A.H.W.M. Schuurs

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.

Sol Particle Immunoassay (SPIA)

We describe the use of inorganic (metal) colloidal particles as a label for immunoassays. Dose-response curves for human placental lactogen (HPL) and human chorionic gonadotrophin (HCG) were obtained with sandwich immunoassays, using conjugates consisting of antibody-coated colloidal gold or silver particles. Several techniques were used to measure the amount of bound conjugate, viz. colorimetry and carbon rod atomic absorption spectrophotometry (CRAAS). At higher antigen concentrations the results of the assay could be read by the naked eye. Using gold particles as label and CRAAS as detection method, we found a detection limit for a sandwich HPL sol particle immunoassay (SPIA) of 1,4 pmol/l, which was equal to that of an optimalized competitive radioimmunoassay. When using a colorimeter the detection limit for HPL of this SPIA was 5,4 pmol/l, which was superior to that of a corresponding sandwich enzyme-immunoassay (EIA). HPL and HCG were also simultaneously determined, using microtitration plates, coated with a mixture of anti-HPL and anti-HCG, and a mixture of silver particle anti-HPL conjugate and gold particle anti-HCG conjugate. CRAAS was used to measure the bound amount of silver and gold conjugate. This simultaneous assay requires more work in order to obtain better sensitivities.

A sol particle agglutination assay for human chorionic gonadotrophin

Conjugates, prepared by adsorption of antibodies on colloidal gold particles, were used in a homogeneous sol particle immunoassay (SPIA) for human chorionic gonadotrophin (HCG). The technique is based on sol particle agglutination, resulting in colour reduction. Optimal results were obtained using buffered conjugates prepared from 50 nm particles. Addition of polyethylene glycol (PEG) 6 000 to the conjugates, up to 15 g/l, increased the agglutination rate considerably. The optimal PEG 6 000 concentration of the conjugate depended on the desired incubation time and measuring range. The influence of temperature on the agglutination was negligible in the temperature range between 4 and 45 degrees C. Higher conjugate concentrations resulted in steeper dose-response curves. However, the measuring range (between 62.5 and 2 000 IU/l HCG) and the detection limit (approx. 50 IU/l HCG) were about the same. The dose-response curves for HCG dissolved in buffer or in urine were almost identical and their reproducibility was satisfactory. In our experience, homogeneous SPIAs have a high practicability, are easy to automate and provide an interesting new tool for the measurement of a variety of analytes.

A homogeneous sol particle immunoassay for human chorionic gonadotrophin using monoclonal antibodies

A mixture of monoclonal antibodies against human chorionic gonadotrophin (HCG) from 2 different clones was used to develop a homogeneous sol particle immunoassay (SPIA) for HCG, with high discrimination against luteinizing hormone (LH). The optimization of this assay is described. The work resulted in a spectrophotometric and a visual reading version. In an evaluation of the tests with 348 urines from pregnant women, 530 urines of non-pregnant women of fertile age and 100 post-menopausal women, the spectrophotometric screening test was 100% correct in the groups of urines from pregnant women and from post-menopausal women, and 99.8% correct in the group of urines from non-pregnant women of fertile age. This spectrophotometric screening test required an incubation period of 1 h and detected 280 IU/1 HCG. The eye reading test required an incubation period of 2 h and was able to detect about 450 IU/1 HCG. This test showed 100% correct results in the group of urines from non-pregnant women and from post-menopausal women, and 99.1% correct results in the group of pregnant women. In both test versions HCG concentrations up to 200,000 IU/1 did not result in false negative reactions. Urines of non-pregnant women, with an added 1000 IU/1 LH, gave correct negative test results.

Radioimmunoassays for human gonadotrophins and insulin employing a ‘double-antibody solid-phase’ technique

Abstract Radioimmunoassays for urinary gonadotrophins and insulin were developed utilizing second-antibody immunosorbents to separate bound and free labelled hormone. By using these immunosorbents the versatility of the double-antibody procedure was combined with the ease and speed of handling of the solid-phase methods. The immunosorbents were prepared by coupling antibodies raised in sheep against rabbit or guinea pig γ-globulins (a-RGG or a-GPGG, respectively) to agarose and cellulose. Solid-phase radioimmunoassays for rabbit and guinea pig γ-globulin (RGG and GPGG, respectively) were used for studying the properties of these immunosorbents. The properties and applicability of a-RGG-immunosorbents were investigated in radioimmunoassays for HCG/LH and FSH. The effect of different incubation times, of varying the concentration of the first antibody, and of second-antibody immunosorbents on the percentage of labelled hormone precipitated was studied. The applicability of a-GPGG-immunosorbents was investigated in an insulin assay system. The specificity of the assays, which utilize this bound/free separation method, is described.

