Preparation of Monoclonal Antibody for Brevetoxin 1 and Development of Ic-ELISA and Colloidal Gold Strip to Detect Brevetoxin 1

• Published in: Toxins 2018-02, Vol.10 (2), p.75
• Main Authors: Ling, Sumei, Xiao, Shiwei, Xie, Chengjie, Wang, Rongzhi, Zeng, Linmao, Wang, Ke, Zhang, Danping, Li, Xiulan, Wang, Shihua
• Format: Article
• Language: English
• Subjects: Ammonium; Ammonium sulfate; Anhydrides; Animals; Antibodies, Monoclonal - immunology; Antigens; Antigens - analysis; Antigens - immunology; Ascites; Brevetoxin-1; Brevetoxins; Cell Line; Chloroformate; colloidal gold strip; Colloids; Cross-reactivity; Economic conditions; ELISA; Enzyme-linked immunosorbent assay; Enzyme-Linked Immunosorbent Assay - methods; Female; Food Contamination - analysis; Gold; Hybridomas; Immunization; Immunoassay; Immunoglobulin G; Marine organisms; Marine Toxins - analysis; Marine Toxins - immunology; Mice; Mice, Inbred BALB C; Monoclonal antibodies; monoclonal antibody; Myeloma; Octanoic acid; Ovalbumin - immunology; Oxocins - analysis; Oxocins - immunology; Seafood - analysis; Serum Albumin, Bovine - immunology; Shellfish; Shellfish - analysis; Spleen; Sulfates
• ISSN: 2072-6651; 2072-6651
• DOI: 10.3390/toxins10020075

Brevetoxin-1 (BTX-1), a marine toxin mostly produced by the dinoflagellatae , has caused the death of marine organisms and has had numerous toxicological effects on human health. Hence, it is very necessary to develop a rapid, economical, and reliable immunoassay method for BTX-1 detection. In this study, two kinds of complete antigen were synthesized using the succinic anhydride and isobutyl chloroformate two-step methods. Conjugate BTX-1-OVA was used as an antigen for mice immunization, and BTX-1-BSA for measuring the titer of the produced antibodies. A hybridoma cell line 6C6 stably secreting monoclonal antibody (mAb) against BTX-1 was obtained by fusing SP2/0 myeloma cells with the spleen cells from the immunized mouse. The hybridoma 6C6 was injected into the abdomen of BALB/c mice to obtain ascites, and the anti-BTX-1 mAb was harvested from ascites by precipitation with caprylic acid/ammonium sulfate (CA-AS). The anti-BTX-1 mAb was identified as an IgG1 subtype, and the cross-reactivity results showed that anti-BTX-1 mAb was highly specific to BTX-1 with the affinity of 1.06 × 10⁸ L/mol. The indirect competitive ELISA results indicated that the linear range for BTX-1 detection was 14-263 ng/mL with IC of 60 ng/mL, and a detection limit of 14 ng/mL. The average recovery rate from the spiked samples was 88 ± 2% in intra-assay and 89 ± 2% in inter-assay. The limit of detection (LOD) using the colloidal gold strip was 200 ng/mL with high specificity. Therefore, the anti-BTX-1 mAb can be used to detect BTX-1 in shellfish and other related samples.