Fast And Efficient Preparation Of IgM Monoclonal Antibody Using DT40 Cells

Monoclonal antibody(mAb)is widely used in basic research,clinical diagnosis,and therapy due to its specificity and high affinity.Preparation technology for mAb includes hybridoma technology,phage display technology and B cell sorting technology,etc.However,these technologies are laborious and time-consuming.In this study,we intended to utilize the DT40 cell line to establish a rapid and efficient mAb preparation system.DT40 cell is a chicken B lymphoma cell line which expresses and secrets immunoglobulin M(IgM).The highly-frequent gene conversions and point mutations of the Ig gene in DT40 make it a natural antibody library,and these processes are regulated by the activation-induced cytidine deaminase(AID)gene.To fix desirable Ig mutants by stopping hypermutation or to resume mutation for further improvement of the antibody affinity,AID must be switched on or off.To this end,we used CRISPR-Cas9 to knock out AID alleles in DT40 cells.DNA sequencing and western blotting were applied to verify the knockout of AID.The sequence of the IgV gene was analyzed to confirm the loss of function.Then,a Tet-off flanked AID construct was stably transfected into the AID-deficient cell line.The mutation of the IgV gene was analyzed to confirm the gain of function.At last,we screened the DT40 cells by magnetic beads coupled with the NS1 protein of Zika virus,and we successfully obtained DT40 clones secreting IgM against NS1.The DT40 monoclonal antibody preparation system we established is much more efficient than the traditional technologies and may have a wide application prospect in research and diagnosis.