Characterisation of monoclonal and polyclonal antibodies to 19-linked conjugates of testosterone with corresponding radioligands

Abstract

Monoclonal antibodies to testosterone T were produced using testosterone 19- O-carboxy-methyl ether (T19C) and testosterone 19-hemisuccinate (T19H) immunogens. All antibodies were characterised with iodinated derivatives of both T19C and T19H. Monoclonal antibodies derived from the T19C immunogen had similar titres and assay sensitivities with both T19-tracers. In contrast antibodies derived from the T19H immunogen bound the homologous but not the heterologous tracer. Individual antibodies showed a wide variation in cross-reactivity with 5α-dihydrotestosterone, DHT (4.4–100%), androstenedione AN (0.5–100%) and progesterone, Po (0.08–5.4%). One antibody 3F11 derived from a T19C immunogen gave 50% displacement of tracer with 180pgT/tube and low cross-reactivity of 12% with DHT, 3.0% with AN and 1.1% with Po. In general, assay sensitivity and antibody specificity were poorer with an [ 125I]-histamine conjugate of T-3-carboxymethyloxime than with T19 tracers. Radioimmunoassays for T in extracted human serum were developed with [ 125I]T19C as tracer and monoclonal antibody 3F11 (T19C immunogen) and rabbit antiserum T19H3R1 (T19H immunogen). Sensitivities of the extracted assays were 43 and 20 pg/tube respectively and results correlated well with those obtained after chromatographic separation of testosterone ( r = 0.97 for both antibodies). We conclude that 19-linked derivatives of T are highly immunogenic for the production of specific testosterone antibodies. Selection of the appropriate iodinated tracer is essential to achieve optimal titre, assay sensitivity and specificity, since these characteristics vary widely with individual monoclonal antibodies, and classical bridge recognition is not observed.