Immunology: Ovalbumin (OVA) Challenge
Chicken OVA is a T Cell-dependent antigen commonly used as a model protein for studying antigen-specific immune responses in mice. It is non-toxic and inert and therefore will not cause harm to the animals even if no immune response is induced. The murine immune response to OVA has been well characterized, to the extent that the immunodominant peptides for eliciting T Cell responses have been identified Anti-OVA antibodies are detectable 8-10 days after immunization using enzyme-linked immunosorbent assay, and determination of different isotypes of antibodies gives further information on the complex processes that may lead to a deficient response in genetically engineered mice. The cyclosporin-mediated suppression of immune response once again demonstrates the similarity of phenotype using the suppressive agent or the genetic knockout mice in this challenge model. Both cyclosporin-treated animals and mice knocked out for calcineurin A, in this case the a-isoform, show deficiency in T Cell dependent antigen response (Puignero et al., 1995; Zhang et al.,1996). Another example is the cytokine tumor necrosis factor a (TNF-α), whose important role in modulating inflammatory and antibody responses is well known. Two novel treatment options are currently available for patients with rheumatoid arthritis, a soluble receptor (Etanercept) and antibody (infliximab), both based on blocking the TNF-α activity. Underlining the effectiveness of drug therapy, mice deficient in TNF-α exhibit impaired humoral response to both T Cell dependent and T Cell-independent antigens (Pasparakis et al., 1996). It is important to note that, even without antigenic challenge, the make-up of the immunoglobulin repertoire in a knockout mouse is highly informative, because isotype switching of immunoglobulins is dependent on the interaction between B and T lymphocytes. Examples of the type of receptors required for normal function of T and B cells are the so-called co-stimulatory molecules, including CD28 and CD40 receptors, both of which are targets of antibody-based therapy with ongoing clinical trials for the treatment of various autoimmune diseases. In this case, mice deficient in either of these receptors register an impairment in immunoglobulin class switching, which is detectable in the serum of the animals (Shahinian et al., 1993; Kawabe et al., 1994). The ability of mice to raise an antigen-specific immune response is assessed by this protocol.
This assay evaluates a mouse's ability to produce antibodies to a specific protein antigen. The mouse is first immunized with chicken OVA, a protein foreign to the mouse. Animals are injected i.p. with 50 μg of OVA emulsified in Complete Feund's Adjuvant. Fourteen days later blood is collected and the serum is tested for the presence of antibodies to OVA by Enzyme Linked Immunosorbent Assay (ELISA). The amount of OVA-specific antibody in the serum sample is proportional to the Optical Density (OD) value generated by an instrument that scans a 96-well sample plate. Data are provided for a set of serial dilutions of each serum sample.
Displayed below is a sample graph of how OVA challenge results are presented. In comprehensive phenotypic data packages graphs are interactive. Raw or calculated data and statistics can be seen by clicking on points in the graph.
Figure illustrates proportion of OVA-specific IgG1 antibody in serum at dilutions of 1:100 (top left), 1:1,000 (top center), 1:10,000 (top right), and OVA-specific IgG2a antibody in serum at dilutions of 1:10 (bottom left), and 1:100 (bottom right). OVA-specific antibodies of wild type littermates (green circle), homozygous (red diamond), and recent historical wild type (purple line) mice are plotted against long-term historical values (± 2 standard deviations) for wild type animals (green shading). Recent wild type values are calculated from data collected within 60 days of current measures and long-term historical values are derived from data collected on more than 10,000 wild type mice.
Kawabe T, Naka T, Yoshida K, Tanaka T, Fujiwara H, Suematsu S, Yoshida N, et al. (1994) The immune responses in CD40-deficient mice: impaired immunoglobulin class switching and germinal center formation, Immunity, 1:167-178.
Pasparakis M, Alexopoulou L, Episkopou V, Kollias G. (1996) Immune and inflammatory responses in TNF alpha-deficient mice: a critical requirement for TNF-alpha in the formation of primary B cell follicles, follicular dendritic cell networks, and germinal centers, and in the maturation of the humoral immune response, J Exp Med, 184:1397-1411.
Puignero V, Salgado J, Queralt J. (1995) Effects of cyclosporine and dexamethasone on IgE antibody response in mice, and on passive cutaneous anaphylaxis in the rat, Int Arch Allergy Immunol, 108:142-147.
Shahinian A, Pfeffer K, Lee KP, Kundig TM, Kishihara K, Wakeham A, Kawai K. (1993) Differential T cell costimulatory requirements in CD28-deficient mice, Science, 261:609-612.
Zhang BW, Zimmer G, Chen J, Ladd D, Li E, Alt FW, Wiederrecht G. (1996) T cell responses in calcineurin A alpha-deficient mice, J Exp Med, 183:413-420.