Heterologous Radioimmunoassay Research Articles

Isolation and structures of alligator gar ( Lepisosteus spatula) insulin and pancreatic polypeptide

Insulin and a 36-residue peptide with homology to pancreatic polypeptide (PP) were isolated from the endocrine pancreas of the alligator gar ( Lepisosteus spatula), a ganoid fish, by gel filtration and HPLC. Heterologous radioimmunoassays were used to detect insulin-like and PP-like immunoreactivities during purification of the two peptides. The sequence of the 36-amino acid peptide containing a C-terminal tyrosinamide was identical at 31 of 36 positions to porcine neuropeptide Y (NPY). The amino acid sequence of this peptide is YPPKPENPGEDAPPEELAKYYSALRHYINLITRQRY-NH 2. The second peptide, gar insulin, contains 52 amino acid residues and is composed of a 21-residue A chain and a 31-residue B chain. The sequence of the A chain is GIVEQCCHKPCTIYELENYCN. The sequence of the B chain is AANQHLCGSHLVEALYLVCGEKGFFYNPNKV.

Characterization of proteins, insoluble at low temperature, produced by clostridium botulinum type C and C. subterminale. Their antigenic relationship with a similar protein synthesized by C. botulinum type G

The production of a protein insoluble at low temperature (“cryoprotein”), by cultures of Clostridium botulinum type G has been shown to be a metabolic characteristic also shared by C. botulinum type C and by C. subterminale. These new cryoproteins have been purified and some of their chemical and immunological properties studied. It was found that both proteins were chemically very similar among themselves and to the cryoprotein isolated from C. botulinum type G. All these proteins are formed by a single polypeptidic chain of approximately Mr = 180000, with closely related amino acid compositions, isoelectric points and do not contain either free cysteine or disulfide bridges. Homologous and heterologous radioimmunoassays established the existence of an antigenic similitude among the cryoproteins from C. botulinum type G and C. subterminale thus becoming the first purified antigens which relate both bacterial species. If the production of cryoproteins can be shown to be a generalized phenomenon within the genus Clostridium these substances would provide an important tool to examine immunological and genetical relatedness between strains in this bacterial group.

Plasma immunoreactive-growth hormone in domestic fowl: Measurement by homologous and heterologous radioimmunoassays

Concentrations of immunoreactive (IR) growth hormone (GH) in the plasma of domestic fowl have been measured by homologous and heterologous radioimmunoassays and the estimates compared. Both assays detected an age-related decline in the circulating IR-GH concentration, an increase in IR-GH secretion following TRH-stimulation or fasting, and a fall in the IR-GH concentration following adrenocorticotropin administration. However, while the overall estimates of IR-GH concentration were significantly correlated, the magnitude of the changes in IR-GH concentration determined by the homologous assay were far greater than those detected by the heterologous system, which failed to show any inhibitory effect of anesthesia or exogenous thyroid hormones on basal or stimulated IR-GH release. These results suggest that the heterologous GH redioimmunoassay lacks the sensitivity of the homologous chicken GH assay and that circulating GH in birds is probably composed of heterogenous moieties with differing immunoreactivities with rat GH antibodies.

Determination of insulin-like growth factor 1 (IGF1) and IGF binding protein levels in swine

A heterologous radioimmunoassay system was validated for the determination of IGF1 concentrations in swine sera. Parallelism, accuracy and response to physiological stimuli were obtained following the incubation of serum samples with 1M glycine-glycine HC1 buffer at a pH of 3.5±0.2 for 24 hours at 37C. Following acidification and neutralization, circulating IGF1 concentrations were significantly (P<.05) reduced in hypophysectomized swine and elevated in swine injected with porcine growth hormone (pGH) when compared to IGF1 levels in control hogs. IGF binding protein levels were also increased following GH administration and reduced by hypophysectomy. In addition, circulating IGF1 concentrations were significantly (P<.05) correlated with body size in three types of swine which differ in growth rate and mature body weight. These data suggest that IGF1 is involved in the regulation of swine growth in vivo and that its physiologic regulation is similar to that in humans.

