Competitive EIA Research Articles

Preliminary Findings on How Different Management Systems and Social Interactions Influence Fecal Glucocorticoid Metabolites in White Rhinoceros (Ceratotherium simum).

Simple SummaryIn recent years, interest in improving the welfare of wild species in captivity has grown. The aim of this investigation was to evaluate the effects of different social environments and management systems on the cortisol secretion of fourteen white rhinoceroses (Ceratotherium simum) living under different conditions by using use noninvasive methods. The fecal glucocorticoid metabolite secretion was found to be affected by both management systems and social interactions. Additionally, sex is another factor that seems to influence. This research provides a deeper understanding of glucocorticoid production in white rhinoceroses, but more studies are needed to fully understand its influence on reproductive biology.White rhinoceroses (Ceratotherium simum) are the most social and gregarious species of all rhinoceroses known worldwide. One of the most critical effects of elevated glucocorticoid concentrations, especially in threatened species, is its relation to chronic stress, which could potentially lead to immunosuppression and reduced reproductive activity. Our aim is to determine how different social environments and management systems might be influencing the secretion of fecal glucocorticoids in white rhinoceroses. We have analyzed the concentration of fecal glucocorticoid metabolites in 658 fecal samples from 14 white rhinoceroses, seven free-ranging rhinos, and seven rhinos from two different managed captive populations. HPLC techniques were used to determine the main glucocorticoid metabolite found in this species, and a competitive EIA was used to establish fecal 5α-pregnan-3 3β, 11β, 21-triol-20-oneglucocorticoid metabolite (fGCM) levels. Our results reveal that management systems and social interactions had an influence on fGCM levels, suggesting that the more restrictive the management and social conditions are, the higher the glucocorticoid concentrations that are found. Additionally, sex was also found to influence fGCM levels, as in females, fGCM concentrations were higher than in males. We conclude that the analysis of glucocorticoids in relation to other factors is a powerful tool to assess adrenocortical response in white rhinoceros in order to broaden the knowledge of their reproductive biology and improve the management of the species.

Positive correlations of age and parity with plasma concentration of macrophage migration inhibitory factor in Japanese black cows.

Plasma Macrophage migration inhibitory factor (MIF) concentration correlates positively with age, and negatively with self-rated health in women, and optimal MIF concentration may promote proper reproductive function. This study was conducted to evaluate the hypotheses that plasma MIF concentration changes with parturition or postpartum first ovulation, and that age in months and parity correlate with plasma MIF concentration in Japanese black cows. Western blotting utilizing an anti-MIF mouse monoclonal antibody of various tissues and plasma from females indicated that MIF expression was stronger in the anterior pituitary than in other tissues. We developed a competitive EIA utilizing the same anti-MIF mouse monoclonal antibody with sufficient sensitivity and reliable performance for measuring bovine plasma samples. We then measured MIF concentrations in bovine plasma collected from 4 weeks before parturition to 4 weeks after postpartum first ovulation. There was no significant difference in plasma MIF concentration pre- and post-parturition, or before and after the postpartum first ovulation. Plasma MIF concentrations were positively correlated (P < 0.01) with parity (r = 0.703), age in months on the day of parturition (r = 0.647), and age in months on the day of the postpartum first ovulation (r = 0.553) when we used almost all data, except for that from a third-parity cow with an abnormally high plasma MIF concentration. We therefore concluded that plasma MIF concentrations may increase with age in months and parity, but do not change either before and after parturition or before and after postpartum first ovulation in Japanese black cows.

