The reduction of nonspecific binding in chemiluminescent sandwich enzyme immunoassays

Abstract

The reduction in nonspecific binding obtained in EIA procedures based on glucose oxidase (GO)-labelled antibody is determined by the origin of the antibodies used for both the solid phase and for GO labelling. We have examined the relationship between nonspecific binding and various antibody combinations for the purpose of establishing highly sensitive chemiluminescent sandwich enzyme immunoassays. It was shown that nonspecific binding could be reduced by the following combination of solid phase and GO labelled antibodies: (i) guinea pig IgG-guinea pig IgG, (ii) goat IgG-rabbit IgG, (iii) goat IgG-rabbit Fab'. On the basis of these results, we succeeded in establishing highly sensitive chemiluminescent sandwich enzyme immunoassays for human chorionic gonadotropin (hCG) and alpha-fetoprotein (AFP). The detection limit was 2.5 mIU/ml for hCG using combination (i), 1 ng/ml for AFP using combination (ii), and 0.05 ng/ml (70 amol/assay) for AFP using combination (iii).