A sensitive and specific radioimmunoassay for arginine vasopressin and its validation

A sensitive and specific radioimmunoassay (RIA) for arginine vasopressin (AVP) has been developed and validated. Synthetic AVP was coupled to bovine serum albumin (BSA) with glutaraldehyde. Antisera against AVP were raised in three rabbits immunized with AVP-BSA complex. After 6 months, at the 16th injection, one of the antisera had a titer high enough to be utilizable for RIA at a final dilution of 1:400,000. The labeling of AVP with 125I Na was performed with the modified chloramine T method, and the purification of iodinated AVP was done with gel filtration chromatography on a Sephadex G-25 fine column (1 X 20 cm) with an elution buffer of 0.01 M acetic acid containing 0.1% BSA. Radioactivities from the Sephadex G-25 were eluted in three peaks. 125I-AVP, which was reactive to the antiserum, was contained in the third peak, and 125I-AVP in the fractions on the down slope of the peak was used for the radioligand in the amount of 1000 cpm. The specific activity of purified 125I-AVP was about 400 muCi/microgram. Diluted antiserum and samples, unlabeled AVP or related peptides were preincubated at 4 degrees C for 24 hr, and then 125I-AVP was added to the mixture and incubated for a further 72 hr. Separation of B and F was done with polyethyleneglycol. The minimal detection limit of AVP, which was 95% of the confidence limit of the mean value of B0, was 0.4 pg/tube. The cross-reactivities with lysine vasopressin, arginine vasotocin, DDAVP and oxytocin were 0.1%, 30%, 1% and 0%, respectively. AVP in plasma was extracted with cold acetone and petroleum ether. The recoveries of synthetic AVP from plasma which was added (2-16 pg) were more than 94%. The intra and inter-assay coefficients of variation determined by plasma of AVP concentration of about 4.8 pg/ml were 8.7% and 11.3%, respectively. The RIA detected AVP of concentration as low as 1 pg/ml following the extraction procedure. AVP immunoreactivity was detected without extraction in urine, and the lyophilized cerebrospinal fluid and acid extract of tissues of the central nervous system, and the reactivities in these samples were demonstrated to be immunologically identical to that of synthetic AVP when diluted serially. The changes of plasma and urinary AVP concentration on water intake, water deprivation and smoking in humans were clearly demonstrated.(ABSTRACT TRUNCATED AT 400 WORDS)     

A specific radioimmunoassay for human beta-lipotropin.

A homologous RIA for human beta-lipotropin (beta hLPH) has been developed. At a final dilution of 1:24,000, the antiserum employed shows cross-reaction with beta hLPH but none with human beta-MSH (beta hMSH), and it is concluded that the antigenic determinant lies within the N-terminal 1-36 region of beta hLPH. With extraction of 3-ml plasma samples, the assay is sufficiently sensitive to measure circulating beta hLPH levels in normal individuals at 0900 h (25-200 pg/ml). There is a circadian variation with levels falling to (less than 20-80 pg/ml) at 2300 h. beta hLPH levels rise after metyrapone and after insulin-induced hypoglycemia, and fall after administration of dexamethasone. In patients with a variety of diseases of the pituitary-adrenal axis, levels of beta hLPH follow immunoreactive ACTH levels, although the two are not always secreted on a 1:1 molar basis.     

A sensitive and precise radioimmunoassay for human thyroid-stimulating hormone.

A high-sensitivity radioimmunoassay for human thyroid-stimulating hormone (hTSH) has been developed utilizing a highly specific rabbit antibody (developed by AFP) to hTSH with an affinity constant of 4.4 x 10(11) M-1. Optimal conditions which minimized both 125I-hTSH radiation damage and 125I dissociation included storage at -60 C in 0.25% albumin. Repetitive freezing and thawing did not cause loss of structural integrity or of full immunochemical reactivity of 125I-hTSH. Effects of human and calf sera on the precipitation of first antibody-bound 125I-hTSH by second antibody have been tabulated; the addition of TSH-free human serum to the standard curve is necessary to compensate for the serum effect. The minimum detectable amount of TSH in the new assay at a 1:1,000,000 final dilution of the hTSH antibody is 0.02 muU/tube. The mean normal hTSH value of 1.5 +/- 1.0 muU/ml (mean +/- SD) fell within the central one-third of the logit (B/Bo) plot where antigen concentrations are measured with highest precision. This assay gives good precision and reproducibility of measurements throughout the normal range of serum TSH concentrations.     

A sensitive radioimmunoassay for quantitation of igm rheumatoid factor

A solid-phase radioimmunoassay capable of detecting nanogram quantities of human IgM rheumatoid factor (RF) in biologic fluids has been developed. Binding curves for monoclonal IgM RF and polyclonal IgM rheumatoid factors were similar under the conditions utilized for the assay. Human IgG did not interfere with the detection of IgM RF by this method. Small quantities (≤ 0.2%) of nonspecific binding by nonRF IgM to the human IgG coated tubes utilized in the assay were corrected for by assaying samples in parallel bovine serum albumin coated control tubes. As expected, patients with seropositive rheumatoid arthritis (RA) had significantly higher concentrations of IgM RF than seronegative RA patients (mean ± 1 SD = 652 ± 553 μg/ml versus 11.3 ± 13.3 μg/ml, P < 0.001). In contrast, all normal control sera assayed to date contained < 0.1 μg/ml of IgM RF. The capacity of the assay to detect nanogram quantities of IgM RF should permit investigation of the cellular mechanisms underlying RF production.     

