No abstract

Brevetoxin-1 (BTX-1), a marine toxin mostly produced by the dinoflagellatae Karenia brevis, 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 IC50 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.

Podoplanin (PDPN), a 38 kDa transmembrane sialoglycoprotein from human, is expressed in lymphatic endothelial cells but not in vascular endothelial cells, and has been considered as a specific marker of lymph. In this study, the gene encoding the extracellular part of PDPN (ePDPN) was synthesized and used to expressed fusion protein ePDPN-His and GST-ePDPN, respectively, in E.coli. The purified GST-ePDPN fusion protein was mixed with QuickAntibody-Mouse5W adjuvant to immune mice, and the antiserum titer was determined by indirect ELISA. A stable cell line named 5B3 generating anti-PDPN monoclonal antibody (mAb) was obtained by hybridoma technology. The isotype of 5B3 cell line was IgG2b, and the chromosome number was 102 ± 4. The 5B3 mAb was purified successfully from ascites fluid through Protein G column, and its affinity constant was 2.94 × 108 L/mol. Besides, excellent specificity of the 5B3 mAb was further demonstrated in ELISA, western blot and immunohistochemistry experiments, suggesting that 5B3 mAb displays similar application value to D2-40, a commercial available antibody. Hence, the current study provides conclusive guidelines for preparation of other mAbs and their applications in immunohistochemistry diagnosis.

Numerous studies have focused on the mechanism of aflatoxin B1 (AFB1) metabolism and its carcinogenic mechanism, but how AFB1 is transported into hepatocytes and how it is transferred inside hepatocytes remains unknown. In this study, the AFB1interacting protein, estradiol 17 beta-dehydrogenase 5 (Akr1c6), was identified with an immobilized affinity chromatography technique and LC-MS/MS. The interaction between Akr1c6 and AFB1 was confirmed with ELISA, and the results showed that Akr1c6 could efficiently bind AFB1. Anti-Akr1c6 polyclonal antibody from rabbit was prepared, and the IC 50 values of AFB1 to BRL (normal big rat liver cells) and NRK (normal rat kidney cells) were detected using the MTT assay. It was found that the Akr1c6 expression level in BRL was significantly affected under the IC 50 value of AFB1 (P < 0.05), but no obvious expression difference of Akr1c6 was observed in NRK. This suggested that Akr1c6 in liver cells participates in the transportation and/or metabolism of AFB1, and though Akr1c6 was expressed in the kidneys, it did not play a role in AFB1 transportation or metabolism. The conclusions of this study lay a foundation for further exploring the role of AFB1 binding proteins in the toxicology of AFB1 to hepatocytes and the pathway through which AFB1 enters hepatocytes and their nuclei.