Abstract:
In order to develop an ELISA KIT for detection of domoic acid (DA) in seafood in Vietnam, DA antibody was produced by immunizing in New Zealand white rabbit using DA-DSS (disuccinimidyl suberate)-BSA (Bovine Serum Albumin) as an antigen. The DA antibody in rabbit’s serum was purified for its use as a material of ELISA KIT. In antibody/antigen purification process, immunoaffinity chromatography (IAC) is considered as the best method by both its specification and ability to enrich antibody. However, DA is a hapten, and it is impossible to apply a regular immunizing principal for making an IAC column with DA as a solid phase to catch DA antibody. This paper presents a trial using polymer sepharose EAH 4B resin (omega-aminohexyl) IAC bound with hapten DA as a solid phase for DA antibody purification from rabbit’s serum. Using DSS as a chemical linker; then, based on the reaction between a carboxyl group of DA and a carbodiimide group of the resin EAH 4B, we were successful to make the IAC column with DA-DSS as the solid phase. Recovery ratio of the made IAC column reached 91.5 ± 1.5% (n = 3) to capture the antibody against DA standard. DA antibody from white rabbit’s serum was eluted successfully using this column with 0.5 M NaCl; 0.1 M PBS and then, 0.1 M Ldy-HCl solutions (pH 2.7). The obtained antibody was qualified by Western Blot using a specific reaction with the 2nd specific antibody HRP-labeled anti-goat IgG and the confirmed result revealed that it can be used as ELISA KIT material.
