Construction Of Anti-A, B Blood Type Single-chain Fv (scFV)

Single-chain antibody (Single chain FV, scFV) is a widely used small molecule weight recombinant antibodies. The antibody heavy chain variable region VH and light chain variable region VL was fused together by a flexible connecting linker. As the smallest functional antigen-binding structural unit, the size of scFV is only one-sixth of a full antibody, and it has all the specificity of parental antibody. Single-chain antibody is easy to penetrate and has low immunogenicity and a short half-life in vivo. It also can be expressed in large quantity, easy to be genetically manipulated.Combined with the concept of personalized cancer therapy, our strategy is to test the feasibility to build a unique single-chain antibody library from individual patient's peripheral blood and to find unique antibodies that can recognize the tumors of each individual.As a proof of concept, we isolated the peripheral blood lymphcyotes from a healthy individual and generated cDNA from that. Based on literature reading and the antibody database, we designed a series of primers that cover the entire antibody genes to PCR amplify VH and VL fragment. Through the random combination of the primers it can also amplified some single-chain antibody (scFV) which didn't exist in vivo due to the immune tolerance. So we can get more types of antibodies than in vivo.A total of 31 primers was designed,75 seperated PCR reactions was conducted to ensure the quality of the scFV library. The heavy chain and light chain fragments were fused together through a flexible linker with sequence of (GGGGS) 3. We used electroporation to prepare single-chain antibody library to increase transformation' efficiency therefore increase the library size. Eventually we constructed a scFV library with at least 2×106 complexities.We used ELISA to screen single-chain antibodies against A, B blood type, so we first need to explore the condition to coat the blood membrane to the high-affinity microplate. Through microplate reader to detect the coat is successful or not, it turns out that we successfully explore the conditions to coat the membrane for ELISA screening.