Oxidized low density lipoprotein (LDL) seems to take a part in atherogenesis through direct interactions with macrophages, endothelial cells, and smooth muscle cells, and is thought to participate in renal glomerular injury. For the purpose of illustrating the role of oxidized LDL in the human diseases, monoclonal antibodies were developed and characterized, recognizing oxidized LDL-specific epitopes that do not exist on native LDL. LDL was oxidized by the incubation with CuSO4, and used as immunogen. Splenocytes from the immunized mouse and mouse myeloma cells were fused to produce hybridomas, which were screened for the secretion of oxidized LDL-specific antibodies. Immunoblot analysis and binding affinity assay showed that these monoclonal antibodies recognize malondialdehyde-conjugated peptide epitopes.
Antibodies, Monoclonal ; Antibody Affinity ; Antibody Specificity ; Enzyme-Linked Immunosorbent Assay ; Epitopes ; Human ; Lipoproteins, LDL/immunology* ; Malondialdehyde/immunology ; Malondialdehyde/analysis ; Peptide Fragments/immunology ; Thiobarbituric Acid Reactive Substances/analysis

OBJECTIVETo screen the severe acute respiratory syndromes (SARS) mimotopes with random phage peptide library and to investigate their immunogenicity.

METHODSUsing SARS sera as selective molecule, a 12 mer phage peptide library was biopanned and positive clones containing the mimic epitopes were selected. The immuno-characteriation of the epitopes were then investigated.

RESULTS2 positive clones that having specific affinity to SARS sera were obtained. The DNA sequencing data showed no homology between the sequences of the deduced amino acid of the two mimic antigen peptides and the sequence of SARS.

CONCLUSIONSARS mimotopes were obtained by phage peptide library screening. This method might provide a new approach for SARS therapy and vaccine development.

Animals ; Antigens, Viral ; Base Sequence ; Biomimetic Materials ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; Epitopes ; genetics ; immunology ; Humans ; Peptide Library ; Sequence Analysis, DNA ; Severe Acute Respiratory Syndrome ; immunology

To evaluate the effectiveness of rabies vaccination, we developed the SPA-ELISA method to detect the antibodies against rabies virus (RV) using the main antigenic determinant of nucleoprotein (RV N1) as antigen. The complete Nucleoprotein (N) gene and the partial N1 gene (1 000-1 353 bp) of RV Flury LEP strain were amplified using RT-PCR and PCR approaches. The two fragments were inserted into pGEX-6P-1 respectively. Then we transformed the recombinant plasmids into Escherichia coli BL21(DE3) strain and expressed them by adding 1 mmol/L of IPTG (isopropyl-beta-D-thiogalactopyranoside). SDS-PAGE analysis showed that both of the two recombinant proteins were presented as inclusion bodies. Compared with the complete nucleoprotein, the partial protein (RV N1) was expressed at a much higher level in E. coli BL21(DE3). The antigenic specificity of the partial N1 protein was confirmed by Western blotting. By coating the plates with purified RV N1 as an antigen, an SPA-ELISA method for the detection of the antibodies against RV was established. By optimizing this method, the optimal concentration of RV N1 coating the ELISA plate was 2 mg/L. The optimal concentration of serum samples and SPA-HRP was 1:100 and 1:4 000 respectively. Compared with a commercially available ELISA kit coating RV as antigen, the coincidence rate of SPA-ELISA was 94.1%. Our results show that the developed SPA-ELISA based on the RV N1 was useful for the detection of the antibodies against RV in the sera of dogs.
Animals ; Antibodies, Viral ; analysis ; immunology ; Dogs ; Enzyme-Linked Immunosorbent Assay ; methods ; Epitopes ; immunology ; Escherichia coli ; genetics ; Genetic Vectors ; genetics ; Nucleocapsid Proteins ; immunology ; Rabies virus ; immunology ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Staphylococcal Protein A

PURPOSE: To identify new immunogenic HLA-A*33;03-restricted epitopes from the human papillomavirus (HPV) 16 E7 protein for immunotherapy against cervical cancer. MATERIALS AND METHODS: We synthesized fourteen overlapping 15-amino acid peptides and measured intracellular interferon-γ (IFN-γ) production in PBMC and CD8+ cytotoxic T lymphocytes (CTLs) after sensitization with these peptides using flow cytometry and ELISpot assay. The immunogenicity of epitopes was verified using a ⁵¹Cr release assay with SNU1299 cells. RESULTS: Among the fourteen 15-amino acid peptides, E7₄₉₋₆₃ (RAHYNIVTFCCKCDS) demonstrated the highest IFN-γ production from peripheral blood mononuclear cells (PBMCs), and CD8+ CTLs sensitized with E7₄₉₋₆₃ showed higher cytotoxic effect against SNU1299 cells than did CD8+ CTLs sensitized with other peptides or a negative control group. Thirteen 9- or 10-amino acid overlapping peptides spanning E7₄₉₋₆₃, E7₅₀₋₅₉ (AHYNIVTFCC), and E7₅₂₋₆₁ (YNIVTFCCKC) induced significantly higher IFN-γ production and cytotoxic effects against SNU1299 cells than the other peptides and negative controls, and the cytotoxicity of E7₅₀₋₅₉- and E7₅₂₋₆₁-sensitized PBMCs was induced via the cytolytic effect of CD8+ CTLs. CONCLUSION: We identified E7₅₀₋₅₉ and E7₅₂₋₆₁ as novel HPV 16 E7 epitopes for HLA-A*33;03. CD8+ CTL sensitized with these peptides result in an antitumor effect against cervical cancer cells. These epitopes could be useful for immune monitoring and immunotherapy for cervical cancer and HPV 16-related diseases including anal cancer and oropharyngeal cancer.
Amino Acid Sequence ; CD8-Positive T-Lymphocytes/immunology/metabolism ; Epitopes/*immunology/therapeutic use ; Female ; *HLA-A Antigens ; Human papillomavirus 16/*immunology ; Humans ; *Immunotherapy ; Interferon-gamma/analysis/*biosynthesis ; Leukocytes, Mononuclear/immunology/metabolism ; T-Lymphocytes, Cytotoxic/immunology/metabolism ; Uterine Cervical Neoplasms/*therapy

