Abstract: In the present study, a sandwich enzyme-linked immunosorbent assay (ELISA) for 3-amino-2-oxazolidnone (AOZ) was developed. A novel derivatizing agent for AOZ was synthesized by linking 2-nitro-4-carboxybenzaldehyde and biotin through 1,6-hexanediol. During the sample pretreatment, the agent was added for AOZ derivatization with an efficiency of 89%. The monoclonal antibody (McAb) against AOZ was coated on the microplate and horseradish peroxidase-labeled avidin or anti-biotin antibody was used as a secondary conjugate in the ELISA method. From a practical perspective, the limit distances between the two epitopes in the double-antibody sandwich and antibody-avidin sandwich modes were 12 and 13 Å, respectively, and the ideal distances were 16 and 17 Å, respectively. The detection limits of the double-antibody sandwich and antibody-avidin sandwich modes were 1.8 and 0.8 pg/mL for AOZ, respectively. Compared with competitive ELISA, a 25-fold improvement in the sensitivity of the developed ELISA method was achieved. The average recoveries and average relative standard deviations (RSDs) were 73%–85% and 9.0%, respectively.

Key words: 3-amino-2-oxazolidinone; sandwich immunoassay; epitope spacing distance; horseradish peroxidase-labeled avidin