The N-terminal sequence of HIV1 gp41 (amino acid residues 584-623) was known to be the immundominant region of HIV1 gp41 protein. In order to determine epitope for gp41 protein of Korean anti-HIV1 positive sera, multiple antigenic peptides (MAPs) for the sequences corresponding to 584-604, 590-612, 604-623 and 584-618 of HIV1 gp41 were synthesized by solid phase method using Fmoc-Lys (Fmoc)-OH and used as coating antigens for ELISA. The reactivities of the synthetic peptides with Korean HIV1 positive (21 samples) and anti-HIV1 negative sera (22 samples) obtained from healthy blood doner were estimated by an indirect ELISA. MAPs for 584-604, 590-612 and 604-623 of gp41 reacted with 62 %, 100 % and 81 % of Korean anti-HIV1 positive sera tested, respectively. The results suggest that the epitope for HIV1 gp 41 for Korean anti-HIV1 positive sera is located in the region of amino acid 590-612 of gp41. MAP for gp41 (584-618) reacted with all (100 %) of anti-HIV1 positive sera tested, but did not react with anti-HlV1 negative sera. In addition, this MAP reacted stronger with seven samples of anti-HIV1 positive sera of anti-HIV1/2 combo performance panel than the mixture of 584-604, 590-612 and 604-623 of gp41, but did not react with anti-HIV negative serum. The high sensitivity and selectivity of MAP of gp41 (584-618) suggest that this peptide as a coating antigen in an ELISA system will be useful for antibody detection of HIV1.
Enzyme-Linked Immunosorbent Assay ; Epitope Mapping* ; Immune Sera* ; Peptides*

To establish a method for identifying protein epitopes recognized by therapeutic monoclonal antibodies, the programmed death receptor-1 (PD-1) was selected as the target protein. Based on the alanine scanning strategy, a rapid expression method of antigen mutants combining site-directed mutagenesis with mammalian cell expression system was established, the conditions for eukaryotic expression element amplification and cell transfection expression were established. 150 PD-1 protein mutants were co-expressed, and the binding ability of these mutants to anti-PD-1 antibody Pembrolizumab was identified. The epitopes of Pembrolizumab were determined based on the binding ability of protein mutants to antibodies and combined with protein structure analysis, which was highly consistent with the reported crystal structure-based epitopes, indicating that this method is simple and accurate and can be used for epitope mapping of therapeutic monoclonal antibodies.
Animals ; Antibodies, Monoclonal ; Antigens ; Epitope Mapping ; Epitopes/genetics*

Computer aid vaccine design (CAVD) is a new area of biomedical engineering. This article reviewed the background of the coming of CAVD and its main research contents, methods and applications. Free tools of the trade in CAVD are listed; the implication and the perspective of CAVD are also suggested.
Animals ; Biomedical Engineering ; instrumentation ; trends ; Computer-Aided Design ; Drug Design ; Epitope Mapping ; Humans ; Software ; Vaccines ; biosynthesis

Cellular prion protein, a membrane protein, is expressed in all mammals. Prion protein is also found in human blood as an anchorless protein, and this protein form is one of the many potential sources of misfolded prion protein replication during transmission. Many studies have suggested that beta-amyloid1-42 oligomer causes neurotoxicity associated with Alzheimer's disease, which is mediated by the prion protein that acts as a receptor and regulates the hippocampal potentiation. The prevention of the binding of these proteins has been proposed as a possible preventative treatment for Alzheimer's disease; therefore, a greater understanding of the binding hot-spots between the two molecules is necessary. In this study, the epitope mapping immunoassay was employed to characterize binding epitopes within the prion protein and complementary epitopes in beta-amyloid. Residues 23-39 and 93-119 in the prion protein were involved in binding to beta-amyloid1-40 and 1-42, and monomers of this protein interacted with prion protein residues 93-113 and 123-166. Furthermore, beta-amyloid antibodies against the C-terminus detected bound beta-amyloid1-42 at residues 23-40, 104-122 and 159-175. beta-Amyloid epitopes necessary for the interaction with prion protein were not determined. In conclusion, charged clusters and hydrophobic regions of the prion protein were involved in binding to beta-amyloid1-40 and 1-42. The 3D structure appears to be necessary for beta-amyloid to interact with prion protein. In the future, these binding sites may be utilized for 3D structure modeling, as well as for the pharmaceutical intervention of Alzheimer's disease.
Amyloid beta-Peptides/*metabolism ; Electrophoresis ; Enzyme-Linked Immunosorbent Assay ; *Epitope Mapping ; Epitopes/*metabolism ; Humans ; *Immunoassay ; Prions/*metabolism ; Protein Binding ; Recombinant Proteins/metabolism

