, which are capable of binding antigen.
A24.1.WP 051287 ~L3~33S~
By the term "analyte" is intended any molecule with an antigenic site capable of binding to an antibody. Such analytes may include but are not limited to proteins, drugs, viruses, cells, haptens, ~ubcellular particles, carbo-hydrates, hormones, vitamins, metabolites and their binding materials.
In one e~bodiment, the first analyte-specific antibody is a polyclonal antibody derived from an animal immunized with the analyte. The second titrating antibody may be heterologous polyclonal antibodies which are specific against the immunoglobulins comprising the firs~ an~ibody. The second antibody may be obtained by isolating the antibodies from a second animal species which has been immunized with antisera from the same species of animal uséd to prepare the first antibody.
In another embodiment, a first IgG monoclonal antibody which is specific to the analyte to be assayed is used. The second titrating antibody comprises a detectably labeled anti-IgG polyclonal antibody specific to the first antibody.
In still another embodiment, a first IgG polyclonal antibody, which is specific to the analyte to be assayed, is used. The second titrating antibody comprises a detectably labeled anti-IgG monoclonal antibody specific to the first antibody.
In a preferred embodiment, a number of different analyte-specific antibodies deriYed from the same animal species may be separately immobilized on defined areas of a solid support which is attached to a dip stick. Thus, the dip stick may be incubated with a sample to assay for many different analytes simultaneously. The unoccupied antibody sites on each defined area can then be titrated with co~mon titrating antibodies which are specific for each antigen-specific antibody. For instance, each analyte-specific A24.1.WP 051287 ~. . ~ , . . - .
13~)~335~) antibody may be of the IgG class. The common titrating antibodies will then be anti-IgG antibodies. As used herein, the term "common titrating antibodiesl' is used in the plu.al, although it will be understood that only one class of antibodies is intended. This asp~ct of the invention provides for the simultaneous detection and quantitation of a multiplicity of antigens by a universal labeling method.
The amount of bound analyte is determined indirectly by measuring the amount of label associated with the second antibody which binds to the unoccupied Pirst antibody. The amount of analyte present in a sample is inversely propor-tional to the amount o~ label present. There are many different labels and methods of labeling known to those of ordinary skill in the art. Examples of the types of labels which can be used in the present invention include, but are not limited to, enzvmes, radioisotopes, dyes, fluorescent compounds, chemiluminescent compounds, bioluminescent compounds and metal chelates. Those of ordinary skill in the art will know of other suitable labels for binding to the antibody, or will be able to ascertain the same by the use of routine experimentation. ~urthermore, the binding of these labels to the antibodies can be accomplished using standard techniques commonly known to those of ordinary skill in the art.
One of the ways in which the titrating antibody of the present invention can be detectably labeled is by linking the same to an enzyme. This enzymeO in turn, when later exposed to its substrate, will react with the substrate in such a manner as to produce a chemical moiety which can be detected as, for example, by spectrophotometric, fluorometric or visual means. Examples of enzymes which can be used to detectably label the antibody of the present invention include malate dehydrogenase, staphylococcal nuclease, delta-A24.1.WP 051287 83~
~2-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphat2 dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholine esterase~ ~vidin-biotin binding may be used to facilitate the enzyme labeling.
The itrating antibody of the present invention can also be labeled with a radioactive i~otope which can then be determined by such means as ~he l~se of a gamma counter or a scintillation counter. Isotopes which are particularly use~ul for the purpose of the present invention are: 3H, 125I 131I 32p, 35S, 14c, 51Cr, 36cl, 57Co, 5~Co, 59Fe and 75Se.
It is also po~sible to label the titrating antibody with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can then be detected due to the fluorescence of the dye. Among the most commonly used fluorescent labelling compounds are fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
The titrating antibody of the invention can also be detectably labeled using fluorescent emitting metals such a~
52Eu, or others of the lanthanide series. These metals can be attached to the antibody molecule using such metal chelating groups as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
The titrating antibody o~ the present inven~ion also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged titrating antibody is then determined by detecting the pre~ence of luminescence that arises during the course o~ a A24.1.WP 051287 ~.3~
chemical reaction. Examples o~ particularly useful chemi-luminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
Likewise, a bioluminescent compound may be used to label the titratiny antibody of the present in~ention. Biolumi-nescence is a type of chemiluminescence ~ound in biological systems in which a catalytic protein increases the Pfficiency of the chemiluminescent reaction. The presence of a biolumi-nescent antibody is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.
