副黏病毒Tianjin株重组血凝素-神经氨酸酶单克隆抗体的制备及在临床检测中的初步应用

目的:建立双抗体夹心ELISA法,调查副黏病毒Tianjin株引起婴幼儿下呼吸道感染情况。方法:用纯化的重组HN片段免疫BALB/c小鼠,按常规方法制备单克隆抗体。以兔抗Tianjin株多克隆抗体为捕获抗体,单抗G7G7E7为检测抗体,建立双抗体夹心ELISA法,检测104例下呼吸道感染患儿支气管肺泡灌洗液(BALF)标本。结果:获得3株能稳定分泌单克隆抗体的杂交瘤细胞株G7H4D3、G7D9G3和G7G7E7。3株单克隆抗体与副黏病毒Tianjin株有较高结合活性,与甲、乙型流感病毒,新城疫病毒(NDV),人副流感病毒(hPIV)1型、3型、肺炎支原体均无交叉反应性。ELISA相加试验和阻断试验表明,3株单抗识别相同或相近表位区域,识别的表位在抗病毒免疫应答中处于免疫优势地位。双抗体夹心ELISA法检测阳性率为1.92%(2/104)。结论:与RT-PCR和间接ELISA法比较,双抗体夹心ELISA法,具有敏感性高、特异性强的优点,适于临床检测使用。副黏病毒Tianjin株是婴幼儿下呼吸道感染的重要致病因子之一。

Preparation of Monoclonal Antibodies against Recombinant Hemagglutinin-Neuraminidase of Paramyxovirus Tianjin Strain and Preliminary Application in Clinical Detection for Respiratory Tract Infection

Objective : To establish a Sandwich ELISA assay for detection of paramyxovirus Tianjin strain in the bronchoalveolar lavage fluid (BALF) samples of the newborns and young children with severe lower respiratory tract infections. Methods: BALB/c mice were immunized with the purified recombinant Hemagglatin in Neuraminidas protein of paramyxovirus Tianjin strain and monoclonal antibodies(mAbs) were prepared by conventional method. A Sandwich ELISA assay was developed in which the rabbit polyclonal antibodies against paramyxovirus Tianjin strain were used as the capture antibody and mAb G7G7E7 as detection antibody. Paramyxovirus Tianjin strain in BALF samples of the newborns and young children with severe lower respiratory tract infections were detected. Results: Three hybridoma cell strains G7H4D3,G7D9G3 and G7G7E7 were obtained. The mAbs secreted by these hybridoma cells showed the ability of specific binding to paramyxovirus Tianjin strain,and no cross -reactions were evidently detected with influenza A and B,New Castle disease virus (NDV),human parainfluenza virus(hPIV) type 1 and 3,mycoplasma pneumoniae. ELISA additivity tests indicated the three mAbs recognized the same or closely adjacent epitope domains which were also suggested immunodominant in the immune response against paramyxovirus Tianjin strain by blocking assay. The Sandwich ELISA assay for detection of paramyxovirus Tianjin strain was set up and 104 BALF samples were detected with the positive rate of 1.92%(2/104). Conclusion: Compared with the methods of RT-PCR and indirected ELISA,the Sandwich ELISA assay for antigen detection of paramyxovirus Tianjin strain is more sensitive and specific. It is suitable for clinical application. In addition,paramyxovirus Tianjin strain most likely causes bronchitis and pneumonia in infants and young children and is one of important causative agents in the lower respiratory tract infections.