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.

Optimization of a sandwich sol particle immunoassay for human chorionic gonadotrophin.

Colloidal gold particles coated with rabbit anti-HCG (Au-(anti-HCG)) were used in a spectrophotometric 'sandwich' SPIA for HCG. The effects of several reagent properties and assay variables were investigated in order to optimize the assay. Best results were obtained with 55 nm particles. Optimum assay conditions involved a sample incubation period of 2 h followed by aspiration of the sample and two washings. A suitable Au-(anti-HCG) concentration corresponded to a value of A1cm540nm = 2.0. The steepest dose-response curves were obtained with an Au-(anti-HCG) incubation period of 16 h at 37 degrees C. The test procedure was suitable for preparing dose-response curves for HCG in buffer, urine and serum. The practical measuring range for HCG dissolved in these media was 30-250 IU/l. The within-run coefficient of variation varied between approx. 4 and 12%. The detection limit for HCG (in buffer) was about 1 IU/l.

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.

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.

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.