WO1991006559A1

WO1991006559A1 - Use of polymerized immunoglobulin to reduce the incidence of false-positive responses in immunoassays

Info

Publication number: WO1991006559A1
Application number: PCT/US1990/005943
Authority: WO
Inventors: Eileen G. Gorman; David Ta-Chih Hua; Richard Lee Tyson; Deborah K. Vickery; Chi-Chin Wang
Original assignee: E.I. Du Pont De Nemours And Company
Priority date: 1989-10-24
Filing date: 1990-10-22
Publication date: 1991-05-16
Also published as: IE903795A1; AU6710790A; PT95669A

Abstract

The present invention relates to a method to reduce false positive results in immunoassays. The method uses polymerized immunoglobulin to overcome binding which is commonly referred to as non-specific binding. The falsely elevated signal generated in the presence of human serum in assays for infectious disease markers, cancer related antigens and peptide hormones is reduced in assays performed in the presence of the polymerized immunoglobulin. The polymerized immunoglobulin can be prepared using different methods of polymerization.

Description

TITLE USE OF POLYMERIZED IMMUNOGLOBULIN TO REDUCE THE INCIDENCE OF FALSE-POSITIVE RESPONSES IN IMMUNOΛSSAYS TECHNICAL FIELD

This invention relates to a method for reducing false positive responses in enzyme immunoassays, more specifically to the method of using polymerized immunoglobulin for removing false positive responses in immunoassays.

BACKGROUND ART Immunometric assays are widely in use for detection of antigens having multiple epitopes . Typically, such an assay involves the use of at least two antibodies, a capture antibody being immobilized onto a solid support and a detector antibody conjugated to a label. One of the most commonly used labels is an enzyme while colorometric, radioactive, fluorescent or chemiluminescent and other labels are available. An enzyme label offers high sensitivity, ready availability and stability compared to a radioactive label. However, the enzyme being a large protein contributes to non-specific protein-protein interactions which induce false positive responses, also refered to as non-specific responses. By a false positive response is meant a response that is not specifically associated with the binding of the capture or the detector antibody to the antigen of interest. Although the problem of false positives is less frequent in immunoradiometric or competitive radioimmunoassays, it manifests itself in high sensitivity assays requiring large sample size and long incubation step [Hunter et al, Lancet, Vol. ii, 1136, 1980] . Thus, it remains a universal problem to be solved. The false positive response can be observed in assays for many different types of clinical parameters, both quantitive and qualitative in nature, including, for example, hormones of peptide nature, such as hCG, LH, somatotropin, FSH, ACTH, LPH, prolactin, MSH, beta- endorphinns, enkephalines, vasopressin, oxitocin, and the like. In addition, false positive results can be observed in determination of markers which are indicators of malignancy, such as, carcinoembryonic antigen (CEA) , alpha-feto protein, prostatic specific antigen, cancer antigens ca 19.9, ca 125, ca 15-3, beta- 2-microglobulin, ferritin, prostatic acid phosphatase, and the like. Assays for specific microbial and viral disease markers are also subject to false positive results. Such markers are hepatitis B surface antigen, hepatitis B core antigen, antibody to hepatitis B core antigen, antibody to HIV, pl7 antigen, ρ24 antigen, HTLV 1 and HTLV 2 antigen and specific peptide markers of infection with HTLV 1 and HTLV 2, markers for hepatitis A, C, D and E.

One form of non-specific interaction involves IgM rheumatoid factor (RF) . Methods for removal of RF interference using non-specific aggregated IgG are well known in the art . It is also well known that aggregated IgG has enhanced reactivity with RF compared to unmodified monomeric IgG [Oreskes et al, Immunology, Vol. 51, 115-121, 1984, and Dissanayake et al, Immunology, Vol. 32, 309-318, 1977]. In fact, Hallgren, US 4,184,847 and US, 4,153,417 discloses a method for detection of RF using labeled aggregated IgG.