Another techni~ue which may also result in greater sensitivity when used in conjunction with the present invention consists of coupling the titrating antibody of the present invention to low molecular weight haptens. The haptens can then be specifically detected by means of a second reaction. For example, it is common to use such haptens as biotin (reacting with avidin) or dinitrophenyl, pyridoxal and fluorescamine (reacting with specific anti-hapten antibodies) in this manner.
In addition, the sensitivity of the assay may be increased by use of amplification strategies including substrate cycling and enzyme channeling as taught by Mosbach, supra, incorporated by reference herein.
For the purposes o~ the present invention, the analyte which is detected by this assay may be present in a sample solution. Normally, the sample is a biological sample such as, for example, saliva, cerebrospinal fluid, blood, serum, urine, water, food and the like. However, the invention is not limited to assays using only these samples, it being possible for one of ordinary skill in the art to determine suitable conditions which allow the use of other samples.
A2401.WP 051287 , ~ ~
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For instance, various chemicals and drugs are also capable of being antigens, and thus may ~e suitable assayed in water, food, or othPr samples by the methods of this invention.
In carrying out the titrating immunoassay of the presPnt invention on a sample containing a multiplicity of analytes, the process comprises:
a~ contacting a sample suspected of containing a ~ultiplicity of analytes with a solid support on which dif~erent analyte-specific first antibodie have been separately immobilized to separately defined areas of said solid support:
b) incubating said sample with said support for a sufficient amount of time to allow the analytes present in the sample to bind to said first antibodies;
c) separating the solid phase support from the incubation mixture obtained in step b~:
d) contacting said solid support with a detectably labeled titrating antibodies which are specific for said first antibodies;
e) incubating the mixture formed in step d) for a time sufficient to allow said titrating antibodies to bind to said first antibodies:
f) separating said solid phase support from the incubation mixture obtained in step e); and g) detecting the analyte in the sample by measuring the amount of bound labeled titrating antibodies to each separately defined area.
Of course, the specific concentration~ of label and analyte, the temperature and timP of incubation, as well as other assay conditions may be varied, depending on various factors including the concentration of antigen in the sample, the n~ture of the sample, and the like. Those skilled in the art will be able to determine operative and optlmal assay A24.1.WP 051287 .
~L3~il33~
conditions for each determina~ion hy employing routine experimentation. In addition, the eluted titrating antibody may be separated from the eluted analyte accoding to means ~nown in the art and also recycled for reuse in the assay.
The eluted analyte may further be recovered.
Detection of the labeled antibody may be accompli~hed by a scintillation count~r, Por example, if the label is a radioactive gamma emitter, or by a fluorometer, for example, if the label is a fluorescent ma~erial. In ~he case of an enzyme label, the detection can be accomplished by colori-metric methods which employ a substra~e for the enzyme.
Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with standards.
Other such stsps as washing, stirring, shaking, filter-ing and the like may of course be added to the assays as is customary or necessary for the particular situation.
The solid phase i~munosorbent may be recycled by elution of the antigen and the titrating antibody with a chaotropic salt such as MgC12. However, the invention is not limited to the use of MgC12, it being possible for one of ordinary skill in the art to determine other chaotropic salts which may be used to recycle the solid phase immunosorbent, without undue experimentation. In addition, the eluted titrating antibody may be separated from the eluted analyte according to means known in the art and may also then be recovered for re-use.
The eluted analyte may further be recovered.
