CA1146067A

CA1146067A - Solid phase immunoassay with labelled anti-hapten antibody

Info

Publication number: CA1146067A
Application number: CA000352140A
Authority: CA
Inventors: A. Robert Neurath; Nathan Strick
Original assignee: New York Blood Center Inc
Current assignee: New York Blood Center Inc
Priority date: 1979-05-21
Filing date: 1980-05-16
Publication date: 1983-05-10
Also published as: US4495296A; AU538814B2; DE3071377D1; AU5855480A; EP0019277A1; EP0019277B1

Abstract

Abstract of the Disclosure A process for detecting the presence of an antigen in a specimen is described, which process comprises:
A) contacting said specimen with a substrate coated with antibodies of said antigen, incubating the contacted substrate and washing the substrate;
B) contacting the washed material of step A) with a hapten conjugated antibody against said antigen, incubating the so-contacted material and washing the so-incubated material;
C) contacting the washed material of step B) with a radioactive material labeled or enzyme containing anti-hapten antibody, incubating the so-contacted material and washing the same; and D) effecting radioimmunoassay if said antibody is radioactive or enzyme labeled immunoassay if said antibody contains an enzyme moiety.
Quantitative determination of the antigen in the specimen is effective by comparing the counts of the radio-immunoassay ir the concentration of enzyme against a standard as by photocolormetric methods.

Description

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BACKGROUND OF THE INVENTION

Field of the Invention . _ .

This invention relates to a process for detecting the presence of an antigen in a specimen. More especially, s this invention relates to the use of a universal labeled or enzyme containing antibody useful in the detection of a wide variety of antigens in a test specimen. ,More especially, this inventlon relates to the use of rsdioactive labeled or enzyme containing anti-hapten antibodies in the detection of the presence of and the amount of antigens in a test specimen.

Discussion of the Prior Art , Radioimmunoassay techniques for biochemical and immunological studies and for clinical research and diagnosis have become an invaluable tool. However, their applicability has been confined to reasonably well characterized antigens which can be purified and used for the preparation of anti-sera serving as a source for isolatior. of immunochemically purified antibodies. Although 12 5~-labeled staphylococcal

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, , ' 1146~67 protein A has becn suggested as a general radioactive reagent for radioim~unoassay, it cannot be used for sandwich type tests with an antibody-coated solid phase. If neither anti8en nor the corresponding antibody are available in relatively purified form, it becomes difficult to prepare radiolabeled reagents for radioimmunoassay (RIA) suitable ior the detection C oi nanogram quantities of antigens.
It therefore became desirable to provide ~ process for the detection of and the quantitative measurement of anti-gens, which process could be used for those antibodies and antigens whose purification into relatively ourified form was not heretofore known. More especially, it became desirable to provide a process by which nanogram quantities of antigens could be detected, which orocess did not rely upon the purifi-~5 cation of antibodies and antigens as source material for the test. Still more especially, it became desirable to provide a proce6s by which one could use a universal reagent for the detection of the presence of a wide variety of different types of antigens.
These and other objects of this invention will become more apparent from the ensuin~ description and claims.

SU~DIARY OF T~E INVENTION

In accordance with this invention, there is provided a process by which the presence of a wide variety of different types of antigen can be detected in test specimens. The pTO-cess comprises: , 1 '' ~ ', ' ~ . ~

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A) contacting a test specimen suspected of containing a given antigen with a substrate coated with ~nt:Lbodies of said antigen, incubating the contacted substrate and wa~ing the substrate;
B) contacting the washed material of step A) with a hapten conjugated antibody against said antigen, incubating the so-contacted material and washing the so-incubated material;
C) contacting the washed material of step ~) with a radioactive material labeled or enzyme containing anti-hapten antibody, incubating the so-contacted material and washing the same; and D) effecting radioimmunoassay if said antibody is radioactive or enzyme labeled immunoassay if said antibody contains an enzyme moiety.
8y conducting the process thusly, the quantitative presence oP antigen can be determined without employing a purifled source of antigen to prepare purlfied antibody.
Qualitative determination of the antigen content in the speci-men is effected by comparing the counts derived from the radioimmunoassay or the enzyme concentration in the case of enzyme labeled immunoassay (ELISA) with a standard known to be free of the antigen. Quantitative determination is effected by.comparing the counts or enzyme concentration against data derived from the same test protocol using samples of known antigen concentrations.
The procedure of the invention takes advantage of the ability of anti-hapten an.tibody to readily react with hapten groups on the antibody employed in step B) which has, in ~urn, reacted with antigen present in t~e test specimen.
This antigen present in the test specimen has previously reacted with the corresponding antibody held on the substrate. By ~his ~,~ , .