Most of the methods for removing RF interference involve a sample pretreatment process. Sakai et al, US 4,680,274, issued July 14, 1987 disclose the use of ultrafine particles of less than 0.2 micrometers in size having immobilized immunoglobulins thereon to absorb non-specific factors from the sample. Vejtorp, J. Virol. Methods, Vol. 1, 1-10, 1980 describes the use of latex particles coated with aggregated human IgG as means for removing RF from the sample in an ELISA based rubella IgM antibody assay. Forghani et al, J. Clin. Microbiol. Vol. 18, 652-657, 1983, also describe a sample pretreatment method using polymerized human IgG. Here, the sample is pretreated with glutaraldehyde- aggregated human IgG followed by centrifugation to remove the interferant . These references do not discuss nor suggest the use of polymerized immunoglobulin, which is of the same animal species of the capture and detector antibodies, in sample or conjugate diluent.

Briantais et al, J. Virol. Methods, Vol. 9, 15-26, 1984, describe the use of a radiolabeled (Fab') 2 antibody to overcome RF interference in rubella and hepatitis B core IgM assays. In their tests for human IgM, they describe specific IgG binding non-specifically with the immobilized IgG antibody. This type of interference is said to be reduced only by the addition to the dilution medium of non-specific human IgG or fetal calf serum (FCS) . They use unmodified IgG to reduce the IgG-IgG interaction in an IgM capture assay. The advantageous use of polymerized Immunoglobulin as in assay milieu is not recognized.

Gerlich et al, J. Med. Virol. Vol. 4, 227-238, 1979, describe the use of excess heat-aggregated human IgG in a sample diluent to remove false positive responses in a hepatitis B core IgM antibody assay. Vejtorp, Acta Path. Microbiol. Scan., B, Vol. 89, 123-128, 1981, also describes the additon of heat- aggregated human IgG to the serum diluent eliminating RF interference in a rubella IgM assay. These are methods for reducing RF interference applicable only in IgM assays. The present invention is useful in immunoassays in which the analyte of interest can be viral markers, hormones or tumor markers, when both RF-positive and RF-negative samples are tested.

A mechanism for false, positive responses in two site immunoassays has recently been proposed by Levinson et al, Clin. Immunol. Letter, Vol. 9, 101-116, 1988. It involves endogeneous immunoglobulins (heterophile antibodies) in a sample having broad specificities toward animal antibodies. These heterophile antibodies are capable of bridging capture antibodies with detector antibodies in the absence of an antigen, producing a non-specific reponse. Removal of this type of interference requires either a sample pretreatment with immunoglobulins from several species or addition of these immunoglobulins to the assay milieu.

The addition of non-specific immunoglobulins derived from the same species as the antigen-specific antibody is commonly used to block non-specific binding caused by heterophile antibodies. For example, the addition of a non-specific murine monoclonal antibody that is of the same subclass as those of the specific monoclonal antibodies is shown to reduce non-specific interactions in a sandwich assay using murine monoclonal antibodies . [Fukuyama et al, JP Application, 63-12,960, published January 20, 1988]

R. C. McCarthy et. al., Arch. Pathol. Lab. Med., Vol. 112, pp. 901-907, 1988, describe the nature of "heterophile antibody" as primarily human IgM having reactivity to mouse IgG. They were able to inhibit this type of interference by adding an excess of unmodified mouse IgG in the sample (final concentration of IgG: 1.3 mg/mL) . They also report that the false positive responses can be reduced, but not completely eliminated, by using Fab' or (Fab')2 of IgG as the detector antibody. The present invention employs polymerized IgG with a defined range of molecular weight in the assay media, which is much more effective in eliminating the false positive responses.

These cited methods do not solve all of the false positive problems. For example, it is often observed in correlating enzyme-immunoassay (EIA) to radio- immunoassay (RIA) that EIA values in certain samples are always higher than the RIA values. Furthermore, due to the complex nature of protein-protein interactions involved, the incidence of false positives is highly dependent on the assay configuration. For example, a forward sandwich assay, wherein a sample is incubated with the capture antibody on a solid support, washed to remove all of the unwanted components, then incubated with the detector antibody, is less prone to non¬ specific interactions.