The assay of the present invention is ideally suited for the preparation of a kit. Such a kit may comprise a carrier means being compartmentalized tc receive in close confinement therewith one or more container mean~ such as vials, tubes and the like, each of ~aid container means comprising the separate elements of the immunoassay. ~or example, there may A24.1.WP 051287 ~L301~35~
be a container means containing the first antibody immobil-ized on a solid phase support, and further container means containing detectably labeled titrating antibodiPs in solution. Further container means may contain standard solutions comprising serial dilutions o~ analytes to be detected. The standard solutions of these an~lytes may be used to prepare a standard curve with the concentration of the analyte plotted on the abscissa and the detection signal on the ordinate. The results obtained from a sample ¢ontain-ing an analyte may be interpolated from such a plot to give the concentration of the analyte.
In another e~bodiment of the ki~, there may be a container means containing a dipstick which comprises a multiplicity of different analyte-specific antibodies separately immobilized to separate defined areas of the solid phase support. Further container means may contain a common titrating antibody which is specific for each analyte-specific antibody. Further container means may contain standard solutions of analytes to be detected. The standard solutions of these analytes may be used to provide a standard reference dipstick for comparison with the sample dipstick.
The various aspects of the invention are further described in the following examples. These examples are not intended to limit the invention in any manner.
EXAMPLES
EXAMPL~ 1 General Procedure For the ImmunoassaY
In the examples that follow, a simple antibody-antigen system illustrates the protocol used for the assay for one antigen by isotopic detection.
A24.1.WP 051287 3~
Sheep anti-rabbit serum (SAR) was induced by sheep by standard procedures and heat inactivated at 56C fsr 30 ~inutes. Rabbit IgG was purchased from Sigma (St. Louis, M0). Iodination was achieved using iodo-beads (Pierce Chemical Company, Rockford, IL) using carrier-free ~odium iodid~ 25 (Dupont N~w England Nuclear, 8Os~on, MA). All other chemicals wer~ reagent grade and were purchased from either 5igma Chemical Corporation (S~. Louis, M0~ or Fisher (Medford, ~A).
Nitrocellulo~e paper (~illipore filter type HAHY 000-lO, O.45 uM pore size, Millipore Corporation, 9edford, MA) was used unless stated otherwise. The other papers te~ted include: Zeta Probe blotting membrane (cationized nylon, BioRad, Ric~mond, C~), mixed cellulose acetate and nitrate paper (GSWP 013-00, O.22 uM pore size3 and Millipore MF
filters (HAWG04750, 0.45 uM pore size, ~illipore Corporation, Bedford, MA), high binding capacity S&S Nytran (positively charged hydrophilic nylon-66, pore siæes 0.2 uM and 0.45 uM), S&S pure 100% hydrophilic nitrocellulose in different pore sizes (no cellulose acetate added, PH70, 0.025 uM pore size and BA83, pore size 0.2 uM, and ~A85, pore size 0.45 uM, Schleicher and Schuell, Xeene, NH), Hydrophilic Hybond-C 87 mm nitrocellulose and Hybond-N 132 mm nylon membranes (Amersham Corporation, Arlington Heights, ~L).
Sev~n millimeter diameter disks of nitrocellulose (Millipore ~A 0.45 uM pore, unless stated otherwise) were made using a standard o~fice hole puncher and transferred by needle to a 15 x 75 millimeter polystyrene tube. Volumes between 50-300 ul o~ diluted antisera (SAR) were incubated with the disXs at room temperature for 10 minu~es. After washing in Tris-EDTA-azide, the samples were inubated in 1-2 ~l of blocker. All ~itration and wash bufPers utilized a base buffer o~ 50 mM Tris, 5 mM EDTA, O. 01% sodium azide at *Trade Marks A24.1.WP 051287 :~ ~
~3~1~3Sg:~
pH 7.4. Blocking buffers included 10% (v/v) bovine serum, 3 BSA, or 5% Carnation non~at milk. The antigen (xabbit IgG) was t~en added (50~300 ul) and ~ncuba~ed for 30-60 minutes, as indicated in the following Examples. After washing three ti~est iodinated non-immune rabbit serum (NIRS-I125) was ~dded (50-300 ul) for the ~ime ~tated (30 minutes minimum).
After a second wash, the ~a~ples were counted for gamma radiation on a 40% efficient Packard Auto-Gamma Scintillation Spectrophotometer Model 5220.