Monoclonal antibodies to the haemagglutinin HA1 subunit of the pandemic influenza A/H1N1 2009 virus and potential application to serodiagnosis

In order to provide specific serological reagents for pandemic influenza A/H1N1 2009 virus, monoclonal antibodies (Mabs) to recombinant haemagglutinin component HA1 (rHA1) were generated after fusing spleen cells from a mouse immunized with rHA1 protein derived from influenza strain A/California/06/09 H1N1 with a mouse myeloma cell line. Five hybridoma clones secreting Mabs specific for the rHA1 protein derived from pandemic influenza A/H1N1 2009 and not for rHA1 from seasonal H1N1 influenza strains A/Brisbane/59/07 and A/Solomon Islands/03/06 were identified by EIA. Mabs 7H4, 9A4, and 9E12 were reactive in Western blots with full length rHA and/or rHA1 subunit derived from A/California/06/09 strain. Only Mab 1F5 inhibited haemagglutination of turkey red blood cells with recombinant NIBRG-121 virus derived from A/California/07/09, but did not react in Western blots. Immunostaining of MDCK cells infected with NIBRG-121 was localized to the membrane/cytoplasm for four of the reactive Mabs. The differing reactivity of the Mabs in Western blots, immunostaining, EIA, and haemagglutination inhibition assay suggest that at least four of the five Mabs recognize different epitopes on HA1 of the pandemic influenza A/H1N1 2009 virus. Ferret antisera to pandemic influenza A/H1N1 2009 (A/England/195/09 and A/California/07/09 strains) and sera from human subjects vaccinated with Influenza A (H1N1) 2009 Monovalent Vaccine (CELTURA®, Novartis Vaccines, Germany), inhibit binding of 1F5-HRP to biotinylated rHA1 derived from A/California/06/09 in a competitive EIA, suggesting that the epitope recognized by this Mab also evokes an antibody response in infected ferrets and vaccinated humans.

Development of Enzyme Linked Immunosorbent Assay for Erythropoietin

The aim of our study was to optimize and establish erythropoietin (EPO) enzyme linked immunosorbent assay (ELISA) system. We prepared several monoclonal and polyclonal antibodies specific to human-EPO. The best combinations of antibodies for coating and detecting antibodies were selected for the establishment of ELISA. We tested several methods such as a competitive EIA and a sandwich ELISA. The best sandwich ELISA was optimized compared to competitive EIA when purified polyclonal antibody (PoAb) was used as a coating antibody and biotinylated PoAb as a detecting antibody. This sandwich ELISA easily detected EPO when PoAb pairs were used compared to the ELISA using monoclonal antibody and PoAb. There were no significant differences between the effects of various blocking solutions on the performance of sandwich ELISA using biotinylated antibody. The ELISA system using PBST containing 3% BSA as a blocking solution can sensitively detect EPO (10 mU/mL) in a broad range of EPO concentrations (10-2,000 mU/mL) and there were cross-reactions with other cytokines). EPO can be easily determined by using biotinylated PoAb as a detecting antibody and another PoAb as a coating antibody.

An Immunochromatographic Assay of 2,4-Dichlorophenoxyacetic Acid and Simazine Using Monoclonal Antibodies Labeled with Colloidal Gold

A method of the competitive immunochromatographic assay of the pesticides 2,4-D (2,4-dichlorophenoxyacetic acid) and simazine (2-chloro-4,6-bis(N-ethylamino)-1,2,5-triazine) in aqueous samples was developed. Monoclonal antibodies to these pesticides labeled with colloidal gold were used to visualize the results. The sensitivity of the 2,4-D and simazine assay is 12 ng/ml, and the time of analysis is 3-7 min. The method does not differ in sensitivity from the competitive EIA using conjugates of monoclonal antibodies to the pesticides with horseradish peroxidase; however, the time of the EIA is 1.5 h. The immunochromatographic method of the pesticide detection is available and simple and may be recommended for the development of assays of any other low-molecular compounds. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 2; see also http://www.maik.ru.

