A. Rees Midgley

Radioimmunoassay for Rat Luteinizing Hormone with Antiovine LH Serum and Ovine LH-131I

Summary A highly sensitive and specific radioimmunoassay for rat LH has been developed utilizing antiovine LH serum and ovine LH for iodination. Estimates of the LH potency of pituitary preparations with widely varying LH to FSH and LH to TSH potencies obtained with this assay agree with estimates obtained with bioassay and with those obtained with a radioimmunoassay utilizing antirat LH serum and rat LH for iodination. With this technique it has been possible to measure the LH in serum obtained from females throughout the estrus cycle and in serum from normal and castrated males.

Autoradiographic Analysis of Gonadotropin Binding to Rat Ovarian Tissue Sections

Autoradiographic analysis of serial frozen tissue sections revealed that radioiodinated protein hormones, applied topically, were bound specifically and selectively to target tissues. To study relationships between the functional state of target tissues and protein hormone binding, autoradiographic analysis was performed on ovaries from rats with different known physiologic states. Changes in ovarian and pituitary function were assessed by radioimmunoassay of progesterone, luteinizing hormone (LH), follicle stimulating hormone (FSH), and prolactin in serum. Corpora lutea and interstitial tissue bound labeled rat growth hormone, ovine prolactin, and human chorionic gonadotropin (hCG). The extent of this binding varied with endogenous hormone concentrations and with the functional state of the ovary. At all times, thecal cells bound labeled hCG and granulosa cells of some moderate to large follicles bound labeled FSH. Granulosa cells bound other hormones variably. These results indicate the existence of specific intraovarian tissue-hormone binding, presumably to receptors, and suggest that this receptor activity varies with the functional state of the intraovarian tissues.

A specific, non-chromatographic radioimmunoassay for human plasma cortisol

A radioimmunoassay technique has been developed for the measurement of cortisol in a single methylene chloride extract of human plasma without chromatography. The antiserum, obtained by immunizing rabbits with cortisol-3-carboxymethyl-oxime conjugated to bovine serum albumin, had a high affinity (KA = 1.8 X 10(9) 1/mole) and capacity (2.3 X 10(-6) moles/L undiluted serum) for cortisol. The minimum detectable amount determined at the lower 95% confidence limit of the buffer control tubes was 8.3 +/- 4.7 pg/tube and a log dose - logit response standard curve was linear between 20 pg and 20 ng/tube. The antiserum was highly specific for cortisol with only corticosterone, cortisone, 11-deoxycortisol and 21-deoxycortisol showing significant cross-reaction (12.4, 6.6, 3.8 and 3.7%, respectively). The cross-reaction for the other tested naturally occurring and synthetic steroids did not exceed 1%. Regression analysis of cortisol concentration estimates obtained on 20 samples before and after Sephadex LH-20 column chromatography gave a coefficient of correlation (r) of 0.995 and a regression coefficient (b) of 1.04. Recovery of cortisol added to plasma samples was quantitative. The intra-assay error was 8.5% and the inter-assay error averaged 5.7%. The method is simple requiring a single solvent extraction of plasma, therefore permitting large numbers of samples to be handled efficiently by a single technician.

Physiologic and pathologic profiles of circulating human prolactin.

Abstract These radioimmunoassay studies were designed to test three hypotheses: (1) prolactin plays a major role in regulation of mammary function; (2) in the normal woman, prolactin is not a major obligatory factor in gonadal and menstrual cycle regulation; and (3) assessment of alterations in circulating prolactin concentration in response to stimulatory agents is useful in the evaluation of the hypothalamic-pituitary axis. (1) During pregnancy, serum prolactin increased progressively to term. Suckling resulted in a prompt increase in prolactin concentrations. (2) Daily prolactin determinations during the menstrual cycle revealed no cyclic pattern and no significant daily variation. (3) Increase in prolactin response to TRF in nine patients with idiopathic galactorrhea, eight of whom had amenorrhea, was similar to that observed in normal control subjects. Administration of l -dopa resulted in a decrease in prolactin in normal women, women with idiopathic galactorrhea, women following hypophysectomy, a patient with primary hypothyroidism, and in a patient with a chromophobe adenoma. These data support the validity of the first two hypotheses and suggest that further studies on the response to administration of hypothalamic-releasing factors may be useful clinical tools to distinguish neoplastic from nonneoplastic states.