EXAMPL~_2 The Effic,iency of ~qG Immobilization The efficiency of IqG immobilization on nitrocellulose disks was determined as follows~ Serial dilutions of SAR
were incubated with a ni~rocellulose disk. The nitrocellu-lose ~ilter were then washed and titrated with iodinated antisera. A plot o~ the amount of bound IgG, as represented by the amount o~ radioactivity, appears sc~ematically in the inset to Figure 2. The competition between IgG and the larger proportion o~ other serum proteins for protein binding sites on nitrocellulo6e results in the affinity displacement (A) of IgG at high serum concentrations. As the antiserum concentration diminishes, the amount of immobilized IgG
increases until the inflection point (X) is reached, where all the sample protein i8 absorb~d equally. Diminishing concentrations of IgG beyond the in~lection point are ~hen reflected by diminishing counts of ti~rating antibody (B).
This ln~lection point can be displaced to higher serum concentration~ i~ desired ~imply by providing more nitrocel-lulose binding 8ite5. Figure 2 shows the relationship between the number of nitrocellulose disks and the amount o~
bound radioactivityO 500 uL of ~heep anti-rabbit serum was *Trade Marks A24~1.WP 051287 ~,, ~3~183S~) incubated ~or 10 minutes with 1-10 disks as shown in Figure 2, washed with Tris buffer, blocked with 1 ml of 10% bovine serum blocker for 20 minutes, wàshed, ~itrated with 1 ml of NIRS-I125 for 60 minutes, washed, and the radioactivity bound to the disk determined. A~ a tenfold increase in nitrocel-lulose surface area, the number oP binding sites was no longer limitiny for that concentrat~on of sample. This illustrates that it is advantageous to use dilute sera for coating the matrix. Alternatively, puri~ied IgG may be used to achieve high coating densities.
Stability of the Immobilized IaG
The stability of immobilized IgG on different immobili-zation matrices, with and without 20% methanol, was deter-mined as follows. Disks of cellulose or nylon papers were incubated with iodinated SAR in Tris-EDTA for 20 minutes, with or without 20% methanol. The disks were then washed with Tris-EDTA and the radioactivity bound to the disks measured. The following papers were tested: 1) BioRad Zeta Probe, 2) Millipore nitrocellulose HA 0.~5 uM pore, 3) Millipore mixed esters 0.22 uM pore, 4) Millipore MF 0.45 uM
pore, 5) S&S Nytran 0.2 uM pore, 6) S&S 100% nitrocellulose 0.025 uM pore, 7) S~S Nytran 0.45 uM pore, 8) S&S 100%
nitrocellulose, 0.2 uM pore, 9) S~S 100% nitrocellulose, 0.45 uM pore, 10) Amersham Hybond Nitrocellose, 11) Amersham Hybond nylon. The amount of bound radioactivity is depicted in Figure 3. The relative abilities of different papers to absorb iodinated SA~ under the best conditions (with (+) or without (-) 20% methanol) is depicted in Figure 4. Millipore 0.45 uM pore nitrocellulose gave the best results and was chosen for all subsequent experiments. Mixed acetate paper A24.1.WP 051287 ~ 3 [)~
also performed well despite reports in the literature that such papers poorly absorbed proteins (Gershoni, J.M., et al., Anal. Biochem. 131:1-15 (1983)~. For S&S pure nitrocellulose paper, smaller pore sizes performed better (0.02 v~rsus 0.45 13M ) . ~
The e~fsct on stability o~ the immobilized IgG was then examined during recycling. Nitrocellulose disks in this experiment, were recycled four times. Nitrocellulose disks containing immobilized sheep anti-rabbit IgG and titrating anti~ody NIRS-I125 were e~uted with 2.5 M MgCl2 and the loss of antibody during the elution cycle determined. Four elutions with MgCl2 resulted in a 20% loss of absorbed antibody (as determined by iodinated sheep anti-rabbit serum3 as shown in Figure 5. To test the ~unctional integrity of the SAR remaining after each cycle of the elution, the disks were inc~bated with 150 uL o~ iodinated NIRS-antigen for 60 minutes, followed by washing in milk bu~fer, and counting the radioactivity remaining on the disk. Background counts were determined using the counts adhering after elution of the iodinated antigen. All data as shown in Figure 5 is normali-zed as a percentage of the starting value. During regenera-tion cycles, nonspecific binding increased gradually, which indicates the absorption of label onto sites vacated by the blocker protein or the sheep anti-rabbit serum.