N-terminal pro-BNP is a possible early biomarker for the cardiac side-effects of anthracyclines

Anthracyclines (ACs) cause cardiac side-effects which may result in cardiomyopathy. Identification of early biomarkers for this condition is thus useful. The N-terminal split product of proBrain Natriuretic Peptide (Nt-pBNP) may be such a biomarker and was included in an exploratory study to identify biomarkers for the cardiac side-effects of ACs. Three groups were studied: patients who completed a full course of AC-based chemotherapy (group I), patients starting AC-based therapy (group II) and a group of gender, age and BMI-matched controls (group III). Blood samples were taken and ECG recordings made. For group I, measurements were done from ≈1 month after the completion of therapy and 3 times thereafter and compared with the controls from whom samples were collected once. In group II samples were drawn pre-dose, at cessation, and after 24 hrs for each course. Nt-pBNP was measured in duplicate using a competitive EIA. Statistical analysis included paired and non-paired t-test to detect differences between the time points and groups. Mean (SD) Nt-pBNP in the patients who completed AC-therapy was 754 (177) pmol/L which was higher (p<0.01) than in the controls 588 (80) pmol/L. There was a tendency for Nt-pro-BNP to slightly decline over time in group I but this was not significant (p=0.21). In group II Ntpro-BNP increased during the 1st AC course from 530 (54) pmol/L pre-dose to 612 (52) pmol/L (p<0.05) immediately post-dose and further to 789 (124) pmol/L at 24 hours (p=0.001). The pre-dose value before the 3rd AC course was 721 (115) pmol/L and thereby higher than the pre-dose value before the 1st AC course (p=0.01), and increased to a maximum 1019 (227) pmol/L (p<0.05). Nt-pBNP seems a promising biomarker for the cardiac effects of ACs and will be explored further. Clinical Pharmacology & Therapeutics (2004) 75, P59–P59; doi: 10.1016/j.clpt.2003.11.224

Type-specific enzyme immunoassay for detection of pneumococcal capsular polysaccharide antigens in nasopharyngeal specimens

We developed a new competitive EIA method for the demonstration of pneumococcal capsular polysaccharides from respiratory samples. The pediatric types 4, 6B, 9V, 14, 18C, 19F and 23F were selected for this study, because these capsular polysaccharides were included in the first heptavalent pneumococcal conjugate vaccines, which were used in the Finnish Otitis Media Vaccine Trial. Sensitivity of the EIA tests for purified polysaccharide antigens varied between 5 and 100 ng/ml, depending on the type. The assays performed well in 100 nasopharyngeal samples (NPS) samples processed through an enrichment culture, with an almost 100% sensitivity compared with routine culture. The method appeared type-specific, except that EIA for 6B capsule also detected 6A. The method is applicable for type-specific identification of pneumococcus in carriage studies.

Characterization of monoclonal antibodies and epitope mapping of the NS4 protein of hepatitis C virus

Recombinant DNA containing sequences of HCV NS4 protein was expressed in Escherichia coli cells. Six hybridoma clones producing monoclonal antibodies (MAB) to recombinant NS4 protein (rNS4), aa 1677–1756, were developed. Mapping with a panel of 33 peptides and reciprocal competitive EIA have shown that MAB obtained revealed five antigen determinants, not described earlier: MAB 3F11 and 3F12-one genotype-independent epitope of NS4A (aa 1700–1707) common for genotypes 1, 2 and 3; MAB 1D11-genotype-independent epitope (aa 1713–1728) and MAB 1D3-genotype (subtype 1b)-specific epitope of NS4B (aa 1711–1731); MAB 6B11 and C1-two conformation-dependent determinants in 5-1-1 region. These data indicate that the 5-1-1 region of NS4 protein has a complex antigenic structure and contains at least eight epitopes, including five, revealed in the present work. MAB obtained recognized native viral protein in the cytoplasm of liver cells of patients with chronic hepatitis C. The positive rates of the immunostaining for NS4 antigen using MAB 6B11, 1D11 and 3F12 were 64, 59 and 50%, respectively. It was found that 6B11 MAB to a conformation-dependent epitope much more actively interacts with native NS4 than with the recombinant protein to which MAB was developed. The epitope recognized by 6B11 MAB is highly immunogenic since it induces the B-cell response in all patients investigated with identified anti-NS4 antibodies in blood serum. The MAB panel obtained in this study may become a useful tool for the diagnostic purposes, for the investigation of NS4B function and for the host-viral interactions at the cell level.