A homogeneous sol particle immunoassay for total oestrogens in urine and serum samples

Conjugates prepared by adsorption of antibodies to colloidal gold particles were used in a homogeneous sol particle immunoassay for total oestrogens. The assay is based on inhibition by free oestrogens in the sample of the agglutination of a reaction mixture, consisting of the sample, the gold particle anti-oestriol conjugate and a suitable amount of oestriol-16/17-monosuccinyl-bovine serum albumin. The effect of different assay conditions is discussed. Under optimum conditions for an assay performed at room temperature the measuring range for E3 in buffer was 2-10 ng/ml. After appropriate pretreatment of samples the assay can be used to quantitate the level of total oestrogens in both urine and serum. The within-run coefficient of variation was 6.3% and over-all 6.9%. This method is also suitable for the assay of other haptens.

Effects of steroids with different endocrine profiles on the development, morphology and function of the bursa of Fabricius in chickens

This paper describes the effect of various steroids on the anlage of the bursa of Fabricius in chickens. The steroids were administered by dipping embryonated eggs on the third day of incubation in ethanolic solutions of these steroids. The results with about 30 different steroids show that the capacity to inhibit the development of the bursa does not correlate with the endocrine properties of these steroids as measured in routine screening tests for androgenic, anabolic, progestational and oestrogenic activities or with the relative binding affinities for various endocrine receptors. More elaborate studies with several representative steroids show that testosterone (10 mg/ml), nandrolone (10 mg/ml), 11 alpha-hydroxynandrolone (10 mg/ml), ethylestrenol (1 mg/ml), lynestrenol (1 mg/ml), and Org OD14 [tibolone] (0.1 mg/ml) induce also histomorphological changes in the remaining bursa tissue still present in 10 day- and 53-day old chickens and in the bursa-dependent sites of their spleens (53-day old chickens only). Testosterone and lynestrenol induced smaller changes than nandrolone or ethylestrenol. Tibolone and 11 alpha-hydroxynandrolone were more effective than nandrolone. All drugs, except testosterone and lynestrenol, imparied antibody formation to Newcastle Disease Virus and decreased the serum levels of total IgG, but not of total IgM. Also these effects were not correlated with endocrine properties. In other studies (for references, see text) we found that several of these steroids, notably tibolone, favourably influence the course of spontaneous autoimmune diseases of NZB/W mice and Obese Strain chickens. Since this autoimmunosuppression is likely to be caused by inhibitory effects on bursa or bursa equivalent, we may use this approach for developing medically useful autoimmunosuppressive steroids with minimal endocrine effects.

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.

Effects of tibolone, lynestrenol, ethylestrenol, and desogestrel on autoimmune disorders in NZB/W mice.

The effects of two progestagens--lynestrenol, desogestrel--an anabolic steroid--ethylestrenol--and a compound with weak progestational, anabolic, androgenic, and estrogenic activities--tibolone--on the development of systemic lupus erythematosus and Sjögrens syndrome-like disorders were studied in the NZB/W mouse. All four compounds inhibited the expression of autoimmune disease. Tibolone was 10-40 times more potent--depending on the parameter used--in preventing symptoms of autoimmunity than the second most effective compound lynestrenol. Ethylestrenol was the third effective compound and desogestrel the least effective compound in this series. Combined with literature data, these results show that steroids with different endocrine profiles can prevent the development of autoimmunity in the NZB/W mice. Since the NZB/W mouse is a good animal model for human systemic lupus erythematosus and Sjögrens syndrome and since tibolone, lynestrenol, and ethylestrenol have endocrinological profiles which are not prohibitive for treatment of male and female patients, investigation whether these compounds have a value in the treatment of human autoimmune diseases seems warranted.

Effects of nandrolone decanoate or testosterone decanoate on murine lupus: further evidence for a dissociation of autoimmunosuppressive and endocrine effects.

Nandrolone decanoate injections (20, 40 or 80 mg/kg/3 weeks) in female or castrated male New Zealand black/white mice, starting at various ages (4, 9, 17 or 26 weeks) prolong survival, reduce proteinuria and affect the weights of various endocrine and non-endocrine organs. Nandrolone decanoate is at least equally potent as testosterone decanoate with respect to its beneficial effects on lupus-associated symptoms; in contrast, its effects on some other androgen-sensitive endocrine parameters are significantly less. These observations show that the autoimmunosuppressive effects of steroids are not quantitatively correlated to endocrine properties.