| Benzylmalonic acid (BA) and 3-(4-Hydroxyphenyl) propionic acid (34-HPA) selected as the hapten of BA and 4-MP, was coupled with bovine serum albumin (BSA) in activation ester method to prepare complete antigen BA-BSA and 34HPA-BSA which was used to immunize 3~4 months old rabbits and get the polyclonal antibodies against BA and 4-MP. After the antibodies were purified by the sedimentation of saturation ammonium sulfate, the titers were characterized in the way of indirect enzyme-linked immunosorbent assay (i-ELISA) which reached 1:102400, 1:204800. Indirect competitive ELISA (ic-ELISA) was developed using antigen coupled with ovalbmin (OVA) in mixed anhydride method as the original coating and BA, 4-MP as competitive antigen. The results of BA showed the best detection conditions for ic-ELISA: the coating antigen BA-OVA concentration was 3.46μg/mL; the antibodies was 6400 diluted; 0.1% gelatin solution blocked 1.0 h; competitive time was 1.0 h; horse radish peroxidase (HRP) labeled goat anti-rabbit IgG was diluted 2000 times and incubated 1.5 h. The linear range of ic-ELISA to detect BA was 1.0×10-2 ng/mL~1.0×103 ng/mL and the regression equation is y = -13.59x + 63.58. The detection limit of the assay was 0.01 ng/mL, the intra-assay and inter-assay coefficient of standard variation is 5.5%, 3.8%~7.8%, the recovery is 87.16%~111.43%. The results of 4-MP showed the best detection conditions for ic-ELISA: the coating antigen 34HPA-OVA concentration was 1.05μg/mL; the antibodies was 12800 diluted; horse radish peroxidase (HRP) labeled goat anti-rabbit IgG was diluted 1000 times; 0.1% gelatin solution blocked 1.5 h; competitive time and incubated time of antibody and HRP-IgG was 1.0 h. The linear range of ic-ELISA to detect 4-MP was 1.0×10-2 ng/mL~1.0×104 ng/mL and the regression equation is y = -9.524x + 64.48. The detection limit of the assay was 0.002 ng/mL, the intra-assay and inter-assay coefficient of standard variation is 6.3%, 4.3%~6.7%, the recovery is 87.52%~110.50%. |
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