Patent ID: 8551722

Claim:
A method of determining amidolytic enzyme activity in a sample over a given period of time based on conversion of a fluorogenic or chromogenic substrate of the enzyme, comprising the steps of: measuring a signal (F diag ) produced from splitting said substrate by enzymatic activity after contact with a determined initially fixed concentration of the enzyme (E) over a time period (t) so as to correctly obtain calibration under initial conditions, and preparing a diagnostic time curve from F diag measured over time; preparing a diagnostic plot from a first derivative of said diagnostic time curve against F diag , said diagnostic plot being a straight line or a parabolic line at least over a portion of said diagnostic plot, wherein the initial rate of substrate conversion (v init ) is where the diagnostic plot intercepts the ordinate and the theoretical upper limit of the signal (α) is where the diagnostic plot intercepts the abscissa, said diagnostic plot providing calibration under initial conditions; measuring an experimental signal produced by the sample (F exp ) resulting from splitting the substrate by enzymatic activity from the enzyme, said enzyme activity generating in and/or disappearing from the sample over a period of time, the F exp and F diag being measured under identical conditions; preparing an experimental time curve F exp =f(t) from the F exp measured over time; transforming the F exp into an ideal value (F transf ) by an equation according to: (i) F transf =−(α ln(1−F exp /α) for a portion of the diagnostic plot that forms a straight line; (ii) F transf =α arctan h(F exp /α) for a portion of the diagnostic plot that forms a parabolic line; and determining the enzyme concentration (E exp ) from E exp =v init F transf /dt so that the enzymatic activity over a given period of time is determined without continuous comparison to an array of values or a calibrator curve simultaneously obtained from a parallel calibration experiment.