Standardization and clinical application of a radioimmunoassay for human calcitonin.

A radioimmunoassay for the measurement of Human Calcitonin (HCT) is described. Serum levels of HCT in normal subjects and in individuals under different pathological conditions have been studied with this method. HCT labelling is performed following the chloramine T method of Hunter and Greenwood. Adding successively Quso G-32 (a finely powdered silica) and an anion exchange resin (AGI-X10 resin), to the tracer, reduces both the damaged fraction and the free isotope which originate during storage. Purification of the labelled hormone is carried out through a Sephadex G-50 gel column. Sera stored at --20 degrees C preserves its immunoreactivity up to 4 months after extraction. The mean basal HCT levels in 110 fasting normal persons is 277 +/- 123 pg/ml (undetectable 8.28%). No significant correlation between HCT levels and various serum ions has been observed. Basal HCT values in seven patients with medullary thyroid carcinoma (MTC), oscillated between 5 and 110 ng/ml, while in six other patients with non medullary thyroid carcinoma the values remained within normal range. Both a calcium infustion and a pentagastrin injection are used to stimulate HCT secretion. The increase of HCT basal levels produced by the latter in normal controls and in patients with MTC, is faster and more intense. Calcium infusion produced a significant correlation between calcium and the increased HCT levels only in patients affected with MTC.     

A radioimmunoassay for human plasma corticosterone.

A radioimmunoassay for human plasma corticosterone has been developed. Antiserum against corticosterone was produced in rabbits immunized with corticosterone-21-hemisuccinate conjugated to bovine serum albumin. The antiserum cross-reacted with progesterone, DOC and dehydrocorticosterone more than 20%. After the extraction with ether, and the separation by Sephadex LH-20 microcolumn chromatography, recovery was 51.2 +/- 12.1% in 50 assays. The mean coefficient of variation between assays was 7.7% and within assays was 8.6%. Human plasma corticosterone is measured readily by assaying aliquots of an ether extract of 0.05 to 0.1 ml of plasma after microcolumn chromatography. The mean plasma corticosterone concentration at 9 a.m. was 7.1 +/- 3.2 ng/ml in 45 normal subjects. Plasma corticosterone increased 5.2 times as much as basal values after ACTH injection, whereas radioimmunoassayed cortisol increased 2.4 times. On the other hand, plasma corticosterone decreased to 22.6% of basal values at four hours after 1 mg dexamethasone, whereas radioimmunoassayed cortisol decreased to 12.3% of basal values.     

Development of an avian calcitonin radioimmunoassay using synthetic chicken calcitonin as immunogen

1. A sequential double antibody radioimmunoassay (RIA) has been developed using synthetic chicken calcitonin (CT) as antigen, tracer and standard. 2. The immunoassay has a minimum detection limit of 0.5 ng and effective dose (ED50) of 7 ng. Serial dilutions of chicken and turkey plasma were parallel to serial dilutions of CT standard. Extracts of chicken and turkey ultimobranchial glands caused parallel displacement of tracer similar to synthetic CT. 3. Primary antisera (anti-chicken CT) was raised in guinea pigs immunized with RIBI: animals treated with Freund's complete adjuvant failed to respond. 4. Chicken CT was determined to have a half-life of 60 sec in the turkey hen. Development of a homologous RIA for avian CT will allow studies to elucidate the role of this hormone in birds.     

A radioimmunoassay for human epidermal growth factor receptor

The development of a radioimmunoassay (RIA) for the human epidermal growth factor receptor solubilized with nonionic detergents which employs iodinated epidermal growth factor (125I-EGF) as the specific ligand is described. A monoclonal antibody (R1) that binds specifically to human EGF receptors [Waterfield, M. D., et al. (1982) J. Cell Biochem. 20, 149-161] was used to separate solubilized receptors saturated with 125I-EGF from free ligand by absorption to protein A-Sepharose, and the bound radioactivity was determined. The RIA was linear when increasing amounts of solubilized membrane protein were added and, when compared to the standard polyethylene glycol assay, was more reproducible. In addition, the background nonspecific binding obtained in the presence of a hundred-fold excess of unlabeled EGF was less in the RIA. Substitution of normal mouse serum for the monoclonal antibody gave very low nonspecific background ligand binding and avoided the use of large amounts of unlabeled EGF in the assay. Two major classes of binding sites for EGF were observed in membrane preparations from the cervical carcinoma cell line A431 or from normal human placental tissue. These were present in approximately equal amounts, with apparent dissociation constants of 4 X 10(-10) and 4 X 10(-9) M. Upon solubilization with the nonionic detergent Triton X-100, only one class of EGF binding sites was detected in both cases, with a dissociation constant of 3 X 10(-8) M. The RIA can be used to monitor receptor purification and for quantitation of receptor number and affinity in various cell types.     

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.     

