单增李斯特菌免疫磁珠的制备研究

目的:制备可高效、特异地分离单增李斯特菌的免疫磁珠。探讨羧基修饰磁珠与多克隆抗体的不同偶联条件和免疫磁珠捕获单增李斯特菌的能力。方法:以单增李斯特菌作为抗原,免疫新西兰兔,获得兔源多克隆抗体,鉴定其与单增李斯特菌体的结合能力;选择6种不同的偶联缓冲溶液,设置了6个主要偶联时间和6组偶联温度,通过比较磁珠与抗体偶联后上清中剩余的抗体量来确定最佳偶联条件。结果:制备的多克隆抗体效价为1.3×105,该抗体与单增李斯特菌体有较好的结合,抗体与1mg羧基修饰磁珠在pH6.0的0.01mol/L一水吗啉乙磺酸缓冲溶液(MES)中,37℃偶联2h,偶联抗体的量为160μg;制得的免疫磁珠的捕获率可达77.0%。结论:获得羧基磁珠与抗体偶联的最佳条件,该免疫磁珠用于食品中单增李斯特菌的检测,与常规的平皿增菌培养显色法比较,检测时间至少缩短20h。

Study on preparation of immunomagnetic beads used for Listeria monocytoenes separation

Objective:To prepare the immunomagnetic beads which separated Listeria monocytogenes efficiently and specifically,and to investigate the optimal conditions of coupling reaction for carboxylate-modified magnetic beads and polyclonal antibody,and to evaluate the capture capacity of immunomagnetic beads.Methods:The cell of Listeria monocytogenes was used as antigen for immunization of rabbit to prepare polyclonal antibody and binding affinity between polyclonal antibody,and Listeria monocytogenes was determined.Three main options were set:6 different coupling buffers,6 main coupling time and 6 groups coupling temperature.The optimal coupling conditions were obtained by comparing the antibody surplus of supernatant after coupling.Results:The titer of purified polyclonal antibody which had high binding affinity with Listeria monocytoenes was about 1.3×105.With the coupling buffer of 0.01mol/L 2-(N-Morpholino) ethanesulfonicacid(MES,pH6.0),purified polyclonal antibody was coupled with 1mg carboxylate-modified magnetic beads for 2h at 37℃,and the amount of coupled antibody was 160μg.The capture ratio of newly produced immunomagnetic beads was about 77%.Conclusion:The optimal conditions of coupled reaction were achieved and the immunomagnetic beads were prepared successfully.Comparing with the traditional culture-based methods,the entire procedure for listeria monocytogenes detection could be reduced at least 20 hours prepared with immunomagnetic beads.