Dengue virus (DENV) is the most widely transmitted arbovirus in the world. Due to the lack of diagnostic technology to quickly identify the virus serotypes in patients, severe dengue hemorrhagic fever cases caused by repeated infections remain high. To realize the rapid differential diagnosis of different serotypes of DENV infection by immunological methods, in this study, four DENV serotype NS1 proteins were expressed and purified in mammalian cells. Monoclonal antibodies (MAbs) against NS1 protein were obtained by hybridoma technology after immunizing BALB/c mice. Enzyme-linked immunosorbent assay, indirect immunofluorescence assay, dot blotting, and Western blotting were used to confirm the reactivity of MAbs to viral native NS1 and recombinant NS1 protein. These MAbs include not only the universal antibodies that recognize all DENV 1-4 serotype NS1, but also serotype-specific antibodies against DENV-1, DENV-2 and DENV-4. Double antibody sandwich ELISA was established based on these antibodies, which can be used to achieve rapid differential diagnosis of serotypes of DENV infection. Preparation of DENV serotype-specific MAbs and establishment of an ELISA technology for identifying DENV serotypes has laid the foundation for the rapid diagnosis of DENV clinical infection.
Animals ; Antibodies, Monoclonal ; Antibodies, Viral/metabolism* ; Dengue/diagnosis* ; Dengue Virus/immunology* ; Enzyme-Linked Immunosorbent Assay ; Humans ; Mice ; Mice, Inbred BALB C ; Sensitivity and Specificity ; Serogroup ; Viral Nonstructural Proteins/immunology*

Monoclonal antibody (mAb) is an important biological macromolecule and widely used in immune detection, in vitro diagnostics, and drug discovery. However, the inherent properties of mAb restrict its further development, such as high molecular weight and complex structure. Therefore, there is an urgent need to develop alternatives for mAb. Various types of miniaturized antibodies have been developed, among which the variable domain of immunoglobulin new antigen receptor (VNAR) is very attractive. The shark single-domain antibody, also known as shark VNAR, is an antigen-binding domain obtained by genetic engineering technology based on the immunoglobulin new antigen receptor (IgNAR) that naturally exists in selachimorpha. It has a molecular weight of 12 kDa, which is the smallest antigen-binding domain found in the known vertebrates at present. Compared with mAb, the shark VNAR exhibits various superiorities, such as low molecular weight, high affinity, tolerance to the harsh environment, good water solubility, strong tissue penetration, and recognition of the hidden epitopes. It has attracted wide attention in the fields of immunochemical reagents and drug discovery. In this review, various aspects of shark VNAR are elaborated, including the structural and functional characteristics, generating and humanization techniques, affinity maturation strategies, application fields, advantages and disadvantages, and prospects.
Animals ; Antibodies, Monoclonal ; immunology ; Antibodies, Monoclonal, Humanized ; immunology ; Antigens ; Epitopes ; metabolism ; Protein Domains ; immunology ; Receptors, Antigen ; chemistry ; immunology ; Sharks

Pepino mosaic virus (PepMV) causes severe disease in tomato and other Solanaceous crops around globe. To effectively study and manage this viral disease, researchers need new, sensitive, and high-throughput approaches for viral detection. In this study, we purified PepMV particles from the infected Nicotiana benthamiana plants and used virions to immunize BALB/c mice to prepare hybridomas secreting anti-PepMV monoclonal antibodies (mAbs). A panel of highly specific and sensitive murine mAbs (15B2, 8H6, 23D11, 20D9, 3A6, and 8E3) could be produced through cell fusion, antibody selection, and cell cloning. Using the mAbs as the detection antibodies, we established double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), Dot-ELISA, and Tissue print-ELISA for detecting PepMV infection in tomato plants. Resulting data on sensitivity analysis assays showed that both DAS-ELISA and Dot-ELISA can efficiently monitor the virus in PepMV-infected tissue crude extracts when diluted at 1:1 310 720 and 1:20 480 (weight/volume ratio (w/v), g/mL), respectively. Among the three methods developed, the Tissue print-ELISA was found to be the most practical detection technique. Survey results from field samples by the established serological approaches were verified by reverse transcription polymerase chain reaction (RT-PCR) and DNA sequencing, demonstrating all three serological methods are reliable and effective for monitoring PepMV. Anti-PepMV mAbs and the newly developed DAS-ELISA, Dot-ELISA, and Tissue print-ELISA can benefit PepMV detection and field epidemiological study, and management of this viral disease, which is already widespread in tomato plants in Yunnan Province of China.
Animals ; Antibodies, Monoclonal/immunology* ; China ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay/methods* ; Female ; Hybridomas ; Solanum lycopersicum/virology* ; Mice ; Mice, Inbred BALB C ; Plant Diseases/virology* ; Potexvirus/metabolism* ; Sensitivity and Specificity ; Nicotiana

Animals ; Antibodies, Monoclonal ; pharmacokinetics ; therapeutic use ; Antibodies, Viral ; therapeutic use ; Hepatitis B virus ; immunology ; Histocompatibility Antigens Class I ; metabolism ; Humans ; Macaca fascicularis ; Mice ; Protein Binding ; Protein Engineering ; Receptors, Fc ; metabolism