Comparison of rabbit epididymal androgen binding protein and serum testosterone estradiol binding globulin—II. Immunological properties

Purified rabbit epididymal androgen binding protein and serum testosterone estradiol binding globulin have been immunologically compared. A comparison using the steady state gel method of Ritzen et al.[1] indicated immunological cross-reactivity. In order to further compare their immunological properties we developed a radioimmunoassay for both rbABP and rbTeBG using specific antisera directed against each. When these assays were compared, the extent or completeness of displacement proved to be the only parameter that was significantly different. This data obtained with homologous and heterologous radioimmunoassays is consistent with the idea that these two proteins contain minor antigenic determinants which are distinct.

Determination of calcitonin in reptilian serum by heterologous radioimmunoassay

A heterologous radioimmunoassay for reptilian calcitonin using a commercially available kit for human calcitonin was used to determine circulating levels and brain levels of calcitonin in the green iguana, Iguana iguana. Cross-reactivity between anti-human calcitonin antibodies and iguana plasma was demonstrated. Removal of reptilian calcitonin from the plasma with affinity chromatography eliminated the cross-reactivity. Assays on blood samples indicated normal circulating levels of calcitonin to range between 600–975 pg/ml. Young female iguanas had higher blood levels than males. The use of this assay permits more detailed study of the secretion of calcitonin in reptiles in response to appropriate stimuli.

F.9 embryonal carcinoma cell calcitonin autocrine system: correlation between immunoreactive calcitonin secretion and calcitonin receptor number.

Mouse teratocarcinoma cells in culture offer an in vitro system to study the initial steps of embryogenesis. It has been suggested that, at such early stages, cell functions are regulated by an autocrine process in which embryonic cells produce factors that in turn act on themselves. F.9 cells possess specific membrane receptors for calcitonin (CT) (120 fmol/mg of protein, Ka, = 3.5 X 10(8) M-1). These cells produce CT detected by heterologous radioimmunoassay in serum-free culture-conditioned medium (75 pg/10(7) cells/12 h). When F.9 cells are incubated in serum-free medium, CT binding and secretion concomitantly drop by 50% within the first 2 h, then increase progressively to an upper plateau after the sixth hour. Preincubation with 10(-6) M CT leads to disappearance of CT receptors and CT secretion in the culture medium up to 6 h. Avoiding accumulation of CT in the medium by a continuous flow rate for 6 h leads to a progressive decrease of CT receptors. In addition, retinoic acid treatment of cells induces a parallel progressive decrease of CT receptor number and of total CT synthesis. These results suggest a reciprocal regulation of CT receptors and CT secretion, or a close relationship between their regulations.

Minimal quantities of FSHβ subunit are released by rat anterior pituitary cells in primary culture

FSHβ in the medium and extracts of cultured rat anterior pituitary cells was quantitated with a heterologous radioimmunoassay utilizing anti-rFSHβ and hFSHß as the standard and tracer. After 4 days in culture, extracts were prepared from washed cells, cells incubated for 6 h or 24 h in fresh medium or cells incubated for 6 h in the presence of 10 −7 M GnRH. FSHß/rFSH molar ratios were approximately 0.05, 0.04, 0.21 and 0.35, respectively. The elevation in FSHß/rFSH molar ratios in 24 h basal and GnRH-teated cultures was due in part to an increase in intracellular FSHß. Both unstimulated and GnRH-treated cultures contained non-detectable quantities of FSHß in the medium (< 0.063 ng/100000 cells; FSHß/rFSH molar ratio < 0.024). Thus, it appears that cultured rat anterior pituitary cells contain a small amount of uncombined FSHß but that minimal quantities are released. Furthermore, GnRH may increase intracellular quantities of FSHß but does not induce its release.

Seasonal variations of plasma prolactin and LH concentrations in the female blue fox (Alopex lagopus).

A heterologous radioimmunoassay system developed for the rabbit and suitable for a wide range of mammalian species has been shown to measure prolactin in the plasma of the blue fox. Evaluation of prolactin levels throughout the year showed the concentrations displayed a circannual rhythm with the highest values occurring in May and June. Prolactin concentrations remained low (approximately 2.5 ng/ml plasma) from July until April with no consistent changes found around oestrus (March-April). In 8 pregnant females, the prolactin increase in late April and May coincided with the last part of gestation and lactation: concentrations (mean +/- s.e.m.) increased to 6.3 +/- 0.6 ng/ml at mid-gestation, 9.7 +/- 2.1 ng/ml at the end of gestation and 26.7 +/- 5.0 ng/ml during lactation. In 10 non-pregnant animals, the mean +/- s.e.m. values were 7.2 +/- 1.2 ng/ml in April, 8.8 +/- 2.2 ng/ml in May and 9.8 +/- 1.3 ng/ml in June. The prolactin profile in 4 ovariectomized females was similar to that observed in non-pregnant animals, but the plasma values tended to be lower during the reproductive season (April-June). In intact females, the only large LH peak (average 28 ng/ml) was observed around oestrus. During pro-oestrus, baseline LH levels were interrupted by elevations of 3.1-10.4 ng/ml. During the rest of the year, basal levels were less than 3 ng/ml. In ovariectomized females, LH concentrations increased within 2 days of ovariectomy and remained high (35-55 ng/ml) at all times of year.

