Chemical control of reaction time in an enzyme assay and feasibility of enzyme spot tests

Abstract

There are many circumstances in which the understanding of a patient's status would be improved by knowing one or more enzyme activities. Such data are routinely produced in clinical laboratories, but simple, noninstrumental tests for enzymes are a rarity, so their extralaboratory determination is also rare. The essential problem is that effective clinical determinations of enzyme activities are typically carried out by measuring reaction rates, so the reaction time needs to be controlled. The reaction time of a sample can be controlled by using a passive, ion exchange-based titration. In this work, OH-, H+, and quinidine have been used to stop the enzymes LDH (EC 1.1.1.27) (with H+ and OH-) and cholinesterase (EC 3.1.1.8) (with quinidine). The ion exchange material containing the enzyme-stopping ion is separated from the sample by a filter. The sample contains ions that can exchange with the enzyme-stopping ion in the ion exchange material, and it may contain species that buffer the enzyme-stopping ion. The reaction time is governed by the exchanging ion's concentration in the sample, the quantity of buffer in the sample, the thickness of the filter between the ion exchange material and the sample, and the temperature. A test for LDH requiring 50 microL of serum and no instrumentation can be made so that results from sera with elevated levels appear different than those with normal levels.