A. H. W. M. Schuurs

Immunoassay using antigen—enzyme conjugates

Most of the enzyme-immunoassays described in the literature employ enzyme-labelled antigen or hapten [l-8]. A few use enzyme-labelled anti-antibody (anti-IgG*) [8,9]. Enzyme-labelled antibodies against the substance to be determined, provide a third tool for enzyme-immunoassay. In this report we describe two methods for the assay of human chorionic gonadotrophin (HCG), which employ anti-HCG labelled with horse-radish peroxidase (HRP). The methods are analogous to the ‘immunoradiometric’ [lo] and the ‘sandwich’ [ 1 I] radioimmunoassays. Their sensitivities and the economy of reagents are compared with those of the Dasp enzyme-immunoassay for HCG, which employs HRPlabelled HCG. The latter method has been shown to be markedly more sensitive than the corresponding solid-phase assay [ 11.

Enzyme-Immunoassay: A Powerful Analytical Tool

This paper reviews various aspects of enzyme-immunoassay (EIA). Firstly it summarizes the principles of tests in which use is made of labelled antigen or antibody. Since these constitute essential reagents in EIA they are discussed next. Many assay principles call for a bound/free separation. The various methods to accomplish this are therefore briefly discussed. Very important are the characteristics of EIA: specificity, sensitivity, precision and practicability, the latter including reagent stability, performance requirements, assay times and automation potential. Finally, fields of application are listed and some more recent developments are mentioned.

Enzyme immunoassay for total oestrogens in pregnancy plasma or serum.

We have developed an enzyme immunoassay for total oestrogens in pregnancy serum of plasma, using horseradish peroxidase (HRP) as the marker enzyme. A test combination consisting of an antiserum against oestriol-16/17-monosuccinyl-albumin and oestriol-16/17-monosuccinyl-HRP yielded a sensitive system, which reacted to approximately the same extent with oestrone, oestradiol, oestriol and their 16- and 17-conjugates. Samples had to be diluted 1 to 10 to avoid interference of plasma factors with the immune reaction. Bound/free separation was achieved with the double antibody solid phase (DASP) method. The HRP activity of the bound fraction was measured, after washing, to eliminate plasma factors disturbing the HRP reaction. The detection limit of the assay system was approx. 0.1 pmol/tube, while the index of precision lambda ranged from 0.02 to 0.06. To measure total oestrogens, including the 3-conjugated ones, we used an enzymatic hydrolysis with an extract of Helix pomatia. Hydrolysis was found to be optimal after 1 h at 50 degrees C and pH 5.0. The method was used on serum samples from normal pregnancies. The results showed a very good correlation (r=0.98) with those obtained by radioimmunoassay. Normal values for total oestrogens during pregnancy were determined in a multicentre clinical trial.

Enzyme-immunoassay of human placental lactogen

An enzyme-immunoassay (EIA) for estimation of human placental lactogen (HPL) in plasma or serum was developed using HPL labelled with horseradish peroxidase (HRP) and anti-HPL sera raised in rabbits. The separation between antiserum-bound and free labelled hormone was accomplished with a double antibody solid phase technique. Interference of substances from the sample with the immunoassay was prevented by dilution of the sample with at least a factor of 20 and measurement of the labelled hormone attached to the solid phase. The peroxidase activity was colorimetrically measured using o-phenyl-enediamine and urea peroxide as substrate. The standard curve of the assay ranged from 3 to 40 ng HPL/ml allowing estimations of HPL in serum or plasma starting from about the 10th week of pregnancy. Normal values were established. Intra- and inter-assay variation coefficients of 6 and 7.5% respectively were found. The EIA showed no cross-reaction with human serum proteins or HCG. The low cross-reaction noted with human growth hormone did not interfere with the assay. An excellent agreement was found with the results of radioimmunoassay (RAI).

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).

Characteristics of Monoclonal Antibodies Against Human Chorionic Gonadotrophin

Abstract Various hybridomas producing antibodies against HCG were tested in five enzyme-immunoassay (EIA) systems. On the basis of their specificity in these systems the hybridomas were divided in three major groups: α (reacting with HCGα),; (reacting with HCGβ) and αβ (reacting with intact HCG but not with either subunit). The EIA systems were also used to check homogeneity of the hybridomas. Antibodies from group αβ were found to be highly specific for HCG. Combinations of these specific antibodies and those from group β led to a highly sensitive and specific EIA and gave also optimal results in agglutination tests using either erythrocytes or gold sol particles coated with monoclonal anti-HCG. The practical use of monoclonal anti-HCG was assessed by comparing these antibodies with conventional rabbit anti-HCG in a pregnancy test based on the haemagglutination inhibition principle. Monoclonal and rabbit anti-HCG gave similar results in the study which included some thousand urine samples. Most clones produced antibodies of the IgG 1 subclass, while affinity constants in the order of 10 9 1/mol were found.

The use of Monoclonal Antibodies Against Prolactin in a Solid Phase Enzyme-Immunoassay

Abstract Spleen cells from mice immunized with crude Prl (4% pure) were used for cell fusion and production of monoclonal antibodies (MCAs) against Prl. The MCAs were characterized and used in a solid phase EIA. Affinity constants of four MCAs, as determined by radio-immunoassay (RIA), were in the order of 10 7 l/mol, against that of a rabbit anti-Prl serum of 10 9 l/mol. Using these four MCAs we obtained detection limits of about 200 ng Prl/ml in competitive RIAs, and of 1 ng Prl/ml in sandwich ElAs.