Screening Of Enterovirus 71 Antibodies And Establishment Of QPCR Detection Method For Common Respiratory Viruses

Hand, foot and mouth disease(HFMD) is an infectious disease caused by enterovirus infection and multiple in infants and children under 5 years of age, some of which by the enterovirus 71(EV71) infection in children can cause nervous system symptoms of infection and lead to heart and lung failure, severe cases can lead to death. The infection of EV71 is a huge number, but only a minority of patients develops severe. Therefore, the development EV71 neutralizing antibodies provide specific treatment for the patients with severe HFMD; provide one ofthe possible solutions for reducing morbidity and mortality of HFMD.Using markers CD19 and CD27 beads screening specific B cells, cultured B cells in the feeder layer cells, with stimulatedand activated the B cells to antibody-secreting cells by cytokines, the positive clone cells were tested by ELISA, extracted the total mRNA, then amplified immunoglobulin heavy and light chain genes by reverse transcription and nested PCR. After sequencing, the sequence was cloned into the eukaryotic expression vector. Recombinant antibody was obtained by instantaneous transmission of 293 T cells, 59 groups of monoclonal antibodies were successfully isolated; theaffinity activity was tested by ammonium thiocyanate elution method, a total of 6 groups of monoclonal antibodies with high affinity were obtained Neutralizing activity was detected by neutralization test. Successfully screening 1 group of anti-EV71 humanized neutralizing antibody. The half maximal inhibitory concentration was 5.5 μg/mL. In this study, we havesuccessfully established the humanized anti-EV71 monoclonal antibody preparation, laid a foundation for the early diagnosis of EV71 and selection of other humanized monoclonal antibodies.High incidence of respiratory diseases was usual causedby a variety of respiratory viruses and bacteria in spring and winter. This study has also established real-time PCR detection method, to detect the 12 kinds of common respiratory viruses: RSV, PIV1, PIV2, ADV, EBV, FluA, FluB in totally 178 samples of throat swab from Wangjing Hospital of CACMS between 2011 and 2012 The percentage of Respiratory Viruses was 36.2%(64/178) in samples of throat swab. The work provideed a reliable technical means for the detection of respiratory pathogens, and provideed basic data for the prevention and control of respiratory diseases.