以热灭活副溶血性弧菌(Vibrio parahaemolyticus,VP ATCC17802)免疫新西兰大白兔制备多克隆抗体,用间接酶联免疫吸附法测定抗血清效价,纯化后的多克隆抗体效价为512 000。该抗体与5株副溶血性弧菌均能特异性结合,但与其他12株非副溶血性弧菌无交叉反应。在此基础之上建立了间接竞争酶联免疫吸附法(indirect competitive enzyme-linked immunosorbent assay,ic-ELISA),并优化了抗原抗体浓度、二抗浓度、封闭液等条件。结果表明,该法的线性范围为5×104~107 CFU/mL,线性回归方程为y=0.21x-0.74(R2=0.99),IC50为106CFU/mL,最低检出限为1.7×104 CFU/mL,板内变异系数为2.61%~4.15%,板间变异系数为4.71%~8.74%。用建立的ic-ELISA检测市场中鱼、贝类养殖水和实验室用水中的VP,实验结果与国标检测方法的结果一致。该方法快速便捷,灵敏度高,准确性好,为进一步研究VP的快速检测奠定了基础。
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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