Monoclonal antibodies against human, bovine and rat prolactin: epitope mapping of human prolactin and development of a two-site immunoradiometric assay

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Abstract
Nine mouse hybridoma cell lines producing monoclonal antibodies (MCA) against human prolactin (hPRL), 19 cell lines against bovine prolactin (bPRL) and one MCA against rat prolactin (rPRL) were established. The MCA were characterized by one- and two-site radioimmunoassays (RIA) as well as indirect immunofluorescence (IIF) and used for epitope mapping of hPRL and immunoradiometric assays (IRMA). Interspecies cross-reactivity studies by RIA revealed two groups of anti-hPRL MCA: seven which reacted only with hPRL and two additionally recognizing bPRL and ovine prolactin (oPRL). The anti-bPRL MCA, which were tested on pituitary sections by IIF could be divided into 17 MCA cross-reacting with the closely related oPRL, and two MCA which showed additional cross-reactions with equine prolactin. The anti-rPRL antibody reacted exclusively with rPRL in direct binding RIA studies. No intraspecies cross-reactions with the closely related protein hormones placental lactogen and GH were detected. To elucidate the antigenic surface of hPRL all MCA directed against hPRL were then used for two-site epitope mapping studies in which pairs of MCA were assessed for simultaneous reaction with the same antigen. The native hormone was incubated with the first, solid-phase bound, so called 'capture MCA', and this complex treated with a second, 125I-labelled 'detection MCA'. Based on the results of these combinations, at least three sterically non-overlapping and (taking RIA cross-reaction studies into consideration) two additional epitopes could be defined. Two antibodies (code numbers INN-hPRL-1 and INN-hPRL-9) recognizing different antigenic determinants were selected and used to elaborate a two-site IRMA with an operating range wider and a reaction time shorter than those obtained with a conventional one-site RIA. J. Endocr. (1987) 114,311–318