Avian antisera to various gangliosides: detection by enzyme immunoassay

We attempted to produce specific antibodies to various gangliosides by immunizing chickens. Antibody activity was determined by enzyme immunoassay (EIA). The optimal conditions for EIA were examined by using chicken anti-GM1 ganglioside (GM1) serum and the final procedure was as follows. Fifty microliters of ethanol solution containing 2.5 micrograms of the glycolipid antigen and 50 micrograms of taurodeoxycholate were added to each well of an EIA microtitration plate and dried at 37 degrees C for 2 h. Nonspecific sites were blocked by incubation with 1% gelatin-containing buffer, then titration of the chicken antisera was carried out in the antigen-coated plate by incubation at 4 degrees C for 12 h. Next, alkaline phosphatase-labeled anti-chicken IgG (specific antibody, 15 ng; enzyme, 0.054 units) was allowed to react at 37 degrees C for 2 h. The enzyme activity which was bound to the plate was assayed with p-nitrophenol phosphate as substrate. Under the above conditions, anti-GM1 serum reacted strongly with GM1 and asialo GM1, and anti-GM2 serum indicated a considerable specificity for GM2. However, we failed to elicit any antibody to GD1a, GD1b, and GT1b. Anti-NeuAc-sialosylparagloboside and anti-NeuGc-sialosylparagloboside sera showed a high specificity for the homologous ganglioside. However, anti-NeuGc-hematoside serum reacted equally with both the homologous antigen and NeuGc-sialosylparagloboside, and anti-NeuAc-hematoside (GM3) serum cross-reacted with both N-acetyl and N-glycolyl types of hematoside and sialosylparagloboside.