We wished to assess the role of chlamydia micro virus capsid protein Vp3 in recombinant molecules, chart its molecular evolution, screen the wild-type strain, and reveal its value in clinical research. Using a protein BLAST multiple-alignment program, we compared various strains of Chlamydia micro virus capsid protein Vp3 sequences. Using a "distance tree" of those results, we created a phylogenetic tree. We applied the Karplus-Schulz method of flexible-region analyses for highly conserved alignments of amino-acid sequences. Gamier-Robson and Chou-Fasman methods were employed to analyze two-level structures of sequences. The Emini method was used for analyses of the accessibility of surface epitopes. Studies of hydrophilic proteins were undertaken using Kyte-Doolittle and Hopp-Woods methods. Analyses of antigen epitopes helped to reveal the antigen index using the Jameson-Wolf method. All sequences in the six strains of chlamydia micro virus capsid protein Vp3 were highly conserved, with the main differences being between Vp3 protein in Chp1 and the other five strains of the micro virus. The viral strain of Vp3 protein was based mainly on micro-alpha helix structures, and multiple epitopes were noted in highly conserved regions. Vp3 protein was highly conserved structurally, and was an important protein of the chlamydiaphage capsid. Vp3 protein has a complicated molecular structure, highly conserved regions with strong immunogenicity, and has considerable research value.
Amino Acid Sequence ; Capsid Proteins ; chemistry ; genetics ; immunology ; Chlamydia ; genetics ; immunology ; Conserved Sequence ; Epitope Mapping ; Evolution, Molecular ; Molecular Sequence Data ; Recombination, Genetic

Ebola virus (EBOV) causes highly lethal hemorrhagic fever in humans and nonhuman primates and has a significant impact on public health. The nucleoprotein (NP) of EBOV (EBOV-NP) plays a central role in virus replication and has been used as a target molecule for disease diagnosis. In this study, we generated a monoclonal antibody (MAb) against EBOV-NP and mapped the epitope motif required for recognition by the MAb. The MAb generated via immunization of mice with prokaryotically expressed recombinant NP of the Zaire Ebola virus (ZEBOV-NP) was specific to ZEBOV-NP and able to recognize ZEBOV-NP expressed in prokaryotic and eukaryotic cells. The MAb cross-reacted with the NP of the Reston Ebola virus (REBOV), the Cote-d'Ivoire Ebola virus (CIEBOV) and the Bundibugyo Ebola virus (BEBOV) but not with the NP of the Sudan Ebola virus (SEBOV) or the Marburg virus (MARV). The minimal epitope sequence required for recognition by the MAb was the motif PPLESD, which is located between amino acid residues 583 and 588 at the C-terminus of ZEBOV-NP and well conserved among all 16 strains of ZEBOV, CIEBOV and BEBOV deposited in GenBank. The epitope motif is conserved in four out of five strains of REBOV.
Animals ; Antibodies, Monoclonal ; immunology ; Ebolavirus ; chemistry ; immunology ; Epitope Mapping ; methods ; Escherichia coli ; genetics ; metabolism ; Mice ; Mice, Inbred BALB C ; Nucleoproteins ; immunology ; Recombinant Proteins ; biosynthesis ; genetics ; immunology

In order to develop monoclonal antibodies (McAbs) against the gp90 protein of reticuloendotheliosis virus (REV), the His-tagged gp90 protein of REV was used to immunize BALB/c mice. Hybridomas were generated by fusing mouse myeloma cells SP2/0 with the splenocytes from the immunized mice. After screening and 3 rounds of cloning process, 3 hybridomas (3G5-B8, 3G5-A10 and 1G12) that stably secreted McAbs against the REV-gp90 were obtained. The isotypes of the McAbs were determined to be IgG1, IgG1 and IgG2b. The McAbs specifically bound to gp90 in REV-infected DF-1 cells, as demonstrated by Western blotting and indirect immunofluorescence assay. The recognition regions on gp90 that were recognized by 3G5-B8/3G5-A10 and 1G12 were located between amino acids 200 to 245 and 230 to 235, respectively, as demonstrated by Western blotting analysis. These McAbs will be useful in the diagnosis and pathogenesis study of REV.
Animals ; Antibodies, Monoclonal ; biosynthesis ; Antibody Specificity ; Blotting, Western ; Epitope Mapping ; Hybridomas ; Immunoglobulin G ; biosynthesis ; Mice ; Mice, Inbred BALB C ; Reticuloendotheliosis virus ; immunology ; Viral Envelope Proteins ; immunology

OBJECTIVETo screen HCV NS5 mimotopes by using monoclonal antibody and phage peptide library.