~1 technique, the initial antibody employed on the substrate need not be particularly pure and the quantity of antigen in the specimen is readily detected owing to magnification of .
test results a8 a result of the described sandwich technique wherein hapten conjugated groups on the antibody are reacted with anti-hapten antibody In accordance with the process, the substrate con-taining the antibody is contacted with test specimen containing the suspected antigen. The suspected antigen. reacts with the .10 antibody on the substrate and, in turn, is available for fur-ther reaction with a hapten conjugated antibody. ~hen, in accordance with step B), the hapten conjugated antibody contacts the antibody-antigen product ~esulting from step A), there is formed a sandwich structure wherein the antigen is sandwiched on one side by the substrate-antibody reagent and on the other side by tlle hapten con~ugated antibody, The sandwich structure which results has available hapten groups, since it is the antibody portion of the hapten conjugated antibody that reacts with the antigen held by the substrate-antibody material used in step A). This makes the hapten groups on the hapten conjugated antibody readily available for reaction with radio- or enzyme-labeled anti-hapten antibody. Since the hapten conjugated antibody can contain a multitude of hapten moieties, subsequent reaction with the labeled anti-hapten antibodies provides a substance which provides a magnified count whether analysis be by radioimmunoassay or ELISA. In other words, since the qoantity of hapten moieties on the hapten conjugated antibody .

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is many multiples of the number of antigens absorbed, a greater nu~ber of anti-hapten antibodies will react with those sites.
This means that the number of counts per antigen is greater than in the ~tandard radioimmunoassay techniques. This magni-fication permits the measurement of nanogram quantities of antigen in the test specimen. It is this magnification by the use of hapten conjugated antibody with the universal labeled anti-hapten antibody that permi.ts use of antibody reagents in -step A) which are not particularly pure.
The process of the invention can be used with respect to any antigen, the presence of which is suspected in a given serum. All that is required is that an antibody of such sus-pected antigen be deposited on a substrate, that the specimen containing the suspected antigen contact the antibody on the substrate, incubation is effected and the so-incubated material is washed. Thereafter, in accordance with the secont proce~ural series of steps, the washed material ~s contacted with hapten con~ugated antibody against said antigen, which contacting is also followed by incubation and washing. lhese steps provide the hapten moieties on the antibody against the suspected antigen, which hapten moieties will react with radioactive labeled or enzyme containing anti-hapten antibody. There-after, the anti-hapten antibody which is either radioactive labeled or contains an enzyme is contacted with the washed material which is followed by incubation and washing. Radio-immunoassay or enzyme labeled immuno~ssay is effected to determine qualitatively the presence of the antigen and quanti-tatively the amount of antigen by comparison with pre-prepared ~ ~ .

I''''' '-'- , ' 1146~167 standards The higher the counts from a ~-counter or the higher concentration of enzyme in an ELISA test, the higher is the quantity of antigen in a test specimen.
Antigcns whose presence and a~ount can be detected ~n accordance with the claimed process include essentially any antigen, for example viral, e.g., hepatitis B, influenza, a~enovirus, and all other viral antigens, as well as bacterial antigens, tumor-specific antigens, serum antigens, enzyme prQteins and all other antigens having at least twc antigenic sites.
The antibodies of these antigens can be hapten conjugated with a wide variety of haptens including those which provide the following hapten moieties: dinitrophenyl, trinitrophenyl, diazotized sulfanilic acid, p-azobenzene arsonate, benzyl penicillin, p-azobenzoate, aspirin, fluorescein, isothiocyanate, p-iodobenzoate, p-~p'-hydroxy-phenylazo) benæoate, pho8phorylcholine and others.
The conju~ation of haptens with proteins and the preparation of anti-hapten antibodies and their properties hsve been extensively reviewed (see, for example: "Advanced Immunochemistry", E.D. Day, Williams E. Williams, Baltimore, 1972; A.L. deWeck, "Low Molecular Weight Antigens" in: THE
ANTIGENS, Ed. M. Sela, Academic Press, New York, 1974, Volume 2, pages 142-249).
Anti-hapten antibodies can be formed which corre-spond, in respect of the hapten moiety, to the hapten moiety on the conjugated antibody. Thus, the labeled anti-hapten antibody used in step C) corresponds with reæpect to its , ~.. .... ....... . . . .

