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肺炎衣原体单克隆抗体的研制和应用 被引量:3

Preparation and Application of the Monoclonal Antibody to the Chlamydia pneumoniae
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摘要 目的:以杂交瘤技术制备抗肺炎衣原体(Cpn)单克隆抗体,用于衣原体感染的诊断及相关疾病的研究。方法:以进口Cpn抗原免疫BALB/c小鼠,将免疫小鼠的脾脏细胞与SP2/0细胞融合,用间接ELISA法筛选抗体阳性杂交瘤细胞。收集接种过杂交瘤细胞的小鼠腹水,分别用ELISA法检测抗体效价、用免疫琼脂扩散试验鉴定单抗的类别、用微量荧光免疫试验(MIF)检测单抗的种属特异性。用克隆表达的主要外膜蛋白(MOMP)通过Dot-ELISA法分析单抗的特异性。通过建立直接免疫荧光法(DIF)检测病人和正常人外周血单核细胞(PBMC)标本,并进行统计处理。结果:小鼠脾脏细胞与SP2/0细胞的融合率为61.46%(236/384),最终获得4株稳定分泌Cpn单抗的细胞株。用ELISA法检测小鼠腹水,效价高者可达1∶100000。免疫琼脂扩散试验鉴定为IgG类单抗,扩散效价达1∶128。自制单抗能与重组MOMP发生结合反应,表明其为抗CpnMOMP抗体。自制单抗与进口单抗类似,即与鹦鹉热衣原体(Cps)出现一定程度的交叉反应,而与沙眼衣原体(Ct)则无交叉反应。对240份PBMC标本用自制单抗和进口单抗同时检测Cpn抗原,2种单抗检测均阳性的共86份,经SPSS软件分析两者具有较好的一致性。DIF检测显示,心血管疾病和呼吸道疾病Cpn抗原阳性检出率分别为69.34%(95/137)和72.06%(49/68),与正常人标本Cpn抗原阳性率相比,均具有显著性差异。结论:获得IgG类抗CpnMOMP单抗,自制Cpn单抗的特异性和敏感性均与进口单抗具有较好的一致性。PBMCCpn抗原检测的统计分析证实,对于动脉粥样硬化等某些疾病的发生和发展,Cpn感染可能是重要的原因之一,但其中的因果关系还有待深入研究。 Objective: The hybridoma technique was used to prepare the anti-Cpn(Chlamydia pneumoniae) monoclonal antibody(McAb), and the McAb was employed for the diagnosis of the Chlamydia-infectious diseases and related researches. Methods: The imported Cpn was used as the antigen for the immunization to male BALB/c mice, and the spleen cells of one mouse were fused with SP2/0 cells. The ELISA was used to analyze and screen the hybrid cells. After collected the ascites of the mice injected with the hybrid, ELISA and agar immunodiffusion were used to detect the titers and identify the type of McAb. The specificity of McAb was identified with the reconstructed Cpn major outer membrane protein(MOMP) by micro-immunofluorescence assay(MIF) and Dot-ELISA. The direct immunofluorescence assay(DIF) assay was estabilished to detect the peripheral blood mononuclear cell(PBMC) samples from patients and healthy person and analyzed with the statistical method. Results: The percent of infusion was 61.46%(236/384) and 4 clones secreted the McAb stably were finally obtained. The highest titer of the ascite was more than 1:105. All these McAbs belong to IgG class and the titer of the diffusion was higher than 1:128. The McAb can react with the reconstructed MOMP which means the McAb is anti-Cpn MOMP antibody. It also showed that there're crossed reactions between the McAb and Chlamydia psittaci (Cps), while not with Chlamydia trachomatis (Ct). All 240 PBMC samples were detected with obtained McAb and the imported McAb at the same time. It was found that among them 86 were both positive and the result through SPSS showed a common extent of consistency between them. The results of DIF showed that the positive rate of the Cpn antigen were 69.34% and 72.06% respectively for the PBMC samples from patients suffered from cardiovascular diseases and respiratory tract diseases. Compared with the positive rate of healthy group, both results showed discrepancy remarkably. Conclusion: The anti-Cpn MOMP
出处 《生物技术通讯》 CAS 2006年第3期362-366,共5页 Letters in Biotechnology
关键词 肺炎衣原体单抗 免疫荧光试验 肺炎衣原体主要外膜蛋白 外周血单核细胞 Chlamydia pneumoniae(Cpn) anti-Cpn monoclonal antibody immunofluorescence assay Cpn major outer membrane protein peripheral blood mononuclear cell
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参考文献10

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