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 non-chromatographic radioimmunoassay of 18-hydroxy-11-deoxycorticosterone in human plasma

A method is described for a non-chromatographic assay of 18 hydroxy-11-deoxycorticosterone (18-OH-DOC) using a sensitive and specific antiserum. This direct measurement is assessed in terms of accuracy and precision. The mean 8a.m. plasma 18-OH-DOC levels in the supine position was 10.1 ± 6.5 ng per 100ml in 20 normal subjects and 9.4 ± 4.2 ng per 100ml after two hours of movement. These values are correlated with those obtained in aldosterone. The ACTH-dependency of 18-OH-DOC is demonstrated by diurnal variation and treatment with dexamethasone.     

A non-chromatographic radioimmunoassay of prugesterone in serum

Convenient methodology based on separation of progesterone from alcoholic neutral steroids by means of a sulfation-procedure has been developed for the radioimmunoassay (RIA) of progesterone in male and female serum. When coordinated with our previously published nonchromatographic procedure for the RIA of estrone and estradiol in serum, all 3 seteroids can be determined in the same specimen. Validation of the procedure was based on: 1. Agreement between results obtained using TLC and sultation to fractionate progesterone (r=0.98; b=0.86), 2. accurate recovery of different quantities of progesterone added to serum, 3. independence of the concentration of progesterone and volume of serum used for assay, 4. low procedural blanks (3.6 ± 1.3 pg), 5. low intraassay (9.7 – 10.3%) and interassay (11.0 – 11.6%) variability and 6. correspondence of observed values for progesterone in male serum (108 ± 20 pg/ml) and in female serum (follicular, 285 ± 149 pg/ml; luteal, 3.46±1.45 ng/ml) with those reported previously by others.     

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.     

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.     

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.     

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 radioimmunoassay for plasma corticosterone

A radioimmunoassay for plasma corticosterone has been developed. Antiserum against corticosterone was produced in rabbits immunized with corticosterone -21- hemisuccinate conjugated to bovine serum albumin. The assay was practical and reliable. The coefficient of variation between assays was ± 23% and among assays was ± 8%. Plasma corticosterone of mice is measured readily by assaying directly aliquots of a methylene chloride extract of 40 μl of plasma. The mean plasma corticosterone concentration of mice was similar to that obtained by other methods.     

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 sensitive radioimmunoassay for budesonide in plasma

Budesonide is a highly potent non-halogenated glucocorticoid with local anti-inflammatory properties. A sensitive radioimmunoassay for the measurement of the drug in unextracted plasma has been developed. Budesonide 21-hemisuccinate and budesonide 3-(O-carboxymethyl)oxime were conjugated to ovalbumin using the mixed anhydride method and antibodies to the haptens produced in sheep. The specificity of the antisera towards cortisol, and possible budesonide metabolites reflected the different sites of the attachment of the haptens to the carrier protein. An antiserum raised against the 3-(O-carboxy methyl)oxime conjugate was more specific (0.001% cross reaction) with respect to cortisol than the antiserum raised against the 21-hemisuccinate conjugate (0.344% cross reaction). Endogenous steroids at concentrations normally encountered in clinical samples, would not interfere with the measurement of budesonide.The radioimmunoassay was developed using the budesonide 3-(O-carboxy methyl)oxime antiserum, [³H]-budesonide and dextran coated charcoal phase separation. The theoretical limit of detection of the assay was 50 pg/ml. Budesonide was quantitatively recovered from normal human drug/free plasma at concentrations above 1 ng/ml (2.32nmol/l) with a between batch variation of 12–15%. Budesonide was measured in the unextracted plasma of volunteers who had inhaled the drug from a pressurised aerosol spray.     

A computerized semi-automated radioimmunoassay for plasma testosterone

A new computer program has been developed for the treatment of data from steroid radioimmunoassays. It uses an iterative technique that solves some 600 quadratic equations to compute the binding parameters for two independent, saturable binding agents from the standard curve data. The accuracy of curve fitting was such that all points on the standard curve were within one standard deviation of perfect fit, and no systematic bias was introduced into the results. Because the binding parameters were independent of the labelled ligand concentration, the methodology could be simplified, partially automated, and made more accurate and precise. The method was rapid, and 112 male plasma samples and 14 quality controls could be analysed (duplicate extracts on 40 μl aliquots with duplicate assays on each extract) by one person in one and a half days. The coefficient of variation between duplicate extracts of a reference plasma sample containing about 1000 ng testosterone/l00ml was 3.1% and the variation between assays on this sample conducted over a 3-month period was 3.8%.     

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.