To understand the HA1 genetic variation characterization of influenza H3N2 virus isolates in Zhu-hai during 2008-2009, we selected 20 of H3N2 Influenza strains cultured in MDCK cell. Viral RNAs were extracted and amplified by using RT-PCR. The amplified products were purified after identified by gel electrophoresis and then the nucleotide sequences of the amplicons were determined. The results were analyzed by the software ClustalX and MEGA4. 1. When compared with the amino acid sequences of the epitopes of HA1 district of H3N2 influenza vaccine recommended by WHO in 2008, changes were found in those of H3N2 influenza strains in Zhuhai in 2008: K140I in all of H3N2 influenza strains, L157S in 08-0343 and 08-0677, K158R in 08-0466, 08-0620 and 08-0667, K173E in 08-0466 and 08-0620, K173N in 08-0667, and I192T in 08-0667. The epitopes of HA1 district of H3N2 influenza strains in Zhuhai in 2009 are different from that of H3N2 influenza vaccine during the same time: K173Q and P194L occur in all of H3N2 influenza strains, N144K, K158N, and N189K occur in the strains except the strain 09-0056. HA1 domain of H3N2 influenza strains in 2009 has lost a glycosylation site at amino acid position 144 while the glycosylation sites of HA1 domain of H3N2 influenza stains isolated in 2008 remained. This study suggested that H3N2 influenza virus in Zhuhai in 2008 was not evolved a novel variant and H3N2 influenza variant in 2009 was attributed to antigenic drift in HA1 district.
Animals ; Antigens, Viral ; immunology ; Cell Line ; China ; Dogs ; Epitopes ; immunology ; Glycosylation ; Hemagglutinin Glycoproteins, Influenza Virus ; chemistry ; genetics ; immunology ; metabolism ; Humans ; Influenza A Virus, H3N2 Subtype ; classification ; genetics ; immunology ; isolation & purification ; Mutation ; Phylogeny ; Sequence Analysis, DNA

Amino Acid Sequence ; Amyloid beta-Peptides ; genetics ; immunology ; Enzyme-Linked Immunosorbent Assay ; Epitope Mapping ; Epitopes ; genetics ; immunology ; Humans ; Immunoglobulin Fragments ; genetics ; immunology ; Immunoglobulin Variable Region ; genetics ; immunology ; Molecular Sequence Data ; Peptide Library ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA

BACKGROUNDTo understand the antigenic and genetic characteristics of Victoria like strain of influenza B virus isolated recently and to provide a scientific evidence for influenza surveillance and monitoring of influenza epidemic in future.

METHODSViruses were passed in embryonated hen eggs and virion RNA was extracted from allantoic fluid and reverse transcribed to synthesize cDNA. cDNA was amplified by PCR and the PCR product was purified with a purification kit. Afterwards RNA sequence analysis was performed by dideoxynucleotide chain termination and a cloning method. Finally, phylogenetic analysis of the sequencing data was performed with MegAlign.

RESULTSB/Sichuan/63/2001 and B/Zhejiang/2/2001 viruses were antigenically different from B/Shandong/7/97 strain. The substitution of nucleotide sequences of HA1genes of them compared with those of B/Shandong/7/97strain resulted in the change of amino acid sequences in antigenic determinants on HA1 protein domain. The phylogenetic analysis also indicated that strains isolated recently were genetically different from B/Shandong/7/97/strain. However, there was neither differences on the antigenicity nor genetic partern between these two isotates.

CONCLUSIONSThe antigenic drift of Victoria-like strain of influenza B virus isolated recently in China has further occurred.

