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.
Highlights
The toxic photosynthetic dinoflagellate Karenia brevis (K. brevis) is widely distributed in the Gulf of Mexico, and has been indicated in other locations [1]
In order to obtain a monoclonal antibody (mAb) against Brevetoxin 1 (BTX-1), BTX-1 should be coupled to a carrier protein
Bovine serum albumin (BSA) and ovalbumin (OVA) are frequently used as carrier proteins for conjugate preparation, and BTX-1-BSA and BTX-1-OVA conjugates were prepared by the succinic anhydride method and isobutyl chloroformate method
Summary
The toxic photosynthetic dinoflagellate Karenia brevis (K. brevis) is widely distributed in the Gulf of Mexico, and has been indicated in other locations [1]. Proliferations of K. brevis occasionally generates large ’red tide’ blooms. Natural toxins from these blooms cause toxicity to fish, other animals, and human health problems. Brevetoxins (BTXs), actively biosynthesized by K. brevis, are the first example of polyether compounds [2]. BTXs consist of more than nine congeners, which are divided into two types (types A and B) on the basis of their polyether backbone structure.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
