The determination of 18-hydroxycorticosterone in saliva and plasma: Comparison with aldosterone and glucocorticoids

A radioimmunoassay (RIA) for 18-hydroxycorticosterone (180HB) is described using antibodies raised against 180HB-3-CMO conjugated to bovine albumen and an iodine-125 labelled ligand. Extracts of plasma and saliva required thin-layer Chromatographic purification prior to RIA. These assays have been validated in terms of precision, accuracy and specificity. The concentration of 180HB in saliva is approx 20% of that in plasma and the two values are correlated. Thus, in a series of 20 healthy subjects the saliva and plasma concentration were 169 ± 92 and 904 ± 621 pmol·1⁻¹ respectively with r = 0.72 (P < 0.001). When the diurnal patterns of 180HB, aldosterone and glucocorticoid (cortisol + cortisone) concentrations in saliva were investigated the patterns of 180HB and aldosterone were usually similar though the 180HB: aldosterone ratio can vary considerably. These and similar data suggest that the secretion of 180HB and aldosterone are not completely synchronous. Our results show that saliva is a suitable body fluid in which to estimate 180HB concentration and this measurement gives a useful reflection of the plasma 180HB concentration. This technique should facilitate the investigation of the physiological role of this compound.     

Elisa for salivary and plasma estriol in pregnancy

A competitive, sensitive, and rapid enzyme-linked immunoadsorbent assay (ELISA) was developed for the determination of estriol in saliva and in plasma. Horseradish peroxidase (HRP) was used as the label enzyme; separation between free and bound steroid was carried out by insolubilized antibody prepared by adsorbing purified IgG of rabbit anti-6-oxoestriol-6-(0-carboxymethyl)oxime-BSA on polystyrene balls. The enzyme activity was measured by a colorimetric reaction using o-phenylenediamine dihydrochloride and hydrogen peroxide as substrate. The sensitivity of the assay was 12 pg/tube.In order to compare ELISA to RIA estriol estimations in different biological fluids, we selected six women during normal pregnancy, from the 30th to the 40th week of gestation. Salivary estriol was assayed by direct and extraction methods, while the corresponding plasma samples of the same subjects were analyzed only for unconjugated estriol by an extraction method.A good agreement was found between the results obtained by RIA and ELISA: r=0.897, p <0.001 between direct RIA and direct ELISA in saliva; r=0.909, p < 0.001 between extraction RIA and direct ELISA in saliva; and r=0.916, p < 0.001 between extraction RIA and extraction ELISA in plasma. A good correlation (r=0.793, p<0.001) was present between plasma samples by RIA and saliva samples by ELISA (direct method).These results indicate that: 1. ELISA is a reliable method for the determination of estriol in plasma and saliva. 2. Saliva samples can be used for the assay of estriol and therefore for the assessment of fetal conditions during pregnancy.     

一种改良的双峰驼血浆促黄体素放射免疫分析方法

本研究用单峰驼LH标准品CamLH、CamLH NZY01和人的LH（hLH)或人绒毛膜促性腺激素（hCG)作为标准品,^125I-hLH或^125I-hCG作为标记品,抗hLH或hCG血清作为抗血清,组成了不同的双峰驼血浆促黄体素放射免疫分析的组合系统,通过血浆样品稀释曲线,比较了hLH和hCG抗原抗体与CamLH,CamLH NZY01的交叉反应,选择出测定双峰驼血浆促黄体素交叉反应较强的异种抗原抗体反应系统,即用CamLH作为标准品,^125I-hLH作为标记品,抗hLH血清作为抗血清组成的测定系统,并通过血浆中添加标准参考品后的回收实验及母驼垂体兴奋实验等证明了该方法的特异性、准确性和可靠性。说明该方法可以用于测定双峰驼血浆促黄体素的浓度,是研究双峰驼生殖内分泌学的可靠手段之一。     

Determination of Serratia protease by radioimmunoassay

A specific, highly sensitive radioimmunoassay has been developed for the determination of Serratia protease. The radioimmunoassay (RIA) was based upon competition of the protease with ¹²⁵I-labeled protease for antiprotease, followed by a second antibody to separate bound enzyme from free enzyme. The RIA provided a range of 1 to 10 ng for determining the enzymes under conditions in which the enzymatic activity could not be measured. The assay was completely inhibited in the presence of human plasma. The inhibition resulted from a complex formation of the enzyme with plasma α₂macroglobulin. By treatment of the complex with acetone, however, the RIA could be achieved.     

