The nature of enzyme activity in immunoassays

The accuracy of the EIA is affected by all parameters which influence the action of the enzyme, and a meaningful test cannot be developed without a real understanding of the effects the test design may have on the enzyme activity. 

The primary interest in EIA is not in the mechanism of enzyme action. It is, nevertheless, essential to understand the elementary nature of enzyme reaction and of the effect of external factors, such as pH, temperature, ionic strength, other molecules and the solid-phase, on enzymic activity. For an optimal assay it is necessary to know (i) the stoichiometric details of the reaction (ii) the mole-cule(s) which should be present or avoided (iii) the kinetic dependence of the reaction on these molecules (iv) the optimization of experimental conditions and, (v) the accurate monitoring of the enzyme activity. Knowledge of the kinetic behaviour makes it possible to estimate the quantity of the immunoreactant present and to compare EIA to other assays. 

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