Neuroendocrine control of follicle-stimulating hormone (FSH) secretion. I. Direct evidence for separate episodic and basal components of FSH secretion

Continuous sampling of hypophyseal portal blood from unrestrained sheep is providing an unprecedented means for measuring and defining the characteristics of the secretory profile of GnRH. With this method, GnRH has been shown to be released in discrete pulses lasting 5–8 min, with the amplitude of some pulses exceeding 50-fold. Although the relationship between these pulses and the accompanying pulses of LH measured in the jugular vein are unambiguous, the relationship of GnRH pulses to the release of FSH has not been well defined due to the longer clearance of FSH. In previous studies we have shown that hypophyseal portal blood, in addition to serving as a source material for hypothalamic secretions, provides a means to define secretory patterns of pituitary hormones. Because of this we hypothesized that the GnRH-FSH secretory relationships would be easier to define in hypophyseal portal than in jugular vein blood before the secretory products are subjected to dispersion and clearance in circulation. To...

Use of antibodies for characterization of gonadotropins and steroids.

Publisher Summary This chapter focuses on the use of antibodies for the characterization of gonadotropins and steroids. Antiserum for immunoprecipitation of rabbit antibodies to hormones—that is, anti-rabbit γ-globulin (anti-RGG)—has been prepared in adult male sheep using the same scheme that is used for immunization of rabbits. Demonstration of the ability of an antiserum to neutralize the activity of a hormone as measured in a specific biological assay provides strong support for the contention that antibodies capable of reacting with the hormone do exist in the antiserum. Immunohistochemical procedures should occupy a position of prime importance in the field of immunoendocrinology. It has been claimed that radioimmunoassay procedures have been used only to demonstrate concepts previously expected on the basis of bioassay data or have only been applied to problems that could have been solved by bioassay. Radioimmunoassays in combination with ultrastructural immunohistochemical procedures and various analytical methods can be used to define the processes of biosynthesis, storage, and release of a hormone within subcellular organelles; to determine the utilization and mechanisms of action of the hormone at target tissues; to study the fate of the hormone at sites of metabolism; and to describe the molecular properties of the hormone at all levels and times.

Administration of LH-releasing hormone to selected subjects

Abstract Administration of highly purified porcine LH-releasing hormone (LRH) to 12 human beings caused a maximum mean increase in serum LH of 460 per cent. Lysine vasopressin, used as a control substance, had almost (10 per cent) no effect. The selected subjects were divided into several groups, so that specific aspects of the effects of LRH could be studied. Postmenopausal women were responsive to LRH regardless of whether or not their serum LH levels had been supressed by pretreatment with an oral contraceptive. Subcutaneous administration of LRH was at least as effective as the intravenous route of administration. Putrescine did not elevate serum FSH or LH levels and did not augment the response to LRH. This study demonstrates the effectiveness of LRH of porcine origin in releasing LH in the human being.

Hapten-Radioimmunoassay for Steroid Hormones

One of the major advantages of using radioimmunoassay techniques for the measurement of steroid hormones is their unique potential for quantitating these hormones in unextracted serum, providing a truly specific antibody can be obtained to a given steroid. However, data regarding the factors which influence the specificity of antibodies to steroid hormones are incomplete. Since the basic structure of all steroid hormones is similar, i.e., they all contain the same basic cyclopentanophenanthrene nucleus, they offer an unequaled opportunity to study how minor alterations of this basic structure influence their immuno-reactivity. The structure of all steroid hormones differs only in the type and location of functional groups which are attached or introduced into this basic four ring nucleus.

Hapten-radioimmunoassay: A general procedure for the estimation of steroidal and other haptenic substances

Abstract Hapten-radioimmunoassay may be used for sensitive and specific quantitation of low molecular weight substances which can be made immunogenic by conjugation to a protein, This method, which involves radioiodination of the protein-portion of the conjugate, has been used for quantitation of non-conjugated estrogenic steroids.

Lack of alteration of serum gonadotropins in men and women following sexual intercourse.

Abstract Serum levels of LH and FSH were determined before and at 10 intervals from 10 minutes to 8½M hours after coitus in 8 men and 5 women. The women were studied between day 8 and 12 of their menstrual cycles. Gonadotropin concentrations did not differ from control means at any interval after sexual intercourse and are therefore, under these circumstances, independent of sexual activity. Testosterone concentrations in men were also unaffected by coitus. In addition, concentrations of LH, FSH, and testosterone during a 2 month interval of sexual abstinence were not different than during periods of intercourse 2 or 3 times a week.