A Protein Microarray-based Strategy For High-throughput Screening Of Specific Nanobodies

Antibodies are a group of immunoglobins,which can bind specifically to given antigens.As a category of key biological reagents,antibodies have already been extensively and successfully applied in basic research,clinical studies as well as therapeutics.However,it is time-consuming for traditional antibody generation,and tedious animal immunization is required.There is no animal immunization for phage display based antibody generation,however,following the current procedure,only one or a few antibodies could generated in one project.We are in an era of-omics,for omics study,it is usually necessary to globally study the proteome of a given organism.Thus,it is needed for us to develop a high-throughput,fast and general antibody generation technology.In order to meet the requirement of high-throughput antibody generation,we have combined nanobody phage display and proteome microarray,tried to screen antibodies for thousands of proteins in a single project.Nanobody is a specific type antibody originally discovered in camel.In comparison to traditional antibody,nanobody is much smaller,soluble and very stable.In this study,we first evaluated our nanobody phage display lib by next generation sequencing,and determined its diversity is 7×108,which is suitable for large-scale screening.After optimization,we then have performed 5 rounds of screening of the nanobody library on the Mycobacterium tuberculosis proteome microarray(4,262 proteins).The microarray results clearly demonstrated that specific and high affinity nanobodies have been significantly enriched after 3 rounds of panning.In order to recover the nanobodies that specifically bound the immobilized proteins on the microarray,a laser capture microdissection(LCM)protocol has been developed.One key for this protocol is the substrate slides,which should suitable for both LCM and microarray protein immobilization.After several rounds of comparison,the PET-GMA(Polyethylene terephthalate-Glycidyl methacrylate)substrate slide was selected because it has the best overall performance.After optimization for positioning and washing,we have established a high-throughput and high efficient protein spot recover procedure.Finally,take the M.tb proteome microarray as a model,we have obtained a full set of nanobodies for every individual M.tb protein.Taken together,taking advantage of two high-through technologies,i.e.,protein microarray and nanobody phage display,we have principally established an efficient protocol for globally discovering of specific antibodies for a given set of proteins or even a proteome.We strongly believe that after further optimization,we can then provide a brand-new technology for antibody generation in the era of-omics.