Effects of different batches of iodine on properties of I-hFSH and characteristics of radioligand-receptor assays

Radioiodination of highly purified human follicle-stimulating hormone (hFSH) (4000 IU/mg) was performed every other week for 23 weeks using 2 mCi carrier free Na ¹²⁵I (Amersham Corp., 15 mCi/μg I₂) in the presence of lactoperoxidase. Incorporation of ¹²⁵I into hFSH was determined by the method of [7.]Biochem. J. 89, 114). Hormone binding was studied in vitro under steady-state conditions (16 h, 20°C) using different calf testis membrane preparations having similar receptor characteristics. Each ¹²⁵I-hFSH preparation was characterized for maximum bindability, specific activity of bindable radioligand as determined by self-displacement analysis, and by determination of K_a and R_t. Incorporation of ¹²⁵I into FSH was relatively constant over the large number of experiments (62.4 ± 6.4 μCi/μg; n = 23). By comparison, however, specific radioactivity of the receptor bindable fraction of ¹²⁵I-hFSH was related to the lot of ¹²⁵I utilized, and was significantly (P ≤ 0.01) lower and more variable (28.7 ± 10.5 μCi/μg). Maximum bindability of ¹²⁵I-hFSH was not correlated to specific activity (r = 0.06) but was negatively correlated to hFSH ¹²⁵I incorporation (r = −0.47; P ≤ 0.05). These observations demonstrate the need to assess the quality of each batch of radioligand before undertaking radioligand-receptor assays and suggest that differences in Na¹²⁵I lots affect specific radioactivity of the radioligand and its receptor binding characteristics.     

Receptor-Purified,Bolton-Hunter Radioiodinated,Recombinant, Human Epidermal Growth Factor: An Improved Radioligand for Receptor Studies

Abstract

We report an assessment of the applicability of the Bolton-Hunter method to the radioiodination of epidermal growth factor (EGF). Recombinant human EGF (hEGF) could be radioiodinated successfully by this method, whereas murine EGF could not. Bolton-Hunter ¹²⁵I-labeled hEGF was compared with commercial ¹²⁵I-labeled hEGF prepared by the chloramine-T radioiodination method. Neither radioligand was sufficiently pure for a detailed characterization of the purportedly heterogeneous pattern of binding of EGF to its receptors. A procedure based on receptor adsorption was thus developed for repurification of the Bolton-Hunter ¹²⁵I-labeled hEGF. This provided a much purer radioligand suitable for detailed studies of receptor-binding heterogeneity.     

Radioimmunoassay of plasma and salivary progesterone during the menstrual cycle

We have developed specific and sensitive radioimmunoassays for progesterone in saliva (SP4) easily applicable in routine practice to assess ovarian function. One of them uses [³H]progesterone, the other 3-CMO-[¹²⁵I]histamin as tracer. The reliability criteria of both techniques have been controlled and the results obtained with the two methods in 50 saliva samples are correlated well. Our clinical study has been chiefly done with [³H]progesterone. In the present study we reported the values obtained daily during the greatest part of the menstrual cycle in 14 normal cases and they are in agreement with previous reports. The use of [¹²⁵I]tracer, by increasing the sensitivity of the RIA and shortening the duration of the assay, improves the use of SP4 as a practical non-invasive sampling alternative for many clinical situations involving evaluation of the ovarian function.     

Radioimmunoassay for human salivary amylase

A radioimmunoassay (RIA) for human salivary amylase was developed. Human salivary amylase was purified from parotid saliva by a combination of Sephadex gel filtration and cation exchange chromatography. Purified salivary amylase was used both as the standard antigen and for the generation of ¹²⁵I-labeled amylase. Antibody to salivary amylase was raised in New Zealand white rabbits and used in a nonequilibrium double-antibody procedure for the RIA. The RIA was sensitive (10 ng/ml) and specific, displaying a limited cross-reactivity for pancreatic amylase (1%, $\text{w}\text{w}$). Analysis of patient sera by RIA shows that salivary amylase constitutes approximately 60% of the total serum amylase, that the salivary amylase found in the serum of patients with Sjögren's syndrome and macroamylasemia is immunologically indistinguishable from that of normal persons, and that salivary amylase can be evaluated by RIA in the serum of patients with pancreatitis.     