The present invention provides a method useful for reducing non-specific interactions in immunoassays wherein the capture antibody and detector antibody are derived from the same or different animal species . It is useful regardless whether the false positive sample is RF-positive or RF-negative. The method can be used either with a whole antibody or (Fab') 2 fragment.

SUMMARY OF INVENTION The present invention provides a method for reducing false positive responses in an immunoassay for detection of an antigen comprising:

(a) forming a heat or chemically polymerized immunoglobulin that has no specific reactivity with said antigen,

(b) treating a sample suspected of containing said antigen with said polymerized immunoglobulin, and

(c) subjecting the treated sample to an immunoassay employing antibodies of the same animal species as the polymeric immunoglobulin. EXAMPLE I: SYNTHESIS OF POLYMERIC IMMUNOGLOBULIN (POLY IG, The polymeric Ig can be generated by heating as described in the references mentioned in Background Art . Alternatively, it can be synthesized under defined chemical conditions as follow. In all cases the procedures are optimized with the goal of maintaining the solubility of the Poly Ig in buffered aqueous solution. 1.1 Reaction with S-Acetylmercaptosuccinic Anhydride (SAMSA) and N,N'-1-4-Phenylenedimaleimide (PPM) :

Sulfhydryl groups are introduced into a monoclonal IgG by treatment with SAMSA at 50 molar excess in phosphate buffer, pH 7.0. The sulfur protecting group is cleaved with hydroxylamine and the protein fraction is obtained by chromatography on Sephadex G-25, eluting with phosphate buffer pH 6.5.

The thiolated IgG is treated with a 20 molar excess of PDM and the reaction mixture is allowed to rock at room temperature. The progress of the polymerization reaction is monitored by HPLC analysis on a Zorbax(R) Bio Series GF-250 column (9.4 X 250 mm) . When the yield of IgG polymer of optimal size reaches a maximum the reaction is quenched with 2-mercaptoethylamine or directly fractionated by preparative HPLC on a Zorbax (R) Bio Series GF-250XL column (21.1 X 250 mm) . 1.2 Reaction with Disuccinimidyl Suberate (DSS) :

A purified monoclonal IgG (IgGl subclass) at approximately 5 mg/ml concentration, is reacted with DSS at 12 to 25 fold molar excess to the antibody. The mixture is stirred magnetically for 2 hours.

A sodium azide solution is added to the reaction mixture in order to achieve a final sodium azide concentration of 0.1%. After stirring for 0.5 hour, the mixture is transferred to a dialysis bag and dialyzed against a buffer solution containing 0.1 M Tris, 0.15 M Sodium chloride and 0.1% azide for 24 hours.

1.3 Reaction with Glutaraldehvde:

Monoclonal IgG solution at a concentration of 4 to 10 mg/ml is reacted with a 0.005% to 0.03% solution of glutaraldehyde at 20 to 24°C for 16 to 24 hours. Preferred conditions are IgG at 5 mg/ml, reacting with 0.02% glutaraldehyde at 22°C for 20 hours.

The solution is dialyzed against phosphate buffered saline overnight .

1.4 Molecular Weight Distribution:

The polymerized IgG preparations are analyzed by molecular sieving chromato raphy. The solution of polymer is fractionated in a glass column (approximately 2.5 cm in diameter and 85 cm in length) packed with a gel such as Sephacryl S300 (Pharmacia Co.) and equilibrated with 0.1 M phosphate buffered saline solution, pH 7.4.

In addition to monomeric IgG, four major polymeric ractions are obtained. By comparing to standard molecular weight markers, the median molecular weights of the four fractions are estimated to be 1,500,000 to 2,000,000; 1,000,000; 700,000 and 400,000 Daltons . These values translate to polymers of 10-15, 6-8, 4-6 and 3-4 IgG molecules respectively. All fractions are soluble in aqueous buffer, such as phosphate buffered saline.

