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

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.

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.

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.

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.