Sorting And Identification Of Single B Lymphocyte-based Antibody Against Viruses

Antibody plays an important role in humoral immune response.Due to its specificity and sensitivity,antibody has been continuously tried to be expressed and produced in vitro,and used for the research and prevention of viruses and pathogenic microorganisms.From the polyclonal antibody technology to monoclonal antibody and genetic engineering antibody technology,it is found that those technologies remain to be difficult to use,time-consuming and heterologous reaction.In order to solve these problems,a new antibody technology based on single B lymphocyte has been developed in recent years.This method relies on FACS,and can sort and express a large number of specific antibodies at the level of single B lymphocyte.In this study,a monoclonal antibody technology based on single B lymphocytes was developed to produce and identify mouse antibodies against type O FMDV.mouse antibody against CPV and human antibody against RABV.B cells was firstly enriched by using CD19,which is the B cell surface marker,magnetic beads from spleen cells suspension of healthy C57BL/6 mice,and then the rate of 19/500000 single antigen specific B cells were sorting via FACS in B220~+/IgG1~+/CD38~+/O FMDV antigen~+.After that the targeted genes of those cells were amplified by single-cell RT-nested PCR.From 19 single antigen specific B cells,8 sequences of variable region of light chain and 19 sequences of variable region of heavy chain were obtained,the positive rate was 33.3%and 79.2%respectively,but there were only 6natural pairs of light and heavy chain genes among them,namely 2-4FAb,11-8FAb,7-8FAb,7-9FAb,12-5FAb and 5-7FAb.Next,the recombinant plasmids containing light/heavy chain from these cells were constructed and then transfected into HEK-293 cells to express 6 monoclonal antibodies.Western Blot was used to identify the expression of antibody light/heavy chain,showing that the molecular weight of light and heavy chain from 5-7FAb,11-8FAb and 12-5FAb were consistent with the expected molecular weight of the antibody light and heavy chain(heavy chain is 55 kD and light chain is 25 kD).ELISA testing for the antibody isotyping showed that the three antibodies belong to IgG1 isotype,indicating used to the developed technology above was able to select IgG1~+B lymphocytes.Immunofluorescence test showed that all the three antibodies could be expressed in cytoplasm.ELISA analysis on those three antibodies showing that all the antibodies could specifically bind the inactivated type O FMDV.The A450nm values from those three antibodies were 3 times higher than that of the negative control.Furthermore,immunofluorescence showed that the three strains of antibodies could specifically bind to the type O FMDV on infected cells.Meanwhile,using the expression vectors in this technology,we constructed the expression plasmids of light/heavy chain of CPV mouse recombinant antibody and anti-RABV human recombinant antibody,and obtained corroessponding recombinant antibodies.The immunofluorescence test with these antibodies showed that the antibodies against RABV and CPV can be expressed in the cytoplasm and could specifically binds to RABV and CPV,respectively.The virus microneutralization test showed that the antibody against RABV could neutralize the virus.There was efficiency of 70%when the antibody in cell culture supernatant was diluted 1 in 32and efficiency of 60%,,when the concentrated antibody diluted 1in168.In conclusion,a method for direct generation of the monoclonal antibody from single B lymphocyte has been developed,which would provide a new way for the development of new assays and approaches for detection and treatment of viruses infection.