Entero virus 71 (EV71) causes hand, foot, and mouth disease (HFMD) and occasionally leads to severe neurological complications and even death. Scavenger receptor class B member 2 (SCARB2) is a functional receptor for EV71, that mediates viral attachment, internalization, and uncoating. However, the exact binding site of EV71 on SCARB2 is unknown. In this study, we generated a monoclonal antibody (mAb) that binds to human but not mouse SCARB2. It is named JL2, and it can effectively inhibit EV71 infection of target cells. Using a set of chimeras of human and mouse SCARB2, we identified that the region containing residues 77-113 of human SCARB2 contributes significantly to JL2 binding. The structure of the SCARB2-JL2 complex revealed that JL2 binds to the apical region of SCARB2 involving α-helices 2, 5, and 14. Our results provide new insights into the potential binding sites for EV71 on SCARB2 and the molecular mechanism of EV71 entry.
Amino Acid Sequence ; Animals ; Antibodies, Monoclonal ; chemistry ; genetics ; metabolism ; Binding Sites ; Cell Line ; Crystallography, X-Ray ; Enterovirus A, Human ; drug effects ; genetics ; growth & development ; immunology ; Fibroblasts ; drug effects ; virology ; Gene Expression ; HEK293 Cells ; Humans ; Immunoglobulin Fab Fragments ; chemistry ; genetics ; metabolism ; Lysosome-Associated Membrane Glycoproteins ; chemistry ; genetics ; immunology ; Mice ; Models, Molecular ; Protein Binding ; Protein Conformation, alpha-Helical ; Protein Conformation, beta-Strand ; Protein Interaction Domains and Motifs ; Receptors, Scavenger ; chemistry ; genetics ; immunology ; Receptors, Virus ; chemistry ; genetics ; immunology ; Recombinant Fusion Proteins ; chemistry ; genetics ; immunology ; Sequence Alignment ; Sequence Homology, Amino Acid ; Sf9 Cells ; Spodoptera ; Thermodynamics

With the application of monoclonal antibody technology more and more widely, its production technology is becoming more and more perfect. Small molecule monoclonal antibody technology is becoming a hot research topic for people. The application of traditional Chinese medicine small molecule monoclonal antibody technology has been more and more widely, the technology for effective Chinese medicine component knockout provide strong technical support. The preparation of monoclonal antibodies and small molecule knockout technology are reviewed in this paper. The preparation of several steps, such as: in the process of preparation of antigen, hapten carrier coupling, coupling ratio determination and identification of artificial antigen and establishment of animal immunization and hybridoma cell lines of monoclonal antibody, the large-scale preparation; small molecule monoclonal antibody on Immune in affinity chromatography column method is discussed in detail. The author believes that this technology will make the traditional Chinese medicine research on a higher level, and improve the level of internationalization of Chinese medicine research.
Animals ; Antibodies, Monoclonal ; chemistry ; genetics ; immunology ; Humans ; Hybridomas ; metabolism ; Immunologic Techniques ; methods ; trends

Antibodies, Monoclonal ; chemistry ; immunology ; Antigens, Viral ; chemistry ; genetics ; immunology ; Binding Sites ; Capsid Proteins ; chemistry ; genetics ; immunology ; Epitopes ; chemistry ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Hepatitis E ; immunology ; prevention & control ; virology ; Hepatitis E virus ; chemistry ; immunology ; Humans ; Molecular Docking Simulation ; Mutagenesis, Site-Directed ; Peptide Mapping ; Protein Binding ; Recombinant Proteins ; chemistry ; genetics ; immunology ; Viral Hepatitis Vaccines ; administration & dosage ; biosynthesis

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

BACKGROUNDPrevious studies showed that anti MHC-II monoclone antibody (MAb) only had partial inhibiting effect of alloreactive mixed lymphocyte reaction (MLR) in vitro and it was unsteady and non-persistent. The aim of this research was to determine whether radioactive isotope (188)Re marked MHC-II antibody could benefit the allograft acceptance in transplantation as compared to normal MHC-II antibody.

METHODS188Re was incorporated to 2E9/13F (ab')(2) which is against swine MHC class II antigen (MAb-(188)Re). Porcine peripheral blood mononuclear (PBMC) cells were examined for proliferation and cytokine mRNA expression after stimulation with MHC-II MAb or MAb-(188)Re.

