抗MRSA-PBP2a转肽酶区蛋白单克隆抗体的制备、鉴定及初步应用

目的:制备和鉴定抗MRSA-PBP2a转肽酶区蛋白单克隆抗体(mAb),初步建立检测PBP2a的乳胶凝集方法。方法:以基因工程重组PBP2a转肽酶区蛋白为抗原,免疫BALB/c小鼠,制备鼠源性mAb,间接ELISA鉴定IgG亚类、腹水效价及亲和常数,Western blot检测mAb的特异性,用所得mAb致敏聚苯乙烯乳胶,建立检测PBP2a的乳胶凝集方法。结果:筛选出2株能稳定分泌抗PBP2a mAb的杂交瘤细胞株F1和F2,免疫球蛋白亚类均为IgG1类;腹水效价为0.5×106~1×106,亲和力常数分别为1.57×108M-1和5.43×109M-1。Western blot显示F1、F2抗体均能有效识别重组PBP2a转肽酶区蛋白及MRSA临床分离株中的天然PBP2a,用mAb F2建立了乳胶凝集法,敏感性达1mg/L。结论:获得2株特异性好、亲和力高、能稳定分泌抗PBP2a mAb的杂交瘤细胞株,为研制MRSA快速鉴定试剂盒奠定了良好的基础。

Preparation, characterization and preliminary application of monoclonal antibody against the transpeptidase domain of MRSA-PBP2a

AIM: To prepare monoclonal antibody (mAb) against the penicillin binding protein 2a (PBP2a) of MRSA and establish a latex agglutination assay to detect PBP2a. METHODS: BALB/c mice were immunized with the recombinant transpeptidase domain of PBP2a expressed by gene-engineering. Mouse mAb against PBP2a was obtained with hybridoma technique. mAb's characteristics (IgG subclasses, titers, specificities, and affinities) were identified and determined by indirect ELISA and Western blot. Polystyrene beads were sensitized with mAb F2 by physical adsorption, and a latex agglutination assay was developed to detect PBP2a. RESULTS: Two strains of hybridoma cell lines, which could stably secret anti-PBP2a mAb were obtained, named F1 and F2. The isotype of F1 and F2 was both IgG1. Ascites titers were 0.5x10(-6)-1x10(-6) and the affinity constants (Kaff) were 1.57 x 10(8) M(-1) and 5.43 x 10(9) M(-1), respectively. Western blot analysis showed that both the mAbs had specific binding abilities with both recombinant protein and clinically isolated MRSA-PBP2a. The sensitivity of the anti-PBP2a latex agglutination assay with serial dilutions of purified PBP2a was found to be 1 mg/L. CONCLUSION: The obtained two strains of hybridoma cell lines can secret anti-PBP2a mAb stably, which lays the foundation for establishment of a simple and rapid anti-PBP2a latex agglutination assay kit.