In thi~ example, the amount of IgG necessary to give a readable ~ignal was determined. Five ul aliquots of serial dilutions of SAR were spotted onto disks o~ nitrocellulose and dried. They were blocked for 30 minutes in 2 ml of 5%
non~at milk, washed in Tris-EDTA, and incubated with 150 ul A24.1.WP 051287 , ~L3~3~q~
of iodinated NIRS in 3~ BSA for 60 minutes. Following thorough washing, the radioactivity bound to the disks was determined. As can be seen in Figure 6, the lowest detect-able amount of bound IgG under these conditions was approxi mately 25 ng. A parallel series of experiment~ were con-ducted by amplifying the detection signal with iodinated S~R.
Again, the lowest detection limit was about 25 ny.
The amount of Ig~ bound to the dis~s after brief incubation with a range of serum dilutions is depicted in Figure 7. 100% efficient absorption was achieved at a titre of approximately 1:500. At lower dilutions, decreasing amounts of sample IgG were absorbed onto the matrix. The capacity of the nitrocellulose is approximately 80 ug/cm2.
The area of the disk used was about 1 cm2. Thus, the amount of specific IgG is far short of saturating the disk. About 50% absorption of total sample IgG was achieved by using 1:128 dilution of serum.
O~timizatiQn_of Incubation Time for Disk Coatinq To achieve maximal immobilization of antisera, serial dilutions of SAR were incubated with nitrocellulose disks for minutes or overnight. A comparison of a 10 minute incubation with an overnight incubation for a wide range of SAR dilutions is shown in Figure 8. A lO-minute incubation bound more IgG than an overnight incubation. In addition, an overnight incubation showed an inflection point. The development of the inflection point only in the longer incubation time is due to the displacement of IgG by other serum proteins, due to either mass action or af~inity differences. At all dilutions o~ SAR, the binding of Ig~ was diminished with the longer incubation time~ After incuba A24.1.WP 051287 ~3~)~335(~
tion, the samples were washed, blocked with 1 ml of 5% nonfat milk for 20 minutes, washed again and incubated with 450 ml of iodinated NI~S for 60 minutes in non~at milk buffer.
After thorough washing, the radioactivity bound to the disks was measured and is presented in Figure 8. ~he relationship of the time of incubation ~or shorter time periods and the radioactivity bound to the disks appears in Table 1.
Radioactivity bound to disk SAR concentration Time of incubation (minutes) Undiluted 1:4 0 (blank) -1 171 + ~0 158 + 12523 ~ 2 155 + 9 199 + 16984 + 14 151 + 33 171 + 26738 + 13 162 ~ 7 318 + 159 1036 + 136 126 ~ 4 172 + 0718 + 147 130 146 + 33 215 + 571190 + 8 .
Undiluted, 1:4, and samples with no SAR were spiked with~
iodinated SA~, and 100 ul incubated with each disk for th~
time shown. After washing with 2 ml volumes of 5% nonfat milk buf~er, the counts remaining on the disks were deter-mined. The total counts added initially were 3610 + 596.
Thus, the percentage of the total that became bound ranged ~rom 14% at one minute to 33% at 130 minutes. The counts are expressed as mPan and SD o~ duplicates. Maximal binding of IgG in a 100 ul sample occurred at 20 minutes at a 1:4 A24.1.WP 051287 . ........... -.
, ` :
~L308350 dilution~ However, this data was no~ significantly dif~erent from the 10 minute incubation. Table 2 depicts the percen-tage of total counts bound to the blank disk for each length of time. The binding plateaus between 10-20 minutes.