The effects of different anaesthetic treatments on the adreno-cortical functions and glucose levels in NZW rabbits.

The effects of five anaesthetics on the corticosterone, cortisol and glucose concentrations were investigated in the NZW rabbit. Sixty animals were assigned to 6 treatment groups (n= 10 per group): control ( iv saline solution injection), ketamine (10 mg/kg iv) with either xylazine (3 mg/kg iv) or diazepam (2 mg/kg iv), pentobarbitone (30 mg/kg iv), thiopentone (20 mg/kg iv) and fentanyl/droperidol (1 mg/kg sc). Plasma glucocorticoids were measured by competitive enzymeimmunoassay EIA and glucose by an autoanalyzer, previously validated for this species in both cases. Blood samples were obtained at 6 time-points: before injection, at 10, 30, 60, 120 min and 24 h after injection of the anaesthetics/saline. A significant decrease of plasma glucocorticoids at 10-60 min was observed in the pentobarbitone and fentanyl/ droperidol groups, whereas the administration of ketamine/diazepam or thiopentone stimulated plasma glucocorticoid release, principally in the recovery period. However, in the ketamine/xylazine group no changes were observed in the glucocorticoid levels, except for a significative increase of cortisol at 60-120 min. Glucose levels significantly increased after ketamine/diazepam administration and principally, after ketamine/xylazine treatment. The present data suggest that ketamine/xylazine has little effect on glucocorticoid levels and provides an adequate level of surgical anaesthesia, hence it would be the anaesthetic of choice, although the hyperglycaemic effect after injection has to be considered for any experimental procedures in rabbits.

Analysis of ultrashort feedback regulation in human placenta: synthesis and secretion of GnRH by human trophoblastic cells.

Gonadotropin-releasing hormone (GnRH) presumably controls placental growth and functions by autocrine/paracrine mechanisms, and is therefore an important part of the neuroendocrine network in human placenta. Our earlier work had indicated that GnRH was expressed in human placenta; in extension to these findings, we wanted to analyse synthesis and release of GnRH by trophoblastic cells. GnRH-associated peptide, co-linearly synthesised with GnRH, was used as indicator of actual peptide synthesis. First, we immunised rabbits with lipopeptides containing partial sequences of GnRH-associated peptide (GAP) and developed antibodies for immunohistochemical staining. Second, we set up a competitive enzyme immunoassay to measure GnRH: Non-biotinylated GnRH, GnRH analogues or trophoblastic cell culture supernatants were used to inhibit binding of biotinylated des-pGlu1-GnRH to a monoclonal anti-GnRH antibody. a) Placental sections stained positive for GAP in the layers of trophoblastic cells. b) GnRH could be detect by a competitive EIA in supernatants of placental cultures in concentrations between 200 and 5 nM. GnRH is synthesised and released by trophoblastic cells.

Comparison of chemiluminescent and chromogenic substrates of alkaline phosphatase in a direct immunoassay for plasma estradiol

The colorimetric and chemiluminescent determination of alkaline phosphatase activity in a direct enzyme immunoassay of the competitive type for determination of estradiol-17β (E 2) in human plasma or serum were compared. In this assay biotin-labeled E 2 and E 2 from standard or patient samples compete for free antibody-binding sites. Antibody-bound E 2-biotin is then reacted with streptavidin-alkaline phosphatase. Enzyme activity in the bound fraction was detected either with the chromogenic substrate p-nitrophenyl phosphate (pNPP) or with the chemiluminescent substrate disodium 3-(4-methoxyspiro l,2-dioxetane-3,2′-(5′-chloro)-tricyclo[3.3.1.1 3,7]decan-4-yl)phenyl phosphate (CSPD). Contrary to expectations, the use of CSPD for chemiluminescent endpoint determination of alkaline phosphatase did not improve assay performance. Both the limit of detection of analyte E 2 (160 vs. 73 pmol ml −1) and overall precision (C.V., %) were less favourable with CSPD than with pNPP. These results and the high cost of CSPD make this chemiluminescent substrate less suited for endpoint determination in competitive EIA for haptens.

