4-Nitrophenethylamine was chemically linked to bovine serum albumin (BSA) or ovalbumin (OVA) by coupling with BSA through glutaraldehyde to form immunizing antigen and coating antigen. The complete antigen was determined by UV spectrometry and MALDI-TOF MS scanning method. Polyclonal antibodies against 4-nitrophenethylamine were produced from rabbits, and the antiserum was determined with an ELISA test, which showed that the titer of the antibodies was 1∶32000. The most appropriate concentration of coating anti¬gen was 0.5 mg/L, and that of antiserum was 1∶6000. Indirect competitive ELISA (icELISA) was established with this antiserum. The curve had a favorable linear relation within the concentration range of 1～1000 μg/L. The curve indicated that IC₅₀ was (52.73±2.67) μg/L, the lowest detection limit was 5.12 μg/L. No analogues of nitroaniline was found to interfere with the analysis of nitroaniline. Thus, the indirect competitive enzyme immuno-chemistry of nitroaniline was developed successfully.

Key words: nitroaniline, complete antigen, polyclonal antibody, enzyme linked immunosorbent assay (ELISA)