期刊
TOXINS
卷 9, 期 8, 页码 -出版社
MDPI
DOI: 10.3390/toxins9080250
关键词
domoic acid; monoclonal antibody; cell fusion; hybridoma technology; ic-ELISA
资金
- Program from Key Scientific and Technology Project of Fujian Province of China [2014YZ0001, 2017Y4002]
- Fujian Provincial Department of Ocean and Fisheries (Fujian Ocean Hi-tech) [[2015] 26]
- Fujian Provincial Development and Reform Commission (the study and invention of a new type of marine biotoxin detection kit) [[2013] 229]
Domoic acid (DA) is a potent toxin, marine biotoxin, and primarily produced by Pseudo-nitzschia. The DA hapten was coupled with bovine serum albumin (BSA), and ovalbumin (OVA) as carrier proteins. DA-BSA conjugate was used as immunogen and DA-OVA as coating antigen. Cell fusion between spleen cells and sp2/0 myeloma cells developed 1C3 hybridoma clone producing 1C3 monoclonal antibody (mAb). Hybridoma was injected into the mice to produce ascites, and further purified by caprylic acid/ammonium sulfate method. The mAb was of IgG3 subclass, and was specific to DA with high affinity (2.5 x 10(8) L/mol). Moreover, western blot exhibited significant specificity to the DA antigens. Indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) showed DA working range of 0.006-0.2 ng/mL. The IC50 was 0.03 ng/mL with low limit of detection (LOD) of 0.006 ng/mL. Average DA recovery from spiked shellfish extract was 100.56 +/- 2.8% with the coefficient variation of 0.01-0.1%. Hence, mAb producing 1C3 hybridoma was successfully developed and could be used to detect DA in contaminated samples.
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