Development of Monoclonal Antibodies for Detection of Conserved and Variable Epitopes of Large Protein of Rabies Virus

Wen Zhao; Jingyin Su; Naiyu Zhao; Jie Liu; Shuo Su

doi:10.3390/v13020220

Development of Monoclonal Antibodies for Detection of Conserved and Variable Epitopes of Large Protein of Rabies Virus

Viruses ◽

10.3390/v13020220 ◽

2021 ◽

Vol 13 (2) ◽

pp. 220

Author(s):

Wen Zhao ◽

Jingyin Su ◽

Naiyu Zhao ◽

Jie Liu ◽

Shuo Su

Keyword(s):

Monoclonal Antibodies ◽

Rabies Virus ◽

Detection Methods ◽

L Protein ◽

Amino Acid Alignment ◽

Linear Epitopes ◽

Asian Lineage ◽

And Function ◽

Death Cases ◽

Human Death

Rabies virus (RABV) causes fatal neurological encephalitis and results in approximately 6000 human death cases worldwide every year. The large (L) protein of RABV, possessing conserved domains, is considered as the target for detection. In this study, three monoclonal antibodies (mAbs), designated as 3F3, 3A6 and L-C, against L protein were generated by using the recombinant truncated L protein (aa 1431–1754) and the epitopes were also identified using a series of overlapping truncated polypeptides for testing the reactivity of mAbs with different RABV strains. The 1479EIFSIP1484, 1659RALSK1663 and 1724VFNSL1728 were identified as the minimal linear epitopes recognized by mAbs 3F3, 3A6 and L-C, respectively. Amino acid alignment showed epitope 1724VFNSL1728 recognized by mAb L-C is completely conserved among RABV strains, indicating that mAb L-C could be used to detect all of the RABV strains. Epitope 1479EIFSIP1484 is highly conserved among RABV strains except for a P1484S substitution in a China I sub-lineage strain of Asian lineage, which eliminated the reactivity of the epitope with mAb 3F3. However, the epitope 1659RALSK1663 was only completely conserved in the Africa-2 and Indian lineages, and a single A1660T substitution, mainly appeared in strains of the China I belonging to Asian lineage and a Cosmopolitan lineage strain, still retained the reactivity of the epitope with mAb 3A6. While both A1660T and K1663R substitutions in a China I lineage strain, single K1663R/Q substitution in some China II strains of Asian lineage and some Arctic-like lineage strains and R1659Q mutation in a strain of Africa-3 lineage eliminated the reactivity of the epitope with mAb 3A6, suggesting mAb 3A6 could be used for differentiation of variable epitopes of some strains in different lineages. Thus, variability and conservation of the three epitopes of L protein showed the reactive difference of mAbs among RABV strains of different lineages. These results may facilitate future studies in development of detection methods for RABV infection, the structure and function of RABV L protein.

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Identification of Mycoplasma hyorhinis P37 protein-specific B cell linear epitopes using monoclonal antibodies against baculovirus-expressed P37 protein

10.21203/rs.2.9644/v1 ◽

2019 ◽

Author(s):

Hongzhen Zhu ◽

Yanwu Wei ◽

Liping Huang ◽

Dan Liu ◽

Yongxing Xie ◽

...

Keyword(s):

Monoclonal Antibodies ◽

B Cell ◽

Critical Role ◽

Diagnostic Techniques ◽

Etiologic Agent ◽

Antigenic Structure ◽

Mycoplasma Hyorhinis ◽

Linear Epitopes ◽

Baculovirus System ◽

And Function

Abstract Background Mycoplasma hyorhinis (Mhr) is the etiologic agent of lameness and polyserositis in swine. Mhr P37 is a membrane protein that may play a critical role in immunity. It is a potential target for diagnostic development, but there is little information concerning its B cell epitopes. To investigate the epitopes of Mhr P37, a recombinant protein was developed in a baculovirus system using monoclonal antibodies (mAbs) prepared against P37 protein. Results Western blot and indirect immunofluorescence assays (IFA) confirmed that the expressed P37 protein was recognized by Mhr-positive porcine and mouse sera. Furthermore, the P37 protein was purified using affinity chromatography and used to immunize mice for hybridoma cell fusion. Four mAbs were found to be positive for Mhr. A panel of truncated P37 proteins was used to identify the minimal B cell linear epitopes of the protein based on these mAbs. The core epitope was determined to be 206KIKKAWNDKDWNTFRNF222. Conclusions This study identified mAbs that could provide useful tools for investigating the antigenic structure and function of the Mhr P37 protein as well as its application to diagnostic techniques.

