摘要：
Penicillic acid is one of the main mycotoxins in moldy feedstuff and has toxic effect on livestock and poultry and probably humans due to food chain transmission. The objective of this study was to generate and characterize a monoclonal antibody to penicillic acid for the efficient detection of penicillic acid from Penicillium cyclopiumby immunological methods. To this end, penicillic acid was conjugated to bovine serum album (BSA) using the Mannich reaction and coupled with ovalbumin (OVA) by the method of 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochloride (EDC) to generate artificial antigens penicillic acid-BSA and penicillic acid-OVA. A hybridoma cell line was obtained after fusion of mouse SP2/0 myeloma cells with spleen cells of BALB/c mice immunized with artificial antigen penicillic acid-BSA. A monoclonal antibody specific against penicillic acid was produced in vivo by this hybridoma cell line. Further analysis revealed that the monoclonal antibody to penicillic acid was of the IgG1 subtype, with a titer of 1: 2.05 × 105. The antibody to penicillic acid had no or less cross-reaction with mycotoxins, including aflatoxin B1, zearalenone, T-2 toxin and fumonisins and more importantly, it assumed an affinity of about 1.54 × 108 liters per mol. Our ability to produce a monoclonal antibody to penicillic acid provides necessary groundwork for the effective detection of penicillic acid in various tissues of animals and human, using the immunocytochemistry, Western blots and enzyme-linked immunosorbent assay (ELISA).
Abbreviation
EDC, 1-Ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochloride; BSA, bovine serum albumin; OVA, ovalbumin; PEG, polyethylene glycol; FCS, fetal calf serum; PBS, phosphate buffer saline; UV, ultraviolet; ELISA,enzyme-linked immunosorbent assay; HRP, horseradish peroxidase; HT,hypoxanthine-thymidine; HAT, hypoxanthine, aminopterin, and thymidine; DMF,dimethylformamide; NHS, N-hydroxysuccinimide; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.
作者机构：
[Wang, Guiping; Yu, Xinglong; Hu, Shifeng] Hunan Agr Univ, Fac Vet, Changsha 410128, Hunan, Peoples R China.
通讯机构：
[Yu, Xinglong] H;Hunan Agr Univ, Fac Vet, Changsha 410128, Hunan, Peoples R China.
关键词：
avian influenza virus;latex agglutination test;matrix 1 protein
摘要：
The matrix 1 (M1) gene, present in all subtypes of avian influenza virus (AIV), was cloned and expressed in Escherichia coli. Reactivity of the expressed protein was confirmed by western blot. Subsequently, the M1 gene expression product was purified and used as the antigen to develop a latex agglutination test (LAT) for detecting antibodies against these conventional subtypes of AIV including AIV H3, H5, H7, and H9 from chicken sera. The LAT is specific for AIV, and no cross-reaction was shown with chicken antisera against other avian viruses. Compared with the hemagglutination inhibition test, the corresponding specificity, sensitivity, and correlation were 95.7%, 88.7%, and 89.0%, respectively, in detecting 491 serum samples from vaccinated chickens.
