副溶血性弧菌免疫磁珠的制备及其应用

研究比较了不同的免疫磁珠(Immunomagnetic Beads,IMBs)富集捕获副溶血性弧菌的能力。采用两种副溶血性弧菌多克隆抗体,分别包被经羧基和甲苯磺酰胺基基团修饰的磁珠,制备完成5个免疫磁珠(IMB-C1、IMB-C2、IMB-T1、IMB-T2和IMB-T3)。研究引入表面抗体浓度作为评价免疫磁珠捕获能力的特征性参数,两种副溶血性弧菌多克隆抗血清包被的羧基磁珠表现出了较高的表面抗体结合能力,分别为34.25fg/μm2和36.73fg/μm2,明显高于甲苯磺酰胺基磁珠。五个免疫磁珠对副溶血性弧菌捕获能力的研究结果表明:在起始菌液浓度为4.32logCFU/mL时(p<0.05),两个羧基磁珠捕获率达到79%和94%,均优于甲苯磺酰胺基磁珠。采用环介导恒温扩增(loop-mediated isothermal amplification,LAMP)技术对免疫磁珠菌体细胞复合物进行体外扩增,结果表明:磁珠的存在并未影响对菌体细胞的后续分子检测。本研究分析比较了不同免疫磁珠的捕获能力,得到羧基磁珠对副溶血性弧菌的捕获能力比甲苯磺酰胺基磁珠强,直接法和间接法偶联方式对免疫磁珠的富集捕获能力无影响。

Preparation and application of Vibrio parahaemolyticus using immunomagnetic beads

The objective of this study was to use Immunomagnetic beads(IMBs)to detect Vibrio parahamolyticus.IMBs were synthesized using anti-V.parahaemolyticus polyclonal antibody(commercial antibody R1 and self-regulating antibody R2)and magnetic beads coated with activated carboxyl groups and tosylated surfaces groups(two carboxyl IMBs IMB-C1 and IMB-C2;three toslated IMBs IMB-T1,IMB-T2 and IMB-T3 which coated by the secondary antibody and polyclonal antibody).After the IMS process,one part of suspension was plated on TCBS to count the colony,the other to extract DNA with thermal cracking for Loop-mediated Isothermal Amplification(LAMP).The antibody concentration per surface area(AC/SA)was used for evaluation the properties of IMBs.The result showed that the AC/SA of the carboxyl IMBs are higher than toslated IMBs(34.25fg/μm2 and 36.73fg/μm2).And the carboxyl IMBs captured more V.parahaemolyticus during the IMS process than toslated IMBs,when the pure culture concentration was about 4.32logCFU/mL(p<0.05).After the IMS process,approximately 79%and 94%of the cells in suspension were captured by the carboxyl beads with commercial and self-regulating antibodies.And,there were not obviously different about the capturing rate between the directed and indirected conjugation methods,compared the IMB-T3 and IMB-T2.The LAMP results showed that the molecule detection method was not influenced by the IMBs-cell complex.