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Objective: To identify the epitope of von Willebrand factor (vWF) recognized by monoclonal antibody SZ-125 (mAb SZ-125) using immune-affinity matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) assay in combination with peptide synthesis and amino acid site-mutagenesis technology., Methods: Recombinant vWF A3 domain (rVWF A3) was directly affinity bound to SZ-125 antibody beads and proselytized by trypsin. The digested peptide fragments were then measured using MALDI-TOF-MS. The detected peptide sequence by MALDI-TOF-MS was synthesized and several amino-acids in it were mutated to test its affinity with mAb SZ-125., Results: The epitope of rVWF A3 recognized by SZ-125 was identified to be the peptide fragment(1001);EGGPSQIGDALGFAVR(1016);. Synthesized peptide NH2;-EGGPSQIGDALGFAVR-COOH could bind to mAb SZ125., Results: of site-directed mutagenesis revealed that amino acids E1001, F1013, V1015 and R1016 played critical roles in the binding between mAb SZ-125 and rVWF A3., Conclusion: The epitope of rVWF A3 recognized by mAb SZ-125 has been accurately confirmed using immune-affinity mass spectrometry in combination with peptide synthesis and site-directed mutagenesis of special amino acids.

Aim: To prepare the monoclonal antibodies associated with hepatocellular carcinoma (HCC) for diagnosis., Methods: 3 BALB/c mice were immunized with high metastasis characteristic HCC cells (HCCLM-6). The splenocytes from the immunized mice were fused with murine myeloma cells (Sp2/0). The hybridoma cells were screened by ELISA after coating with the total protein and the culture supernatant of HCCLM-6 cells. The mAbs were characterized by immunohistochemical staining in the HCC tissues, and by indirect immunofIuorescent staining in different cell lines. The antigen and epitope recognized by the mAbs were identified by the screening premade Uni-ZAP human liver cDNA expression library., Results: Twenty-eight hybridoma cells secreting mAbs were established. One clone of the hybridomas, QGA062, secreted specific mAb associated with HCC. The antigen recognized by the mAb QGA062 was identified as fibronectin (FN), and the epitope was localized among the peptide YTVSLVAIKGNQESPK., Conclusion: The mAb against a HCC-associated epitope in FN is established and characterized, will be a very useful reagent for diagnosis of HCC.

Aim: To obtain mimic epitopes of monoclonal antibody (mAb) against Hantaan virus, and identify preliminarily their immunological characteristics., Methods: Group-specific mAbs F3 and B11 were used as selective molecules for biopanning. After biopanning, the positive phages were identified by ELISA and DNA sequencing. Among the positive phages, F3-8, B11-18, B11-20 and B11-24 were selected to immunize BALB/c mice three times, respectively. The antiserum was then titered and confirmed by sandwich ELISA and competition ELISA., Results: After 3 to 4 rounds of effective screening, the majority of the selected clones were found able to react to mAb F3 or B11 in a dose-dependent manner, but not to BSA, rHBsAg or other unrelated mAbs. The amino acid sequences of the clones binding to mAb F3 contained an identical sequence MHGPTKNQMWHT, which had higher homology to 750-759 amino acids between a pair of cysteines within glycoprotein G2 of HTNV/SEOV, while those of clones binding to mAb B11 had no evident homologous regions within HTNV/SEOV proteins. The specific antibodies increased significantly following immunization with phage peptides, which indicated that the phage-displayed peptides had not only good antigenicity, but also strong immunoreactivity., Conclusion: The phage-displayed peptides could mimic the epitope of HFRSV antigen, which would provide the potential for preparing more effective epitope-based vaccines and specific diagnostic reagents.

Aim: To analyze epitope recognized by anti-HCV antibodies; patients suffered from hepatitis C., Methods: Anti-HCV Abs were purified from the patients serum through an affinity chromatography column which was prepared with sepharose 4B coupled with protein A. These Abs were used for biopanning of a phage-displayed random 15-peptide library., Results: After 3 rounds of biopanning, the ratio of output to input increased to 3.3 x 10(3) and the false positive rate reduced to 0.2%, suggesting that the enrichment was effective. After the third round of biopanning, sixteen clones were selected to conduct binding test to Abs from the patients and normal person's sera. Nine of them were proved to react specifically to the sera from the patients. From the deduced insert sequence in the coat protein VIII, the core sequence of WPWS was found in 8 clones. The positive phage clones could react to different patients' and not react to normal person's sera., Conclusion: These findings indicate that WPWS motif in the short peptide may mimic the HCV epitope recognized by anti-HCV Abs.

A method for studying virus epidemiology in epitope level was established via phage random peptide library and thioredoxin surface display technique and the method was proved by test with core protein of HCV.

A novel method was designed for disease-specific B cell epitope mapping and epitope expression in E. coli. A phage library displaying random dodecamers was biopanned first with human total IgG antibodies against HIV-1, and then non-specific phages were subtracted by HIV(-) polyclonal antibodies. After three rounds of screening, the positive phages were tested in an ELISA for their reactivity with HIV(+)-IgG and HIV(-)-IgG antibodies. Phages that showed positive reactivity with HIV(+)-IgG, but negative to HIV(-)-IgG, were selected and their displayed peptides were determined by DNA sequencing. All the 13 positive clones sequenced displayed five kinds of peptides (SPKCLGKLLCAF, THQCLGKLQCGV, SCSAKFTCTTQI, KSDCSARFMCSV, DCLKQWACEWSR) that have homology to the HIV-1 gp41(602GCSGKLICTTNV613), demonstrating there is a dominant epitope in the region.