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 μ1 of ethanol solution containing 2.5 μg of the glycolipid antigen and 50 μg of taurodeoxycholate were added to each well of an EIA microtitration plate and dried at 37°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°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°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 GD_1a, GD_1b, and GT_1b. 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 (GM₂) serum cross-reacted with both N-acetyl and N-glycolyl types of hematoside and sialosylparagloboside.