A double-antibody radioimmunoassay for serum progesterone using progesterone-3-(O-carboxymethyl) oximino-[125I]-iodo-histamine as radioligand.

A reliable, convenient and economical radioimmunoassay (RIA) for serum progesterone has been established and tested. This procedure employs diethyl ether extraction followed by RIA utilizing rabbit anti-11 alpha-hydroxyprogesterone 11-hemisuccinyl-bovine serum albumin (progesterone-11 alpha-BSA) serum, progresterone-3-(O-carboxymethyl) oximino-[125I]-iodohistamine (progesterone-3-[125I]) as radioligand and goat anti-rabbit gamma globulin as second antibody. In conjunction with antiprogesterone-11 alpha-BSA serum, the overall assay specificity of the progesterone-3-[125I] RIA is similar to that of the [3H]-progesterone method using dextran-coated charcoal. The results of serum progesterone measurements during the menstrual cycle obtained by the progesterone-3-[125I] RIA appear comparable to those of [3H]-progesterone assays which employ similar anti-progesterone-11 alpha-BSA sera. The progesterone-3-[125I] double-antibody RIA, however, is more convenient and less expensive than the [3H]-progesterone RIA method.     

Characteristics of antibodies against 17 beta-oestradiol in homologous and heterologous systems of radioimmunoassay

Directly iodinated oestradiol-2(4)-iodo-[125I] and oestradiol-6-O-(CMO)-[125I]iodohistamine were prepared and used in conjunction with anti-oestradiol-2(4)-azo and anti-oestradiol-6-O-(CMO) sera for the development of various radioimmunoassay (RIA) systems which showed marked differences in sensitivity and relatively small differences in specificity. Whereas the heterologous combination of oestradiol-6-O-(CMO)-[125I]iodohistamine and anti-oestradiol-2(4)-azo-serum showed a sensitivity expressed in femtograms, the homologous combination using oestradiol-6-O-(CMO)-[125I]iodohistamine radioligand exhibited a sensitivity two orders lower. The specificity of both the heterologous and homologous system did not differ significantly from the RIA system using tritiated radioligand. The combination of directly iodinated oestradiol and anti-oestradiol-2(4)-azo-serum showed a higher sensitivity and a lower specificity as compared with tritiated radioligand.     

Comparison of a solid-phase,no-extraction radioimmunoassay for progesterone with an extraction assay for monitoring luteal function in the mare,bitch, and cow

Equine, canine, and bovine plasma samples were assayed for progesterone (P₄) using extracted plasma in a liquid-phase assay and unextracted plasma in a solid-phase ¹²⁵I direct assay developed for human serum. The direct assay was also used to monitor P₄ levels in defatted milk. Results indicated that the direct-assay method was as reliable as the extraction assay for monitoring changes in plasma P₄ during the estrous cycle and early pregnancy. The regression equation specifying the relationship for the two methods was exponential. In this study, correlation coefficients from data subsets ranged from 0.93 to 0.99. The direct assay for P₄ did result in higher values than the extraction assay in plasma from diestrous animals. The use of diluted standards with stripped plasma from the species being assayed gave values that correspond more closely to the extraction assay. The comparisons in this study indicate that the direct radioimmunoassay (RIA) method offered a convenient, reliable method for monitoring luteal function in the species that were evaluated.     

A re-investigation of saliva collection procedures that highlights the risk of potential positive interference in cortisol immunoassay

Background

The use of saliva for measurement of cortisol permits non-invasive study of adrenal function, but collection can be technically difficult, particularly in small infants. Saliva collection can be assisted by citric acid to increase saliva flow, or by the use of cotton or polyester swabs in the mouth.

Aim

To determine whether different methods of saliva collection affect cortisol radioimmunoassay (RIA) performance.

Experimental

Cortisol was measured in saliva collected from 16 adults using intra-oral cotton swabs or polyester swabs, compared with saliva dribbled directly into a pot either alone (plain saliva) or after citric acid had been placed on the tongue. An in-house RIA, without prior extraction, was used to measure cortisol with an encapsulated sheep antibody.

