Developing a Z-domain based latex agglutination assay for C. difficile toxins

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 DESCRIPTION (provided by applicant): The most common diarrhea in hospitals and nursing care facilities are attributed to toxins secreted by Clostridium difficile. These toxins, called Toin A (TcdA) and Toxin B (TcdB), are the primary markers for the diagnosis of C. difficile associated diarrhea (CDAD). The gold standard for diagnosis of CDAD is isolation of the bacteria followed by cytotoxin detection using cultured human cells. Due to time, labor and sophisticated setup, these procedures are not routinely deployed in many hospital settings. EIA-based assays for toxins, while specific, is not very sensitive. RT-PCR assay for toxin genes is very sensitive but cannot differentiate colonizers from those with active disease. As a result, there is over-diagnosis and treatment of CDAD. Accordingly, there is an unmet need for a rapid and cheaper diagnostic assay for detecting C. difficile toxins with high sensitivity and specificity at hospitas or nursing care facilities. Our company, Saureus Inc., has the expertise and technology to enable compilation of a simple, cheap and rapid assay for C. difficile toxins. This technology is found on an agglutination platform of optimized latex beads (size, charge density and coupling chemistry) that has covalently-linked unique IgG-binding domain protein which binds rabbit IgG better mouse IgG. Rabbit monoclonal antibodies against TcdB or TcdA would bind to Z-domain protein on the surface of optimized latex beads via the Fc. By using several monoclonal antibodies against divergent epitopes of TcdA and TcdB, cognate toxins in stool filtrates will bind to immobilized antibodies, leading to an agglutination reaction. We predict that our assay, based on anti-toxin antibodies immobilized by IgG-binding domain protein on the surface of optimized latex beads, will be rapid (< 10 min), cheap and highly specific for TcdB and TcdA. The development of a novel agglutination platform to detect C. difficile toxins will eliminate the need for expensive equipment and high operator skill required for most PCR-based assays. For development of this assay, we have two specific aims: I) optimize the agglutination platform based on rabbit monoclonal antibodies to C. difficile toxins A and B attached to unique IgG-binding domain protein that is covalently linked to optimized latex bead; II) optimize the lead prototype agglutination kit for the diagnosis of C. difficile toxins A and B (i.e. sensitivity, specificity, quantitation, reagent stability and ease of use). Our goal is to develop an agglutination reaction that is more sensitive than existing EIA. Upon completion of these aims, we expect to apply for Phase II SBIR funding to complete an optical reading device which will be used to evaluate clinical studies on diarrheal samples from patients. Completion of these clinical trials will enable us to apply to the FDA-CLIA for approval of the diagnostic kit.