| Objective To find the mimic epitope of Rh(D) blood group antigen from the phagepeptide library which has been screened by pooled anti-Rh(D) polyclonal antibody ofhuman-oriented purification. This method would provide an experimental basis for theresearch of new-type diagnostic antigen for the RhD-Hemolytic disease of newborn(RhD-HDN), for preparation of molecular vaccine and exploration of novel therapeuticagent.Methods1. Five cases of Rh(D)-negative sera that had been indentified to be positiveand contain anti-Rh(D) antibodies through screening by polyclonal antibodies werecollected and mixed in the same ratio. Rh(D)(-)A and RhD(-)B type packederythrocytes were used to absorb anti-A and anti-B antibodies in the sera respectively at4℃overnight. The ghost cells were prepared from the RhD-positive and recombinantexpression of type CCDee erythrocytes of O-type, which were released from the mixedsera with osmotic concentration of30mmol/L PB as the lysates. These cells were usedas the antigens to adsorb anti-Rh(D) antibodies from the mixture. Anti-Rh(D) antibodieswere eluted, separated and purified by low pH buffer solution. The IgG fraction ofanti-Rh(D) antibody was extracted by DEAE-Sephadex A-50chromatography, the stripcomponents were identified by Western blot, and the titer were detected by microcolumn technique.2. The purified anti-Rh(D) polyclonal antibody was used to screen the phage display12-mer random peptide library three times, and the enrichment ratio of each round wascalculated. Affinities of eluate with anti-Rh(D) antibody and affinities of anti-Rh(D) sera with26phage clones from round3were detected by ELISA. The16phage clonesin the test with an A450value above0.5were screened out preliminarily to detect theaffinities of each phage clone with the antibody. The DNA of positive clones wasextracted and sequenced. The the amino acids of the foreign polypeptide were deduced.The simulative effect of affinities of the phage polypeptide with Rh(D) blood groupantigen was tested by agglutination inhibition test.Results1. The sera from all5investigated subjects contained anti-Rh(D) antibody, andthe titer of the antibody in the mixed sera was detected by micro column method is32.The anti-A and anti-B type antibodies in the sera were entirely absorbed. After theabsorption, the titer of both anti-A and anti-B was below1:2. The prepared ghost cellsshowed a strong antigenicity. After purification of the anti-Rh(D) antibody, the titer ofIgG components reached1:32.2. After3rounds of screening, the enrichment ratio rised from (4.45×10-6)%to(4.27×10-5)%. As the screening rounds increase, the affinities of the phage elutionwith anti-Rh(D) antibody increase gradually. The26phage clones from round3havedifferent affinities with anti-Rh(D) antibody, the2,8,12,17,21,22clones show a highaffinity with the antibody. The titer of elution from the serum by preliminary screeningshows that clones2,8,12,17,21,22have a higher content after the combination withthe antibody, and the content in the elution after BSA reaction was very low. In theabove6positive clones, the4phage clones show the identical DNA sequence of5’-CCGAGCCAGGGGTACGTGATACTGCTGGACGAGAAG-3’ and a same aminoacid sequence (PSQGYVILLDEK), named C2. The other2phage clones share theDNA sequence of5’-ACAATGTTAAATCGTGCTCATGATTTTTCTGAATGT-3’ andthe same amino acid sequence (TMLNRAHDFSEC), named C21. The agglutinationinhibition test results showed that the phage of the two polypeptides could inhibit theagglutination of positive Rh(D) antigen erythrocytes. Conclusion1. The study successfully purified the anti-Rh(D) polyclonal antibody andobtained the IgG antibodies with high titer, which layed the research foundation for thefurther use of phage peptide library to get Rh(D) blood group antigen epitope.2. Polypeptides of PSQGYVILLDEK and TMLNRAHDFSEC can simulate the Rh(D)blood group antigen epitope, which has potential in development of new diagnosticantigen and preparation of candidate vaccines for RhD-HDN. |
PDF Full Text Request