Poly Ig prepared by different procedures described above consists of somewhat different proportions of the four fractions. However, the effectiveness in reducing false positivity can not be differentiated. The preferred poly Ig preparation therefore consists of 3 to 15 IgG molecules . EXAMPLE II: USE OF POLYMERIZED IMMUNOGLOBULIN IN AN ENZYME IMMUNOASSAY FOR HUMAN ANTIGEN (HBSAG ASSAY) An enzyme immunoassay for human hepatitis B surface antigen is used to demonstrate the efficacy of Poly Ig in reducing the incidence of false-positive responses. The assay steps are described as follows: II.1 TWO-STEP FORWARD FORMAT:

1. Antiαen Capture: In a plastic test tube, serum sample (200 ul) is first incubated with chromium dioxide particles (100-200 ug) , that are coated with anti-HBsAg monoclonal antibodies (IgGl subclass) , and a sample diluent (100 ul) containing 0.1 M sodium citrate, and 0.15 M^" sodium chloride. The incubation is allowed to proceed, for 30 minutes in a 37°C heating block.

2. Wash:

A wash solution containing 0.1 M Tris and 0.15 M sodium chloride, pH 7.8 was added to the reaction mixture. After mixing the slurry by vortexing, the test tube is placed in a rack equipped with small cylindrical magnets situated at both sides of the tube. The chromium dioxide particles are attracted to the side walls of the tube. The wash solution is then removed by suction. The wash, particle separation and aspiration steps are repeated two more times .

3. Enzyme Labelling:

A solution (50-250 ul) containing covalently conjugate of anti-HBsAg monoclonal antibody (or its (Fab')2 fragment, IgGl subclass) and calf intestinal alkaline phosphatase, 0.1 M Tris and 5% bovine serum albumin, pH 7.8 is added to the washed particles. The mixture is allowed to incubate at 37°C for 30 minutes. 4 . Wash :

The same wash steps as described in Step (2) are repeated.

5. Substrate Reaction and Measurement: A substrate solution (240 ul) containing

4-methylumbelliferyl phosphate in diethyleneamine, pH 8.9, is added to the washed chromium dioxide particles. The substrate reacts with alkaline phosphatase to generate, fluorescent 4-methylumbelliferone. The reaction is allowed to proceed for precisely 5 minutes at which time 0.5 M EDTA solution is added to stop the reaction.

The particles are separated from the solution in the magnetic rack and the clear supernatant is transferred to a IMMUNLON-2 (R) microtiter plate. The fluorescent signals are measured on a Dynatech MicroFLUOR(R) reader equipped with a 365 nm excitation filter and a 450 nm emission filter.

Poly IgG, when used, is added either in the sample diluent (Step 1) or in the conjugate reagent (Step 3) at a concentration specified by each experiment . II.2 SIMULTANEOUS FORMAT:

Alternately, the assay can be performed by the simultaneous incubation of sample, sample diluent, antibody-coated chromium dioxide particles and conjugate reagent. This format necessarily omits the first wash step (Step 2 of II.1) . After the incubation, the assay proceeds to Steps 4 and 5 as described above.

Again, Poly Ig, when used, is added either in the sample diluent or in the conjugate reagent at a concentration specified by each experiment. EXAMPLE III: USE OF SAMSA/PDM POLYMERIZED MONOCLONAL IGG (Polv IσG) IN HBSAG ASSAY III.l Efficacy on normal serum samples:

Table 1 exhibits correlative decrease of the fluorescent signals produced by sample No. 630 with the increasing concentration of Poly IgG added. This sample is rheumatoid factor (RF) negative. However, it produces a false-positive response with pepsin- fragmented anti-HBsAg/alkaline phosphatase conjugate [ (Fab¹)2-AP] . When other normal HBsAg-negative serum samples are tested with the inclusion of Poly IgG no significant effect is observed.

The response of a HBsAg-containing positive control is not affected by poly IgG (see control values in Tables 1) . This is also apparent by the results shown in Table 3.

Table 1 also indicates that a minimum of 7 ug/test Poly IgG is needed to reduce the assay response to negative, but additional amounts do not induce much further change. The preferred quantity of Poly IgG used in this type of heterogeneous enzyme immunoassay is 7 to 15 ug/test (200 ul serum or plasma sample per test) .