Adequate coating oP disks was achieved using 100 ul of SAR at a dilution of 1:200 for 10 minutes.
TABLE Z
Time (minutes) ~ of Total Counts Bound to Blank Disk _ 1 14.5 + 0.3 27.2 + 2 20.4 + 2 28.7 ~ 13 19.9 + 2 134 32O9 + 0.6 LRgend: Timecourse of SAR adsorption onto nitrocellulose disks. The data is derived from Table 1.
a~
Optimization of Incubation Time With Titratin~ Antibody ~ he relationship between the time of incubation of the titrating labeled antibody (60 or 140 minutes) and the amount of bound label for various serial dilutions of NIRS appears Figure 10. The disks were coated with SAR at a 1:~ dilution (100 ul for 20 minutes), blocked for 20 minutes in 1 ml of 5%
nonat milk buffer, and incubated with 200 ul of serial ~24.1.WP 051287 ' .
3~C~
-2~-dilutions of NIRS serum overnight at 4C. After thorough washing, the free sites were titrated with 100 ul of iodinated NIRS for ~he ~ime specified. After washing, ~he radioactivity bound to the disk was determined.
The 140 minute incubation did increase the amount of bound label and the amplitude of ~he signal, although the error bars were grea~er. This indicated that there was an increased amount of nonspecific bin~ing with longer incuba-tion times. The ampli~ication was grea~est at low concentra-tions of sample NIRS, where the n~mber of ~ree IgG binding sites was greatest. The titrating antibody may have a greater affinity for the immo~ilized IgG than the sample antigen.
~AMPLE 7 ~mplificatlon bv Two Iodinated Titratinq I~G's The effect of amplification of the signal was then examined. Antigen-free IgG molecules were incubated with iodinated NIRS followed by binding the remaining unbound IgG
molecules with an excess of unlabeled anti-IgG molecules to provide a carpet for the subsequent binding of a second labeled anti-IgG molecule. Amplification occurs as a result of the ability of each immobilized primary antibody to bind to more than one labeled IgG molecule. This second layer of molecules allows a higher binding of a second labeled titrating antibody by virtue of mass action. Further, there is a higher probability of collision between the titrating IgG molecule and the "carpet" target IgG molecules than collision with primary immobiliz~d antibody dua to their number. Amplification resulted in an increase in the signal, especially where there was less antigen-bound IgG.
A24.1.WP 051287 s~
~25-Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters and concentrations and conditions withou~ departing from the spirit and scope of the invention or any embodiment thereof.
The descriptions disclosed herein refer only to a model system. Any antibody-antigen combination may be determined by one o~ ordinary skill in ~he art without undue experimentation.
A24.1.~P 051287
-2~-dilutions of NIRS serum overnight at 4C. After thorough washing, the free sites were titrated with 100 ul of iodinated NIRS for ~he ~ime specified. After washing, ~he radioactivity bound to the disk was determined.
The 140 minute incubation did increase the amount of bound label and the amplitude of ~he signal, although the error bars were grea~er. This indicated that there was an increased amount of nonspecific bin~ing with longer incuba-tion times. The ampli~ication was grea~est at low concentra-tions of sample NIRS, where the n~mber of ~ree IgG binding sites was greatest. The titrating antibody may have a greater affinity for the immo~ilized IgG than the sample antigen.
~AMPLE 7 ~mplificatlon bv Two Iodinated Titratinq I~G's The effect of amplification of the signal was then examined. Antigen-free IgG molecules were incubated with iodinated NIRS followed by binding the remaining unbound IgG
molecules with an excess of unlabeled anti-IgG molecules to provide a carpet for the subsequent binding of a second labeled anti-IgG molecule. Amplification occurs as a result of the ability of each immobilized primary antibody to bind to more than one labeled IgG molecule. This second layer of molecules allows a higher binding of a second labeled titrating antibody by virtue of mass action. Further, there is a higher probability of collision between the titrating IgG molecule and the "carpet" target IgG molecules than collision with primary immobiliz~d antibody dua to their number. Amplification resulted in an increase in the signal, especially where there was less antigen-bound IgG.