Amino Acid Sequence ; Antigenic Variation ; China ; Epitopes ; Genetic Variation ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; immunology ; Humans ; Influenza B virus ; genetics ; immunology ; Influenza, Human ; virology ; Molecular Sequence Data ; RNA, Viral ; analysis ; Sequence Analysis, RNA

Identification of expressed protein profiles and antigenic determination are some of the most challenging aspects of proteomics. Two-dimensional gel electrophoresis (2-DE) combined with immunoblot analysis were employed to study the N. caninum proteome. Protein sample preparation was carried out by first conducting sonication, followed by adding lysis buffer containing 7M urea plus 2M thiourea to the purified tachyzoites in order to complete disruption. A total of 335 differentially expressed protein spots were detected using pH 4-7 IPG strip (7 cm) that were run in a 56 kVh isoelectric focusing (IEF) system. Of the spots analyzed, 64 were identified as antigenic spots on immunoblot profile. Major antigenic spots appeared at 65 kDa (pI 5.2-5.3), 51 kDa (pI 5.5), 38 kDa (pI 5.1), 33 kDa (pI 4.4), 29 kDa (pI 5.6) and 15.5 kDa (pI 5.0) were observed to be significantly distinct compared to the rest of the antigenic spots. The results indicate that combination of 2-DE and immunoblotting methods were thought as very useful tools in defining both proteins and antigens of N. caninum tachyzoites. Additionally, present 2-DE profiles may be valuable in further proteomic approaches and study of the pathogen.
Animals ; Antibody Formation ; Antigens, Protozoan/*analysis/isolation&purification ; Electrophoresis, Gel, Two-Dimensional/methods ; Electrophoresis, Polyacrylamide Gel ; Epitopes/analysis ; Image Processing, Computer-Assisted ; Immunoblotting/methods ; Isoelectric Focusing ; Neospora/*chemistry/immunology ; Proteome/analysis ; Proteomics ; Protozoan Proteins/*analysis/isolation&purification

Swine influenza viruses (SwIVs) cause considerable morbidity and mortality in domestic pigs, resulting in a significant economic burden. Moreover, pigs have been considered to be a possible mixing vessel in which novel strains loom. Here, we developed and evaluated a novel M2e-multiple antigenic peptide (M2e-MAP) as a supplemental antigen for inactivated H3N2 vaccine to provide cross-protection against two main subtypes of SwIVs, H1N1 and H3N2. The novel tetra-branched MAP was constructed by fusing four copies of M2e to one copy of foreign T helper cell epitopes. A high-yield reassortant H3N2 virus was generated by plasmid based reverse genetics. The efficacy of the novel H3N2 inactivated vaccines with or without M2e-MAP supplementation was evaluated in a mouse model. M2e-MAP conjugated vaccine induced strong antibody responses in mice. Complete protection against the heterologous swine H1N1 virus was observed in mice vaccinated with M2e-MAP combined vaccine. Moreover, this novel peptide confers protection against lethal challenge of A/Puerto Rico/8/34 (H1N1). Taken together, our results suggest the combined immunization of reassortant inactivated H3N2 vaccine and the novel M2e-MAP provided cross-protection against swine and human viruses and may serve as a promising approach for influenza vaccine development.
Animals ; Antibodies, Viral/blood ; Antigens, Viral/genetics/*immunology ; Body Weight ; Cross Protection/*immunology ; Disease Models, Animal ; Epitopes, T-Lymphocyte/genetics/immunology ; Female ; Influenza A Virus, H3N2 Subtype/genetics/*immunology ; Influenza Vaccines/*immunology ; Mice ; Mice, Inbred BALB C ; Orthomyxoviridae Infections/*immunology/mortality/pathology/prevention & control ; Peptides/genetics/*immunology ; Random Allocation ; Survival Analysis ; Vaccines, Synthetic/immunology ; Virus Replication

A highly specific antigenic protein of 31 kDa from plerocercoid of Spirometra mansoni (sparganum) was obtained by gelatin affinity and Mono Q anion-exchange column chromatography. The purified 31 kDa protein was subjected to N-glycan enzymatic digestion for structural analysis. The relative electrophoretic mobility was analyzed by SDS-PAGE, before and after digestion. On SDS-PAGE after enzymatic digestion, the 31 kDa protein showed a molecular shift of approximately 2 kDa, which indicated the possession of complex N-linked oligosaccharides (N-glycosidase F sensitive) but not of high-mannose oligosaccharides (endo-beta-N-acetylglucosaminidase H, non-sensitive). Chemically periodated 31 kDa protein showed statistically non-significant changes with human sparganosis sera by enzyme linked immunosorbent assay (ELISA). Therefore, the dominant epitopes of the 31 kDa molecule in human sparganosis were found to be mainly polypeptide, while N-glycans of the antigenic molecule in sparganum was minimal in anti-carbohydrate antibody production.
Animals ; Antigens, Helminth/*analysis/chemistry/immunology/metabolism ; Carbohydrates/analysis/immunology ; Chromatography, Affinity ; Chromatography, Ion Exchange ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Epitopes/analysis/immunology ; Glucosaminidase/metabolism ; Human ; Peptide-N4- (N-acetyl-beta-glucosaminyl) Asparagine Amidase/metabolism ; Periodic Acid/chemistry ; Sparganosis/*parasitology ; Sparganum/immunology/metabolism ; Spirometra/immunology/*metabolism ; Support, Non-U.S. Gov't