Radioimmunoassay of Luteinizing Hormone in the Baboon (Papio hamadryas)

A heterologous radioimmunoassay (RIA) for luteinizing hormone (LH) consisting of a cynomolgus LH tracer and an antiserum raised against human chorionic gonadotropin (cynLH:anti-hCG) fulfilled the recognized criteria of reliability when applied to baboon (Papio hamadryas) plasma and pituitary extracts obtained in different endocrine conditions. This RIA is 5.5 times more sensitive than the ovine (oLH:anti-oLH) system, yields estimates of baboon LH (bLH) fairly close to those obtained by in vitro bioassay, and recognizes all bioactive molecular species of bLH present in male and female pituitary extracts. However, the system yields slightly but significantly lower estimates of bLH than the in vitro bioassay.

Release of atrial natriuretic peptide by atrial distension

A heterologous radioimmunoassay was used to measure the concentration of immunoreactive atrial natriuretic peptide (iANP) in plasma from the femoral artery of eight chloralose anaesthetized dogs. Mitral obstruction which increased left atrial pressure by 11 cmH2O increased plasma iANP from 97 +/- 10.3 (mean +/- SE) to 135 +/- 14.3 pg/mL. Pulmonary vein distension increased heart rate but did not increase plasma iANP. Bilateral cervical vagotomy and administration of atenolol (2 mg/kg) did not prevent the increase in iANP with mitral obstruction. Samples of blood from the coronary sinus had plasma iANP significantly higher than simultaneous samples from the femoral artery confirming the cardiac origin of the iANP. Release of iANP depends on direct stretch of the atrium rather than on a reflex involving left atrial receptors.

Heterologous radioimmunoassay of LH in two seasonally breeding animals: hare (Lepus europaeus) and mink (Mustela vison)

A heterologous radioimmunoassay using ovine LH as the labelled hormone, canine LH as the standard, and a rabbit antiovine LH serum (GDN 15) was developed and validated for LH measurement in the plasma of two seasonally breeding animals (induced ovulators). Physiological validation of the assay was evidenced by the increase in LH levels after LHRH injections and castration. In the hare, the longer the time after castration, the higher the cumulative response of LH after LHRH stimulation.

Coital stimuli controlling luteinizing hormone secretion and ovulation in the female ferret.

A series of experiments focused on the masculine coital behaviors controlling pituitary luteinizing hormone (LH) secretion and reflex ovulation in the estrous female ferret. An initial experiment investigated which coital stimuli from the male are required to induce ovulation. It was found that corpus luteum formation, which served as an index of ovulation, occurred in estrous female ferrets only if the male achieved a penile intromission. Neck gripping, mounting, and pelvic thrusting behavior without intromission by the male failed to induce ovulation. A second experiment investigated the timing and magnitude of the coitus-induced LH surge associated with ovulation. Blood was obtained via jugular catheters from estrous females in various mating situations. Plasma LH concentrations were measured by a heterologous radioimmunoassay that was validated for use in the ferret. A significant surge in plasma LH occurred only when an intromission was achieved by the stud male. Plasma LH was significantly elevated 2.0 h after the introduction of the male, peak values were reached 6.0 h later, and this elevation lasted on average 5.7 hours (5/5 females). No LH rise occurred in 2/2 female ferrets in which only neck gripping, mounting, and pelvic thrusting, but no intromission, were allowed to occur. The ferret mating pattern and the resultant LH response differ from those seen in three other induced ovulators (cat, vole, and rabbit) in which the male's intromission latency and duration are much shorter than in the ferret, and in which a distinctive peak in plasma LH often occurs within 1 h after mating.

Relaxin concentrations in endometrial, placental, and ovarian tissues and in sera from ewes during middle and late pregnancy.