Results

Mean (median) salivary cortisol was 10.9 (10.5) nmol L⁻¹ in plain saliva, 10.4 (8.4) nmol L⁻¹ in citric acid stimulated saliva; 25.3 (25.1) nmol L⁻¹ in saliva collected on cotton swabs, and 27.9 (27.3) nmol L⁻¹ collected on polyester swabs. Cortisol in saliva collected using citric acid was not significantly different from plain saliva (p = 0.997), but cortisol in saliva collected using cotton and polyester swabs was significantly higher than that of plain saliva (p < 0.01).

Conclusion

The use of cotton or polyester swabs for collection of saliva can result in spuriously high levels of cortisol when measured by RIA.     

Simultaneous quantitation of methotrexate and its two main metabolites in biological fluids by a novel solid-phase extraction procedure using high-performance liquid chromatography

We have developed an assay for the simultaneous determination of methotrexate (MTX) and its main metabolites, 7-hydroxymethotrexate (7-OHMTX) and 2,4-diamino-N¹⁰-methylpteroic acid (DAMPA) in plasma, urine and saliva meeting the requirement of rapidity for routine use in high-dose MTX therapy and the requirement of sensitivity for its potential use in therapeutic drug monitoring in low-dose MTX therapy. Sample preparation is based on solid-phase extraction using C₈ Isolute cartridges. Chromatographic separation was achieved with a reversed-phase column (C₁₈), and quantitation by subsequent exposure to UV light of 254 nm, which converted MTX and its two metabolites by photolytic oxidation to fluorescent products. The recoveries of MTX, 7-OHMTX and DAMPA from plasma at 100 nmol/1 were 85.8, 91.1 and 102.3%, respectively. The limits of detection for MTX, 7-OHMTX and DAMPA in plasma and saliva were 0.1 nmol/1. In urine the limit of detection was 10 nmol/1 for all compounds. The limits of quantitation in plasma and saliva were 0.5 nmol/1 for all compounds.     

The talc-resin-TCA test: rapid screening of radioiodinated polypeptide hormones for radioimmunoassay

The talc-resin-TCA test is a rapid, reliable method for predicting the behavior of ¹²⁵I-hormones in radioimmunoassays (RIA). This test is based on the assessment of three different physicochemical properties of iodinated hormones: adsorption to talc, exclusion from ion exchange resin, and precipitation by TCA. These three parameters produce test patterns that are unique for monomeric ¹²⁵I-hormone, aggregated ¹²⁵I-hormone, and ¹²⁵I-salts. Different iodination methods were used to prepare radioiodinated human and rat PRL, GH, TSH, and LH, and rat FSH. Purified monomeric ¹²⁵I-hormones that gave reliable RIA results all showed a characteristic talc-resin-TCA profile of >90% talc adsorption, <25% resin binding, and >90% TCA precipitation. This profile has successfully predicted reliable RIA results for ¹²⁵I-hormones prepared from more than 80 iodinations.     

Identification and Characterization of a Specific Receptor for Cholecystokinin on Isolated Fundic Glands from Guinea Pig Gastric Mucosa Using a Biologically Active 125I-CCK-8 Probe

Abstract

Specific binding sites for cholecystokinin (CCK) have been identified and characterized in fundic glands isolated by collagenase treatment from guinea pig gastric mucosa using a biologically active ¹²⁵I-labeled derivative of the C-terminal octa-peptide of CCK (¹²⁵IIE-CCK-8). The time course of binding to these glands was rapid, temperature dependent and saturable. At 24, 30 and 37° C, half-maximal binding was reached at 5 min and full binding at 30 min. The addition of a large excess of CCK-8 after 15 and 30 min of binding at 24° C caused a prompt and rapid decline in radioligand bound showing that the interaction was reversible. There was a progressive decline in the amount of ¹²⁵IIE-CCK-8 bound to fundic glands with increasing concentrations of CCK-8 and other structurally related peptides. Gastrin II displaced 50% of the radioligand at 1.6nM, CCK-8 at 3.2nM, gastrin I at 16nM, and desulfated-CCK-8 and pentagastrin at 59nM. Secretin did not displace the radioligand from fundic glands at 1.0uM. The binding was also tissue specific as glands isolated from the antral mucosa did not contain specific binding sites for ¹²⁵IIE-CCK-8. This data provides evidence for specific receptors for CCK on gastric fundic glands that may be involved in the control of acid and pepsinogen secretion.     