Bispecific monoclonal antibodies to human interleukin 2 and horseradish peroxidase.

Hybrid hybridomas (tetradomas), producing bispecific monoclonal antibodies (bmabs) binding to both interleukin 2 and horseradish peroxidase were obtained by fusing IL 2-specific and HRP-specific hybridomas. Parental hybridomas were labelled prior to the fusion with fluorescein isothiocyanate and tetramethylrhodamine isothiocyanate. Bifluorescent (tetradoma) cells were sorted out using fluorescence activated cell sorter. Two clones, designated D8C1/G and H7C11/H were shown to secrete bmabs over at least 6 months growth in culture. Bmabs have been purified from mouse ascites by ammonium sulfate precipitation and affinity chromatography on immobilized peptide, modelling corresponding IL 2 epitope. The purity of antibodies obtained was characterized by capillary electrophoresis. The possibility of using these antibody preparations in rIL 2 analysis was evaluated in two types of EIA: direct and competitive solid-phase EIA. Both assays had similar sensitivity of about 1.5-4 ng IL 2 per ml.

Competitive EIA for anti‐HIV‐2 detection in the Gambia: Use as a screening assay and to identify possible dual infections

The performance of a competitive EIA for the detection of HIV-2-specific antibody utilising a viral lysate antigen was assessed over a 3 year period in The Gambia, West Africa, and compared with a commercially available assay, ELAVIA-2, using three panels of sera. An immunodominant region of the transmembrane glycoprotein of an HIV-2 isolate (ANT 53) was also cloned and expressed in E. coli as a beta-galactosidase fusion protein and the resulting recombinant protein substituted in place of the existing viral lysate antigen. Competitive EIAs were found to be both a specific and sensitive means of reliably determining the HIV-2 status of an individual with a high predictive value, particularly when a strategy of concordant positive results in the two EIAs was used. When either anti-HIV-2 competitive EIAs were used in conjunction with a competitive EIA for anti-HIV-1 detection it was possible in the vast majority of cases to identify the virus-type infecting an individual and speciate HIV-1 and HIV-2 infections. A few sera which showed similar regression profiles when diluted over a serial tenfold dilution steps were identified as possible dual infections.

A comparison of four enzyme immunoassays for the simultaneous detection of HIV-1- and HIV-2-specific antibody

The performance of four HIV 1 and 2 combined assays has been compared with current type-specific assays using three panels of sera. The first panel comprised single samples from 19 HIV-1-infected persons; the second panel comprised 19 sera from 16 HIV-2-infected persons. Samples from both these panels were titrated across interpolated end points of detectability. The third panel comprised sera from 5200 consecutive blood donors. The four combined assays, manufactured by Abbott Laboratories, Behring Laboratories, Diagnostics Pasteur and Wellcome Diagnostics Laboratories detected all anti-HIV-1 sera at high dilution; the immunometric assay from Wellcome was particularly proficient. All assays were broadly similar in their ability to detect sera from recently infected persons. The same assays were also effective in detecting anti-HIV 2 in sera, both from seropositive individuals and from a single recently-infected person, though none was as sensitive as an inhouse competitive EIA. When used for donor screening the repeat reactive rates for donors negative for HIV 1 and 2 antibodies ranged between 1.80% for Elavia Mixt from Pasteur, 0.27% Abbott combined and 0.15% for the Wellcome combined assays.

