| This study aimed at immunological rapid determination of AMP residues in animal food, including the immunogenicity of AMP, synthesis of artificial antigen, preparation and special properties of AMP polyclonal antibody (AMP pAb), AMP monoclonal antibody(AMP mAb)and reagent kits.The main contents and experimental results of this study were as follows:Basis on analysis of molecular structure and immunogenicity of AMP, the carboxy group and amino group of AMP was used to conjugate AMP to bovine serum albumin(BSA) and albumin egg(OVA), and obtained artificial antigen(BSA-EDC-AMP), the coating antigen OVA-EDC-AMP was obtained respectively by EDC and GA, BSA-EDC-AMP and BSA-GA-AMP was synthesized successfully and its conjugation ratio of AMP to BSA was about 8:1 and 15:1 by identification with ultraviolet and SDS-PAGE, the high-titer, sensitive and specific AMP antiserum of Balb/C mice immunized with BSA-EDC-AMP and BSA-GA-AMP had been produced by identification with indirect ELISA. However, after detection of the two types of the artificial antigen by blocking ELISA, BSA-EDC-AMP is superior to the inhibitory effect of BSA-GA-AMP.New Zealand white rabbits were immunized with BSA-EDC-AMP and AMP pAb had been produced. The AMP pAb had the high titers of 1:6.4×105 by indirect ELISA, a good sensitivity with 50% inhibitive concentration (IC50) of 31.6μg/mL and 39.4% cross-reactivity (CR) to cloxacillin and below 0.5% CR to other compounds by blocking ELISA. The AMP pAb had a good specificity by experiment of agar bidirectional pervasion.One Balb/C mice were chosen from five Balb/C mice immunized with BSA-EDC-AMP for cell fusion by the titer of indirect ELISA and blocking ELISA. Four hybridoma lines of 1C8,2H6,3F9和3G7 that secrete AMP mAb were screened by indirect ELISA and blocking ELISA. The indirect ELISA titers of them were 1:6.4×102,1:3.2×102,1:1.28×103,1:3.2×102 in supernatant, 1:3.2×105,1:5.12×105,1:8.1×105,1:6.4×105 in ascites respectively, the isotype of them was IgG2a/λ,IgG2a/κ,IgG1/κ,IgG1/λand the affinity constant(Ka)was 4.49×108,5.16×108,2.06×109 and 9.81×108(L/mol)respectively. The best one of them was AMP mAb of 3F9 showed good sensitivity with an IC50 of 5.2 ng/mL to cloxacillin and 18.6% CR to cloxacillin and below 0.5% CR to other compounds by blocking ELISA. AMP pAb and AMP mAb obtained were both used to establish immunoassay of AMP residues in animal food, but AMP mAb was better than AMP pAb.A blocking ELISA kit for determination of AMP(AMP-Kit) were developed with AMP mAb of 3F9.The calibration curve of AMP-Kit with standard AMP inhibitor was typical sigmoid curve fitted to the four parameters logistic equation with the linear determination of 1.7 to 165.0 ng/mL, and the determination limit of 1.7 ng/mL.The recoveries of AMP spiked in milk were 101.3%, in pig urine were 102.4%, in pig feed were 106.7%.The precision and accuracy of the assay as determined by inter-assay and intra-assay coefficient variation were below 15%.The AMP-Kit generally had 21.1% CR to cloxacillin and below 0.5% CR to other compounds. The dilution solution of AMP had no effect on results of AMP-Kit. The validity of AMP-Kit in 4℃was above six months.
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