Novel biologics that redirect cytotoxic T lymphocytes (CTLs) to kill tumor cells bearing a tumor associated antigen hold great promise in the clinic. However, the ability to safely and potently target CD3 on CTL toward tumor associated antigens (TAA) expressed on tumor cells remains a challenge of both technology and biology. Herein we describe the use of a Half DVD-Ig format that can redirect CTL to kill tumor cells. Notably, Half DVD-Ig molecules that are monovalent for each specificity demonstrated reduced non-specific CTL activation and conditional CTL activation upon binding to TAA compared to intact tetravalent DVD-Ig molecules that are bivalent for each specificity, while maintaining good drug like properties and appropriate PK properties.
Animals ; Antibodies, Bispecific ; immunology ; Antibodies, Monoclonal ; immunology ; pharmacokinetics ; CD3 Complex ; metabolism ; Cell Line, Tumor ; Cytotoxicity, Immunologic ; ErbB Receptors ; metabolism ; Female ; Humans ; Lymphocyte Activation ; immunology ; Mice, SCID ; Neoplasms ; immunology ; pathology ; Rats, Sprague-Dawley ; T-Lymphocytes, Cytotoxic ; immunology

Antibodies, Monoclonal ; immunology ; Glycosylation ; Half-Life ; Humans ; Immunity ; Immunotherapy ; Protein Engineering ; T-Lymphocytes ; metabolism

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

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

OBJECTIVETo investigate the effects of inhibiting TIM-4 function in Kupffer cells (KCs) on liver graft rejection in mice and explore the underlying mechanism.

METHODSMouse models of orthotopic liver transplantation were treated with a control mAb group and TIM-4 mAb. The activated KCs were assayed with immunohistochemistry after operation. The expression of TIM-4 in KCs were assayed with Western blotting and RT-PCR and the levels of AST, ALT, TBIL, TNF-α, IFN-γ and CCL2 were assayed detected. The expression of TIM-4 in KCs was observed with laser confocal microscopy. HE staining was used to observe the microstructure of the liver tissues, and the number of CD25Foxp3T cells was determined using with flow cytometry; the proteins levels of p-P65and p-P38 were assayed with Western blotting. The donor mice were treated with clodronate liposomes to destroy the KCs in the liver before transplantation, and the liver grafts were examined for graft rejection.

RESULTSThe number of activated KCs in the liver graft increased progressively over time. Compared with the sham-operated group, the liver graft showed significantly increased TIM-4 protein and mRNA levels at 1, 3, and 7 days after transplantation (P<0.05) and increased levels of AST, ALT, TBIL, TNF-α, IFN-γ and CCL2 at 7 days (P<0.05). The graft in TIM-4 mAb group showed mild pathological changes with a mean RAI score of 2.67∓0.75, which was significantly lower than that in control mAb group (P<0.05). The mean survival time of the recipient mice was 53.8∓6.4 days in TIM-4 mAb group, significantly longer than that in the control mAB group (14.5∓2.9 days, P<0.05). Donor treatment with clodronate liposomes resulted in comparable RAI scores in TIM-4 mAb and control mAb groups (8.01∓0.64 vs 7.93∓0.56, P>0.05). The protein levels of p-P65 and p-P38 in TIM-4 mAb group were significantly lower than those in control mAb group (P<0.05), and CD25Foxp3T cells in the liver graft increased significantly in TIM-4 mAb group.

CONCLUSIONInhibition of TIM-4 function in KCs reduces the production of inflammatory factors after liver transplantation possibly by inhibiting the NF-κB and MAPK signaling pathways and promoting the proliferation of Foxp3Treg cells to induce allograft tolerance.

Animals ; Antibodies, Monoclonal ; pharmacology ; Graft Rejection ; Immunohistochemistry ; Kupffer Cells ; drug effects ; metabolism ; Liver ; surgery ; Liver Transplantation ; Male ; Membrane Proteins ; antagonists & inhibitors ; Mice ; NF-kappa B ; metabolism ; T-Lymphocytes, Regulatory ; immunology

Inflammatory bowel disease (IBD) is a chronic progressive idiopathic inflammatory disorder that involves the digestive tract from the mouth to the anus. Over the past decades, many therapeutic strategies have been developed to manage IBD, but therapeutic strategies based only on relief of clinical symptoms have not changed the natural history of this disease entity. This underlines the importance of understanding the natural history of IBD itself. When we look at the natural history of Crohn's disease (CD), it first begins with inflammation of the intestinal mucosa and this inflammatory reaction proceeds to stenosing or penetrating reaction if not adequately controlled. However, it takes a considerable amount of time before mucosal inflammation proceeds to stenosis of the intestinal lumen or penetration into the adjacent bowel. Therefore, it can be expected that if proper care is given during that period, progression of CD to such a complicated disease could be prevented. Even though the concept of mucosal healing was introduced in the early 1990s, no correlation could be observed between healing of mucosal lesions and relief of clinical symptoms. However, the introduction of biologic agents targeting tumor necrosis factor has changed the way to treat IBD that is refractory to standard medications and has allowed us to aim for a new therapeutic goal, 'deep remission'. Further advances in biologic agents have provided highly effective treatments for IBD, making deep remission a realistic goal. Whether IBD patients may benefit by experiencing a 'deep' remission beyond the control of clinical symptoms need to be evaluated in further investigation. Nevertheless, it can be anticipated that attaining deep remission might ultimately have an impact on important outcomes such as the need for surgery and the quality of life.
Anti-Inflammatory Agents, Non-Steroidal/therapeutic use ; Antibodies, Monoclonal/therapeutic use ; Colitis, Ulcerative/drug therapy/metabolism/pathology ; Crohn Disease/drug therapy/metabolism/pathology ; Humans ; Inflammatory Bowel Diseases/drug therapy/metabolism/*pathology ; Intestinal Mucosa/metabolism/pathology ; Mesalamine/therapeutic use ; Tumor Necrosis Factor-alpha/immunology/metabolism