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.
摘要：
Very little information is known about the toxic effects of cadmium on somatic cells in mammalian testis. The objective of this study is to explore the toxicity of cadmium on piglet Sertoli cells. Sertoli cells were isolated from piglet testes using a two-step enzyme digestion and followed by differential plating. Piglet Sertoli cells were identified by oil red O staining and Fas ligand (FasL) expression as assayed by immunocytochemistry and expression of transferrin and androgen binding protein by RT-PCR. Sertoli cells were cultured in DMEM/F12 supplemented with 10% fetal calf serum in the absence or presence of various concentrations of cadmium chloride, or treatment with p38 MAPK inhibitor SB202190 and with cadmium chloride exposure. Apoptotic cells in seminiferous tubules of piglets were also performed using TUNEL assay in vivo. Cadmium chloride inhibited the proliferation of Piglet Sertoli cells as shown by MTT assay, and it increased malondialdehyde (MDA) but reduced superoxide dismutase (SOD) and Glutathione peroxidase (GSH-Px) activity. Inhibitor SB202190 alleviated the proliferation inhibition of cadmium on piglet Sertoli cells. Comet assay revealed that cadmium chloride caused DNA damage of Piglet Sertoli cells and resulted in cell apoptosis as assayed by flow cytometry. The in vivo study confirmed that cadmium induced cell apoptosis in seminiferous tubules of piglets. Transmission electronic microscopy showed abnormal and apoptotic ultrastructure in Piglet Sertoli cells treated with cadmium chloride compared to the control. cadmium has obvious adverse effects on the proliferation of piglet Sertoli cells and causes their DNA damage, cell apoptosis, and aberrant morphology. This study thus offers novel insights into the toxicology of cadmium on male reproduction.
