A non-chromatographic radioimmunoassay for serum dehydroepiandrosterone using a mixture of antisera

A simplified method for evaluating serum dehydro-epiandrosterone (DHEA) without chromatography has been developed, using mixtures of two different anti-DHEA antisera, anti-3β-hydroxy-Δ⁵ antiserum and anti-11-deoxy-17-ketosteroid antiserum, in which cross-reactivity of each antiserum is reduced to a negligible amount. Serum (20 μ1) was extracted with 1 ml of n-hexane. One milliliter of 80% methanol was added to the n-hexane extract which was stirred and centrifuged. The n-hexane layer was discarded, and the methanol layer was evaporated to dryness. The residue was incubated with an antiserum mixture containing DHEA-7α-³H, pepsin-treated human immune serum globulin and bovine serum albumin. Ammonium sulfate was used to separate free from bound DHEA-7α- ³H. The accuracy, precision, sensitivity and specificity were satisfactory. Good agreement was found between the serum DHEA levels obtained by the present radioimmunoassay and those obtained by radioimmunoassay with paper chromatography, making this method suitable for routine use.     

Solid phase radioimmunoassay of plasma aldosterone

A solid phase radioimmunoassay for the measurement of aldosterone in plasma is described. The antiserum was produced by immunizing rabbits with 3-carboxymethyloxime of aldosterone-18–21-diacetate coupled to bovine serum albumin. This antiserum was covalently linked to an iminocellulose according to the procedure of Wide and used in the assay at a $\text{1}\text{1050}$ final dilution. It contained antibodies with association-constant of 1.1 × 10¹⁰ M^?1 and was fairly specific since with the exception of aldosterone acetates, none of the tested steroids cross-reacted more than 0.05 per cent.Aldosterone was extracted with dichloromethane, purified by paper chromatography, then submitted to the assay. The intra-assay reproducibility varied between 4 and 13 % and the inter-assay reproducibility between 13 and 21 %. The least detectable amount was 5 pg per tube. This method is very simple and, with the exception of the chromatographic step, can be completed in half a working day.     

Solid phase radioimmunoassay of plasma aldosterone

A solid phase radioimmunpassay for the measurement of aldosterone in plasma is described. The antiserum was produced by immunizing rabbits with 3-carboxymethyloxime of aldosterone-18-21-diacetate coupled to bovine serum albumin, This antiserum was covalently linked to an iminocellulose according to the procedure of Wide and used in the assay at a $\text{1}\text{1050}$ final dilution. It contained antibodies with association-constant of 1.1 × 10¹⁰M^?1 and was fairly specific since with the exception of aldosterone acetates, none of the tested steroids cross-reacted more than 0.05 per cent.Aldosterone was extracted with dichloromethane, purified by paper chromatography, then submitted to the assay. The intra-assay reproducibility varied between 4 and 13 % and the inter-assay reproducibility between 13 and 21 %. The least detectable amount was 5 pg per tube. This method is very simple and, with the exception of the Chromatographie step, can be completed in half a working day.     

A non-chromatographic radioimmunoassay of estrone and estradiol-17beta serum

A sensitive and efficient non-chromatographic procedure employing the Girard reagent and solvent-partitioning has been developed for the accurate radioimmunoassay (RIA) of estrone (E1) and estradiol-17β(E2) in a single 1.0 ml specimen of male or female serum. Using standard curves which permitted the discrimination of zero from 0.75–1.5 pg (p=0.05), the following mean procedural blanks (pg ± S.D.) were determined (1.0 ml water, n= 24): estrone, 2. 1 ± 1.1 (range 0–4.1); estradiol 1.0± 1.1 (range 0–3.6).A comparison of RIA of estrogens (1) in serum after separation by the Girard procedure and by TLC yielded correlation coefficients of 0.99 and 0.98 for E1 and E2 respectively. The following results (pg/ml ± S.D.) were obtained on RIA of E1 and E2 in 12 different 1.0 ml specimens of male and female serum using the Girard procedure: male. E1 (32.0 ± 9.2), E2 (24.1 ± 10.9); female, E1 (108.5 ± 60.8), E2 (126.4 ± 63.2).The intra-assay variability (c.v.) was found to be 12.6% for E1 and 9.4% for E2. The interassay variability was 14.2% for both estrogens.Twenty-four assays of E1 and E2 can be completed by one person in 2 working days.     