These studies were designed to determine the tissue source of ovine relaxin and to determine the feasibility of using the pregnant ewe for study of relaxin production and secretion. On Day 4 of gestation, ewes were laparotomized, the nonpregnant uterine horn was ligated, and the ovary not containing the corpus luteum was removed. During a second surgery at Day 45 (n = 8) or 140 (n = 9) of gestation, 10-ml blood samples were drawn from a uterine artery, the ovarian vein, and veins draining the pregnant and nonpregnant uterine horns. Endometrial, placental, and luteal tissues were obtained for immunocytochemistry and extraction. Relaxin was detected by a heterologous porcine radioimmunoassay (RIA) in 3 of 54 serum samples (701.3 +/- 25.4 pg/ml, mean +/- SEM). Relaxin was not detected in crude tissue extracts, but low quantities were detected by RIA following Sephadex G-50 column chromatography of tissue extracts. Total relaxin activity for all tissues was equivalent to 0.57 +/- 0.13 ng of porcine relaxin/g tissue (w.w.). Relaxin was not detected immunocytochemically by light or electron microscopy. These data indicate that low quantities of relaxin are present in tissues and sera of pregnant ewes.

Immunological analysis of alpha 1-microglobulin in different mammalian and chicken serum. alpha 1-Microglobulin is 5-8 kilodaltons larger in primates.

Heterologous radioimmunoassays for a semiquantitative analysis of alpha 1-microglobulin were developed, exploiting the binding between polyclonal rabbit or goat antisera against human, guinea pig, or rat alpha 1-microglobulin and 125I-labeled human, guinea pig, or rat alpha 1-microglobulin. Homologues of this protein were detected in human, guinea pig, Rhesus monkey, rat, mouse, rabbit, goat, horse, and cow serum by inhibition of a set of heterologous radioimmunoassays. Serum proteins were separated by gel chromatography, and fractions were pooled, concentrated, and radiolabeled with 125I. By immunoprecipitation of the radioiodinated serum pools with heterologous anti-alpha 1-microglobulin-sera, and separating the precipitates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, analogues of alpha 1-microglobulin were isolated from serum of man, guinea pig, Rhesus monkey, rat, mouse, horse, and chicken. The apparent molecular weight of alpha 1-microglobulin was 31,000-32,000 in human and monkey serum and 24,000-26,000 in guinea pig, rat, mouse, horse, and chicken serum. The possibility of an addition of a 5,000-8,000-Da peptide in primate alpha 1-microglobulin is discussed.

Seasonal variations in plasma luteinizing hormone and testosterone levels in the European badger Meles meles L

Although active spermatogenesis occurs throughout the year in the male European badger Meles meles L., seasonal fluctuations of testicular activity were observed. The present study compared the pattern of luteinizing hormone (LH) and testosterone (T) secretions throughout the annual cycle. LH secretion was investigated by means of a heterologous radioimmunoassay using anti-ovine LHβ antiserum and canine reference standard. Seasonal and nycthemeral changes in LH and T secretions were observed. During the year a period of maximal values for both hormones is well correlated with the breeding. Moreover, episodic LH releases occurs during the period of minimal testosterone values, indicating resumption of hypothalamo-pituitary activity well before the breeding period.

Plasma concentrations of somatomedin-C in hypophysectomized, dwarf and intact growing domestic fowl as determined by heterologous radioimmunoassay.

The application of a human somatomedin-C radioimmunoassay for the determination of somatomedin-C in chicken plasma has been examined. Parallel inhibition of binding of 125I-labelled somatomedin-C to antisera raised against somatomedin-C was observed with acid-treated human and chicken plasma. The concentration of immunoreactive (IR)-somatomedin-C in the plasma of the domestic fowl appears to be GH dependent. Plasma concentrations of IR-somatomedin-C were reduced after hypophysectomy and partially restored by replacement therapy with chicken GH. The age/development pattern of circulating concentrations of IR-somatomedin-C has been determined in normal and dwarf strains of domestic fowl. Increases in the plasma concentration of IR-somatomedin-C were observed between 1 and 6 weeks of age in control male domestic fowl of either heavy (broiler type) or light (White Leghorn) strains. Thereafter, the plasma concentrations of IR-somatomedin-C remained constant in the heavy strain birds but declined in White Leghorn chicks. Plasma concentrations of IR-somatomedin-C were reduced in sex-linked dwarf chickens, in both light and heavy strains of fowl, but were unaffected in autosomal dwarf chickens.