Reversed-phase high-performance liquid chromatography of salmon calcitonin and its degradation products in biological samples using column switching and flow-through radioisotope detection

For the determination of salmon calcitonin and its degradation products in biological samples, a reversed-phase HPLC method with column switching and flow-through radioisotope detection has been developed using high specific activity [¹²⁵I]salmon calcitonin. Effects of the precolumn packing material and washing solvent were examined in terms of [¹²⁵I]salmon calcitonin recovery. Spiked samples of [¹²⁵I]salmon calcitonin in plasma and kidney homogenate were injected onto a LiChroprep RP-8 precolumn after dilution with 0.1% trifluoroacetic acid. After washing the polar interfering compounds with 0.1% trifluoroacetic acid, the concentrated [¹²⁵I]salmon calcitonin and its degradation products were eluted and separated on a W-Porex C₁₈ column with a gradient of 0.1% trifluoroacetic acid in acetonitrile-water. Detection and calibration of [¹²⁵I]salmon calcitonin were possible down to picogram levels. Reproducible kinetic data for the degradation of intact [¹²⁵I]salmon calcitonin by rat kidney homogenate could be traced.     

Generation of novel radiolabeled opiates through site-selective iodination

Tritiated opioid radioligands have proven valuable in exploring opioid binding sites. However, tritium has many limitations. Its low specific activity and limited counting efficiency makes it difficult to examine low abundant, high affinity sites and its disposal is problematic due to the need to use organic scintillants and its relatively long half-life. To overcome these issues, we have synthesized both unlabeled and carrier-free radioiodinated iodobenzoyl derivatives of 6β-naltrexamine (¹²⁵I-BNtxA, 18), 6β-naloxamine (¹²⁵I-BNalA, 19) and 6β-oxymorphamine (¹²⁵I-BOxyA, 20) with specific activities of 2100 Ci/mmol. To optimize the utility of the radioligand, we designed a synthesis in which the radiolabel is incorporated in the last synthetic step, which required the selective iodination of the benzoyl moiety without incorporation into the phenolic A ring. Competition studies demonstrated high affinity of the unlabelled compounds for opioid receptors in transfected cell lines, as did the direct binding of the ¹²⁵I-ligands to the opioid receptors. The radioligand displayed very high sensitivity, enabling a marked reduction in tissue, as well as excellent signal/noise characteristics. These new ¹²⁵I-radioligands should prove valuable in future studies of opioid binding sites.     

A solid-phase immunoassay for human immunoglobulin E: use of 125I-labeled protein A as the tracer

A solid-phase immunoassay has been developed for human immunoglobulin (Ig) E. The specific binding of ¹²⁵I-labeled protein A (¹²⁵I-PA) to the Fc region of rabbit IgG anti-IgE served as a quantitative measure of specific anti-IgE antibody bound to the IgE beads under optimal assay conditions. Inhibition of antibody binding by known amounts of standard IgE was reflected in a decreased binding of ¹²⁵I-PA. The degree of inhibition of ¹²⁵I-PA binding was related to the amount of fluid-phase IgE present and gave a standard curve which was used to determine the concentration of IgE in test samples. The sensitivity of this method and a double antibody radioimmunoassay (RIA), which was developed using the same IgE preparation and anti-IgE antibody, was approximately the same. These assays gave similar results when used to determine levels of IgE in normal human sera that had been absorbed with protein A—Sepharose to remove components responsible for specific and nonspecific interference in the assays.     

A radioimmunoassay using labeled antibody for polyhedron protein from a nuclear polyhedrosis virus of Wiseana cervinata

A radioimmunoassay (RIA) for polyhedron protein based on precipitation of the antibody-antigen complex by sodium acetate buffer, pH 5.0, was studied. The effects of varying assay conditions and of using [³H]acetate- or ¹²⁵I-labeled antibodies were examined. The RIA could detect specific differences between the polyhedron protein from the nuclear polyhedrosis viruses of Bombyx mori and Wiseana cervinata at the level of 3 × 10⁴ polyhedral/ml when ¹²⁵I-labeled antibodies were used. This procedure could provide the basis for making serological comparisons of polyhedron proteins and for detecting viruses in samples such as soil and bird feces.