Abstract: The neomycin (NEO) was coupled to Ovalbumin (OVA) to form coating antigen NEO-OVA by the EDC methods, determined by SDS-PAGE, and then indirect competitive ELISA (ic-ELISA) method was established; The NEO was coupled to horseradish peroxidase (HRP) by NaIO4 method，making enzyme tracer NEO-HRP and direct competitive ELISA . Then, the direct ELISA was developed and optimized in many parameters, such as coating liquids, pH value, incubation time , incubation temperature and so on. At last, the IC20 value (20% inhibitory concentration) was less than 1 ng/mL and IC50 value (50% inhibitory concentration) was 7.6 ng/mL . The formation is y = -0.2798x + 0.74 56, R2= 0.991. The direct ELISA could run in only about 1 h while the indirect assay duration was at least 1.75 h.

Key words: neomycin, indirect competitive enzyme-linked immunosorbent assay, direct competitive enzyme-linked immunosorbent assay