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Strategic Aspects of NPY-Based Monoclonal Antibodies for Diagnosis and Treatment of Breast Cancer

Current Protein and Peptide Science ◽

10.2174/1389203721666200918151604 ◽

2020 ◽

Vol 21 (11) ◽

pp. 1097-1102

Author(s):

Drashti Desai ◽

Pravin Shende

Keyword(s):

Breast Cancer ◽

Monoclonal Antibodies ◽

Diagnosis And Treatment ◽

Detection Methods ◽

Active Immunotherapy ◽

Immune Checkpoints ◽

Proliferation Index ◽

Protein Fusion ◽

Ki 67 ◽

Cost Efficient

: Immunotherapy emerges as a treatment strategy for breast cancer marker, diagnosis and treatment. In this review, monoclonal antibodies (mAbs)-based passive and peptide vaccines as active immunotherapy approaches like activation of B-cells and T-cells are studied. Passive immunotherapy is mAbs-based therapy effective against tumor cells, which acts by targeting HER2, IGF 1R, VEGF, BCSC and immune checkpoints. Neuropeptide Y (NPY) and GPCR are the areas of interest to target BC metastases for on-targeting therapeutic action. Neuropeptide S (NPS) or NPS receptor 1, acts as a biomarker for Neuroendocrine tumors (NET), mostly characterized by synaptophysin and chromogranin-A expression or Ki-67 proliferation index. The protein fusion technologies arise as a promising avenue in plant expression systems for increased recombinant Ab accumulation and cost-efficient purification. Recently, mAbs-based immunotherapy effectiveness is appreciated as a novel therapeutic combination of chemotherapy and immunotherapy to reduce the side effects and improve therapeutic responsiveness. Synthetic drug resistance will be overcome by mAbs-based therapy through several clinical trials and detection methods need to be optimized for accuracy and precision. Pharmacokinetic attributes need to be accessed for preferred receptor-agonist activity without ligand accumulation.

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Characterization of conformation-specific monoclonal antibodies against rabies virus nucleoprotein

Archives of Virology ◽

10.1007/s00705-010-0709-x ◽

2010 ◽

Vol 155 (8) ◽

pp. 1187-1192 ◽

Cited By ~ 14

Author(s):

Yan Jiang ◽

Yonghuang Luo ◽

Frank Michel ◽

Robert J. Hogan ◽

Ying He ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Rabies Virus

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Regulation of human lung fibroblast phenotype and function by vitronectin and vitronectin integrins

Journal of Cell Science ◽

10.1242/jcs.114.19.3507 ◽

2001 ◽

Vol 114 (19) ◽

pp. 3507-3516 ◽

Cited By ~ 1

Author(s):

Amelia K. Scaffidi ◽

Yuben P. Moodley ◽

Markus Weichselbaum ◽

Philip J. Thompson ◽

Darryl A. Knight

Keyword(s):

