Repeated injections of synthetic LH RH decapeptide, adsorbed on polyvinylpyrrolidone and emulsified with complete Freund's adjuvant, resulted in the production of a specific antiserum to LH RH in 2 of 3 rabbits. The animals which produced this antiserum showed a reduction of pituitary LH content and marked atrophy of the testes. The antiserum antibody complex was detected by the complement fixation test. The antiserum was capable of binding 125I labeled LH RH. After iodination of LH RH (using 125I and either the chloramine T or lactoperoxidase method) separation of the iodination products on CMC yielded 3 main peaks of radioactivity: The first was free iodide, the second was labeled peptide with low immunoreactivity, and the third was immunoreactive peptide. This 3rd peak consisted of 2 or 3 subpeaks; the leading subpeak(s) were more readily bound by antiserum than the trailing one(s). Binding of these fractions to antiserum was increased in the presence of small amounts of unlabeled LH RH (a phenomenon called 'paradoxical binding' or 'hock effect') but inhibited by larger amounts. Both the augmentation and the inhibition effects were dose related, allowing the development of 2 different radioimmunoassay (RIA) systems for LH RH. An ordinary (competitive) type of RIA was developed in which a small amount (0.31 ng/assay tube) of unlabeled LH RH was added to the labeled peptide. This saturated the antiserum's capacity for paradoxical binding, so that further addition of LH RH (from 0.04 to 2.5 ng/tube) inhibited binding of labeled LH RH. The assay developed using paradoxical binding omitted the premixing of labeled and unlabeled LH RH; in this assay addition of very small amounts (0.5 to 310 pg) of unlabeled LH RH to the assay tubes increased the amount of label bound to antiserum and allowed construction of a parabolic curve of positive slope when B/T was plotted against arithmetic dose. A number of other hypothalamic and pituitary hormones (thyrotropin releasing hormone, MSH release inhibiting hormone, the peptide proposed as GH releasing hormone, MSH, arginine vasopressin, ACTH, oxytocin, ovine LH, rat FSH and GH, human GH, LH, FSH and TSH, and bovine TSH) failed to interfere in these assays, suggesting that the assays are highly specific for LH RH, although both polymers and degradation products of LH RH appeared to have some immunoreactivity. Extracts of rat and human hypothalamus, tested at various doses prepared by serial dilution, produced dose response curves indistinguishable from synthetic LH RH decapeptide. This supports but does not prove the proposition that LH RH decapeptide is the only immunoreactive molecule in these extracts.
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