Analysis and testing of equine immunologic reagents

The roles cytokines play in equine immune responses has been the focus of much research. The cDNA for a number equine cytokines have been cloned and sequenced and a variety of molecular detection assays have been developed t< measure cytokine-specific mRNA expression in a variety of clinical and experimental settings. Thus it has been possible to demonstrate the presence of particular patterns of T cell cytokine gene expression in infectious, parasitic diseases and non-infectious disease. The major drawback of these molecular assays is the technical difficulty and hig cost associated with their performance. Thus these efforts have been limited to a small number of laboratories owing the complexity of the methodology. Further advances, including direct clinical applications, will depend upon the availability of equine-specific reagents, including cytokine-specific monoclonal antibodies. The importance of developing these reagents for equine researchers, clinicians and, ultimately, the equine industry cannot be overstated. As part of a multi-institutional effort, my laboratory will test and characterized monoclonal antibodies generated against key equine cytokines and other regulatory proteins. This reagents will be developed in the laboratories of Drs Baldwin and Bettina Wagner (Cornell) and in collaboration with scientists at Endogen. We anticipate screening approximately 10 equine-specific reagents over the course of this project. SCOPE OF WORK We will pursue the development of three specific immunoassays for the targeted equine cytokines; ELISA, ELI SPOT and intracellular staining for F ACS analysis. These three approaches allow for the characterization and quantitation of each cytokine in biological fluids and culture supernatants (ELISA), and cytokine production by individual cells (ELISPOT and FACS analysis). These reagents will be provided to my laboratory by Dr. Baldwin. These will include both equine-specific reagents as well as those generated for other species that may be cross-reactiv' with the equine cells. The first two approaches (ELISA, ELI SPOT) require two monoclonal antibodies where one of the antibodies is used to capture the specific cytokine while the second is used to detect the cytokine bound to the first antibody. The ELISA assay involves the following steps: (1) the wells of microtiter plates are coated with specific (capture) antibody, (2) the plates are blocked to prevent noon-specific protein binding, (3) addition of test solutions containing antigen (cytokine), (3) unbound antigen is washed out, (4) a different antigen-specific antibody conjugated to enzyme (Le., developing reagent) is added, (4) unbound conjugate is washed out and (5) substrate is added and the degree of substrate hydrolysis is measured. The amount of substrate hydrolyzed is proportional to the amount of antigen in the test solution. Several considerations must be met for successfully antibody pairing in capture ELISAs. In addition to the antibodies having sufficient specificity and affinity for the antigen (cytokine), the antibody pair must also recognize different epitopes on the antigen such that the binding of one antibody to the antigen does not interfere with the subsequent binding of the second antibody. Since a third antibody is to be used to detect the bound secondaI') antibody, the first two antibodies must be of different isotypes (or subisotypes). Alternatively the second antibody can be directly conjugated to an enzyme (horse radish peroxidase, alkaline phosphatase) or biotinylated. Sufficient monoclonal antibodies or the hybridomas producing them will be provided by Dr. Baldwin for this purpose. As this will require the production and screening of multiple monoclonal antibodies in pairs in order to identify those combinations with the ideal specificity and sensitivity, it is expected that this process will take the four years allotted for this project. The ELI SPOT assay involves five specific steps: (1) coating a purified cytokine-specific antibody (-1 0 ~g/ml) to a nitrocellulose-backed microtiter plate; (2) blocking the plate to prevent nonspecific absorption of any other proteins; (3) incubating the cytokine-secreting cells at several different dilutions; (4) adding a labeled second anticytokine antibody; and (5) detecting the antibody-cytokine complex. Again the same considerations regarding the ELISA method apply here, as well. This multiple pairs of cytokine-specific reagents will have to be screened and in, some cases, conjugated. It is likewise expected that this process will take the four years allotted for this project. To stain intracellular proteins, antibodies have to be able to cross cellular membranes. An antibody's coupling with an intracellular antigen depends on its affinity for the antigen and how easily it can access the appropriate epitope~ inside the cell. It is also sometimes preferable to stain intracellular proteins using polyclonal antisera because they represent mixtures of antibodies of different affinities that are directed against a variety of epitopes. Again we will initially be limited to the use of monoclonal antibodies until purified cytokines are available for polyclonal sera generation. Our approach will be to screen pools of monoclonal antibodies for their ability to stain the cells (PMAstimulated PBMC, cell-lines expressing the recombinant protein) expressing the cytokine of interest. The cells will b processed for intracellular staining using standard techniques for permeabilizing the cells and analyzed using a Becto Dickinson F ACSCalibre flow cytometer. This method is currently in use in my laboratory using an anti-bovine interferon-gamma antibody (Serotec) to detect equine gamma-interferon producing PBMC. In all cases, screening will initially be performed with recombinant proteins or cell-lines producing the recombinant proteins (CHO cells are currently available in my laboratory that express equine IL-4 and IL-5, and we will obtain others from Dr. Wagner) and confirmed using native equine cytokines from mitogen-stimulated PBMCs.