Preparation and application of polyclonal antibody against Vibrio parahaemolyticus
WEI Chunhao1,2, CHI Hai1,2, YANG Guangxin2*, TAO Leren1
1 (School of Medical Instrument and Food Engineering, University of Shanghai for Science and Technology,Shanghai 200093,China) 2 (East China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Shanghai 200090, China)
Abstract: The current study presents a high specific polyclonal antibody for the determination of the Vibrio parahaemolyticus (VP) using indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). The antibody was obtained by immunizing New Zealand white rabbits with heat-killed V. parahaemolyticus ATCC17802. The titer of the final purified antibody was 512 000. The antibody possessed high selectivity to 5 strains of VP, but no cross-reaction to 12 non-VP strains. Several conditions such as antigen-antibody concentration, secondary antibody and blocking solution were optimized to construct the new method. Under the optimal conditions, good linearity was achieved within a range of 5 × 104-107 CFU/mL under the linear regression equation of y=0.21x-0.74(R2=0.99). The observed half-maximal inhibition concentration (IC50) was 106 CFU/mL, and the limit of detection was 1.7×104 CFU/mL. The intra-plate variation coefficient was 2.61%-4.15% and the coefficient of variation between plates was 4.71%-8.74%. The developed ic-ELISA was used to detect VP in real water samples collected from laboratory and aquatic product holding ponds in the market. The results showed good correlation with the results obtained by standard methods. The method was simple, specific, sensitive, and less time-consuming, and this study made a good foundation for the rapid determination of VP in real samples.
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