Characterization of a genetically segregating determinant of chicken fetal antigen by a new hapten inhibition of microcytotoxicity (HIM) assay.

The immunodominant structure of a chicken fetal antigen (CFA) determinant was investigated using a new hapten inhibition of microcytotoxicity (HIM) assay. This HIM assay employing avian erythrocytes was shown to be a highly sensitive, economical technique and was verified in a separate experiment using bovine serum albumin (BSA) coupled to Japanese quail peripheral red blood cells (QPRBCs). Inhibition of microcytotoxicity was measured following preincubation of 1 microliter of specific antiserum with 1 microliter of antigen solution. A concentration of 21.1 nM BSA was found to produce effective (50%) inhibition of microcytotoxicity of BSA-coated QPRBCs. The percentage cytotoxicity was determined by estimating the proportion of intact RBCs to free nuclei using an inverted microscope. Staining of the reaction mixtures was not required for scoring. Application of this technique for the characterization of immunodominant structures was demonstrated by the analysis of a CFA determinant known to exhibit both developmental expression and genetic segregation. R-anti-CFA-10 was effectively inhibited only by D-galactose (35.5 mM) and the galactose-containing sugars lactose (28.2 mM), raffinose (29.9 mM), and stachyose (39.8 mM). Implications of the carbohydrate nature of CFA are discussed.