Development of a rapid, microplate-based kinetic assay for measuring adenosine deaminase activity in body fluids

摘要

Background: Adenosine deaminase (ADA) catalyzes the deamination of adenosine to inosine. The activity of ADA in body fluids has clinical utility in the assessment of suspected tuberculosis. Methods: The conversion of adenosine to inosine was monitored in 96-well microplates as a continuous decrease at 265 nm for 15 min at ambient temperature. Analytical precision, sensitivity, linearity, accuracy, and enzyme stability were validated. Reference intervals were established from >120 tuberculosis-negative pleural, peritoneal, and cerebrospinal fluid samples. Results: The molar extinction coefficients of adenosine and inosine at 265 nm were 12,715 and 4,918 l/mol cm and their difference was used to calculate ADA activity. Maximum within-day imprecision was <11% and maximum total precision was <19%. Analytical sensitivity was 0.5 U/l and the assay was linear to 40 U/l. ADA recovery was 96-110% over an activity range of 11.7-25.3. U/l. ADA was stable for 1, 7 and 30 days at ~25 °C, 4-8 °C, and -20 °C storage, respectively. Upper reference limits were 9.4, 7.3, and 1.5. U/l for pleural, peritoneal, and cerebrospinal fluid, respectively. Conclusions: The microplate-based kinetic ADA assay has favorable performance characteristics. This method eliminates the need for assay calibration and allows 96 samples to be tested simultaneously.