Production of seven monoclonal equine immunoglobulins isotyped by multiplex analysis.
Abstract: Horses have 11 immunoglobulin isotypes: IgM, IgD, IgA, IgE, and seven IgG subclasses designated as IgG1-IgG7, each of which are distinguished by separate genes encoding the constant heavy chain regions. Immunoglobulin (Ig) isotypes have different functions during the immune response and pathogen-specific isotypes can be used as indicators for immunity and protection from disease. In addition to existing monoclonal antibodies to various equine Igs, quantification of the individual isotypes requires pure isotype standards. In this report, we describe a fusion between X63-Ag8.653 mouse myeloma cells and horse PBMC to create equine-murine heterohybridomas. Initial screening for Ig production was performed by ELISA. Further testing was performed by a new 5-plex fluorescent bead-based assay able to simultaneously detect equine IgM, IgG1, IgG4/7, IgG5, and IgG6. Production of IgG3 and IgE was tested by separate bead assays. Seven stable heterohybridoma clones producing monoclonal equine IgM, IgG1, IgG3, IgG4/7, IgG5, IgG6 and IgE were created. Purified Ig isotypes were then tested by SDS-PAGE. The pure, monoclonal equine Ig isotypes and the new equine Ig multiplex testing developed here are valuable tools to quantify antibody responses and to accurately determine individual isotypes concentrations in horses.
Copyright © 2013 Elsevier B.V. All rights reserved.
Publication Date: 2013-02-19 PubMed ID: 23541920DOI: 10.1016/j.vetimm.2013.02.010Google Scholar: Lookup
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Summary
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This research is focused on the creation of hybrid cell lines (heterohybridomas) that produce equine monoclonal immunoglobulins (Ig), helping to measure specific immune responses and concentration of individual immunoglobulin isotypes in horses.
Introduction
- The study revolves around horses, which are known to have 11 immunoglobulin isotypes (IgM, IgD, IgA, IgE, and seven IgG subclasses- IgG1-IgG7). These isotypes are differentiated by separate genes that encode the constant heavy chain regions.
- Each immunoglobulin (Ig) isotype has distinct roles within the immune response and pathogen-specific isotypes may be used as indicators for immunity as well as protection from disease.
- Quantifying individual isotypes necessitates pure isotype standards.
Methodology
- The researchers used a X63-Ag8.653 mouse myeloma (a type of cancer cell) fused with horse peripheral blood mononuclear cells (PBMC) to create heterohybridomas (a hybrid cell line).
- A preliminary screening was carried out for Ig production using the Enzyme-Linked Immunosorbent Assay (ELISA) method.
- A new 5-plex fluorescent bead-based assay was created to simultaneously detect equine IgM, IgG1, IgG4/7, IgG5, and IgG6.
- IgG3 and IgE production were tested by separate bead assays.
Results
- The study successfully established seven stable heterohybridoma clones. These clones produce different equine immunoglobulins – IgM, IgG1, IgG3, IgG4/7, IgG5, IgG6, and IgE.
- The immunoglobulin isotypes were purified and tested using SDS-PAGE (Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis), an analytical technique used to separate protein based on their electrophoretic mobility.
Conclusion
- The pure, monoclonal equine Ig isotypes which have been produced, along with the new multiplex testing technique, showed promising results in quantifying antibody responses and accurately determining individual isotype concentrations in horses.
- This study, therefore, provides valuable mechanisms to study the immune response of horses, thus becoming essential tools in equine medicine and research.
Cite This Article
APA
Keggan A, Freer H, Rollins A, Wagner B.
(2013).
Production of seven monoclonal equine immunoglobulins isotyped by multiplex analysis.
Vet Immunol Immunopathol, 153(3-4), 187-193.
https://doi.org/10.1016/j.vetimm.2013.02.010 Publication
Researcher Affiliations
- Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA.
MeSH Terms
- Animals
- Antibodies, Monoclonal / biosynthesis
- Antibodies, Monoclonal / immunology
- Cell Fusion
- Horses
- Hybridomas / immunology
- Immunoglobulin Isotypes / biosynthesis
- Immunoglobulin Isotypes / immunology
- Mice
- Molecular Weight
Citations
This article has been cited 8 times.- Badenhorst M, Saalmüller A, Daly JM, Ertl R, Stadler M, Puff C, de le Roi M, Baumgärtner W, Engelmann M, Brandner S, Junge HK, Pratscher B, Volz A, Saunier B, Krey T, Wittmann J, Heelemann S, Delarocque J, Wagner B, Todt D, Steinmann E, Cavalleri JV. An Equine Model for Vaccination against a Hepacivirus: Insights into Host Responses to E2 Recombinant Protein Vaccination and Subsequent Equine Hepacivirus Inoculation.. Viruses 2022 Jun 27;14(7).
- Schnabel CL, Fletemeyer B, Lübke S, Marti E, Wagner B, Alber G. CD154 Expression Indicates T Cell Activation Following Tetanus Toxoid Vaccination of Horses.. Front Immunol 2022;13:805026.
- Raza F, Ivanek R, Freer H, Reiche D, Rose H, Torsteinsdóttir S, Svansson V, Björnsdóttir S, Wagner B. Cul o 2 specific IgG3/5 antibodies predicted Culicoides hypersensitivity in a group imported Icelandic horses.. BMC Vet Res 2020 Aug 10;16(1):283.
- Larson EM, Babasyan S, Wagner B. Phenotype and function of IgE-binding monocytes in equine Culicoides hypersensitivity.. PLoS One 2020;15(5):e0233537.
- Jonsdottir S, Fettelschoss V, Olomski F, Talker SC, Mirkovitch J, Rhiner T, Birkmann K, Thoms F, Wagner B, Bachmann MF, Kündig TM, Marti E, Fettelschoss-Gabriel A. Safety Profile of a Virus-Like Particle-Based Vaccine Targeting Self-Protein Interleukin-5 in Horses.. Vaccines (Basel) 2020 May 9;8(2).
- Schnabel CL, Wimer CL, Perkins G, Babasyan S, Freer H, Watts C, Rollins A, Osterrieder N, Wagner B. Deletion of the ORF2 gene of the neuropathogenic equine herpesvirus type 1 strain Ab4 reduces virulence while maintaining strong immunogenicity.. BMC Vet Res 2018 Aug 22;14(1):245.
- Carossino M, Wagner B, Loynachan AT, Cook RF, Canisso IF, Chelvarajan L, Edwards CL, Nam B, Timoney JF, Timoney PJ, Balasuriya UBR. Equine Arteritis Virus Elicits a Mucosal Antibody Response in the Reproductive Tract of Persistently Infected Stallions.. Clin Vaccine Immunol 2017 Oct;24(10).
- Wagner B, Perkins G, Babasyan S, Freer H, Keggan A, Goodman LB, Glaser A, Torsteinsdóttir S, Svansson V, Björnsdóttir S. Neonatal Immunization with a Single IL-4/Antigen Dose Induces Increased Antibody Responses after Challenge Infection with Equine Herpesvirus Type 1 (EHV-1) at Weanling Age.. PLoS One 2017;12(1):e0169072.
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