基于NHS基团修饰的副溶血性弧菌免疫磁珠制备及其富集性能评价

摘要

[目的]制备NHS基团修饰的副溶血弧菌免疫磁珠并评价其富集性能.[方法]采用偶联率确定免疫磁珠最佳制备条件(磁珠粒径、偶联时间和抗体加入量),采用捕捉率确定免疫磁珠最佳富集条件(菌悬液pH、孵育时间和免疫磁珠加入量),并通过方法的特异性、灵敏度、精密度和稳定性评价免疫磁珠富集性能.[结果]0.1 mLN2型NHS磁珠可与0.2mL副溶血孤菌抗血清偶联2h制备IMB,,加入到副溶血弧菌菌悬液(pH7.0～7.4)中孵育2h,IMBvp最佳工作液体积为0.4mL.该方法特异性好、灵敏度高(15 cfu/50 mL)、精密度高(批内变异系数和批间变异系数均在3％ ～6％)、稳定性好(准确度大于91％).[结论]自制副溶血弧菌免疫磁珠富集性能好,方法工作效率高,成本低,为副溶血弧菌高效快速检测提供了可靠的富集手段.%[Objective] The aim was to prepare Vibrio parahaemolyticus immunomagnetic beads (IMBvp) based on NHS group and evaluate the enrichment capacity.[Method] The best coupling conditions were decided by conjugate ratio,like conditions range from magnetic beads particle diameter,coupling time and the adding amount of antibody.The best enrichment conditions were decided by capture rates,like conditions range from pH,incubation time and the adding amount of IMBvp.The enrichment capacity of IMBvp was evaluated according to specificity,sensitivity,precision and stability.[Result] The IMBvp was prepared by chemical bond,which combined 0.1 mL of NHS bead with size N2 and 0.2 mL of Vibrio parahaemolyticus antiserum.IMBvp was placed into the bacterial suspension which pH was 7.0-7.4.Volume addition of was IMBvp 0.4 mL.They were incubation 2 h together.The method was involved in some advantages,high specificity,sensitivity and accuracy.The detection limit was 15 cfu/50 mL.The CVs of intra and inter assay were between 3％ and 6％.The accuracy was greater than 91％.[Conclusion] The above results demonstrate that homemade IMBvp has good enrichment performance.Experiment results show that it is an effective method,convenience,low cost and quickness.