A new radioimmunoassay for human chorionic gonadotropin using monoclonal antibody

A new radioimmunoassay (RIA) for human Chorionic Gonadotropin (hCG) was developed using murine monoclonal antibody to the beta-subunit of hCG (beta-hCG). The IgG fraction of the monoclonal antibody which did not react with 125I-beta-hCG was purified from hybridoma ascites, and covalently coupled to Sepharose 4B. This solid-phase antibody was incubated with standard hCG or serum sampled for 48 hours. The reaction medium was then removed by centrifugation and 125I-beta-hCG and anti-beta-hCG rabbit polyclonal antibody were added to the precipitate. The alcohol precipitation method was used for separating "bound" and "free" forms in the second reaction. The sensitivity for hCG in this assay system was 0.5 mIU/ml serum and the cross-reactivity with human Luteinizing Hormone (hLH) was 0.4%. This assay system was shown to be clinically applicable. Serial serum samples from two patients with trophoblastic disease were assayed and minute amounts of hCG, which could not be determined by conventional assay methods, could be assayed by this new RIA.     

A radioimmunoassay for 7 alpha-hydroxy dehydroepiandrosterone in human plasma.

A radioimmunoassay for plasma 3 beta, 7 alpha-dihydroxy-5-androsten-17-one (7 alpha-hydroxy DHA) has been developed using anti-sera raised against 3 beta, 7 alpha-dihydroxy-5-androstene-17 beta-carboxyl-bovine serum albumin conjugate and [1, 2 (n) - 3H] 7 alpha-hydroxy DHA as the radioligand. Significant cross reactivity was found with 3 beta, 7 alpha-dihydroxy-5-pregnen-20-one (44%), 3 beta, 7 beta-dihydroxy-5-androsten-17-one (6%), 3 beta, 6 beta-dihydroxy-4-androsten-17-one (2.5%), 3 beta-hydroxy-5-androsten-17-one (DHA, 2%), 3 beta, 7 beta-dihydroxy-5-pregnen-20-one (2%) and 7 alpha-hydroxy-4-androstene-3, 20-dione (1%). 7 alpha-Hydroxy DHA was extracted from plasma and separated from cross-reacting factors using alumina micro-columns. The separation of bound and free steroid was achieved using dextran-coated charcoal. The concentration of 7 alpha-hydroxy DHA in the plasma of breast cancer patients was significantly lower than the concentrations in the plasma of normal women, hospitalized women suffering from non-endocrine diseases and patients with benign breast disease. The decrease in the concentration of 7 alpha-hydroxy DHA in the plasma of pregnant women was not significant.     

A second human calcitonin/CGRP gene

The calcitonin (CT) gene is alternatively expressed in a tissue-specific fashion producing either the calcium regulatory hormone CT in the thyroid or the neuropeptide calcitonin gene related peptide (CGRP) in the brain. In medullary carcinoma of the thyroid both peptides are produced. We present here evidence for the existence in the human genome of a second CT gene, which is also expressed in human medullary thyroid carcinoma. This gene encodes a second human CGRP, differing from the known human CGRP in 3 of the 37 amino acids.     

A radioimmunoassay of human circulatory dopamine-β-hydroxylase

A procedure for the radioimmunoassay of human circulatory dopamine-β-hydroxylase (DβH) using antibodies directed toward human DβH is described. A significant correlation between serum DβH activity and the amount of serum immunoreactive (IR) DβH was found in the analyzed population. The radioimmunoassay of serum DβH could be used in monitoring changes of sympathetic activity in various physiological and pathological states.     

A radioimmunoassay for lithocholic acid conjugates in human serum and liver tissue

A sensitive and specific radioimmunoassay for glycine and taurine conjugates of lithocholic acid (CLCA) has been developed. ³H-glycolithocholic acid (S.A. = 17Ci/mmol) was used as tracer. Separation of free from antibody-bound bile acid was carried out using ammonium sulphate (saturated solution). The antiserum showed high specificity for both glyco and tauro conjugated lithocholate (100% cross reaction) and lithocholic acid (25% cross reaction). The sensitivity of the assay (1 pmole/tube), was adequate for measuring CLCA in peripheral blood and hepatic tissue in man.     

A radioimmunoassay for chironomid hemoglobin

A double antibody, competitive radioimmunoassay was developed for the quantitation of stage-specific hemoglobin from the insect Chironomus thummi. The radioimmunoassay will detect as little as 150 picograms of Hb 3, a hemoglobin synthesized and secreted into the hemolymph of larvae during the 4th, but not the 3rd instar. The assay also detects cross-reacting hemoglobins purified from 4th instar larvae and in freshly laid eggs. Application of this radioimmunoassay is discussed in light of the cross-reacting Hbs in the insect.     

A coated-tube radioimmunoassay for beta-thromboglobulin

A solid phase radioimmunoassay for beta-thromboglobulin is presented which has the advantage of easy technical performance. The antibody was absorbed onto polystyrene tubes and the separation of free and bound radiolabelled tracer was performed by simply sucking the tubes empty after incubation. The assay is precise and accurate but not particularly rapid, since an overnight incubation is needed. Maximum assay sensitivity was found to be 5.6 ng/ml beta-thromboglobulin for plasma samples. Preliminary studies showed that the sensitivity can be increased by the use of a more dilute antiserum. The plasma concentration of beta-thromboglobulin in 37 normal subjects was 17 +/- 4 ng/ml. The analytical performance of the assay corresponds to a commercial liquid-phase kit used for reference.