ABSTRACT
In order to investigate peptide mimics of carbohydrate blood group A antigen, a phage display 12-mer peptide library was screened with a monoclonal antibody against blood group A antigen, NaM87-1F6. The antibody-binding properties of the selected phage peptides were evaluated by phage ELISA and phage capture assay. The peptides were co-expressed as glutathione S-transferase (GST) fusion proteins. RBC agglutination inhibition assay was performed to assess the natural blood group A antigen-mimicking ability of the fusion proteins. The results showed that seven phage clones selected bound to NaM87-1F6 specifically, among which, 6 clones bore the same peptide sequence, EYWYCGMNRTGC and another harbored a different one QIWYERTLPFTF. The two peptides were successfully expressed at the N terminal of GST protein. Both of the fusion proteins inhibited the RBC agglutination mediated by anti-A serum in a concentration-dependent manner. These results suggested that the fusion proteins based on the selected peptides could mimic the blood group A antigen and might be used as anti-A antibody-adsorbing materials when immunoabsorption was applied in ABO incompatible transplantation.
Subject(s)
Adsorption , Bacteriophages , Blood Group Antigens/chemistry , Enzyme-Linked Immunosorbent Assay , Epitopes/chemistry , Glutathione Transferase/metabolism , Peptide Library , Peptides/chemistry , Protein Structure, Tertiary , Recombinant Fusion Proteins/chemistryABSTRACT
Objective To explore the peptides that can mimic the blood type A antigen and evaluate the anti-A antibody detection value of these peptides.Methods The anti-A monoclonal antibodv (NaM87-1F6)was used to panning the phage clones from a phage display 12-mer peptide library.Positive clones were identified by phage ELISA,phage mieropanning methods.Phage DNA Was sequenced and the corresponding peptide sequences were deduced.Agglutination inhibition test WaS performed to assess the ability of phage clones to inhibit the binding between the type A red blood cell and the anti-A antibody. ABO-ELISA based on the selected peptides was compared with classical haemagglutination test jn the detection of senlm anti-A antibody.Results Seven positive clones were chosen after panning,phage ELISA and phage micropanning.Six clones displayed peptide EYWYCGMNRTGC(C5),the other one displayed peptide QIWYERTLPFTF(C17).The phages displaying the selected peptides could specifically inhibit agglutination of type A red blood cells(RBCs)by anti-A antibodies.In the ABO-ELISA based on C5 and C17,the receiver operating characteristic(ROC)Curve showed that area under curve(AUC)were 0.889 (P=0.000),0.75l(P=0.000)respectively.The Spearman correlation Coeffieient between the ABO-EliSA value and the antibody titer derived from haemagglutination assay were 0.743(P<0.01),0.664(P<0.01)respectively.As for C5,0.300 was the best cut-off for ABO-ELISA with 82.2% sensitivity and 83.3% specificity.As for C17,the sensitivity and specificity of ABO-ELISA was 68.9% and 63.3% respectively when the cut-off value was 0.250.Conclusions The peptides EYWYCGMNRTGC and QIWYERTLPFTF can mimic the blood type A antigenic epitope.ABO-ELISA based on these peptides has the potential for the detection of anti-A antibody.