RESULTSThe proliferative response of recipient PBMCs in mixed lymphocyte reaction (MLR) to donor alloantigen showed that the stimulation index of MAb-(188)Re group was significantly lower than the MHC-II MAb group and control (P < 0.05). mRNA expression of interleukin 2, interferon Υ and tumor necrosis factor α (type 1 cytokines) was lower in MAb-(188)Re group than the MHC-II MAb group, while interleukin 10 (type 2 cytokines) was higher in MAb-(188)Re group in the first 24 hours.

CONCLUSIONMAb-(188)Re could help the graft acceptance by inhibiting T cell proliferation, lowering the expression of type 1 cytokines and elevating the type 2 cytokines produced by PBMC.

Animals ; Antibodies, Monoclonal ; chemistry ; pharmacology ; Cell Proliferation ; drug effects ; Interleukin-10 ; genetics ; Interleukin-2 ; genetics ; Isoantigens ; immunology ; Leukocytes, Mononuclear ; drug effects ; radiation effects ; Lymphocyte Culture Test, Mixed ; Mitomycin ; pharmacology ; Radioisotopes ; Reverse Transcriptase Polymerase Chain Reaction ; Rhenium ; Swine ; Tumor Necrosis Factor-alpha ; genetics

A mouse-anti-human monoclonal antibody was produced by using the membrane proteins of human lung carcinoma cell line A549 as the immunogen to generate monoclonal antibodies against lung carcinoma with the use of hybridoma techniques. McAb4E7 was prepared successfully. To identify its antigen, proteomic technologies such as two-dimenstional electrophoresis, western blotting and mass spectrometry were employed. The targeting antigen of McAb4E7 expressed positive in human lung cancer cell lines A549 and human hepatocarcinoma cell line HepG2, moreover, the expression of the antigen was stronger in A549 cells. Finally, we obtained one positive protein in A549 cell line that has strong affinity and specificity for McAb4E7, which was identified to be ATP synthase beta subunit. We identified ATP synthase beta subunit as the targeting antigen of lung carcinoma special monoclonal antibody McAb4E7.
Animals ; Antibodies, Monoclonal ; biosynthesis ; chemistry ; immunology ; Antibodies, Neoplasm ; immunology ; Antibody Specificity ; Antigens, Neoplasm ; genetics ; immunology ; Cell Line, Tumor ; Humans ; Lung Neoplasms ; immunology ; Membrane Proteins ; immunology ; Mice ; Mice, Inbred BALB C ; Mitochondrial Proton-Translocating ATPases ; immunology

OBJECTIVETo examine inhibition action of multi-glycoside of Tripterygium wilfordii (GTW) on infiltration of inflammatory cell in glomeruli with anti-Thy1.1 glomerulonephritis (anti-Thy1.1 GN), and to clarify its effects on inflammatory in vitro.

METHODTwo types of anti-Thy1.1 GN were induced in rats by a single or two intravenous injections with 500 microg of anti-Thy1.1 mAb 1-22-3. Rats were randomly divided into two groups, the GTW group and control group, and sacrificed on day 7 or on day 42 after induction of anti-Thy1.1 GN. Daily oral administration of different dose of GTW and distilled water as a control was started from 3 days before injection or at the same time of injection till the day of sacrifice. Proteinuria was determined during days 7 or during days 42. Infiltration of macrophage and T lymphocyte in glomeruli and mRNA expression of interleukin (IL)-2 and interferon (IFN)-gamma in renal tissue were examined.

RESULTIncrease of infiltration of macrophage in reversible anti-Thy1.1 GN model, glomerular macrophage infiltration and IL-2 mRNA expansion were attenuated by higher dose of GTW (75 mg x kg(-1) x d(-1)), and increased accumulation of activated macrophage and T lymphocyte in irreversible anti-Thy1.1 GN model, accumulation of macrophage and T lymphocyte in glomeruli and mRNA expansion of IL-2 and IFN-gamma were decreased by middling dose of GTW (50 mg x kg(-1) x d(-1)) as well. Proteinuria was significantly ameliorated after GTW administration.

CONCLUSIONThe findings suggested that different dose of GTW can ameliorate infiltration of inflammatory cell in glomeruli with anti-Thy1.1 glomerulonephritis in vitro by decreasing the expression of IL-2 and IFN-gamma.

