Pop. Med. & Diag. Sci.
Non Technical Summary
The understanding of immune responses of the horse is essential for infectious disease research, vaccine development, and testing new treatments for acute and chronic inflammatory diseases. Immunological research requires specific antibodies, typically monoclonal antibodies (mAbs).MAbs to more than 250 different cell surface markers and over 60 cytokines/chemokines are available for humans or mice. For horses, the number of available mAbs to immune system molecules has grown in recent years due to NIFA-funded reagent development projects from about 30 to presently >60 mAbs. However, many immune reagents for horses are still lacking. The hypothesis of this proposal is that new mAbs will improve the evaluation of host immunity during infectious and inflammatory diseases of the horse. The project preceding this renewal proposal focused on mAbs to soluble immune markers such as cytokines/chemokines.
Animal Health Component
Research Effort Categories
Goals / Objectives
The goals of this project are:Objective 1 - to finish the characterization and assay development for cytokine/chemokine mAbs, including generation of two additional mAbs and assays to improve the analysis of cell-mediated immunity and inflammationObjective 2 - is the development of ten mAbs to cell surface molecules defined by equine researchers for an improved phenotypic characterization of major equine immune cell populations. A preliminary mAb priority list has been developedObjective 3 - to further simplify the distribution of the mAbs and develop a sustainable access system for the equine immune reagents produced under the NIFA umbrella. We will continue to distribute high quality mAbs and provide validated immune marker assays to the entire equine research community.
Monoclonal antibody productionThe purified recombinant equine proteins are used for immunization of mice. The protocol has been established in the PD's group in 2003 and has been used with very good success since then. An IACUC approved animal protocol exists for all procedures outlined here. Balb/C and/or Black6 mice will be immunized using 50-100 μg purified recombinant protein for the first injection and 25-50 μg for all following booster injections. The increase of serum anti-target protein titers in mouse serum will be confirmed by ELISA. Mouse spleen cells will be fused to X63-Ag8.653 myeloma cells, and plated into 24 well plates in selection media (cell culture medium containing HAT for selection of hybridoma clones, toxic for non-fused myeloma cells).After 2 weeks, the cell culture supernatants will be tested for mAbs to the recombinant target protein by ELISA. Single clones will be picked from positive wells and transferred into individual wells of fresh 24 well plates. While the clones are growing up, a more comprehensive, initial characterization of mAbs will be performed using recombinant and native equine target proteins (see 5.2.). After 4-5 weeks of passaging, cell cultures will be tested for clonality by murine isotype ELISA and flow cytometric analysis using a FITC conjugated goat anti-mouse IgG(H+L) antibody. The first step defines that mAb cell cultures produce a single isotype. The latter step makes sure that mAb cultures do not include non-secreting clones. Cultures will be cloned by limiting dilution until they secrete a single mouse isotype and show a uniform Ig positive population by intracellular staining with anti-mouse Ig (true mAb clones). Clones willbe weaned from HAT to HT selection medium, and then to normal cell culture medium with 10% fetal calf serum. Aliquots of mAb clones that are specific for target (see 5.2.) will be stored in liquid nitrogen as soon as possible and at 5-6 later time points.