Table 1: Effect of the Quantity of Poly IgG on HBSAG Assay Results**

Poly IgG added** 0 ug/test 7 ug/test 14 ug/test

Pos. Control(RFU) # 1882 1907 1885

Neg. Control(RFU) 45 42 49

Cut Off* 165 162 169

No. 630 (+/-) 264 (+) 91 (-) 61 (-)

Footnotes:

## A two-step forward format and (Fab')2-AP conjugate are used in this experiment. ** Poly IgG is synthesized by SAMSA/DPM procedure.

# RFU: Relative Fluorescent Unit

* Cut off = Neg^". Control + 120, Values greater than Cut off are assigned as positive.

Table 2 summarizes a study comparing no addition, addition of monomeric IgG and addition of poly IgG in the HBSAG assay. While a sample (#34) with normal response is not affected, false positive samples are reduced to negative much more effectively by Poly IgG than monomeric IgG.

Table 2: Comparison of Monomeric IgG to Poly IgG in Reducing False Positive Responses in HBSAG Assay*

Footnotes:

* A two-step forward format and intact IgG-alkaline phosphatase conjugate are used in this study.

** Twenty (20) ug/test of either monomeric or polymeric IgG is used. Poly IgG is synthesized by the SAMSA/DPM Procedures.

# Cut off = Neg. Control+120, Values greater than Cut off are assigned as positive.

III.2 Efficacy of Poly IσG on rheumatoid factor positive samples:

Rheumatoid factors are autoimmune antibodies which are known to interfer with enzyme-linked immunoassays and cause sporadic false-positive responses. Such false-positive responses are not directly correlated to the RF titers. The use of pepsin-fragmented IgG

[(Fab*)2] to prepare the antibody-enzyme conjugate has been reported to help reduce the incidence of false- positive responses associated with RF-positivity.

Data presented in Table 3 indicate that certain RF-positive samples still produce false-positive results, even with the use of (Fab' ) 2-enzyme conjugate. These interferences are eliminated by the use of Poly IgG.

Table 3: Effect of Poly IgG on Rheumatoid Factor Positive Samples when (Fab')2-ΛP is Used as Enzyme

Conjugate

CONTROL VALUES:

Pos Control* Neg Control Cut-Off(Neg+120)

1189 92 212

+/-

False positive 2/12 0/12

Footnotes:

* A two-step forward format and a (Fab')2-AP conjugate are used.

* Poly IgG prepared by SAMSA/DPM Procedure, 15 ug/test used. EXAMPLE IV: EFFECT OF DSS-POLYMERIZED IGG IN HBSAG ASSAY Table 4 summarizes a study involving 80 negative samples that are identified as likely to cause false positive responses and 7 HBsAg-positive samples .

When the assay is conducted without Poly IgG, 24 samples exhibit falsely positive responses. With the addition of DSS-polymerized Poly IgG, all samples are statistically indistinguishable from the negative control. The positive samples are not affected by the Poly IgG.

Table 4: Effect of DSS-polymerized IgG on HBSAG Assay

No Poly IgG Poly IgG

Total Negative Samples Tested** Total False Positive Percent False Positive

False Positive Mean (St. Dev.) Negative Control (St. Dev.) Delta

(False Positive mean-Neg Control)

Total Positives Tested 7 7

Total True Positive (T/P) 7 7

T/P Mean (St. Dev.) 3162(976) 3115(1009)

Footnotes:

* A two-step forward format and an intact IgG-Alkaline Phosphatase conjugate are used in this study. Poly IgG is used at 15 ug/test. ** These are samples identified as likely to cause false positive responses. EXAMPLE V: EFFECT OF GLUTARALDEHYDE- POLYMERIZED IGG ON HBSAG ASSAY Table 5 exhibits the results of using two Poly IgG preparations in the HBSAG assay with five identified false positive samples. These two lots were synthesized from IgG generated from two mouse monoclonal cell lines. Both preparations reduce the false positive responses to negative range. This study indicates that glutaraldehyde- polymerized IgG performs as well as the SAMSA/DPM- and DSS- polymerized material in reducing the false positive responses . It also shows that Poly IgG prepared from two lots of IgG perform equally well.

