G.F. Read

SOME ASPECTS OF THE USE OF 125I-LABELLED LIGANDS FOR STEROID RADIOIMMUNOASSAY

Abstract Steroid-[ 125 I]-iodohistamine radioligands were synthesized and used to investigate a number of potential radioimmunoassay systems for the assay of oestradiol, progesterone, testosterone, norethisterone, norethisterone acetate, D -norgestrel, testosterone- 17β) -glucuronoside, androsterone-3α-glucuronoside and aetiocholanolone-3α-glucuronoside. Viable systems were predicted for example using anti-oestradiol-6-(O-carboxymethyl) imino-BSA with either oestradiol-3-hemisuccinyl-[ 125 I]-iodohistamine or oestradiol17β-hemisuccinyl-[ 125 I]-iodohistamine radioligands. Similarly, the free 4-en-3-oxosteroids could be assayed using anti-steroid-3- (O-carboxymethyl) imino-BSA sera with a steroid-3- (O-carboxymethyl)-imino-[ 125 I]-iodohistamine (steroid-3- 125 I) radioligand (homologous system), or antisteroid-11α-hemisuccinyl-BSA sera with a steroid-3- 125 I radioligand (heterologous system). All such systems are less specific than those employing a 3 H-labelled tracer but certain homologous systems using rare antisera are more specific still and therefore retain the advantages of using a radio-iodine label. The radio-iodine labelled ligands may also be used to investigate the nature, size and shape of antibody binding sites. For example, the occurrence of binding of a particular 125 I-labelled radioligand to an antiserum indicates that the serum cannot “see” that part of the molecule bearing the radio-iodine labelled side-chain. Absence of binding conversely may indicate that the position of attachment of the label is involved in the binding of the unlabelled steroid.

Some observations on the determination op cortisol in human plasma by radioimmunoassay using antisera against cortisol-3-BSA

Antisera were obtained from rabbits immunised against cortisol-3-BSA with a view to examining their application in a radioimmunoassay of the steroid. One such antiserum was studied in detail; cross-reactivity with other C21 steroids normally present in human plasma was negligible and it proved possible to establish a radioimmunoassay which satisfied all criteria of reliability. The specificity of the cortisol determination achieved in human plasma was examined by performing measurements with and without including an initial Sephadex LH-20 column chromatographic purification step; the values obtained were in excellent agreement both for normal plasma and for that obtained following adrenal stimulation with ACTH.

Evaluation of luteal-phase salivary progesterone levels in women with benign breast disease or primary breast cancer.

Salivary progesterone concentrations were determined in premenopausal parous women with a mean age of ca. 40 yr who had a history of either benign breast disease (n = 15) or primary breast cancer (n = 15) and in a group of age-matched healthy women (n = 15). Saliva samples were collected at 09.00 and 21.00 hr daily for one complete menstrual cycle and progesterone concentration was measured by radioimmunoassay. Characteristic luteal-phase progesterone profiles were observed in all subjects in each of the three groups but no statistical intergroup differences could be demonstrated for age-matched subjects in each group. These studies indicated that ovarian dysfunction, as judged from salivary progesterone concentrations, was not apparent in older premenopausal women with a history of benign breast disease or primary breast cancer when compared with age-matched controls.

A radioimmunoassay for ethinyl oestradiol in plasma incorporating an immunosorbent, pre-assay purification procedure

A radioimmunoassay for plasma ethinyl oestradiol, featuring an immunosorbent extraction procedure, is described. Ethinyl oestradiol (EE2) was extracted using a non-specific, anti-oestrogen serum, raised to an oestradiol-17-hemisuccinate conjugate. The antiserum, coupled to microcrystalline cellulose, selectively extracted EE2 but not norethisterone (NE), thus conferring specificity on a radioimmunoassay which has previously exhibited unacceptably high cross-reactivity with the synthetic progestagen, norethisterone, often used concomitantly with ethinyl oestradiol. This radioimmunoassay was shown to fulfil accepted assay validation criteria. Levels in subjects not receiving EE2 were less than 25 pmol/l. Circulating concentrations of EE2 could therefore be accurately determined in patients receiving low-dose combined preparations (EE2 35 μg; NE 500 μg).

A Simple Robust Assay for Testosterone in Male Plasma Using an 125I-Radioligand and a Solid-Phase Separation Technique

A radioimmunoassay for testosterone in male plasma utilising a gamma-emitting radioligand and a solid-phase antiserum is described. The radioligand is testosterone-3-(O-carboxymethyl)-oxime coupled to 125I-iodohistamine, and the solid-phase antiserum is prepared by coupling antitestosterone-3-bovine serum albumin to cyanogen bromide activated cellulose. The new procedure retains much of the specificity associated with a published, specific radioimmunoassay using an antiserum raised against testosterone-11α-BSA and a tritium radioligand and incorporating a dextran-coated charcoal separation procedure; values obtained by the two procedures are in excellent agreement (r = 0·98, n = 20). The combination of an 125I-radioligand and a solid-phase separation technique greatly increases sample throughput and has the further advantage of reduced running costs and a greater potential for automation. The method gives satisfactory levels of sensitivity, precision, and accuracy.