~146Q67 hapten moicty to the hapten moiety of the hapten conjugated antibody against the suspected antigen. The same can be pre-pared in known manner, as by haptenating an antigen and in,troducing the so-haptenated antigen in~o a test animal, such as a rabbit, to effect an antibody response. As a result thereof, as i5 known, there is formed the antibody of the antigen and an anti-hapten antibody. The resultant serum is recovered and the anti-hapten antibody is separated from the other serum proteins including the antibody of the original antigen.
The anti-hapten antibody is thereafter labeled, either with a radioactive material such as I 12 5 or I 131 or is conjugated with an enzyme whereby there is formed an enzyme-containing anti-hapten antibody. This enæyme-cGntaining anti-hapten antibody can then be used as a "labeled" anti-hapten antibody - labeled in the sense that it contains an enzyme, but is not radioactive. Detection of the absorption of the "labeled" anti-hapten antibody can be by RIA or ELISA in accordance with known techniques. RIA involves the use of a , 20 radiation detection means, whereas ELISA inv,olves a measurement of the concentration of enzyme. The higher the enzyme con-centration, the higher is the concentration of antigen adsorbed and the concentration of antigen in the original test specimen.
The incubation required in accordance with steps A), B), and C) can be effected in known manner, such as under the following conditions: 1-8 hours at 37-50C or 16-72 hours at 18-30C.
Washing is typically effected using an aqueous . .
' solution such as one ~uffered at a pH of 6-8, preferably at a pH of about 7, employing an isotonic saline solution.

BRIEF DESCRIPTION OF DRAWINGS

Referring to the drawings herein:
Fig. 1 shows the results of radioimmynoassay tests for ferritin and ovalbumin using a dinitrophenylated anti-body. Normal human and sheep serum were used as diluent for ferritin and ovalbumin;
Fig. 2 shows the test results for adenovirus group-specific antigen using dinitrophenylated antibodies.
Normal goat serum was used as diluent. The control corresponds to a 1:5 dilution of tissue culture medium from non-infected cells. The fluid harvested from infected cells had a CF titer of 1:32; and Fig. 3 show6 a comparison of RIA tests for hepat$tis B e-antigen (HBeAg). Dilutions of HBeAg-positive human serum in normal human serum ( - control ) were tested.
In order to more fully illustrate the nature of the invention and the manner of practicing the same, the following examples are presented:

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1146~67 EX~`IPLES

Horse spleen ferritin and apoferritin were obtaihed from Sigma, St. Louis, Missouri; ovalbumin (5x crystallized), rabbit anti-ferritin and rabbit anti-DNP-bovine serum albumin (BSA) were obtained from Miles Laboratories, Elkhart, Indiana;
goat anti-ovalbumin was from Research Products International Corp., Elk Grove Village, Illinois. Tissue culture medium containing adenovirus group-specific complement-fixing (CF) antigen and the corresponding goat antiserum (GF titer 1:128) were obtained from Microbiological Associates, Walkersville, Maryland.
IgG isolated from the antisera by chromatography on DEAE-cellulose (H.H. Fudenberg, ~ethods in Immunolo~y and Immunochemistry, Academic Press, New York, Volume 1, pages 321-324, 1967) was uscd to coat polystyrene beads (diametcr 6 mm; Precision Plastic Ball Co., Chicago, Illinois) at a concentra~ion of 100 ~g/ml in 0.1 M tris-(hydroxymethyl) aminomethane, pH 8.8 (Neurath et al, J Gen. Virol., 38, 549-559, 1978). Aliquots of IgG (50 tolO0 ~g in 200 ~1), dialyzed against 0.05 M borate pH 8.5 were labeled with 0.5 to 1.0 mCi of 12 5I-Bolton-Hunter reagent (Amersham, Arlington Heights, Illinois) overnight at ODC. After addition of 200 ~1 of 1.0 M glycine-0.1 M borate pH 8.5 for 30 minutes, the labeled IgG was separated from other radioactive products by gel C filtration on 0.7 x 20 cm columns of SephadeY G-75 using as eluant 0.05 M phosphate pH 7.5 containing 2.5 mg/ml of gelatine.

