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目的:制备抗铜绿假单胞菌外膜蛋白F(OprF)的单克隆抗体(mAb),并建立双mAb夹心ELISA检测方法。方法:利用分子生物学方法克隆OprF基因,诱导表达并纯化OprF。用OprF免疫BALB/c小鼠后通过杂交瘤技术制备特异性的mAb,并用ELISA法测定mAb的免疫活性;建立检测铜绿假单胞菌的双mAb夹心ELISA方法,并对其敏感性、特异性进行初步评价。结果:成功地克隆OprF基因,经诱导表达获得铜绿假单胞菌的OprF。通过杂交瘤技术筛选出4株mAb:6D8F7、2H2B6、3C2F5和7B5D9,经鉴定mAb 6D8F7可作为捕获抗体,mAb 7B5D9被HRP标记后可作为检测抗体,以这2株mAb建立的双mAb夹心ELISA方法重复性好、特异性强,敏感性高达到1×103集落形成单位(CFU/mL)。检测的线性范围为1×103～108CFU/mL。与传统的细菌分离方法比较,检测临床标本的符合率高达94.7%。结论:通过杂交瘤技术制备抗铜绿假单胞菌OprF的mAb,并建立了高特异性、高敏感性检测铜绿假单胞菌抗原的双mAb夹心ELISA方法,可用于临床检测铜绿假单胞菌的感染。
Objective: To prepare a monoclonal antibody (mAb) against outer membrane protein F (OprF) of Pseudomonas aeruginosa and establish a double-antibody sandwich ELISA assay. Methods: The OprF gene was cloned by molecular biology and induced to express and purify OprF. BALB / c mice were immunized with OprF, specific mAbs were prepared by hybridoma technique, and the immunological activity of mAb was determined by ELISA. A double-antibody sandwich ELISA for detecting Pseudomonas aeruginosa was established and its sensitivity and specificity Make a preliminary assessment. Results: OprF gene was successfully cloned and OprF of Pseudomonas aeruginosa was obtained by induced expression. Four mAbs: 6D8F7, 2H2B6, 3C2F5 and 7B5D9 were screened by the hybridoma technique. The mAb 6D8F7 was identified as the capture antibody and the mAb 7B5D9 was labeled with HRP as the detection antibody. The two mAbs sandwich ELISA Repeatability, specificity and sensitivity up to 1 × 103 colony forming units (CFU / mL). The linear range of detection was 1 × 103 ~ 108CFU / mL. Compared with the traditional method of bacterial separation, the coincidence rate of clinical specimens tested up to 94.7%. Conclusion: The mAb against Pseudomonas aeruginosa OprF was prepared by hybridoma technique and a double-mAb sandwich ELISA method was established to detect the high specificity and sensitivity of Pseudomonas aeruginosa antigen. It can be used in the clinical detection of Pseudomonas aeruginosa Infection.