Production of antiserum to LH releasing hormone (LH-RH) associated with gonadal atrophy in rabbits: development of radioimmunoassays for LH-RH

A. Arimura, H. Sato, T. Kumasaka, R. B. Worobec, L. Debeljuk, J. Dunn, Andrew V Schally

Research output: Contribution to journalArticle

158 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)1092-1103
Number of pages12
JournalEndocrinology
Volume93
Issue number5
StatePublished - Dec 1 1973
Externally publishedYes

Fingerprint

Gonadotropin-Releasing Hormone
Atrophy
Radioimmunoassay
Immune Sera
Rabbits
Peptides
Halogenation
Animal Tarsus
MSH Release-Inhibiting Hormone
Human Follicle Stimulating Hormone
Hypothalamic Hormones
Pituitary Hormone-Releasing Hormones
Lactoperoxidase
Melanocyte-Stimulating Hormones
Complement Fixation Tests
Povidone
Thyrotropin-Releasing Hormone
Freund's Adjuvant
Arginine Vasopressin
Iodides

ASJC Scopus subject areas

  • Endocrinology
  • Endocrinology, Diabetes and Metabolism

Cite this

Production of antiserum to LH releasing hormone (LH-RH) associated with gonadal atrophy in rabbits : development of radioimmunoassays for LH-RH. / Arimura, A.; Sato, H.; Kumasaka, T.; Worobec, R. B.; Debeljuk, L.; Dunn, J.; Schally, Andrew V.

In: Endocrinology, Vol. 93, No. 5, 01.12.1973, p. 1092-1103.

Research output: Contribution to journalArticle

Arimura, A. ; Sato, H. ; Kumasaka, T. ; Worobec, R. B. ; Debeljuk, L. ; Dunn, J. ; Schally, Andrew V. / Production of antiserum to LH releasing hormone (LH-RH) associated with gonadal atrophy in rabbits : development of radioimmunoassays for LH-RH. In: Endocrinology. 1973 ; Vol. 93, No. 5. pp. 1092-1103.
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AU - Arimura, A.

AU - Sato, H.

AU - Kumasaka, T.

AU - Worobec, R. B.

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AU - Schally, Andrew V

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N2 - 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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