The active group carboxyl was introduced to Chloramphenicol(CAP) and formed CAP half succinaate(CAP-HS) which has hapten structure by chemical modification. The method of mixed anhydride(MA) was used to conjugate CAP-HS to BSA and obtained artificial immunogen BSA-CAP-HS，the coating antigen OVA-CAP-HS was obtained in the same way. UV and SDS-PAGE were used to identify BSA-CAP-HS. BALB/C mice were immunized with BSA-CAP-HS, the titre of monoclonal antibody (mAb) was detected by indirect ELISA and blocking ELISA. The hybridoma lines that secrete CAP mAb were established with using hybridoma technology. The immunological traits such as titer, affinity, and specificity of the mAb were characterized. The results showed that CAP artificial immunogen was synthesized successfully and its the conjugation ratio of CAP-HS to BSA was about 15.6∶1. Three hybridoma cell lines of 1A10、2B5 and 4F9 were screened for specificity to CAP，the indirect ELISA titer of the mAb were 1∶6.4×102、1∶3.2×102、1∶1.28×103 in supernatant，1∶1.6×105, 1∶8×104, 1∶6.4×105 in ascites, the isotypes of the mAb were IgG2a/κ、IgG2a/λ、IgG1/κ and the affinity constant（Ka） was 2.14×108, 1.68×109, 1.84×1010(L/mol) respectively，the mAb of 4F9 showed good sensitivity with an IC50 of 7.54ng/ml to CAP，specificity integrated with BSA-CAP-HS that determined by WestGold assay，150% cross-reactivity to CAP succinate sodium and little or no cress-reactivity to other compounds. CAP mAb of high-titer, sensitivity and specificity had been generated and made it possible to establish immunoassay of CAP residues in feed and animal food.
