Preparation of human hepatitis B virus monoclonal antibodies using single cell RT-PCR

Objective To establish the system for the isolation of peripheral single antibody- secreting cells( ASCs) and single cell RT-PCR system,and to clone highly specific human hepatitis B monoclonal antibody efficiently and rapidly. Methods Three patients with HBV infection in the recovery stage were enrolled. Peripheral B cells were collected and activated by HBc Ag Peptide pool. Flow sorting was performed to obtain memory ASCs( CD19~+CD10~-IgG~+CD27~+),the limiting dilution method was used to obtain single cells,single cell RT-PCR was used to obtain cDNA,and nested PCR was used for the amplification of the fragment in the constant region of the heavy chain of antibody. PCR product was purified and cloned to pEASY~- T1 simple cloning vector for sequencing and identification. Results The results of ELISA showed that a high level of IgG was observed in all three patients with HBV infection in the recovery stage. There were significant differences in peripheral IgG level between patients and healthy volunteers( both P < 0. 05). The results of flow sorting showed that the proportion of B cells was higher than 94% and memory ASCs were isolated. A total of 24 ASCs containing at least one cell with normal morphology were selected,and after identification,14 were successfully amplified,with a size of about 200 bp,which was consistent with expected results. The analysis of the sequence of the heavy chain of antibody showed successful amplification of the fragment in the constant region of the antibody. Conclusion The system for the isolation of single ASCs and single cell RT- PCR system are successfully established and the sequence of the heavy chain of antibody is obtained,which lays a solid foundation for the high- throughput production of human hepatitis B monoclonal antibodies in future.