The voltage-gated Na channel subtype Nav1.7 is important for pain and itch in rodents and humans. We previously showed that a Nav1.7-targeting monoclonal antibody (SVmab) reduces Na currents and pain and itch responses in mice. Here, we investigated whether recombinant SVmab (rSVmab) binds to and blocks Nav1.7 similar to SVmab. ELISA tests revealed that SVmab was capable of binding to Nav1.7-expressing HEK293 cells, mouse DRG neurons, human nerve tissue, and the voltage-sensor domain II of Nav1.7. In contrast, rSVmab showed no or weak binding to Nav1.7 in these tests. Patch-clamp recordings showed that SVmab, but not rSVmab, markedly inhibited Na currents in Nav1.7-expressing HEK293 cells. Notably, electrical field stimulation increased the blocking activity of SVmab and rSVmab in Nav1.7-expressing HEK293 cells. SVmab was more effective than rSVmab in inhibiting paclitaxel-induced mechanical allodynia. SVmab also bound to human DRG neurons and inhibited their Na currents. Finally, potential reasons for the differential efficacy of SVmab and rSVmab and future directions are discussed.
Animals ; Antibodies, Monoclonal ; therapeutic use ; Biotin ; metabolism ; Cells, Cultured ; Disease Models, Animal ; Female ; Ganglia, Spinal ; cytology ; HEK293 Cells ; Humans ; Hybridomas ; chemistry ; Hyperalgesia ; drug therapy ; Male ; Mice ; Mice, Inbred C57BL ; NAV1.5 Voltage-Gated Sodium Channel ; metabolism ; NAV1.7 Voltage-Gated Sodium Channel ; chemistry ; immunology ; metabolism ; Neuralgia ; drug therapy ; metabolism ; Protein Binding ; drug effects ; Recombinant Proteins ; biosynthesis ; therapeutic use ; Sensory Receptor Cells ; drug effects ; physiology

OBJECTIVETo prepare rabbit monoclonal antibody (RabMab) against guanosine 3', 5'-cyclic monophosphate (cGMP) and to develop a competitive ELISA for the detection of cGMP.

METHODSNew Zealand white rabbits were immunized with synthesized cGMP-keyhole limpet hemoeyanin (cGMP-KLH) to prepared a RabMAb with monoclonal antibody technique of Epitomics. A competitive ELISA kit was produced with cGMP RabMAb. The specificity, the precision and the recoveries of the method were determined.

RESULTSThe RabMAb with high sensitivity towards cGMP were prepared with an antibody timer of 3.1 ng/mL and 50% inhibitive concentration (IC50) of 12.57 ng/mL. The cGMP RabMAb had 33% cross-reactivity to inosine 3', 5'-cyclic monophosphate (cIMP) and little or no cross-reactivity to other compounds. A competitive ELISA was developed for detection of cGMP. The range of detection was 0~120 ng/mL with a minimal limit of 1.95 ng/mL. The recovery of assay was 89%~103%. The inter-assay and intra-assay coefficient variations were below 11.68% and 13.85%, respectively.

CONCLUSIONThe RabMab against cGMP with high affinity and high specificity has been generated successfully, and a competitive ELISA for detection of cGMP has been developed with the prepared cGMP RabMAb.

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

Infectious bursal disease virus (IBDV) VP4 plays an important role in immunosuppression of host. In order to develop monoclonal antibodies (McAbs) against VP4, we vaccinated BALB/c mice with His-VP4, screened and subcloned positive clones. We established 4 hybridoma cell lines that stably secreted McAbs against VP4 and named these cell lines 3B3, 3H11, 4C8 and 4G6, respectively. We tested the dissociation constant (Kd) of these McAbs, and found that their K(d)s were 4.61 x 10(-11), 1.71 x 10(-10), 4.26 x 10(-11), 5.02 x 10(-11), respectively. The isotypes of these McAbs were determined to be IgG1, IgG1, IgG2b and IgG1. These McAbs specifically bound to VP4 in IBDV infected DF-1 cells as demonstrated by Western blotting analysis and fluorescence antibody assay. These McAbs would help to detect IBDV infection and to analyze the biological activities of IBDV VP4.
Animals ; Antibodies, Monoclonal ; biosynthesis ; Blotting, Western ; Cell Line ; Fluorescent Antibody Technique ; Hybridomas ; Infectious bursal disease virus ; Mice ; Mice, Inbred BALB C ; Viral Structural Proteins ; immunology

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

Adult ; Antibodies, Monoclonal ; pharmacology ; Bronchoalveolar Lavage Fluid ; cytology ; Cells, Cultured ; Cytokines ; biosynthesis ; blood ; secretion ; Fas Ligand Protein ; antagonists & inhibitors ; metabolism ; Humans ; Macrophages, Alveolar ; immunology ; metabolism ; Middle Aged ; Occupational Exposure ; analysis ; Signal Transduction ; Silicon Dioxide ; adverse effects ; Silicosis ; blood ; immunology ; fas Receptor ; antagonists & inhibitors ; metabolism

