A New and Rapid Immunoassay for the Detection of 11-Dehydro-Thromboxane B2.

Abstract Aspirin inactivates cyclooxygenase, inhibiting synthesis of thromboxane A2 (TxA2) and other molecules relevant in the development of atherothrombotic vascular events. There is recent evidence suggesting that many individuals are resistant to aspirin’s effects, resulting in increased TxA2 levels. Current methods for detecting aspirin resistance include a number of blood-based assays that measure in vitro platelet aggregation. However, these methods are not quantitative and can be affected by factors that are unrelated to aspirin sensitivity. There is a quantitative commercial immunoassay available for 11-dehydro-thromboxane B2 (11-dh-TxB2), a stable metabolite of TxA2. However, this assay uses a polyclonal antibody to 11-dh-TxB2 and requires an overnight incubation. In order to more quickly and accurately detect aspirin resistance in at-risk patients, we are developing a rapid, quantitative competitive immunoassay for 11-dh-TxB2 using a monoclonal antibody. This new assay can be completed in three hours and utilizes urine as the sample matrix. We compared the monoclonal-based assay with the polyclonal assay using twelve urine controls run multiple times over a period of three months. The controls were derived from aspirin users as well as non-users. The %CV range in the monoclonal assay was 0.3 −12.9% with an average of 4.0%. The %CV range in the polyclonal assay was 0–35.8% with an average of 10.1%. In comparing standard curves, the R2 value of the monoclonal assay averaged 0.9967 compared to 0.9894 for the polyclonal assay. Additionally, although the polyclonal assay was slightly more sensitive (limit of detection was 11.9pg/ml vs. 64.5pg/ml in the monoclonal assay), the monoclonal’s sensitivity is sufficient to detect the lower levels of 11-dh-TxB2 in patients that respond well to aspirin. To validate this, an additional urine panel was established consisting of 10 samples from both aspirin users and non-users, and 11-dh-TxB2 values from the two assays were compared. The correlation (r) between the methods was 0.944, confirming the clinical relevance of the monoclonal assay. Thus, this sensitive, urine-based monoclonal ELISA for 11-dh-TxB2 detection eliminates the potential for analytical variables that can occur in the blood-based assays while offering the sensitivity and quantitation of the ELISA.