High-throughput antibody generation and characterization

A phage display library has been constructed containing over 1010 human antibodies, allowing the large-scale generation of antibodies. Over 38,000 recombinant antibodies against 292 antigens were selected, screened and sequenced, and 4,400 resultant unique clones characterized further.

Background: Isolation of recombinant antibody fragments from antibody libraries is well established using technologies such as phage display. Phage display vectors are ideal for efficient display of antibody fragments on the surface of bacteriophage particles. However, they are often inefficient for expression of soluble antibody fragments, and sub-cloning of selected antibody populations into dedicated soluble antibody fragment expression vectors can enhance expression.

S U M M A R YTissue microarray (TMA) technology allows the miniaturization and characterization of multiple tissue samples on a single slide and commonly uses formalin-fixed paraffin-embedded (FFPE) tissue or acetone-fixed frozen tissue. The former provides good morphology but can compromise antigenicity, whereas the latter provides compromised morphology with good antigenicity. Here, we report the development of TMAs in glycol methacrylate resin, which combine the advantages of both methods in one embedding format. Freshly collected tissue fixed in Ϫ 20C acetone or 10% neutral buffered formaldehyde were cored and arrayed into an intermediary medium of 2% agarose before infiltration of the agarose array with glycol methacrylate resin. Acetone-fixed resin TMA demonstrated improved morphology over acetone-fixed frozen TMA, with no loss of antigenicity. Staining for extracellular, cell surface, and nuclear antigens could be realized with monoclonal and polyclonal antibodies as well as with monomeric single-chain Fv preparations. In addition, when compared with FFPE TMA, formalin-fixed tissue in a resin TMA gave enhanced morphology and subcellular detail. Therefore, resin provides a universal format for the construction of TMAs, providing improved tissue morphology while retaining antigenicity, allows thin-section preparation, and could be used to replace preparation of frozen and FFPE TMAs for freshly collected tissue.