Enzyme Immunoassay of Human Chorionic Gonadotropin Employing β-Galactosidase as Label

Abstract

An enzyme-linked immunoassay was applied to the determination of hCG, a glycoprotein hormone usually assayed by RIA. For this purpose, an enzyme hormone conjugate was prepared by reacting hCG with beta-D-galactosidase (beta-Gal.) of E. coli in the presence of N-(m-maleimidobenzoyloxy) succinimide (MBS) as coupling reagent. The conjugate, after purification by affinity and gel chromatographies, was shown to exhibit sufficient enzyme activity and immunoactivity. The immunoassay of hCG was performed by the double antibody method and, using this assay, 0.4-250 mIU/ml hCG were detectable. This was about 10 times as sensitive as the RIA. Difficulty was experienced when this method was utilized for the determination of hCG in plasma samples from patients. Since the presence of the plasma may have affected this assay method, the following improvements were made: 1) the same volume of hormone-free plasma was added to the standard solutions of hCG, and 2) the volume of plasma sample was 10 microliter. The performance and validity of this assay were comparable to the RIA using [125I]hCG as tracer. The dose-response curves of both assay have the same slope and there was no significant difference between the values (correlation coefficient, Y = 0.96X + 1.53).

Specific enzyme immunoassay for human chorionic gonadotrophin

“…The enzyme immunoassay for hCG we describe is as sensitive and accurate as the radioimmunoassay, and is able to measure a low level of hCG (6 mIU/ml) in serum with little cross-reaction with hUH and hFSH. Kikutani et al (1978) reported a competitive enzyme immunoassay for hCG using the ß-D-galac¬ tosidase for labelled hCG. Their assay was as sensi-tive as the present method, but not specific for hCG, since antiserum to the whole hCG molecule was used for the assay.…”

Measurement of Adult Hemoglobin in Fetal Blood Samples by High-Performance Liquid Chromatography as Purity Control for the Prenatal Diagnosis of Chromosomal Abnormalities

“…The automated determination of the mean red cell volume (MCV) and red cell distribu tion width (RDW) allows a quick comparison of fetal and maternal hematological indices [2,3], Other means include the KleihauerBetke test for F cells [4], measurements of the human chorionic gonadotropic hormone Psubunit [5] and of a-fetoprotein [6], identi fication of anti-I and anti-i cold agglutinins [7], and determination of the ratio of adult to acetylated fetal hemoglobins (HbA/HbFi) by isoelectric focusing [8]. However, none of them are entirely satisfactory either because of a lack of precision or because they take long to perform and do not allow an immediate second sampling when the first one is contam inated.…”