Preparation and application of monoclonal antibody against Coxsackievirus A16
LIU Yang1, ZHAO Xiang-rong1, YU Peng-bo2, GUO Chun-yan1, LI Yan1, ZHAO Peng-hua1, HU Jun1
1.Central Laboratory, Shaanxi Provincial People’s Hospital, Third Affiliated Hospital of School of Medicine, Xi’an Jiaotong University,Xi’an 710068, China; 2.Shaanxi Provincial Centre for Disease Control and Prevention, Xi’an 710054, China
Abstract：To prepare the monoclonal antibodies (mAbs) against Coxsackievirus A16 (CA16), and to assess application of mAbs in cell-based ELISA and cell immunochemical staining. Colloidal gold immunochromatographic assay for rapid diagnosis CA16 was established. CA16 virus was cultivated using RD cells. CA16 was purified by cesium chloride density gradient ultracentrifugation. Purification of CA16 particles were identified by transmission electron microscopy (TEM). Balb/c mice were immunized with inactivated CA16 and hybridoma cell strain secreting mAb against CA16 were objected to screening. Characterization of the prepared mAbs were analyzed by ELISA and Immunofluorescent assay (IFA) respectively. The mAbs were used in development of cell-based ELISA and cell immunochemical staining. Colloidal gold-based immunochromatographic assay was established using monoclonal antibodies against CA16. TEM analysis showed that CA16 particles had icosahedral structure. A hybridoma cell strain secreting mAb against CA16 was obtained, named as CA16-19, the subtypes of the mAb was IgG2a, IFA analysis found that mAb specifically bind to RD cells infected with CA16. Cell-based ELISA indicated that the absorbance value of ELISA increased with the titers of viral TCID50. Cell immunochemical staining showed that mAb also could bind to RD cells infected with CA16 and couldn’t bind to RD cells infected with EV71 or RD cells control. The detection limit of establishment of colloidal gold assay using mAbs against CA16 was 6.25×106.25TCID50/0.1 mL. It is indicated that CA16 particles were purified by cesium chloride density gradient ultracentrifugation, the mAb specifically against CA16 were prepared, which was used in IFA, cell-based ELISA and cell immunochemical staining respectively. Meanwhile, establishment of colloidal gold immunochromatographic assay also provides important method for rapid detection CA16.
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