A24.1.WP 051287 s~
~25-Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters and concentrations and conditions withou~ departing from the spirit and scope of the invention or any embodiment thereof.
The descriptions disclosed herein refer only to a model system. Any antibody-antigen combination may be determined by one o~ ordinary skill in ~he art without undue experimentation.
A24.1.~P 051287
Claims (10)
1. A method for determining the presence of an analyte in a sample, comprising:
(a) contacting a sample suspected of containing the analyte with a solid phase support onto which an analyte-specific first antibody has been immobilized;
(b) incubating said sample with said support for a sufficient amount of time to allow the analyte present in the sample to bind to said first antibody;
(c) separating said solid phase support from the incubation mixture obtained in step (b);
(d) contacting said solid phase support with a second detectably labeled titrating antibody which is specific for said first antibody;
(e) incubating the mixture formed in step (d) for a time sufficient to allow said titrating antibody to bind to said first antibody;
(f) separating said solid phase support from the incubation mixture obtained in step (e); and (g) detecting the analyte in the sample by measuring the amount of bound labeled antibody, wherein the quantity of analyte is inversely proportional to the amount of bound labeled antibody.
(a) contacting a sample suspected of containing the analyte with a solid phase support onto which an analyte-specific first antibody has been immobilized;
(b) incubating said sample with said support for a sufficient amount of time to allow the analyte present in the sample to bind to said first antibody;
(c) separating said solid phase support from the incubation mixture obtained in step (b);
(d) contacting said solid phase support with a second detectably labeled titrating antibody which is specific for said first antibody;
(e) incubating the mixture formed in step (d) for a time sufficient to allow said titrating antibody to bind to said first antibody;
(f) separating said solid phase support from the incubation mixture obtained in step (e); and (g) detecting the analyte in the sample by measuring the amount of bound labeled antibody, wherein the quantity of analyte is inversely proportional to the amount of bound labeled antibody.
2. The method of claim 1 wherein said solid phase support is selected from the group consisting of nitrocellulose, diazocellulose, microtitre plates, glass, polystyrene, polypropylene, polyethylene, dextran, Sepharose, agar, starch and nylon.
3. The method of claim 1, wherein said solid phase support is selected from the group consisting of nitro-cellulose and diazocellulose.
4. The method of claim 1 wherein said detectable label is selected from the group consisting of a radioactive isotope,a dye, a fluorescent label, a bioluminescent compound, and an enzyme.
5. The method of claim 1 wherein said first antibody is of the IgM, IgA, IgD, IgE or IgG immunoglobulin class.
6. The method of claim 5 wherein said first antibody is of the IgG immunoglobulin class.
7. The method of claim 1, wherein said solid phase support is recycled after step (g) and said analyte and titrating antibody recovered by elution with a chaotropic salt.
8. The method of claim 7, wherein said chaotropic salt if MgC12.
9. The method of claim 1 wherein said solid phase support contains a multiplicity of different analyte-specific first antibodies, each immobilized to separately defined areas and said second, detectably labeled titrating antibody is specific for each analyte-specific first antibody.
10. A method for determining the presence of a multiplicity of analytes in a sample, comprising:
(a) contacting a sample suspected of containing a multiplicity of analytes with a solid support on which different analyte-specific first antibodies have been separately immobilized to separately defined areas of said solid support;
(b) incubating said sample with said support for a sufficient amount of time to allow the analytes present in the sample to bind to said first antibodies;
(c) separating the solid phase support from the incubation mixture obtained in step (b);
(d) contacting said solid support with second detectably labeled titrating antibodies which are specific for said first antibodies;
(e) incubating the mixture formed in step (d) for a time sufficient to allow said titrating antibodies to bind to said first antibodies;
(f) separating said solid phase support from the incubation mixture obtained in step (e); and (g) detecting the analyte in the sample by measuring the amount of bound labeled titrating antibodies to each separately defined area, wherein the quantity of analyte is inversely proportional to the amount of bound labeled antibody.