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Dinitrophenylation of proteins was carried out as described ~A.W. Wheeler and P.M. Hatcher, J. Immunol.
Methods, 13, 29-37, 1976) except that the final concentration of sodium 2,4-dinitrophenylsulfonate was 10 2 and the pH
was 9.5.
To isolate anti-DNP from anti-DNP-BSA, 2 ml of the antiserum were mixed with 10 mg of DNP-apoferrin. The mixture was incubated 1 hour at 37C, overnight at 4C, and centrifuged for 1 hour at 90,000 x g. The pellet was dis-solved in 1 ml of 8 M urea-0.01 M phosphate pH 8.0-0.1 percent r Nonidet P40 (BDH Chemicals, Ltd., Poole, England) (UPN) and ~" applied to a 2 ml column of DEAE-cellulose (DE 52; Whatman, Springfield Mill, Maidstone, Kent, England) prewashed with UPN. Anti-DNP IgG recovered in the void volume of the column after elution with UPN was dialyzed first against 0.01 M
tris (hydroxymethyl) aminomethane-0.14 M NaCl-0.02 percent NaN3 (TS) contain~ng 1 mg/ml of Nonidet P40 and then against 0.05 M borate pH 8.5 for labeling with 12 5I-Bolton-Hunter reagent.
For RIA tests, antibody coated beads were incubated overnight at 20C with dilutions of tle corresponding antigens in normal sera (400 ~1). The beads were washed with TS and then incubated with dinitrophenylated immunoglobulins (0.6 to 2.5 ~g IgG per test), diluted in the same normal sera used for the first incubation (except in assays for HBeAg, for which normal human serum diluted 10-fold in fetal calf serum was used) for 2 hours at 37C. The beads were washed with TS, incubated for 2 hours at 37C with 12 sI-anti-DNP (0.1 ~Ci per ~ ~r~de ma~

~ 1146~67 , test; specific activity 1.2 ~Ci/~g) in the proper normal sera as before, washed with TS and counted in a y-counter. In the direct RIA for ~IBeAg, beads were incubated with 12 sI-Bolton-Hunter reagent labeled antibodies to H~eAg (anti-HBe) instead of dinitrophenylated anti-HBe.
Results of RIA tests in which dinitrophenylated immunoglobulins were used are summarized in Figures 1-3. The sensitivity limit for detection of ovalbumin and ferritin was approximately 0.8 and 0.2 ng/ml, respectively (Figure 1).
This corresponds to a 4-15-fold increase in sensitivity as compared with direct RIA ~ests in which the corresponding immunochemically purified 12 sI-labeled antibodies were used.
The RIA for adenovirus group-specific antigen (Figure 2) was approximately 80x more sensitive than the CF test. Comparative RIA test8 ~or H~eAg uslng either ~2sI-labeled IgG from anti-HBe-positive human 8erum or tinitrophenylated anti-UBe IgG
followed by ~ 2 5I-labeled anti-DNP (Figure 3) provide evidence that haptens attached to immunoglobulins may serve as ampli-fiers in RIA tests. Such amplification is expected to facilitate the development of RIA tests for antigens which are inadequately characterized, difficult to purify or not available in sufficient quantities to allow the immunochemical purification of the corresponding antibodies.
The possibility of using 12 sI-labeled anti-DNP
(or other labeled anti-hapten antibodies) as universal re-agents may simplify the development of RIA tests and widen their application to various areas of research and clinical diagnosis.

Claims

Community Blood 216-JFW/ss WHAT IS CLAIMED IS:

1. A process for detecting the presence of an antigen in a specimen which comprises:
A) contacting said specimen with a substrate coated with antibodies of said antigen, incubating the so-contacted substrate and washing the substrate;
B) contacting the washed material of step A) with a hapten conjugated antibody against said antigen, incubating the so-contacted material and washing the so-incubated material;
C) contacting the washed material of step B) with a radioactive material labeled or enzyme containing anti-hapten antibody, incubating the so-contacted material and washing the same; and D) effecting radioimmunoassay if said antibody is radioactive or enzyme labeled immunoassay if said antibody is enzyme labeled.

2. A process according to claim 1 wherein the results of said radioimmunoassay or enzyme labeled immunoassay are compared with a known standard or standards to determine the concentration of antigen in the test specimen.

3. A process according to claim 1 wherein said hapten conjugated antibody is dinitrophenyl conjugated antibody and said anti-hapten antibody is anti-dinitrophenyl antibody.

4. A process according to claim 1. wherein said anti-hapten antibody is a radioactive labeled antibody and the washed material of step C) is subjected to radioimmuno-assay.

5. A process according to claim 1 wherein said anti-hapten antibody is an enzyme-containing anti-hapten antibody and the washed material of step C) is subjected to enzyme labeled immunoassay.