Enzyme-linked immunoassay was applied to the determination of human chorionic gonadotropin (hCG), a glycoprotein hormone usually assayed with radioimmunoassay. For this purpose, enzyme hormone conjugate was prepared by reacting hCG with β-D-galactosidase (β-Gal.) of E. Coli in the presence of meta maleimidebenzoyl N-hydroxysuccinimide ester (MBS) as coupling reagent. The reaction mixture was then passed through a Con A-Sepharose 4B column to remove unreacted β-Gal. followed by the removal of intact hormone by means of gel filtration through Ultrogel AcA 44. The conjugate thus purified was shown to exhibit sufficient enzyme activity and immunoactivity. The immunoassay of hCG was performed by the double antibody method and using assay 0.4-250mIU/ml of hCG were detectable. This was about ten times as sensitive as radioimmunoassay.
However, difficulty was experienced when this method was utilized for the determination of hCG of plasma samples from patients. Since it was considered that the presence of the plasma affected this assay method, the following improvements were made; 1) the same volume of hormone free plasma was added to standard solutions of hCG, 2) volume of plasma sample was desirable as much as 10μl which is twenty times less than that for radioimmunoassay. The performance and validity of this assay were comparable to radioimmunoassay using 125I-hCG astracer. The dose/responce curves of both assays have the same slope and there was no significant differences between the values (correlation coefficient=Y=0.96X+1.53).