Animals ; Antibodies, Monoclonal ; immunology ; Dose-Response Relationship, Drug ; Female ; Gene Expression Regulation ; drug effects ; Glomerulonephritis ; immunology ; metabolism ; pathology ; physiopathology ; Glycosides ; pharmacology ; Inflammation ; metabolism ; pathology ; physiopathology ; Interferon-alpha ; genetics ; Interleukin-2 ; genetics ; Kidney Glomerulus ; drug effects ; pathology ; Macrophages ; drug effects ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats ; T-Lymphocytes ; drug effects ; metabolism ; Tripterygium ; chemistry

Antibodies, Monoclonal ; biosynthesis ; immunology ; Antibodies, Neoplasm ; biosynthesis ; immunology ; Antibody Affinity ; genetics ; Bacteriophages ; immunology ; Base Sequence ; Cell Line, Tumor ; Cells, Cultured ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Genetic Vectors ; Humans ; Immunohistochemistry ; Liver Neoplasms ; immunology ; therapy ; Plasmids ; genetics ; Single-Chain Antibodies ; chemistry ; immunology

In order to develop an anti-human TNF-alpha mAb, mice were immunized with recombinant human TNF-alpha. A murine mAb, TSK114, which showed the highest binding activity for human TNF-alpha was selected and characterized. TSK114 specifically bound to human TNF-alpha without cross-reactivity with the homologous murine TNF-alpha and human TNF-beta TSK114 was found to be of IgG1 isotype with kappa light chain. The nucleotide sequences of the variable regions of TSK114 heavy and light chains were determined and analyzed for the usage of gene families for the variable (V), diversity (D), and joining (J) segments. Kinetic analysis of TSK114 binding to human TNF-alpha by surface plasmon resonance technique revealed a binding affinity (KD) of ~5.3 pM, which is about 1,000- and 100-fold higher than those of clinically relevant infliximab (Remicade) and adalimumab (Humira) mAbs, respectively. TSK114 neutralized human TNF-alpha-mediated cytotoxicity in proportion to the concentration, exhibiting about 4-fold greater efficiency than those of infliximab and adalimumab in WEHI 164 cells used as an in vitro model system. These results suggest that TSK114 has the potential to be developed into a therapeutic TNF-alpha-neutralizing antibody with picomolar affinity.
Amino Acid Sequence ; Animals ; Antibodies, Monoclonal/chemistry/genetics/*immunology ; Antibody Affinity/*immunology ; Antibody Specificity ; Base Sequence ; Blotting, Western ; Cell Line ; Cytotoxicity, Immunologic ; Enzyme-Linked Immunosorbent Assay ; Humans ; Immunoglobulin Variable Region/genetics ; Kinetics ; Mice ; Mice, Inbred BALB C ; Molecular Sequence Data ; Neutralization Tests ; Sequence Analysis, Protein ; Tumor Necrosis Factor-alpha/*immunology

A monoclonal antibody (8H5), which showed strong neutralization activity against 33 strains of H5N1 viruses isolated from hosts at various regions from 2002 to 2006, was characterized in our lab recently. This result indicated the presence of highly conserved neutralizing site on hemagglutinin (HA) of various H5N1 subtypes. In the present study, the peptide phage display technique was applied to generate mimotope of the conserved neutralizing epitope recognized by 8H5 mAb. Five peptides displayed on phage were identified to specifically bind to 8H5 mAb. One of the five peptides, 123, was further displayed on the virus-like particle assembled from aa 1-149 fragment of HBcAg. The chimeric particle HBc-T123 conserved the specific binding to 8H5 mAb, and competed with H5N1 viruses for 8H5 mAb. The antiserum induced by HBc-T123 intensively stained on SF21 cells infected by recombinant baculovirus containing HA gene of YU22 virus, indicating the production of cross-reactive antibody to H5N1 HA.
Amino Acid Sequence ; Animals ; Antibodies, Monoclonal ; immunology ; Epitopes ; chemistry ; genetics ; immunology ; Hemagglutinin Glycoproteins, Influenza Virus ; chemistry ; genetics ; immunology ; Humans ; Influenza A Virus, H5N1 Subtype ; chemistry ; genetics ; immunology ; Influenza, Human ; virology ; Mice ; Mice, Inbred BALB C ; Molecular Sequence Data ; Peptide Library