Bauke K. Van Weemen

The influence of heterologous combinations of antiserum and enzyme-labeled estrogen on the characteristics of estrogen enzyme-immunoassays

Abstract The sensitivity of enzyme-immunoassays for estrogens strongly depends on the particular combination of antiserum and estrogen-enzyme conjugate being used. Homologous assays were relatively insensitive. Using different estrogen derivatives to prepare the immunogen and the estrogen-enzyme conjugate, more sensitive assays could be obtained, with little adverse effect on the specificity. The estrogen derivatives differed in type of estrogen, nature and position of the side chain. The general implications of these findings for hapten immunoassay are discussed.

A simple and efficient method for raising steroid antibodies in rabbits

The production of estradiol antibodies by two immunization procedures was monitored by means of an enzyme-immunoassay. Procedure A consisted of three intramuscular injections given at two-week intervals, followed by five intravenous booster injections and procedure B consisted of multiple intradermal injections given once. Procedure A gave much higher antibody titer. In both procedures the sensitivities of assays using the antisera increased initially and reached a plateau after three to four months of immunization. Consistent changes in specificities were observed. A shortened procedure A is proposed as a simple and efficient procedure for raising steroid antibodies in rabbits.

Specificities of antisera against estrogens linked to albumin at different positions (c6, c11, c16, c17)

Antisera were raised in rabbits using conjugates of albumin with 11-hemisuccinates of 11α-OH E1 and 11α-OH E2. These antisera were compared with antisera to 6-oxo E2 6-CMO-, E2 17-succinyl- and E3 16, 17-disuccinyl-BSA by radioimmunoassay using a statistically designed three-point assay. Sheep anti-(rabbit γ-globulin) coupled to cellulose was used for the separation of antibody-bound and free labeled hapten. Antisera obtained with haptens linked to the carrier at the 17(16) position poorly discriminated between the various estrogens. Antisera obtained with 6- and 11-conjugates showed a much better specificity. In addition the specificity was influenced by using either estrone, estradiol or estriol as tritiated labels. This gives the possibility to determine different parameters by employing different labeled hormones.

ELISA: Highlights of the present state of the art

Abstract ELISA is now very widely used. Many variations on the theme have been described. The search for further improvements is ongoing, mainly in the areas of sensitivity, specificity and practicability. Of course, the application of monoclonal antibodies is important in this respect. Next to that, further improvements in the detection of the enzyme label have come to the fore. Examples regarding the use of tetra-methylbenzidineas a peroxidase-substrate and details on its safety, a novel amplification method for detecting alkaline phosphatase and an enhanced luminescence method to detect peroxidase are given.

ENZYME-IMMUNOASSAY OF STEROIDS: POSSIBILITIES AND PITFALLS

Abstract Enzyme-immunoassay for steroids can be based on either a combination of insolubilized steroid and labelled antibody or a combination of labelled steroid and antibody. Only the latter possibility has been worked out. Two types can be distinguished: heterogeneous (in which a bound/free separation is required) and homogeneous. Heterogeneous assays have been developed for estrogens, progesterone, cortisol and testosterone. They can be equally sensitive, precise and accurate as radioimmunoassay, and are therefore useful alternatives for the endocrinological laboratory.

Enzyme Immunoassay of Hormones

Whereas radioimmunoassay (RIA) has been applied almost exclusively in endocrinology during the first 10 years after its development, enzyme immunoassay (EIA) has found immediate application in many disciplines. Only a few of the early EIA reports described assays for hormones : human chorionic gonadotrophin (HCG) [26], insulin [12], and oestrogens [27]. Subsequently, reports on EIAs for the following hormones have been published: Protein hormones: insulin [lo, 13, 15, 161, HCG [28, 311, human placental lactogen (HPL) [3, 4, 22, 251, and thyroid-stimulating hormone (TSH) [17]. Steroid hormones: oestrogens [4, 8, 19, 25, 29, 301, progesterone [7, 9, 121, cortisol [6, 201, testosterone [5, 231. Thyroid hormones: thyroxin (T4) [ l l , 241. All assays described were of the heterogeneous type, except those for T, [ l l , 241. Nearly all of the heterogeneous hormone EIAs were of the competitive type; sandwich assays were described for HCG [28] and HPL [22] and ‘immunoenzymonetric’ assays for HCG [28, 311. Various enzymes were used as labels: horseradish peroxidase (HRP) [3-5, 12, 15, 19, 22, 35-31], alkaline phosphatase [16, 17, 201, glucoamylase [ 10, 231, p-galactosidase [&9, 13, 141 and malate dehydrogenase [ l l , 241. In our opinion, the variation in hormones assayed, in assay techniques and in enzymes used demonstrates that EIA can in principle be used for measuring any hormone, although practical problems, such as the sensitivity of a given EIA system, may sometimes be limiting. In addition, a hormone EIA will not pose many methodological problems that are not encountered when using EIAs for other substances. The problems usually encountered will be of a general nature (for example, specificity, accuracy) and apply to all immunoassays rather than being typical of EIA. In this contribution we shall assess the present state of the art of EIA as a tool in endocrinology, drawing on our personal experience with EIA of HCG, HPL, oestrogens and testosterone, as well as on the literature. We shall refrain from dealing with homogeneous EIA, because of lack of personal experience with this type of EIA and because of its inherent limitations (for example, molecular weight of the substance to be assayed and, possibly, sensitivity).