A specific radioimmunoassay for 5''-deoxyadenosyl cobalamin in serum

A specific radioimmunoassay for 5'-deoxyadenosyl cobalamin (ado-Cbl) has been developed using an antiserum raised to this cobalamin (Cbl). At a 1:100 or greater dilution the antiserum did not bind radiolabelled methyl-, hydroxo-, sulphito- or cyano-Cbl and these Cbl analogues did not compete in the radioimmunoassay even at 100-fold higher concentration. The serum concentration (range and mean +/- SD) of ado-Cbl in 30 normal subjects was 47-134 and 81 +/- 16 pg/ml. The corresponding values for total Cbl in these sera were 189-610 and 355 +/- 144 pg/ml, and the computed values for methyl-Cbl were 142-476 and 274 +/- 127 pg/ml. The coefficient of variation was substantially greater for methyl-Cbl than for ado-Cbl (46% v. 21%, respectively). The ado-Cbl concentration was in the normal range (75-95 pg/ml) in five healthy subjects with a low normal concentration (189-217 pg/ml) of total Cbl. In two subjects with low total Cbl (118 and 170 pg/ml) and without any clinical evidence of Cbl deficiency, ado-Cbl was normal (63 and 95 pg/ml, respectively). Thus, in this group, low methyl-Cbl accounted for the lower total Cbl. The concentration (mean +/- SD) of serum ado-Cbl and methyl-Cbl in six patients with low total Cbl (57 +/- 25 pg/ml) and clinical evidence for Cbl deficiency was 35 +/- 12 pg/ml and 22 +/- 22 pg/ml, respectively. This difference from the normal mean for each cofactor was highly significant (P less than 0.001). The decrease in the concentration of methyl-Cbl in Cbl deficiency was relatively greater than the decrease in ado-Cbl (92% v. 57%, respectively) which raised the relative concentration of ado-Cbl in Cbl deficiency to 61% of the total Cbl. Although a low serum methyl-Cbl is a sensitive indicator of Cbl deficiency, it may not be as specific as a decrease in serum ado-Cbl. Factor(s) other than tissue stores of Cbl may lower serum methyl-Cbl below the 95% confidence limit in the absence of clinical Cbl deficiency.     

Plasma thromboplastin antecedent (PTA, factor XI): a specific and sensitive radioimmunoassay

A specific, sensitive, and reproducible radioimmunoassay for human plasma thromboplastin antecedent (PTA, factor XI) has been developed with purified PTA and monospecific rabbit antiserum. Precise measurements of PTA antigen were possible for concentrations as low as 0.3% of that in normal pooled plasma. Normal plasma contained approximately 6 microgram PTA/ml. A good correlation (correlation coefficient 0.68) existed between the PTA procoagulant assays and radioimmunoassays among 50 normal adults (25 males and 25 females). PTA antigen was markedly reduced in plasma of 13 patients with congenital homozygous PTA deficiency (range less than 0.003-0.128 U/ml) and 9 patients with hepatic cirrhosis (0.35+/-0.17 U/ml), but was normal in those of 9 patients under treatment with warfarin, 8 patients with disseminated intravascular coagulation and 16 patients with other congenital clotting factor abnormalities, including prekallikrein deficiency (Fletcher trait) and high molecular weight kininogen deficiency (Fitzgerald trait).     

A radioimmunoassay for rat pancreatic secretory trypsin inhibitor

A radioimmunoassay for the detection of pancreatic secretory trypsin inhibitor (PSTI) in the rat is described. The sensitivity of the assay enables the specific measurement of this inhibitor in the serum of normal rats. The average base-line value for multiple animal serum specimens taken from fasting female Wistar rats being fed conventional diets was 11.6 +/- 6.2 micrograms/l. The inhibitor existed in its free form in serum, and PSTI immunoreactivity increased significantly within 2 h of the induction of experimental pancreatitis. The present assay will facilitate the study of PSTI in experimental diseases of the pancreas.     

A specific radioimmunoassay for estrone sulfate in plasma and urine without hydrolysis.