Carbohydrate-enzyme conjugates for competitive EIA

Enzyme glycoconjugates prepared from oligosaccharides or polysaccharides are efficient and durable reagents for direct, competitive enzyme immunoassays based on solid phase antibody. This assay format facilitates simple measurement of association constants and relative inhibitory power of oligosaccharides, while also providing a sensitive quantitative assay of bacterial polysaccharide antigens. Enzyme-saccharide conjugates were prepared by established glycoconjugate methodologies, and protocols for their optimal use were established by investigating the effects of solid phase antibody concentration on the kinetic equilibrium of binding for different antigen-enzyme conjugate concentrations, in the presence and absence of inhibitor. Four discrete monoclonal antibody-carbohydrate antigen systems were studied and it is demonstrated for each, that the relative inhibitory power of oligosaccharides may be readily measured in a convenient assay format, and that association constants can be extrapolated from the same assay data.

Preparation of polysaccharide-enzyme conjugates for competitive binding assays

A variety of bacterial O-polysaccharides were covalently linked to enzymes and it was demonstrated with three discrete monoclonal antibodies that enzyme-glycoconjugates function as convenient labelled antigens in direct enzyme immunoassays, particularly competitive assays that quantify bacterial O-antigens. Two strategies, each involving reductive amination, were used to couple O-polysaccharides to enzymes, while retaining high enzymic activity. Reduction of the Schiff base formed between, 1,3-diaminopropane and the terminal reducing ketodeoxyoctanoic acid (KDO) residue present in the majority of the lipopolysaccharide (LPS) core domains, following mild acid removal of Lipid A, offered the most direct route to mono-aminated polysaccharide. Alternatively, mild periodate oxidation of KDO and heptose residues generated multiple aldehyde targets for Schiff base formation, without affecting the O-antigenic determinant. Hetero- and homobifunctional coupling reagents, sulphosuccinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate and discuccinimidyl suberate, activated polysaccharide for coupling to enzymes at amino and sulphydryl sites and produced conjugates that retained at least 95% of the original enzymic activity. The most suitable enzyme conjugates, especially for competitive inhibition EIA were those bearing one polysaccharide chain, and these were easily prepared from horse-radish peroxidase. Although the extent of conjugation of activated polysaccharide to β-galactosidase and alkaline phosphatase could be controlled by reaction stoichiometry, the use of these enzymes was a less effective utilization of valuable antigen and enzyme.

Detection of anti-HIV antibodies in preparations of human immunoglobulins. A collaborative group study

This paper reports the results of a study organized by the Istituto Superiore di Sanità (Rome, Italy) and carried out in collaboration with several national laboratories. Its aim was to ascertain whether diagnostic kits for detecting anti-HIV antibodies in human serum can also be used to detect the same antibodies in human immunoglobulins. Thirty-three lots of immunoglobulins supplied by six pharmaceutical companies present in Italy were examined using different procedures. On the basis of the results of this study it can be concluded that i. anti-HIV antibodies can be detected in immunoglobulins by means of commercial reagents; ii. a preliminary dilution of immunoglobulin samples should not be made; iii. Western blot method appears to be the 'reference' test; however, competitive EIA tests are equally valuable if a negative control made up of anti-HIV-negative Igs is employed; iv. Igs examination for anti-HIV antibody represents an indirect control on a correct donors' screening procedure.

First documented case of human immunodeficiency virus type 2 infection in an asymptomatic Swiss subject

Few cases of human immunodeficiency virus type 2 (HIV-2) infection have been reported in individuals other than of West African origin. The first well documented case of HIV-2 infection observed in a Swiss subject is presented here. The 50-year-old woman had a sexual relationship with a Senegalese man, who was later shown to be HIV seropositive. Initially, the subject's serum was tested using a routine screening assay for the detection of HIV-1 antibodies. This assay elicited a borderline positive result. A confirmatory competitive EIA and a Western blot test for anti-HIV-1 antibodies showed a positive reaction with gag and pol proteins of HIV-1, but not with env proteins. Thus, HIV-2 infection was suspected and subsequently confirmed by three different methods, including Western blot analysis and an HIV-1/HIV-2 differentiation test. This case emphasizes the need for screening with combined HIV-1/HIV-2 tests.

Production of antibodies and development of an immunoassay for the anticoagulant, diphacinone.