Extracellular Matrix ◽

Smooth Muscle ◽

Monoclonal Antibodies ◽

Human Lung ◽

Stress Fibers ◽

Collagen Gels ◽

Human Lung Fibroblast ◽

Myofibroblast Phenotype ◽

And Function ◽

Blocking Antibodies

Myofibroblasts, characterised by high expression of α-smooth muscle actin (α-SMA), are important and transient cells in normal wound healing but are found in increased number in various pathological conditions of the lung including asthma and pulmonary fibrosis. The mechanisms that regulate the myofibroblast phenotype are unknown but are likely to involve signals from the extracellular matrix transmitted via specific integrins. Vitronectin is a glycoprotein released during inflammation and has been shown to regulate the phenotype of vascular smooth muscle cells via αv and β1 integrins. In the current study we have examined whether vitronectin influences the phenotype and function of normal human lung fibroblasts (HFL-1). Incubation of HFL-1 cells with vitronectin induced a concentration-dependent reduction in α-SMA expression. By contrast, function-blocking monoclonal antibodies to the vitronectin integrins αv, β1, αvβ3 and αvβ5 induced the expression of α-SMA and its organization into stress fibers. Expression of α-SMA induced by all function-blocking monoclonal antibodies was abrogated by inhibition of protein kinase C and phosphatidylinositol-3 kinase, but the effects of inhibition of other signalling pathways was integrin dependent. Exposure to other extracellular matrix proteins such as fibronectin, collagen or their integrins did not influence expression of α-SMA. The expression and organization of α-SMA induced by exposure to function-blocking antibodies was translated into an augmented capacity of HFL-1 cells to contract fibroblast populated collagen gels. By contrast, contraction of collagen gels following incubation with vitronectin was not significantly different to control. This study has shown that vitronectin influences the phenotype and behaviour of HFL-1 cells by downregulating the expression of α-SMA and reducing their contractile ability. By contrast, occupancy of specific integrins by function-blocking antibodies upregulated the expression of α-SMA and induced the formation of functional stress fibers capable of contracting collagen gels. These results suggest that vitronectin modulates the fibroblast-myofibroblast phenotype, implying an important role in the remodelling process during lung development or response to injury.

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The Integrated Mechanical Function Test System of Material and Structural Fatigue Damage

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.532-533.398 ◽

2012 ◽

Vol 532-533 ◽

pp. 398-402

Author(s):

Yu Lan Wei ◽

Bing Li ◽

Sui Ying Jin ◽

Kai Kai Chen

Keyword(s):

Optimal Design ◽

Test System ◽

Integrated System ◽

Detection Methods ◽

Signal Acquisition ◽

Mechanical Function ◽

Structural Fatigue ◽

Material Functions ◽

Function Test ◽

And Function

An integrated system to measure mechanical functions of material or structure is introduced. This system is able to provide more detection methods and experiment environments. And it can discover the characteristics and mechanisms of damnification and breakage in materials, considering the effects of loading and environments. Material functions were analyzed in many aspects, including loading, strain, light, sound, temperature and infrared to ensure the safety of materials and configuration of unmanned plane. Current study has laid a foundation for realizing optimal design of unmanned plane. In this paper, theory, components, and function of the system were discussed, as well as signal acquisition and analysis.

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Antipeptide monoclonal antibodies inhibit the binding of rabies virus glycoprotein and alpha-bungarotoxin to the nicotinic acetylcholine receptor

Molecular Immunology ◽

10.1016/0161-5890(88)90125-3 ◽

1988 ◽

Vol 25 (9) ◽

pp. 881-888 ◽

Cited By ~ 32

Author(s):

Luisa Bracci ◽

Guido Antoni ◽

Maria Grazia Cusi ◽

Luisa Lozzi ◽

Neri Niccolai ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Acetylcholine Receptor ◽

Nicotinic Acetylcholine Receptor ◽

Rabies Virus ◽

Nicotinic Acetylcholine ◽

Rabies Virus Glycoprotein ◽

Virus Glycoprotein ◽

Alpha Bungarotoxin

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Biological characterization of human monoclonal antibodies to rabies virus.