Determination of Cortisol in Human Plasma by Radioimmunoassay Use of the 125I-labelled Radioligand

A radioimmunoassay for plasma cortisol featuring the gamma-emitting radioligand 125I-iodohistamine, coupled to cortisol-3-(O-carboxymethyl)-oxime, is described. The new procedure retains much of the specificity associated with the use of anti-cortisol-3-BSA sera with tritium-labelled radioligands, and has the further advantages that running costs are lower and there is a greater potential for automation. Cortisol values obtained by this procedure agree well with those obtained by a published specific radioimmunoassay using the tritiated cortisol radioligand. Specificity of the procedure was checked by comparing values obtained with and without thin-layer chromatography purification: correlation was excellent (r = 0·96). Satisfactory levels of sensitivity, precision, and accuracy were obtained.

A sensitive solid phase enzymeimmunoassay for norethisterone (norethindrone) in saliva and plasma.

A sensitive, solid phase enzymeimmunoassay suitable for determining norethisterone in small aliquots of plasma (10 microliters) and saliva (100 microliters) has been developed. A solid phase antiserum raised against a norethisterone-11 alpha-hemisuccinyl/bovine serum albumin conjugate was prepared by coupling to cyanogen bromide activated cellulose. A norethisterone/horseradish peroxidase conjugate was used as enzyme label, o-phenylenediamine/hydrogen peroxide being the substrate for colour development. The assay had a lower limit of sensitivity of 3 pg/assay tube and satisfied accepted validation criteria. Norethisterone concentrations determined by enzymeimmunoassay and by a well established radioimmunoassay were in excellent agreement in both plasma (r = 0.993, n = 20) and saliva (r = 0.989, n = 15). Plasma and salivary norethisterone concentrations determined in healthy volunteers reached peak values at about 1 hour after administering a norethisterone-containing oral contraceptive preparation. The maximum values achieved in saliva (775--1430 pmol/l) were only approximately 3% of those observed in plama. Since salivary norethisterone concentrations reflected those in plasma, they may be useful in fertility control programmes and pharmacokinetic studies.

The use of multivariable standard curves in the radioimmunoassay of testosterone and 5α-dihydrotestosterone

Multivariable calibration curves have been used to enable testosterone and 5alpha-dihydrostestosterone to be assayed directly in plasma extracts without further pre-purification of the sample. Two antisera were used, both with relatively high, but different affinities for the substances measured, and with relatively low affinity towards all other substances tested. The antisera were obtained from rabbits immunized against testosterone-3-BSA and 5alpha-dihydrostestosterone-3-BSA. The technique was of adequate precision, accuracy and specificity. The last was examined by comparison of values obtained by the present method and those obtained following prepurification by thin layer chromatography.

Improvement of antisera for cortisol immunoassay by reduction of endogenous hormone concentrations

Markedly elevated concentrations of endogenous steroids in antisera may reduce the potential sensitivity of immunoassays. Attempts to raise antisera containing reduced amounts of cortisol by adrenalectomy, or concomitant administration of synthetic corticosteroid during immunisation, have met with indifferent success. Removal of cortisol, using dextran-coated charcoal in buffer of optimum molarity, may increase the average affinity constant of the antiserum, and improve the assay sensitivity and specificity.

A simple direct solid-phase enzymeimmunoassay for cortisol in plasma

A simple, direct solid-phase enzyme-labelled immunoassay for plasma cortisol was established using horseradish peroxidase/cortisol 21-hemisuccinate conjugate as ‘enzyme label’. The antiserum, raised against a cortisol 21-hemisuccinate/bovine serum albumin conjugate, was coupled to cellulose to facilitate separation of free and bound steroid. Solvent extraction was avoided by the use of heat denaturation of the cortisol-binding globulin. This assay had a lower limit of sensitivity of 16·6 nmol/l and satisfied the standard criteria of accuracy and precision. Cortisol concentrations determined by enzymeimmunoassay were in excellent agreement with a gas liquid chromatography/mass spectrometry procedure (r = 0·98, n = 19) and also with the radioimmunoassay in current use (r = 0·95, n = 20). Cortisol levels after ACTH stimulation and dexamethasone suppression in various subjects are presented. This enzymeimmunoassay is particularly applicable to the routine determination of plasma cortisol in small clinical laboratories or in those with a fluctuating workload.