The central aim of this proposal is to improve a technique we have developed, the Cincinnati cytokine capture assay (CCCA), for measurement of in vivo cytokine production by mice. We will improve this technique by increasing its sensitivity, efficiency and speed, decreasing its cost and extending it to additional cytokines. In addition, we will test its ability to identify immunodeficient and autoimmune mice. The CCCA is based on an observation that the in vivo binding of secreted cytokine by an anti-cytokine mAb can greatly extend the in vivo lifespan of that cytokine, causing it to accumulate in serum as a cytokine-anti-cytokine mAb complex. These complexes can be identified in serum by an ELISA. CCCAs have already been developed for the detection of in vivo secretion of 1L-4 and IFN-gamma. These assays increase the sensitivity of cytokine detection by a factor of at least 1,000 are highly specific and can be performed 3-4 times on the same mouse without damaging the animal or interfering with the course of an ongoing immune response. As presently performed, it takes approximately 6 days to assay production of one cytokine for 100 mice. Studies proposed here would allow us to detect even lower levels of cytokine production and to increase the throughput of our assay to approximately 100 mice in 2 days by switching from an absorbance-based ELISA to a luminescence based ELISA. In addition, we would extend the versatility of the CCCA by adapting it to measure additional cytokines (IL-1 beta, IL-2, IL-3, IL-5, IL-9, IL-13, GM-CSF and TNF-alpha) and further increase assay efficiency and throughput by developing it to measure more than one cytokine in a single mouse serum sample. Finally, we will test the ability of this assay to identify mice with known single gene abnormalities that cause autoimmunity or immunodeficiency, by using it to measure cytokine levels before and after in vivo immune stimulation in congenitally athymic mice (T cell-deficient), SCID mice (B and T cell deficient), muMT mice (B cell-deficient), gammac-deficient mice (few B cells and T cells, no NK cells), CD28-deficient mice (defective T cell costimulation), CD40 ligand-deficient mice (defective antigen-presenting cell costimulation), C3H/HeJ mice (no response to LIPS), NZB mice (autoimmune hemolytic anemia), MRL/lpr mice (SLE-like disease with lymphoproliferation), and viable motheaten mice (SLE-like disease), and, where possible, """"""""normal"""""""" mice of the same background strains. LPS, goat anti-mouse lgD antibody and anti-CD3 mAb will be used as immune stimuli in these experiments. We believe that further development of the CCCA, in the manner proposed, will provide immunologists with a sensitive, specific, and efficient technique that will have broad usefulness for immunological research and will be particularly useful for identifying autoimmune and immunodeficient mice.
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