Development of Monoclonal Antibodies for Analysis of Immune Responses in Livestock
Foreign Animal Disease Research
2013 Annual Report
1a.Objectives (from AD-416):
The objective of this agreement is to develop a number of monoclonal antibodies specific for T cell derived cytokines in cattle and swine and to develop a series of monoclonal antibodies to bovine leukocyte antigen (BoLA) gene products in order to develop a rapid method to tissue type cattle.
The specific objectives are:
1. Produce paired monoclonal antibodies to porcine cytokines IL-13 and IL-15.
2. Produce paired mononclonal antibodies to bovine cytokines IL-13, IL-15, IL-17 and IL-21.
3. Produce a collection of monoclonal antibodies that will differentially bind alleles of the BoLA Class I and Class II major histocompatibility complex (MHC) proteins that can be used to rapidly analyze blood samples and determine MHC gene expression in a given cow.
4. Produce monoclonal antibodies to porcine immunoglobulins.
1b.Approach (from AD-416):
ARS, PIADC and Green Mountain Antibodies will exchange reagents and produce monoclonal antibodies to cattle and swine proteins. Genes encoded with specific proteins will be cloned into the replication defective human adenovirus 5 vector (huAd5). Animal trials (mice, rabbit, cattle and swine) will be conducted and subsequent protein analysis conducted. Mice will then be immunized with Ad5 vectors expressing the protein. Mice will be tested for production of antibody reactive with the protein and positive mice will be tested for antibody production. In vitro screening will be conducted and the proteins generated will be purified.
During FY 2013 activities were focused in three areas:
1. The development of anti-porcine immunoglobulin gamma (IgG). Multiple hybridoma antibody producing lines for each porcine IgG isotype have been produced, subcloned and/or frozen based on specificity results from screening done at the University of Iowa. We have produced 3 hybridoma antibody lines specific for IgG1, and 4 lines specific to IgG3. The remaining lines appear to bind multiple porcine IgG isotypes, but most of these lines will require additional rounds of subcloning to insure monoclonality. Assays used to select hybridoma antibodies are inconsistent and there will need to improve these assays. Antibody from several lines have been produced and these are now available for further characterization.
2. Summary of antibodies to bovine IL-13. A total of 8 hybridoma cell lines have been generated producing antibodies that bind bovine interleukin (bIL)-13. Further, a quantitative sandwich ELISA test for bovine IL-13 has been developed for detection of this cytokine in blood and tissue culture supernatants. All 8 antibodies bind commercial bovine IL-13 prepared in yeast and have tested positive at ARS, PIADC for intracellular staining in flow cytometry. Thus, in our collection 8 hybridomas producing antibody to bIL-13, we have every activity needed for characterization of T cells producing this important cytokine.
3. Summary of antibodies to bovine IL-17. A total of 8 antibodies have been produced that bind bIL-17 in solid-phase ELISA. One antibody binds reduced bIL-17and 6 of the antibodies bind non-reduced bIL-17 in Western blots. Additional characterization of these antibodies in various assay formats will continue as was accomplished for bIL-13.