Table 5: Effect of Poly IgG Synthesized by Glutaraldehyde Procedures on HBSAG Assay**

* These are samples identified as likely to cause false positive responses in the HBSAG assay. # These two lots of Poly IgG are synthesized from two mouse monoclonal cell lines using the Glutaraldehyde Procedure. Poly IgG is used at 15 ug/test. ** A simultaneous format and intact IgG-Alkaline Phosphatase conjugate are used in this study.

EXAMPLE VI: EFFECT OF POLY IGG IN A HETEROGENEOUS IMMUNOASSAY FOR HUMAN CARCINOEMBRYONIC ANTIGEN (CEA)

The heterogeneous immunoassay for CEA is based on the same principle as outlined in Example II. In the assay, 50 ul of serum sample, 50 ul of anti-CEA-alkalirie phosphatase conjugate solution, and 25 ul of an anti-CEA antibody coated chromium dioxide particle suspension are incubated at 37°C for 30 minutes. The particles are then washed and treated with the fluorescent substrate solution in the same manner as described in Example II. Fluorescent signals are then measured at an excitation wavelength of 365 nm and an emission wavelength of 450 nm.

Many serum samples possess interfering material which resulted in falsely elevated CEA results. In the case of a quantitative determination such as measurement of CEA, this false positive result is determined by comparison of the result in a commercially available assay with that obtained in the new assay. Since normal humans will have from 5 to 8 ng/ml of CEA in their sera, values which are greater than this are indicative of disease. Discrepancies between two assays where the value is above the normal range will be considered to be the result of an interferring substance . The interference is usually seen as a value higher in analyte level that the reference, commercially available assay. Inclusion of Poly IgG in the conjugate solution at a concentration of 19 ug/test effectively eliminated such false-positive responses. Table 6 exhibits a summary of CEA tests which demonstrate such effect.

Table 6: Effect of Poly IgG on False-Positive Samples in CEA Assay

* Manufactured and sold by Hybritech Inc. San Diego, CA, EXAMPLE VII: EFFECT OF POLY IGG IN A HETEROGENEOUS IMMUNOASSAY FOR HUMAN THYROID STIMULATING HORMONE (TSH) The immunoassay for human TSH is based on the same principle described in Example II. It is performed by a simultaneous incubation of 50 ul of serum sample, 50 ul of anti-TSH-alkaline phosphatase conjugate solution and 25 ul of anti-TSH coated chromium dioxide particles. The washing, substrate reaction and fluorescent measurement are performed in the same manner as described in Example II.

Table 7 shows the effect of including Poly IgG in the TSH test, when false positive samples are involved. While true TSH concentrations are correctly measured in samples 8523, L(10) and L(50), those exhibiting falsely high results are corrected by the use of Poly IgG.

Table 7: Effect of Poly IgG on False-Positive Samples in TSH assay Sample ID [TSH] [TSH] No PolylgG With PolylgG*

0.85 mlU/L 1.69 2.66 1.64

10.02

50.02

* Calibrators which have been spiked with purified TSH to concentrations of 10 mlU/L and 50 mlU/L, respectively.

# Poly IgG used at 16 ug/test.

Claims

CLAIMS ;

1. A method for reducing false positive responses in an immunoassay for detection of an antigen in a sample, said method comprising not in any order, the steps of: forming a polymerized immunoglobulin from an immunoglobulin that has no specific reactivity with said antigen; contacting said sample suspected of containing said antigen with said polymerized immunoglobulin; and subjecting the sample to an immunoassay employing antibodies derived from the same animal species as that of the polymeric immunoglobulin.

2. The method of Claim 1 wherein the immunoglobuln is IgG.

3. The method of Claim 2 wherein the polymerized IgG comprises about 3 to 15 IgG molecules .

4. The method of Claim 3 wherein the polymerized IgG is present in an amount sufficient to provide about 7 to 20 μg per immunoassay.