Journal of Virology ◽

10.1128/jvi.64.6.3087-3090.1990 ◽

1990 ◽

Vol 64 (6) ◽

pp. 3087-3090 ◽

Cited By ~ 64

Author(s):

B Dietzschold ◽

M Gore ◽

P Casali ◽

Y Ueki ◽

C E Rupprecht ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Rabies Virus ◽

Biological Characterization ◽

Human Monoclonal Antibodies

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Monoclonal Antibodies Generated against Glycoconjugates Recognize Chemical Linkers

Antibodies ◽

10.3390/antib9030048 ◽

2020 ◽

Vol 9 (3) ◽

pp. 48

Author(s):

Jessica Ramadhin ◽

Vanessa Silva-Moraes ◽

Thomas Norberg ◽

Donald Harn

Keyword(s):

Drug Delivery ◽

Flow Cytometry ◽

Monoclonal Antibodies ◽

Structure And Function ◽

Enzyme Linked Immunosorbent Assay ◽

Incomplete Freund’S Adjuvant ◽

Delivery Platform ◽

Igg1 Mabs ◽

Human Milk Oligosaccharide ◽

And Function

Monoclonal antibodies (mAbs) that recognize glycans are useful tools to assess carbohydrates’ structure and function. We sought to produce IgG mAbs to the human milk oligosaccharide (HMO), lacto-N-fucopentaose III (LNFPIII). LNFPIII contains the Lewisx antigen, which is found on the surface of schistosome parasites. mAbs binding the Lewisx antigen are well-reported in the literature, but mAbs recognizing HMO structures are rare. To generate mAbs, mice were immunized with LNFPIII-DEX (P3DEX) plus CpGs in VacSIM®, a novel vaccine/drug delivery platform. Mice were boosted with LNFPIII-HSA (P3HSA) plus CpGs in Incomplete Freund’s Adjuvant (IFA). Splenocytes from immunized mice were used to generate hybridomas and were screened against LNFPIII conjugates via enzyme-linked immunosorbent assay (ELISA). Three positive hybridomas were expanded, and one hybridoma, producing IgG and IgM antibodies, was cloned via flow cytometry. Clone F1P2H4D8D5 was selected because it produced IgG1 mAbs, but rescreening unexpectedly showed binding to both LNFPIII and lacto-N-neotetraose (LNnT) conjugates. To further assess the specificity of the mAb, we screened it on two glycan microarrays and found no significant binding. This finding suggests that the mAb binds to the acetylphenylenediamine (APD) linker-spacer structure of the conjugate. We present the results herein, suggesting that our new mAb could be a useful probe for conjugates using similar linker spacer structures.

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Rational design of peptides for identification of linear epitopes and generation of neutralizing monoclonal antibodies against DKK2 for cancer therapy

Antibody Therapeutics ◽

10.1093/abt/tbaa004 ◽

2020 ◽

Vol 3 (2) ◽

pp. 63-70 ◽

Cited By ~ 1

Author(s):

Rongqing Zhao ◽

Qian Xiao ◽

Maohua Li ◽

Wenlin Ren ◽

Chenxi Xia ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Tumor Growth ◽

Wnt Signaling ◽

Rational Design ◽

Polyclonal Antibodies ◽

Wnt Signaling Pathway ◽

Keyhole Limpet Hemocyanin ◽

Linear Epitopes

Abstract Dickkopf-related protein 2 (DKK2)is a member of the Dickkopf family in Wnt signaling pathway. Recently, we found that antibodies against DKK2 could activate natural killer (NK) and CD8+ T cells in tumors and inhibit tumor growth. In this paper, we report the rational design of peptides for identification of linear epitopes and generation of neutralizing monoclonal anti-DKK2 antibodies. To break the immune tolerance, we designed and chemically synthesized six peptides corresponding to different regions of DKK2 as immunogens and found five of them could generate mouse polyclonal antibodies that can bind to the active recombinant human DKK2 protein. Neutralizing mouse monoclonal antibodies (5F8 and 1A10) against human DKK2 were successfully developed by immunizing the mice with two different peptides (34KLNSIKSSL42 and 240KVWKDATYS248) conjugated to Keyhole limpet hemocyanin (KLH). The monoclonal antibodies not only abolish DKK2’s suppression of Wnt signaling in vitro but also inhibits tumor growth in vivo. Currently, those two mAbs are undergoing humanization as immunotherapy candidates and may offer a new drug for treatment of human cancers.

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