(a) contacting a sample suspected of containing a multiplicity of analytes with a solid support on which different analyte-specific first antibodies have been separately immobilized to separately defined areas of said solid support;
(b) incubating said sample with said support for a sufficient amount of time to allow the analytes present in the sample to bind to said first antibodies;
(c) separating the solid phase support from the incubation mixture obtained in step (b);
(d) contacting said solid support with second detectably labeled titrating antibodies which are specific for said first antibodies;
(e) incubating the mixture formed in step (d) for a time sufficient to allow said titrating antibodies to bind to said first antibodies;
(f) separating said solid phase support from the incubation mixture obtained in step (e); and (g) detecting the analyte in the sample by measuring the amount of bound labeled titrating antibodies to each separately defined area, wherein the quantity of analyte is inversely proportional to the amount of bound labeled antibody.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US4937587A | 1987-05-14 | 1987-05-14 | |
| US049,375 | 1987-05-14 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1308350C true CA1308350C (en) | 1992-10-06 |
Family
ID=21959487
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000566734A Expired - Fee Related CA1308350C (en) | 1987-05-14 | 1988-05-13 | Multiple antigen immunoassay |
Country Status (3)
| Country | Link |
|---|---|
| EP (1) | EP0362284A4 (en) |
| CA (1) | CA1308350C (en) |
| WO (1) | WO1988008978A1 (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2233450B (en) * | 1989-06-24 | 1993-06-30 | Univ Wales Medicine | Detecting or quantifing multiple analytes with luminescent reagents |
| WO1992002818A1 (en) * | 1990-08-10 | 1992-02-20 | Purdue Research Foundation | Matrix sequential addition immunoassay |
| CA2104596A1 (en) * | 1992-01-06 | 1993-07-07 | Dade International Inc. | Multi-test immunochemical reagent and method to use same |
| EP0653065B1 (en) * | 1992-08-03 | 2002-10-30 | Marconi Optical Components Limited | Separation method |
| US5573911A (en) * | 1994-10-03 | 1996-11-12 | Lifecodes Corp. | Methods and materials for detecting autoimmune antibodies |
| GB0129776D0 (en) * | 2001-12-13 | 2002-01-30 | Sec Dep For Environment Food & | Assay device and method |
| GB0415860D0 (en) * | 2004-07-15 | 2004-08-18 | Oxford Immunotec Ltd | Solid supports |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4380580A (en) * | 1978-04-10 | 1983-04-19 | Miles Laboratories, Inc. | Heterogenous chemiluminescent specific binding assay |
| NZ190452A (en) * | 1978-05-19 | 1981-04-24 | Becton Dickinson Co | Solid phase assay receptor contacted with analyte and then with labeled analyte |
| US4305924A (en) * | 1979-08-08 | 1981-12-15 | Ventrex Laboratories, Inc. | Method and apparatus for performing in vitro clinical diagnostic tests using a solid phase assay system |
| US4254096A (en) * | 1979-10-04 | 1981-03-03 | Bio-Rad Laboratories, Inc. | Reagent combination for solid phase immunofluorescent assay |
| US4514508A (en) * | 1982-07-06 | 1985-04-30 | Biond Inc. | Assaying for a multiplicity of antigens or antibodies with a detection compound |
| US4427580A (en) * | 1982-09-01 | 1984-01-24 | Cornell Research Foundation, Inc. | Method for separation and recovery of proteins and nucleic acids from nucleoproteins using water destructuring salts |
| US4693985A (en) * | 1984-08-21 | 1987-09-15 | Pall Corporation | Methods of concentrating ligands and active membranes used therefor |
-
1988
- 1988-05-13 CA CA000566734A patent/CA1308350C/en not_active Expired - Fee Related
- 1988-05-16 WO PCT/US1988/001565 patent/WO1988008978A1/en not_active Application Discontinuation
- 1988-05-16 EP EP19880905482 patent/EP0362284A4/en not_active Ceased
Also Published As
| Publication number | Publication date |
|---|---|
| WO1988008978A1 (en) | 1988-11-17 |
| EP0362284A4 (en) | 1990-12-05 |
| EP0362284A1 (en) | 1990-04-11 |
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