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

Abstract

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.