Anti-human Erythrocyte Antigens A And Rh (d) Phage Antibody Library And Identification

The ABO blood group system and the Rh blood group system were the most important systems in clinical transfusion medicine and organ transplantation because of their immunogenicity. So it was important to study blood group system and to get anti-ABO and anti-Rh antibodies for the prevention of haemolytic disease of the newborn, et al.Phage display library had been used in medical and biological fields recently. It was a valid approach to get antibody to study, diagnose and treat, through constructing phage display library and aim to have antibodies by panning the library.At first, the paper used EB virus transformation technique to construct immortal cell lines, which could provide cells to construct phage display library of anti-A. B lymphocytes were isolated of healthy people and transformed by EB virus. When the B cells were transformed, their supertant were assayed by agglutination. The paper had transformaed 12 samples, the eighty percent samples were successed. The supertant of transformed displayed agglutination with A red cells, several of which could agglutinate O red cells.Combined EB virus transformation technique with phage display library techniques, isolation of total RNA from B lymphoblastoid cell lines secreting anti-A antibody, synthesis of first strand cDNA, amplified V_h and V_k gene by PCR, constructed single-chain variable fragment (scFv) gene by splicing overlap extension (SOE), scFv genes were cloned into vector pCANTAB5E, transformed of E.coli TG1 cells and rescued with M13KO7 helper phage to construct the scFv-displaying phage library. Phage display library, with size being 1.2 × 10⁶, was obstained. The percentage of full-length scFv gene inserted into phage DNA was 0.90. Rescued by helper phage, a phage scFv library with titer of 2.52 × 10¹⁰pfu/mL was established. Specific phage scFv were acquired after 4 rounds of panning, 2 clones exhibited specific binding to A cell were identified by cell ELISA and agglutination.After constructing anti-A phage display library, the paper had alao constructed a anti-Rh(D) phage display library. It's size was 1.2 × 10⁷ , the percentage of full-length scFv gene inserted into phage DNA was 0.80. Rescued by helper phage, a phage scFv library with titer of 3×10⁸ pfu/mL was established. Specific phage scFv were acquired after 4 rounds of panning used intact red cells, 17 clones exhibited specific binding to Rh⁺ cell were identified by Dot Blotting. The positive recombinant phages were expressed by soluble. The positive phage were assayed for specificity by cell ELISA, Dot-blotting assay and agglutination, 9 positive phage were aquired. One clone with highest activity was chosen, DNA sequence of its scFv genes were determined.