Preparation and identification of monoclonal antibody against Citreoviridin and development of detection by Ic-ELISA

Abstract

Citreoviridin (CIT), a neurotoxic mycotoxin produced by Penicillium citreonigrum is generally detected in cereal grains and agricultural products worldwide, and has numerous toxicological effects on human and animal health. Therefore, it is necessary to develop a rapid, sensitive, and reliable immunoassay method for CIT. In this study, artificial antigen CIT-KLH and CIT-BSA was successfully prepared via succinic anhydride and carbodiimide two-step method. CIT-KLH conjugates were injected into Balb/c mice, and titer of the antiserum against CIT was determined using CIT-BSA as coating antigen by ELISA method. A hybridoma cell line 8D8 stably secreting monoclonal antibody against CIT was generated by fusing SP2/0 myeloma cells with the splenocytes from the immunized mice. The titer of 8D8 mAb reached 1: 1.28 × 105 after purified by caprylic/ammonium sulfate precipitation (CA–AS) method. The 8D8 mAb was identified as IgG1 subtype. The cross-reactivity results indicated that anti-CIT mAb was highly specific to Citreoviridin, and the average affinity 4.57 × 108 L/mol. A sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for CIT was established. Under optimal condition, the linear range to detect CIT was 11.02–2370.48 ng/mL with IC50 of 161.66 ng/mL and the limit of detection of the ic-ELISA was 11.86 ng/mL. With the mean coefficient of variation lowing 5%, the mean recovery in intra-assay and inter-assay were (90.06 ± 1.60)% and (89.65 ± 1.69)%, respectively. Therefore, the anti-CIT mAb secreted by 8D8 hybridoma cell line is useful for analysis of food contaminated with CIT.