| The obstacle for pig-to-human transplantation is hyperacute rejection (HAR) triggered by the interaction between human natural antibodies and the antigenic epitope Gal-α1,3-Gal. The a-Gal structure is considered to be the major enoantigenic epitopes present on porcine tissues. Evidences indicate that HAR may be prevented by blocking the interaction of human natural anti-α Gal antibodies with antigenic epitope Gal-α1,3-Gal on pig tissues. The prospect of finding peptides that are able to block or remove human natural antibodies may simplify the development of potent therapeutic agents.Random peptides displayed on the phage surface offer a rich source of molecular diversity in search for ligand peptides that bind to an antibody, receptor or other binding proteins We attempted to identify peptides using a phage-displayed library XCX15. A peptide was identified that appears to bind the monoclonal anti-B antibody (mAB anti-B, which can bind Gal-α-l,3-Gal structure) at the binding site of Gal-αl,3-Gal. Our experiments have demonstrated that the positive phage bearing the peptide can block the human natural antibody-mediated agglutination of pig RBCs. The peptide can also block the agglution of pig RBCs caused by lectin BS-I-B4 (which binds the structure ofGal-α-l,3-Gal).(1) Selection of antibody-binding phage(Biopanning)mAB anti-B (titer 1:128) was coated on microtiter wells at 4癈 overnight. Wells were saturatedwith 5 % BSA. In the first panning, the phage library was incubated at room temperature for 1 hour with slowly shaking. Nonbinding phages were removed by extensive washing with TBST [Tris-buffered saline (TBS) supplemented with Tween-20 (TBST)]. The bound phages were eluted with 100 nig/ml of melibiose(which has Gal-α,6-Glc structure). Phages were amplified in Escherichici coli (kank91). Affinity selection was repeated for four times. The plate was washed with 0.1 % TBST in first two rounds, and with 0.2 and 0.5 % TBST in round 3 and 4, respectively. Although the concentration of TBST increased very much, the enrichment of positive phage clones was observed. The yield of the fourth round was approximately 5-time more than that of the first round.(2) Detection of binding of phage to antibody by ELISAA flat-bottomed microtiter plate was coated overnight at 4℃ with 100 ul of theanti-B antibody. Wells were then saturated with 5% BSA and then washed with 5 mM TBS for three times. Purified phages (50 ul) was added into each well. The wells were washed with 0.2 % TBST to remove unbound phages, and then rabbit anti-M13-HRP was added into each well and incubated at room temperature for 1 hour. Unbound rabbit anti-Mi3-HRP was removed by washing with 0.1% TBST for 10 times. The substrate 3,3',5,5'-tetramethylbenzidine (TMB) was used for development, and the reaction was carried on for 30 minutes, and then terminated by adding 50 ul of 2 M H2SO4. Solute optical density in each well was measured at 450 nm by a microplate reader. After biopanning, 17 clones were randomly picked and screened. 5 out of them were performed the ELISA again to compare the binding activity. All clones selected were able to bind the antibody. Among them, clone 35 had strongest binding ability, and therefore was chosen for the competitive binding assay with melibiose.(3) Inhibition of binding of antibody to peptide by melibioseIn order to test the melibiose(Gal-α 1,3-Glc) inhibition of peptide binding to anti-B antibody, different concentrations of melibiose were mixed with the phage solution before the phage was added into the antibody-coated wells. All other procedures were same as the above ELISA method.The binding to monoclonal anti-B antibody of phage 35 was competitively inhibited by melibiose at the final concentration range of 0.003-300 mM. This result suggested that the phage was bound to monoclonal anti-B antibody at the melibiose binding site. (4) sequence to identify consensus motifsThe positive phage single-stranded DNA was extracted and purified according to Molecular Clonin... |
PDF Full Text Request