Diphacinone, a commonly used anticoagulant rodenticide, was coupled to protein via an (O-carboxymethyl) oxime bridge. Immunization in rabbits produced antibodies with good ability to recognize the hapten as demonstrated by indirect EIA and affinity column adsorption. A competitive EIA was developed which clearly measured 10 micrograms/L diphacinone concentrations showing the sensitivity of the assay. Cross-reactivity study with chlorophacinone showed that the antisera possessed a high degree of diphacinone specificity.

Determination of epitope specificities of monoclonal and polyclonal anti-CEA antibodies by a solid phase competitive enzyme immunoassay: Use of the avidin-biotin system

Biotin bound to antibodies is still available for high affinity interaction with avidin. In a previous study, we described the use of avidin for the precipitation of monoclonal, biotin labeled antibodies against carcinoembryonic antigen (CEA) in a homogeneous phase, competitive radioimmunoassay [1]. In the present investigation, we used the avidin-biotin system for the determination ofepitope specificities ofmonoclonal and polyclonal anti-CEA antibodies in a solid phase competitive enzyme immunoassay. The monoclonal antibodies were produced and characterised as described [2]. The biotinylation of monoclonal antibodyIgG was performed by established procedures [l]. The wells of 96-well polyvinylchloride microtiter plates were coated by incubation with 1.5-2.0 gg CEA in 100 gl of 0.2 mol/1 carbonate buffer, prig.3, overnight at room temperature. Unspecific binding sites of the wells were blocked with a solution of 1% BSA in PBS (PBS-BSA) for 2 h at 37 ~ C. A constant volume of 50 pl of an appropriate dilution of biotinylated antibody was added to equal volumes of doubling dilutions of unlabeled monoclonal or polyclonal antibodies in PBS-BSA. 50 pl of this mixture was added to the wells ofmicrotiter plates coated with CEA. After 2 h at 37 ~ C, the wells were washed 3 times with PBS. 100 gl of avidinperoxidase conjugate (Sigma, Miinchen, FRG) diluted 1:100 in 0.2tool/1 phosphate buffer, pH6.5, containing 20% normal rabbit serum, was added to the wells. After 2 h at 37 ~ C, the plates were washed 5 times with 0.1 tool/1 citrate buffer, pH 5.0. 100 ~tl of substrate solution containing 6 mmol/1 hydrogen peroxide and 40mmol/1 o-phenylenediamine (Sigma) in 0.1 mol/1 citrate buffer, pH 5.0, was added to the wells and incubated for 30rain at RT in the dark. After addition of 100 gl of 3 mol/1 HCI, the OD was read at 492 nm. 6 unlabeled monoclonal anti-CEA antibodies and a polyclonal rabbit anti-CEA antiserum were tested with respect to their epitope specificities using 5 different monoclonal anti-CEA antibodies. The results are demonstrated in the Table 1. Capitals are used instead of the original designations of the antibodies in the text. The binding of labeled antibody B was inhibited by the corresponding unlabeled antibody only. Labeled antibody E was completely inhibited by unlabeled antibodies E and F, indicating identical epitope specificities. The binding of labeled antibodies C and D was inhibited by the corresponding unlabeled antibodies; at high antibody excess, antibody A showed some binding inhibition. The binding of labeled antibody A was partly inhibited by antibodies C, D and F, however was completely inhibited by the corresponding unlabeled antibody A. It is tentatively concluded that antibody A recognizes repetitive epitopes on CEA. The CEA-binding of each of the 5 monoclonal antibodies was inhibited by the polyclonal rabbit antiserum indicating that antibodies with related epitope specificities are present. The results obtained by the competitive solid phase EIA as described here are well comparable with those obtained by the use of a solid phase radioimmunoassay described previously [2]. In comparison with the biosynthetic or chemical introduction of radioactive labels, the competitive solid phase EIA has several distinct advantages: (1)In contrast to radioactive labels, the biotin labeled antibodies are stable over a long period of time, (2) the biotin labeling is easy to perform, (3) precautions due to the use of radioactive compounds are unnecessary.