A solid-phase radioimmunoassay for plasma progesterone

A detailed procedure is presented for the assay of plasma progesterone. The routine assay is based on the use of antiserum which is covalently linked to microcrystalline cellulose, the double-antibody method being used as a reference separation system. This procedure gives high precision accompanied by small and acceptable losses of antiserum titre but without loss of sensitivity when compared with the double-antibody method. Ethanol is first added to the plasma (10vol. of plasma+1vol. of ethanol) after which a single extraction with light petroleum yields a constant recovery [92.4+/-1.2 (s.d.)% of added [(3)H]progesterone] and obviates the need for tracer recoveries on each sample being assayed. Distortions of the response curve owing to solvent residues have been almost eliminated. The assay can measure progesterone at all stages of the menstrual cycle when volumes of 200mul of plasma are used and this permits the detection of the periovulatory rise at its inception. Detailed specificity studies are presented for the assay end point itself and these are related to the responses to be expected in extracts of plasma. Progesterone-like activity was found in urine and a fourfold increase in excretion rates was observed between the follicular and luteal phase of the normal menstrual cycle.     

A plasma dexamethasone radioimmunoassay

A double antibody radioimmunoassay for estimation of plasma dexamethasone is reported. Dexamethasone antiserum was produced by immunization of rabbits with dexamethasone-3-carboxymethyloxime-bovine serum albumin conjugate. All the endogenous steroids tested cross reacted less than 1%. Cortisol with a cross reaction of 0.4% gave significant interference in some plasma samples. This Interference could be removed by chromatography. The recoveries of dexamethasone added to plasma and corrected for procedural losses were 99 ± 9% after dichloromethane extraction and 98 ± 10% after paper chromatography. After dichloromethane extraction and after paper chromatography, the intraassay and inter-assay coefficients of variation were less than 11%. The peak dexamethasone levels were observed between 30 and 60 minutes after a single 1 mg oral dose in two normal subjects. The half-times of disappearance from plasma were 4 and 4.5 hours. During a constant infusion (50 μg/70 kg BW/hr) of dexamethasone phosphate, the plasma dexamethasone level reached a level of 250 ng/dl at 8 hours. It is concluded that plasma dexamethasone levels after either oral or intravenous administration may be measured specifically by radioimmunoassay.     

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.     

A radioimmunoassay for epinephrine and norepinephrine in tissues and plasma

An I¹²⁵ radioimmunoassay (RIA) has been developed for the measurement of plasma and tissue epinephrine (E) and norepinephrine (NE). The assay utilizes an antibody which specifically binds metanephrine. E and NE are detected by conversion to metanephrine with the enzymes catechol-0-methyltransferase and phenylethanol-amine-N-methyltransferase. The assay is very specific and will allow the measurement of E and NE in less than 500 μl of normal human plasma. E and NE concentrations were determined by both the RIA and a radioenzymatic assay in canine, human and rat biologic samples. The correlation coefficients between the two assays were .962 for E and .956 for NE. The RIA is sensitive, specific, precise and significantly less costly and time consuming than present radioenzymatic methods.     

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 rapid radioimmunoassay for determining plasma concentrations of LH in dogs

A heterologous dog LH radioimmunoassay was modified to provide accurate results for LH concentrations in blood plasma of dogs within 3-4 h. This assay utilizes radioiodinated ovine LH (LER-1056-C2), antiserum against ovine LH (GDN-15) at a final dilution of 1:48,000 and dog LH (LER-1685-1) as standard. A 60-min incubation, including a 30-min delay in the addition of tracer, was carried out at 37 degrees C. The free and antibody-bound hormone were separated by addition of a Micro Sepharose bead suspension containing anti-gamma-globulin, followed by incubation at room temperature for 30 min. The minimum detectable concentration in this assay, calculated from the precision profile, was 1.5 micrograms/l. The amount of dog LH needed to cause 50% reduction of the initial binding was 1.57 +/- 0.13 ng/tube (15.7 micrograms/l for 100-microliters samples). Daily blood samples were collected in heparinized tubes from the cephalic vein of 5 pointer and 7 beagle bitches from the onset of pro-oestrus until 3-4 days after either the last mating or artificial insemination with frozen semen or until metoestrus. Samples were assayed for LH content by the short and normal incubations as well as for progesterone and oestradiol-17 beta content. In all bitches plasma concentrations of progesterone increased rapidly within 1 week after the LH peak which indicates that they had ovulated. Comparison of the short (1.5 h) with the normal (24 h) incubation system resulted in a regression equation: y = 1.0 + 0.7 x (r = 0.95, n = 153 samples).(ABSTRACT TRUNCATED AT 250 WORDS)