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

Clin Chim Acta. 2012 Oct 9;413(19-20):1637-40. doi: 10.1016/j.cca.2012.05.001. Epub 2012 May 10.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Adenosine / chemistry
  • Adenosine Deaminase / analysis*
  • Ascitic Fluid / chemistry*
  • Biological Assay / standards*
  • Body Fluids / chemistry*
  • Cerebrospinal Fluid / chemistry*
  • Enzyme Stability
  • Humans
  • Inosine / chemistry
  • Kinetics
  • Pleural Effusion
  • Reference Standards
  • Reference Values
  • Reproducibility of Results
  • Sensitivity and Specificity

Substances

  • Inosine
  • Adenosine Deaminase
  • Adenosine