METHODSBy using HCV NS5 monoclonal antibody as selective molecule, a 7 peptide phage library was biopanned and positive clones were selected by ELISA, competition assay and DNA sequencing.

RESULTSTwelve positive clones were chosen for DNA sequencing. From the experiment and sequencing comparison results, one epitope was confirmed as the mimotope of HCV NS5.

CONCLUSIONSHCV mimotope is obtained by phage peptide library screening. The result provides a new approach for HCV therapy and vaccine development.

Amino Acid Sequence ; Antibodies, Monoclonal ; immunology ; Enzyme-Linked Immunosorbent Assay ; Epitope Mapping ; Hepatitis C ; therapy ; Peptide Library ; Viral Nonstructural Proteins ; chemistry ; genetics ; immunology ; Viral Vaccines ; immunology

Newcastle disease (ND) caused by virulent Newcastle disease virus (NDV) is a highly contagious viral disease of poultry. Virulent NDVs characteristically have a multibasic amino acid sequence (virulence motif) such as (112)RRQKRF(117) at the cleavage site of the precusor fusion (F0) protein. The antigenic and immunogenic characteristics of the virulence motif (112)RRQKRF(117) in the F0 protein of virulent NDVs were investigated. Epitope mapping analysis revealed that a RRQKRF-specific monoclonal antibody 4G2 recognized the KRF section of the motif. A synthetic peptide bearing the RRQKRF motif reacted strongly with sera from virulent NDV (with RRQKRF motif)-infected chickens. These sera also showed reactivity to peptides bearing other virulence motifs ((112)KRQKRF(117), (112)RRQRRF(117) and (112)RRRKRF(117)) but not an avirulence motif ((112)GRQGRL(117)) by ELISA. The synthetic bearing RRQKRF motif reacted with 60% to 91% of sera taken from surviving chickens on ND outbreak farms but not with sera from vaccinated birds, even though most of the sera had antibody to NDV due to vaccination. This indicates that the virulence motif has the potential to differentiate virulent NDV infected birds from vaccinated birds.
Amino Acid Motifs/*immunology ; Amino Acid Sequence ; Animals ; Chickens ; Enzyme-Linked Immunosorbent Assay/veterinary ; Epitope Mapping/veterinary ; Newcastle Disease/*immunology ; Newcastle disease virus/*genetics/pathogenicity ; Poultry Diseases/*immunology/*virology ; Serologic Tests/veterinary ; Viral Fusion Proteins/*genetics/immunology ; Virulence/genetics

CD99 plays an critical role in the diapedesis of monocytes, T cell differentiation, and the transport of MHC molecules. Engagement of CD99 by agonistic monoclonal antibodies has been reported to trigger multifactorial events including T cell activation as well as cell-cell adhesion during hematopoietic cell differentiation. In this study, to identify the functional domains participating in the cellular events, we mapped the epitopes of CD99, which are recognized by two agonistic CD99 monoclonal antibodies, DN16 and YG32. Using recombinant fusion proteins of GST with whole or parts of CD99, we found that both antibodies interact with CD99 molecules independently of sugar moieties. DN16 mAb detected a linear epitope located in the amino terminal region of CD99 while YG32 mAb bound another linear epitope in the center of the extracellular domain. To confirm that the identified epitopes of CD99 are actually recognized by the two mAbs, we showed the presence of physical interaction between the mAbs and the fusion proteins or synthetic peptides containing the corresponding epitopes using surface plasmon resonance analyses. The dissociation constants of DN16 and YG32 mAbs for the antigen were calculated as 1.27 X 10(-7) and 7.08 X 10(-9) M, respectively. These studies will help understand the functional domains and the subsequent signaling mechanism of CD99.
Amino Acid Sequence ; Antibodies, Monoclonal/*immunology ; Antigens, CD/*chemistry/*immunology ; Blotting, Western ; Cell Adhesion Molecules/*chemistry/*immunology ; *Epitope Mapping ; Epitopes/*chemistry/*immunology ; Glutathione Transferase ; Human ; Molecular Sequence Data ; Peptide Fragments/chemistry/immunology ; Recombinant Fusion Proteins/chemistry/immunology