Rapid Screening And Identification Of Antigenic Peptides And Their Applications In Immunoassay And Antibody Preparation

The epitopes or antigenic determinants is the core parts of the antigen involved in its recognition by an antibody. Nowadays, there are lots of methods to investigate and confirm epitopes of antigens. Such as proteolytic cleavage of antigen-monoclonal antibody (MAb) complexes proteolytic or chemical cleavage fragments method Western blotting PEPSCAN method chemical modification or mutation analyses and chemosynthesis of peptide or surface-displayed peptide libraries and random fragment expression libraries, X-ray crystallographic assay and nuclear magnetic resonance spectroscopy assay, and so on. However, most of these methods are complicated difficult to perform and with low efficiency. With the development of computer technology and bioinformation, the research works show that the epitopes arestrongly associated with the structure features of amino acid sequences of protein antigen. As a result, a sets of methods for epitope predicting were developed based on the structure property, these methods can predict antigenic determinants with about 75% accuracy. These epitope predicting methods offer a chance for rapid epitope determination, for predict can reduce the range of possible epitope, so analyzing epitopes by experiments following predicts, will bring us much less workloads.In order to investigate the epitopes of two main antigen protein Urease B (UreB) and Cytotoxin associated gene A(CagA) from Helicobacter pylori (Hp), the second structure of the protein sequences were predicted by Garnier-Robson's method and Chou-Fasman's method, the flexible regions were analyzed by Karplus-Schulz's method, the hydrophilicity regions were predicted by Kyte-Doolittle's method, the surface probability was analyzed by Emini's method, the antigenic index was analyzed by Jameson-Wolf's method, and the antigenic determinants were predicted by Kolaskar-Tongaonkar's method. After comparison of these multiple parameters assay results, 11 amino acid fragments from UreB protein and 9 amino acid fragments from CagA protein were predicted as possible epitopes.In order to analyze and determine the antigenicity of designed epitopes, we synthesize linear peptide antigens by Fmoc solid phase peptide synthesis strategy according to the amino acid sequences of epitopes, and all the synthetic peptides were labeled FITC dye. The productions of antigenic peptides were purified by highperformance liquid chromatography (HPLC) with 99% purity, and the molecular weights of antigenic peptides were coherence with the expectant ones. The fluorescence polarization (FP) assay is one effective technique for research the interaction of molecules in solution. Using FP technique, the antigenicity of antigenic peptides can be determined by analyzing the recognition and combination between antigenic peptides and standard antibody samples. The results show that 9 of 11 UreB synthetic peptides and 6 of 9 CagA synthetic peptides have antigenicity.Commonly, there are several epitopes in one protein antigen, the antigenicity are different among these epitopes. The dominant antigenic peptides of UreB and CagA were screened using 159 UreB antibody positive antiserums and 106 CagA antibody positive antiserums respectively by FP technique. The results show that No.2 No.5 No.11 antigenic peptides of UreB and No.1 No.5 antigenic peptides of CagA were dominant among the samples. Using these dominant antigenic peptides, a new method for UreB and CagA antibody detection based on fluorescence polarization (FP) technique was developed. Using this method to detect Hp infection, the results show that the antibody positive ratio was 51.9% assay by 3 UreB antigenic peptides, the sensitivity and specificity were 84.3% and 98.2% separately; the antibody positive ratio was 33.1% analyzed by 2 CagA antigenic peptides, the sensitivity and specificity were 53.9% and 99.1% separately. CagA antibody assay can be used for virulent Hp identification.Antibody is a very important tool for researches of proteins...