5. The method of Claim 1 wherein the immunoglobulin in IgM.

6. The method of Claim 2 wherein said sample is pretreated with the polymerized IgG.

7. The method of Claim 1 wherein the polymerized immunoglobulin is a component of the reagents used in the assay.

8. The method of Claim 1 wherein the antigen to be detected is selected from the group consisting of hCG, LH, TSH, FSH, alpha-feto protein, prostatic specific antigen, carcinoembrionic antigen (CEA) , cancer antigens ca 19.9, ca 125 and ca 15-3, human hepatitis B surface antigen, hepatitis B core antigen and hepatitis C, D and E.

9. The method of Claim 1 wherein the antigen to be detected is a virus or viral marker.

10. The method of Claim 1 wherein the antigen to be detected is a macromolecular hormone.

11. The method of Claim 1 wherein the antigen to be detected is a molecular component of human malignant tumor cells.

12. The method of Claim 8 wherein the antigen to be detected is human hepatitis B surface antigen.

13. The method of Claim 8 wherein the antigen to be detected is human thyroid stimulating hormone (TSH) .

14. The method of Claim 8 wherein the antigen to be detected is carcinoembryonic antigen (CEA) .

15. A method of performing an enzyme immunoassay employing two antibodies of the same animal species for capture and detection of an antigen in a sample to be assayed, said method comprising the steps of: a) forming a polymerized immunoglobulin of the same species as those in the assay, said polymerized immunoglobulin having no reactivity to the antigen to be detected; b) contacting said sample with a solution containing the polymerized immunoglobulin of step a) while performing said assay; c) performing the assay to determine the presence or absence of antigen in said sample.

16. The method of Claim 15 wherein the immunoglobulin is IgG.

17. The method of Claim 16 wherein said sample is pretreated with the polymerized IgG.

18. The method of Claim 15 wherein the polymerized immunoglobulin is a component of the reagents used in the assay.

19. The method of Claim 15 wherein the detector antibody to enzyme conjugate is comprised of a pepsin- hydrolysis produced antibody fragment.

20. The method of Claim 15 wherein the detector antibody to enzyme conjugate is comprised of a papain produced antibody fragment.

21. The method of Claim 19 wherein the fragment is (Fab')2, F(ab)2 or Fab.

22. The method of Claim 15 wherein the antigen to be detected is a virus or viral marker.

23. The method of Claim 15 wherein the antigen to be detected is a macromolecular hormone.

2 . The method of Claim 1 wherein the antigen to be detected is selected from the group consisting of hCG, LH, TSH, FSH, alpha-feto protein, prostatic specific antigen, carcinoembryonic antigen (CEA) , cancer antigens ca 19.9, ca 125 and ca 15-3, human hepatitis B surface antigen, hepatitis B core antigen and hepatitis C, D and E.

25. The method of Claim 24 wherein the antigen to be detected is human hepatitis B surface antigen.

26. The method of Claim 24 wherein the antigen to be detected is human thyroid stimulation hormone (TSH) .

27. The method of Claim 24 wherein the antigen to be detected is carcinoembryonic antigen (CEA) .

28. The method of Claim 15 wherein the antigen to be detected is a molecular component of human malignant tumor cells.

29. A method for forming polymeric immunoglobulin comprising treating immunoglobulin with a heterobifunctional or a homobifunctional chemical linking reagent whereby the resulting polymeric molecules exist in soluble form; and wherein the polymeric molecules are comprised of 3 to 15 monomeric IgG molecules.

30. The method of Claim 18 wherein the heterobifunctional reagents is S-acetylmercaptosuccinic anhydride (SAMSA) .

31. The method of Claim 18 wherein the homobifunctional agent is disuccinimidyl suberate (DSS) or glutaraldehyde or their derivatives.

32. The method of Claim 7 wherein the amount of polymeric immunoglobulin in the assay is in the range of about 2 μg to 25 μg per test.

33. A diagnostic test kit for conducting an immunoassay for the detection of an antigen and for reducing false positive responses in said immunoassay, said test kit comprising: a selected quantity of polymerized IgG comprising about 3 to 15 IgG molecules in an amount sufficient to provide a minimum of about 7 μg per test, said polymerized IgG having no specific reactivity with the antigen to be detected; and a labelled antibody to the antigen to be detected.