This research was to construct the lentiviral expression vector for anti- p185(erbB2) mouse/human chimeric antibody and to determine the expression of the chimeric antibody gene in 293T cells transfected with this vector. The genes (vL and vH) coding light and heavy chain of variable regions of anti-p185(erbB2) mAb and the constant regions of human IgG1 (kappa and gamma1) were cloned with PCR method. The target genes were assembled by three-primers PCR method to obtain the chimeric light chain (L) and the chimeric heavy chain (H). Both chains inserted into the down stream and upper stream of IRES gene of the plasmid pVAX1/IRES respectively. We digested the plasmid pVAX1/ H-IRES-L with endoenzyme and subcloned H-IRES-L into the lentiviral vector pWPI. The enzyme digestion and sequence analysis showed that the lentiviral expression vector pWPI/H-IRES-L was constructed correctly. Then, it was transfected into 293T cells and after 48h, GFP protein expression in 293T cells were detected by fluorescent microscope and the chimeric antibody expression was detected by RT-PCR and direct ELISA. The results showed that after 293T cells were transfected with recombination plasmid, both light and heavy chains of the chimeric antibody genes could express together. The chimeric antibody expressed could bind to p185(erbB2) specifically. This research may lay a sound foundation for further study of anti-p185(erbB2) engineered antibody.
Animals ; Antibodies, Monoclonal ; biosynthesis ; genetics ; Cell Line ; Chimera ; Cloning, Molecular ; Humans ; Lentivirus ; genetics ; metabolism ; Mice ; Receptor, ErbB-2 ; biosynthesis ; genetics ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Transfection

Chicken anemia virus (CAV) is an important viral pathogen that causes anemia and severe immunodeficiency syndrome in chickens worldwide. In this study, a potential diagnostic monoclonal antibody against the CAV VP1 protein was developed which can precisely recognize the CAV antigen for diagnostic and virus recovery purposes. The VP1 gene of CAV encoding the N-terminus-deleted VP1 protein, VP1Nd129, was cloned into an Escherichia (E.) coli expression vector. After isopropyl-beta-D-thiogalactopyronoside induction, VP1Nd129 protein was shown to be successfully expressed in the E. coli. By performing an enzyme-linked immunoabsorbent assay using two coating antigens, purified VP1Nd129 and CAV-infected liver tissue lysate, E3 monoclonal antibody (mAb) was found to have higher reactivity against VP1 protein than the other positive clones according to the result of limiting dilution method from 64 clones. Using immunohistochemistry, the presence of the VP1-specific mAb, E3, was confirmed using CAV-infected liver and thymus tissues as positive-infected samples. Additionally, CAV particle purification was also performed using an immunoaffinity column containing E3 mAb. The monoclonal E3 mAb developed in this study will not only be very useful for detecting CAV infection and performing histopathology studies of infected chickens, but may also be used to purify CAV particles in the future.
Animals ; Antibodies, Monoclonal/biosynthesis/genetics/*immunology ; Antigens, Viral/analysis ; Capsid Proteins/genetics/*immunology ; Chicken anemia virus/genetics/*immunology ; *Chickens ; Circoviridae Infections/blood/immunology/*veterinary/virology ; Escherichia coli/genetics ; Immunohistochemistry/veterinary ; Liver/virology ; Mice ; Mice, Inbred BALB C ; Microscopy, Fluorescence/veterinary ; Poultry Diseases/blood/immunology/*virology ; Specific Pathogen-Free Organisms ; Thymus Gland/virology

To develop a method based on immunoreactions for detection of Ectropis obliqua Nucleopolyhedrovirus (EoNPV), the polyhedra of the virus were purified and used to immunize the mouse BALB/c. The spleen cells from the immunized mice were then fused with the myeloma cell line Sp2/0. A hybridoma cell line which can stably secrete the monoclonal antibody against EoNPV was achieved by using indirect ELISA screening and cloning methods, and was named as 7D3. Meanwhile, the polyhedrin gene was cloned from EoNPV and expressed in E. coli. Western blotting analysis showed that the monoclonal antibody prepared from 7D3 could specifically react with the recombinant polyhedrin. An indirect ELISA method based on this monoclonal antibody for detecting EoNPV in infected tea looper was developed.
Animals ; Antibodies, Monoclonal ; biosynthesis ; genetics ; immunology ; Antibody Specificity ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; methods ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Hybridomas ; secretion ; Lepidoptera ; growth & development ; virology ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Viral Structural Proteins ; biosynthesis ; genetics ; immunology

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