B Cell Cloning Technology For Screening Of CGAS Monoclonal Antibody

Antibody is a glycoprotein secreted by B lymphocytes upon antigen stimulation which are then differentiated into plasma cells.It is an important effector molecule in humoral immunity.Antibodies mainly include monoclonal antibodies and polyclonal antibodies.Monoclonal antibodies are antibodies produced against a specific antigen-determining cluster and have strong specificity and high sensitivity.The most common antibodies in the market are derived from mouse and rabbit.Rabbit monoclonal antibody performs better than mouse monoclonal antibody in terms of affinity and specificity.Thus,rabbit monoclonal antibody becomes increasingly popular in research and medical fields.The common preparation methods of monoclonal antibodies include hybridoma technology,but this method requires a long preparation cycle and has a low screening efficiency.A new generation of monoclonal antibody development technology based on monoclonal cells overcomes the disadvantage of antibody preparation by hybridoma cells.Monoclonal antibody preparation using this method can efficiently and quickly screen out the needed antibodies.This method using flow cytometry screening can produce specific antibodies of B cells.Through the B cell clone technology,B cell antibody heavy chain and light chain can be recovered by gene cloning.The efficacy of rabbit monoclonal antibody is determined by reacting with its specific antigen.cGAS,a cyclic guanosine-adenosine synthase,is a major DNA receptor.When dsDNA exist in the cytoplasm,DNA can function as a danger signal,which binds and activate cGAS.cGAS then uses ATP and GTP as substrates to synthesize cGAMP.cGAMP can in turn function as a second messenger leading the STING activation followed by type I interferon expression and further immune responses.The study of cGAS protein requires antibodies with high efficiency.However,the cGAS antibodies on the market are expensive and not effective.Therefore,we generated new cGAS monoclonal antibodies by using the B cell cloning technology.To achieve the above goal,human cGAS(aa 161-522)protein was immunized to New Zealand male rabbits.PBMC(peripheral blood mononuclear cell)was obtained from fresh blood of New Zealand male rabbits after three immunonizations.Flow cytometry was used to isolate B cells capable of producing human cGAS(aa 161-522)protein.Heavy chain and light chain genes that can recognize human cGAS(aa 161-522)protein in B cells were cloned through B cell cloning technology,and the recombinant rabbit anti-human cGAS antibody expression vector was identified.Finally,the rabbit anti-human cGAS monoclonal antibodies were expressed in the eukaryotic system and the antibody titer was determined.We finally obtain five effective rabbit anti cGAS monoclonal antibodies.This research has four innovation points described as follows.First,to improve the filtering efficiency of B cells,the anthropogenic cGAS(aa 161-522)protein was coupled with Dylight 650 Antibody.Second,three times repeated screening were performed which led 10 times increase in selecting the positive B cells.Third,to increase the cloning efficiency of heavy chain and light chain genes of B cell antibodies,nested PCR technology was adopted and the success rate for fishing antibody genes had reach to more than 95%.Fourth,to increase the output of antibody expression,the Expi293 Epression System was used to replace the 293 T cell expression system for the antibody expression,and the antibody output was 20 times higher than that of the 293 T cell.Our study establish the B-cell cloning based antibody production system which will be essential for the future therapeutic monoclonal antibody production.