Establishment Of Highthroughput MAbs Production Using Multiplexed Immunization And Antigen Microarray Assay

Mouse derived monoclonal antibodies continue to be the affinity reagentof choice in proteomics analyses, but their production against noveltargets remained restricted by high tissue culture load and lowthroughputscreening methods. Microarray technology has become a crucial tool forlarge-scale and highthroughput biology. It allows fast, easy and paralleldetection of thousands of addressable elements in a single experiment.Here we present a method of hybridoma detection using antigen microarrayassay (AMA), which simultaneously detects antibody-antigen binding. Wewant to find a highthroughput method to produce mAb.Firstly, We expressed 4 proteins with two vectors in E. coil. One forimmunization, the other for detection. We immunised a single Balb/c mousewith four antigens compared to the traditional method which immunize onekind of antigen to one mice one time and form hybridomas using the standardprotocols. Lastly, We detected the hybridoma by WB, IIF and AMA, andcompared the consistancy between ELISA and AMA.The 8 recombinant proteins were highly expressed in E.coil; our system isolated 24 monoclonal hybridomas against total 4 targets in a single batch run; 7 of the 24 could be used in WB, 5 could be used in IIF; We screened 24 monoclonal hybridomas with AMA, and found it was highly compatible between ELISA and AMA. In conclusion, we have developed a novel method for the highthroughput production of mouse derived mAbs using multiplexed immunizations and a parallel screening protocol based on antigen coated microarrays.