The overall goal of this proposal is to develop a solid phase, non radioactive immunoassay for the detection and characterization of HLA alloantibodies. The most widely used screening assay in the current use is a complement-dependent microlymphocytotoxicity (CDC) assay. Unfortunately, CDC labor-intensive, expensive, and utilizes several biological reagents that have limited shelf lives and displays. Solid phase immunoassays based on the use of purified HLA antigens is a logical format for HLA antibody analysis. Because the Class I alleles are so polymorphic (more than 75 serologically-defined alleles can be detected), it is not within the scope of a clinical laboratory to isolate and purify such a large number of HLA molecular species. Thus, it is appropriate to develop and market this clinical assay through commercial vendors. We propose to develop cost-effective, precise and accurate screening assays based on microparticles agglutination, and particle immunoassay. Microparticle-based immunoassays are widely employed to detect a variety of antigens and the assays can achieve assay sensitivities similar to RIA. Adaptation of this technology to HLA antibody detection and analysis should provide excellent sensitivity at reasonable costs. The following test parameters will be addressed: 1) the assays will utilized purified, single-species HLC Class I antigens as targets; 2) the assay will be complement- independent and based on solid phase technology; 3) manual labor will be minimized; 4) the assay must be cost-effective
