Antibody-nucleic acid complexes. Antigenic domains within nucleosides as defined by solid-phase immunoassay

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Abstract
The usefulness of solid-phase immunoassays for the characterization of anti-nucleoside antibodies was investigated. Antibodies specific for guanosine (G), 7-methylguanosine (m7G), and cytidine (C) were obtained from the serum of rabbits immunized with nucleoside-KLH (keyhole limpet hemocyanin) conjugates. Solid-phase assays consisted of measuring the ability of these antibodies to be retained by microtiter wells containing immobilized nucleoside-BSA (bovine serum albumin) conjugates. Nucleosides employed as haptens included adenosine (A), N6-methyl-A (m6A), guanosine (G), N2,N2-dimethyl-G .**GRAPHIC**. 1-methyl-G (m1G), O6-methyl-G (m6G), 7-methyl-G (m7G), cytidine (C), 5-methyl-C (m5C), uridine (U), and ribothymidine (T). Spectral analysis of these conjugates revealed that 15-20 nucleosides were coupled to each BSA molecule. Quantitative information regarding the various reactions associated with these assays was obtained by employing antigen and antibody (IgG) preparations radiochemically labeled via reductive methylation using NaB3H4 and formaldehyde (specific activities 0.6-2.1 .times. 106 cpm/.mu.g). Data obtained with 3H-labeled antigens indicated that the adsorption of all nucleoside-BSA conjugates was uniform and irreversible with respect to the assay conditions used. Assays designed to measure antibody binding in the presence of excess antigen revealed that nonspecific binding to immobilized BSA was negligible, as little as 0.5 ng of bound antibody could be detected, antibody retention was directly proportional to antibody concentration, and each anti-nucleoside antibody cross-reacted to a considerable extent with nonhomologous haptens. At all serum concentrations tested, anti-G antibody cross-reacted with A-, C-, and U-BSA antigens to an extent of 30, 60 and 80%, respectively. Cross-reactivity was diminished and, in most instances, abolished by reducing the quantity of immobilized nucleoside-BSA conjugates by 1-3 orders of magnitude (i.e., from 60 to 0.06 ng). Data obtained by plotting cross-reactivity as a function of the quantity of antigen immobilized suggested that each homologous hapten possesses a major epitope (antigenic surface) within its heterocyclic ring system. Anti-G antibody cross-reacted poorly with m6G, moderately with m7G and m1G, and excessively with .**GRAPHIC**. This indicated that the major epitope of guanine encompassed the N-1, C-6, O-6 and N-7 atoms. A similar analysis of anti-C antibodies suggested that the C-4 and N-4 atoms dominate the major structural determinant. The unprecedented sensitivity of this solid-phase assay to detect antibody binding to previously unrecognized structurally related haptens is illustrated. By progressive reduction of the amount of immobilized haptens, sufficient information is obtained to make logical conclusions regarding the identity of the major antigenic domain within a low MW hapten. The application of this immunoadsorbent assay and its conversion to enzyme-linked immunoadsorbent assays (ELISA) for characterizing and selecting specific monoclonal anti-m7G antibodies is examined.
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