Estrone sulfate, quantitatively the most important estrogen in plasma, has previously been determined only after hydrolysis and chromatography. An antiserum raised against estrone glucosiduronate-bovine thyroglobulin was found to be suitable for the specific RIA of estrone sulfate both in plasma and urine. Plasma levels were measured after solvent extraction without hydrolysis or chromatography. The mean (+/-SE) was 972 +/- 79 pg/ml (range, 537-1581) in 15 women in the follicular phase, 1806 +/- 160 pg/ml (range, 814-3358) in 15 women in the luteal phase, and 922 +/- 62 pg/ml (range, 461-1238) in 13 men. The urinary excretion of estrone sulfate, measured after simple chromatographic separation, ranged from 0.8-7.9 micrograms/24 h in men and 5.1-18.7 micrograms/24h in nonpregnant women. This was generally one-seventh to one-half the simultaneous estrone glucosiduronate excretion rate. An approximate mean renal clearance of estrone sulfate calculated from the above values was 2.7 ml/min. The low clearance rate is taken to reflect extensive binding of estrone sulfate by plasma proteins. The splanchnic extraction of estrone sulfate measured in 6 patients undergoing hepatic vein catheterization for diagnostic purposes was 29.8 +/- 11.1%, indicating net uptake of this compound by the splanchnic area.     

Direct radioiodination of estradiol for a sensitive radioimmunoassay.

17 beta-estradiol (E2) was iodinated with the chloramine-T method. The 125I-labelled material was purified with thin-layer chromatography. The major fraction was applied to a radioimmunoassay with an antiserum against estradiol-6-(0-carboxymethyl)oxime-BSA. The sensitivity of the assay was comparable to assays with 3H-E2 or 125I-histamine-E2 as tracers. The working antiserum dilution (1:1 000 000) was the same with 125I-E2 and 3H-E2 but lower than that with 125I-histamine-E2. The results indicate that iodination of the A-ring of estrogens will not cause significant reduction of the immunoreactivity when combined with an antiserum to a B-ring conjugate.     

A sensitive and specific assay for 5-methoxytryptophol in plasma.

5-Methoxytryptophol (ML) is found in the pineal gland and is known to have biological activity especially as an antigonadotrophic agent, but methods have been lacking for its measurement in the circulation. A gas chromatography-mass spectrometry assay using a trimethylsilyl derivative has been developed for the routine measurement of ML in plasma. The assay is of great specificity and has a sensitivity of 20 pmol/l. Studies on the levels of pineal indoles in the circulation, however, have been hampered by the possibility that extraneous compounds are being cross-measured. Thus the specificity of the routine assay has been further validated by comparing it with an alternative assay system where all the major parameters were changed, i.e. derivatizing reagent, internal standard and mass number. Results that were obtained using both assay systems were closely comparable.     

Highly sensitive radioimmunoassay for chorionic gonadotropin in human urine.

The value of RIAs that measure hCG levels in human urine has been limited principally because of cross-reactivity with human LH. Recently, antisera generated to antigenic determinants on the intact hCG beta subunit and its carboxyl-terminal peptide have been shown to exhibit substantially reduced human LH cross-reactivity. To take maximal advantage of these antisera and to minimize interference by nonspecific substances in urine, a procedure for extracting and concentrating hCG from 24-h urine samples was developed. The procedure involves preparation of a standard kaolin-acetone urine concentrate and adsorption of the hCG in the concentrate to Concanavalin A covalently linked to agarose for purification and subsequent RIA. In urine samples obtained from patients with gestational trophoblastic disease, there was a direct correlation between hCG levels measured by RIA and those estimated by mouse uterine weight bioassay. In individual subjects, hCG levels were determined in serum and urine obtained the same day. When hCG was clearly detectable in the serum at levels greater than 1 ng/ml, the quantity of hCG measured in the urine concentrate exceeded 500 ng/24 h. The concentrates prepared from the urine of normal persons contained an hCG-like glycoprotein substance with antigenic determinants similar to those of the carboxyl-terminal peptide of hCG beta. As the range of hCG immunoreactivity measured in the urine concentrates of normal subjects was 6-52 ng/24 h, specific and sensitive detection of urinary hCG could be accomplished in patients whose sera contained hCG undetectable by conventional RIA. Partial purification and concentration of urinary hCG by this procedure with subsequent RIA provides a sensitive and reliable method for detecting hCG in urine.     

A sensitive radioimmunoassay for the determination of plasma angiotensin II in human subjects.

A sensitive and specific radioimmunoassay for the determination of plasma angiotensin II was developed by using the antisera against synthetic angiotensin II in combination with labeled angiotensin II. This assay employs an acetone extraction procedure and detects as little as 0.8 pg per tube of angiotensin II. The mean (+/- S.E.) plasma angiotensin II concentration in 19 normal subjects was 14.4 +/- 1.8 pg/ml in a state of overnight fasting and recumbency. In 13 normal subjects, in whom 40 mg of furosemide was injected intravenously, plasma angiotensin II concentration before and after 30 and 120 minutes in an upright position was 14.6 +/- 2.2, 56.6 +/- 5.7 and 74.3 +/- 9.0 pg/ml, respectively. In 6 normal subjects, an infusion of isotonic saline, angiotensin II concentration reduced from 14.1 +/- 3.7 to 9.2 +/- 1.7 pg/ml. Thus, it was ascertained that the simplified radioimmunoassay method reported here using an acetone-petroleum ether extraction method was specific and highly sensitive.