Production and characterization of antisera to synthetic thyrotropin-releasing hormone (TRH).

To generate anti-thyrotropin-releasing hormone (TRH) antibodies TRH was rendered antigenic presumably by reaction of its histidine residue with bis-diazotized benzidine (BDB) coupled to bovine serum albumin (BSA). Six California white rabbits were each injected with 330 micrograms protein/ml of emulsified immunogen by the multisite intradermal immunization technique. Seven repeated injections were given at 30-day intervals using half of the original quantity of antigen. Antibodies binding 125I-TRH appeared in the serum of four of the six rabbits three months after the first injection. Five months later the sera of two rabbits bound 50% of the labeled TRH at 1:6000 final dilution. Using this antiserum a radioimmunoassay for TRH was developed in which as little as 10 pg/300 microliter unlabeled TRH can be detected. The linear range of detectable TRH was 10 to 10000 pg. No cross-reaction with various hypothalamic and pituitary hormones, neurotransmitters, neuropeptides, and BSA were detected in this immunoassay. Extracts from rat and frog hypothalami produced 125I-TRH-binding inhibition curves parallel to synthetic TRH. Samples from elutes of rat medio-basal hypothalami superfused in vitro were examined by using this antiserum. Serial dilution of superfusate showed a similar inhibitions curve. Stimulatory effect of K+ depolarization on TRH release from superfused hypothalami was inhibited in Ca2+-free ethylenediaminetetraacetic acid medium. Ouchterlony double diffusion test of the TRH antisera revealed that low levels of antibodies against albumin but not immunoglobulin G or ovalbumin were also produced. However, the immunoprecipitates could only be detected with undiluted serum. In conclusion, this antiserum generated one of the most sensitive TRH radioimmunoassay currently available in the literature. The anti-TRH serum produced by this method can be used to examine both content of TRH from several tissues as well as release from hypothalamic tissue in vitro and might be useful to trace brain TRH pathways by immunocytochemistry.