Method for accurate measurement of enzyme activities

Abstract

This invention relates to a novel method for the use of specialized equipment to significantly improve the accuracy with which enzyme activities are measured. The method establishes an internal standard mathematical curve for the measurement of relative values for each unit in a complex mixture. The novel feature of the method is that the control is an integral part of each and every sample, so that any variation in test conditions affects the control in exactly the same manner as the sample. Therefore, this novel method forces each measured sample to have a separate control internal to each sample that is exactly matched as to all possible variables. Although some prior methods claim an internal control, the prior methods provide only a rough calibration to an external control, which results in substantial deficiencies in accuracy, reliability, and efficiency.

1 . A method for accurate measurement utilizing specialized equipment, comprising the steps of:
 (a) Establish a foundation for comparison in form of measured internal standards for each individual experimental segment, through accurate measurement of each standard segment, strata consisting of a group of segments, and individual samples within each segment.   (b) Separate measurements of the number of samples and the attained score, for each standard segment, each test segment, and each strata including a specific group of segments.   (c) Separate calculation of the mean and the confidence interval, with modification based on the specific sample size for each segment based on the data for each segment and each strata, for both the standard and test circumstances.   (d) Calculation of the mathematical curve that describes the test results, based on a least squares fit, correlation coefficient, or other accepted criteria.   (e) Accurate measurement of the result of the change for each sample.   (f) Calculation of the statistical significance of the test results, to establish standards that prevent distortion from the effect of factors or circumstances that are uncontrolled.   
     
     
         2 . A method according to  claim 1  wherein:
 (a) the internal standards are based on a strata of more than one point per sample, and the standard circumstance is a known percentage of expected product; 
 (b) the attained score is the measured amount of readout from a detector; 
 (c) each segment is an individual test; 
 (d) each strata is a group of segments that are intended to be identical, except for uncontrolled variables 
 (e) the score that is measured is the individual readout from each individual product in a test 
 (f) components of the sample are each individual products monitored within a single test; 
 (g) the equipment used has the basic capabilities to support the accuracy and particularity of measurement necessary for the method described herein. 
 
     
     
         3 . A method according to  claim 1  wherein:
 (a) the internal standard curves established as controls are based on measured amounts for a known standard condition for application with alternative specialized equipment; 
 (b) the alternative equipment may include, but is not limited to, mass spectrometry, Western blot, chromatography, nuclear magnetic resonance, or various other chemical or biochemical equipment, instruments, fixtures, or ingredients; 
 (c) the amount of change is measured by observation of change in the sample from the control, including but not limited to changes in mass, function, fluorescence, radioactivity, intensity in arbitrary units, or other observable information. 
 (d) the test samples are material with amounts and characteristics suitable for measurement by the selected method, 
 (e) the data results are calculated by any suitable method, software, or procedure, that would produce results that provide meaningful data useful for inference, including but not limited to a measured amount for each control, and for each segment, each strata, and each sample, with separate identification of each group of samples and each sample, and a physical count of the number of samples tested.