PBP2a多克隆抗体制备及其乳胶凝集检测法的建立

Preparation of polyclonal antibody against PBP2a and establishment of a latex agglutination test

目的制备兔抗青霉素结合蛋白2a(penicillin binding protein 2a,PBP2a)抗体,建立检测PBP2a的乳胶凝集法。方法以重组PBP2a转肽酶区蛋白免疫家兔制备多克隆抗体,ELISA和Western blot法检测所制备的抗血清效价和特异性,用纯化的多抗建立乳胶凝集法。结果纯化的重组蛋白免疫家兔能有效地刺激特异性抗体的产生,抗血清的效价达1∶25 600,Western blot显示该抗体能有效识别原核表达及MRSA临床分离株中的PBP2a,建立了乳胶凝集法,敏感性及特异性良好。结论成功制备了抗PBP2a抗体血清,初步建立了检测PBP2a的乳胶凝集法,为有效制备高特异的单克隆抗体进而研制MRSA快速鉴定试剂盒奠定了良好的基础。

Objective To prepare rabbit antibody against penicillin binding protein 2a （PBP2a） and develop a latex agglutination test（LAT） to detect PBP2a. Methods New Zealand rabbit was immunized with the E. coli expressed and purified recombinant PBP2a protein. The titer and the specificity of the rabbit anti-PBP2a antibodies were detected by ELISA and Western blot, a LAT was established for rapid diagnosis of PBP2a. Results The titer of anti-PBP2a antibodies was 1：25 600. Western blot analysis showed that the polyclonal anti-PBP2a antibodies could detect both prokaryotic ex- pressed PBP2a, and clinical isolated MRSA-PBP2a, the sensitivity and specificity of the LAT in detecting the PBP2a were high. Conclusions Polyclonal anti-PBP2a antibodies with high specificity and affinity were prepared by using purified recombinant PBP2a as immunogen, a latex agglutination test（LAT） for detecting PBP2a was successfully established, which lays the foundation for the making of LAT kit as well as serving as an useful reference for the prepareration of monoclonal antibody.