Purification and radioimmunoassays for superoxide dismutases in the mouse: Tissue concentrations in different strains

1. 1. Both murine cuprozinc and manganosuperoxide dismutases were purified to electrophoretic homogeneity from liver; the former (dimer) had a sp. act. of 2600 U/mg and a subunit mol. wt of 17,000. the latter (tetramer) 2300 U/mg and mol. wt 23,000. 2. 2. Heterologous radioimmunoassays had sensitivities of 3.1 ng/ml for the former enzyme and 2.5 ng/ml for the latter, and were applicable across murine genetic lines ( Balb/c, ob/ob, db/db). 3. 3. Islets had the lowest concentrations of both enzymes among the tissues studied, and this could explain their vulnerability to free-radical damage.

The gastro-entero-pancreatic system of the turtle, Chrysemys picta

Pancreatic endocrine cells were stained immunocytochemically for insulin, glucagon, somatostatin and pancreatic polypeptide by the PAP technique or sequentially for two hormones by the PAP followed by an indirect immunogold procedure. Pancreatic endocrine cells of Chrysemys are found scattered as single cells or small aggregates throughout the exocrine parenchyma; only the splenic region shows islets consisting of a B cell core surrounded by a loose mantle of A cells and occasional D cells. PP cells were not found in this splenic portion but were found scattered throughout the remainder of the pancreas. In contrast to the typical vertebrate islet, Chrysemys pancreatic endocrine cells are characterized by a lack of preferential association of one cell type with another and suggests that paracrine regulatory mechanisms may not be operable in this species. Insulin secretion from pieces of Chrysemys pancreas has been measured in incubation and perifusion systems employing a heterologous radioimmunoassay. Insulin release by Chrysemys B cells is enhanced by elevated levels of glucose (300 mg/dl), however, response appears to be somewhat slower compared to other vertebrate B cells. Gastrin, secretin. neurotensin, motilin, serotonin, PYY, glucagon, gastric inhibitory polypeptide. somatostatin and insulin were demonstrated immunocytochemically in open-type GEP cells of the mucosal epithelium of the Chrysemys intestine. Of these cells, gastrin, neurotensin and insulin cells appear to be the most numerous while the other types appear less frequently. Cells containing PP, bombesin, cholecystokinin and substance P could not be demonstrated. The localization of insulin to GEP cells of the turtle intestine is an unusual finding but has been confirmed by radioimmunoassay of extracts of the intestinal mucosa.

An immunological approach to study gonadotropin action at the cellular and molecular level

The last two decades have seen a rapid development of a variety of new techniques and probes to understand the regulatory functions of hormones. One such tool is antibodies against hormones. Antibodies against the gonadotropins Lutropin (LH) and Follitropin (FSH), in addition to showing hormonal specificity, react with LH and FSH of a variety of other species (1-3). This property has been found extremely useful in developing heterologous radioimmunoassay (RIA) systems (4) and also for neutralizing endogenous LH or FSH at critical periods of reproduction in a variety of animals (5, 6). The classical approach to study the action of gonadotropins has been to use the hypophysectomized animal model and administer as required hormones having LH or FSH activity. Such an approach would result in the removal, at a stroke, of all the pituitary hormones other than the supplemented gonadotropin. In this procedure the permissive or antagonistic effect that the other pituitary hormones may have on the gonadotropin action when animals are in a normal physiological state is mostly overlooked. In contrast, use of an antibody, whose specificity can readily be manipulated, ensures that a physiologically normal animal can be deprived of a single hormone at any given time for a fixed period. This approach has been taken advantage of in elucidating, beyond reasonable doubt, the luteotropic activity o f L H in the cycling and pregnant rat, hamster, ~nonkey and the rabbit (7,8). Use of a specific FSH antibody similarly helped to establish, for the first time, the need for this hormone in maintaining spermatogenesis and fertility in the primate (9-12). Further, specific gonadotropin antibodies have also been employed successfully in studying events like follicular maturation, ovulation, implantation and also as a vaccine to prevent or terminate pregnancy. This aspect has been reviewed in depth earlier (7, 13,14). In the present paper emphasis will be laid on discussing in some depth how specific LH and FSH antibodies have been used to further our knowledge of the mechanism of action of these hormones at the cellular level.