4.33: Enzyme Analysis

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A laboratory sets up an assay for total alkaline phosphatase (ALP) by a modified AACC method. This kinetic method uses 0.30 mL of reagent, 0.05 mL of sample, and 0.15 mL of diluent in the final reaction mixture. A month after beginning to use this assay, the clinical chemist notes that a large percentage (15%) of specimens require dilution before analysis because the results exceed the assay’s upper limit of linearity. The clinical chemist asks a senior medical technologist to reformat the ALP assay to give it a wider linear range and reduce the number of required dilutions.

QUESTIONS

How can the technologist modify the assay to increase its linear range?
The technologist decides to reduce the sample volume to 0.04 mL and then determines that the linearity of the assay is now satisfactory. However, when comparing results for the ALP assay with 0.05 mL and 0.04 mL sample volumes, the technologist finds that the assay with the smaller sample volume gives consistently lower results than the original assay. What can the technologist do to have the modified assay yield the same results as the original assay?

Questions to Consider

What components of an enzymatic assay determine its linear range?
How would decreasing only the temperature of the assay affect the assay’s linearity?
How would decreasing only the reagent volume affect the assay’s linearity?
How would decreasing only the sample volume affect the assay’s linearity?
How would decreasing only the sample volume affect the assay’s precision?
How are the results of an enzyme assay calculated?
How does an automated instrument calculate the final answer for an enzyme
measurement?

Answer

The technologist can increase the linear range af the ALP assay by monitoring the reaction over a shorter time period, increasing the reagent volume, incubating the reaction at a lower temperature, or decreasing the sample. volume. The ease in which these changes can be made and the impact of any change on analytical performance would have to be monitored.
The technologist merely has to recalculate the instrument’s multiplication “factor,” substituting the new sample volume for the old value. The “factor” is $$\frac{\text{Total Volume} \times \text{Sample Volume}}{\text{extinction coefficient}\; (\epsilon) \times \text{path length}} \ldotp$$For example, the calculation for ALP (Link to Methods CD Alkaline Phosphatase) has the “factor” as 3245.

Answers to Questions to Consider

The components of an enzyme assay that determine linear range are the timing of measurements of the assay and the final concentration of substrates in the reaction mixture (Chapter 54). Both are related to the need to monitor the enzyme reaction under conditions of zero order kinetics (Chapter 54). If the reaction is monitored for too long a time, substrate may become exhausted. If the reaction is monitored for too short a time, the change in absorbance ($\Delta$A) may be so small that precision of analysis is decreased. The greater the amount of substrate per amount of added enzyme analyte, the greater the linear range of the assay.
Decreasing the temperature of the reaction will decrease the apparent enzyme activity (Chapter 54). This means that, for a given time interval for measurement, more substrate will be available for the enzyme. Thus, the linear range of the assay should increase. However, it is often not desirable, or even possible, to change the incubation temperature of a multi-test chemistry analyzer, especially for a single assay.
Decreasing the reagent volume will decrease the substrate in the final reaction mixture and thus decrease the linearity of the assay.
Decreasing the sample volume will decrease the analyte (enzyme) in the final reaction mixture and thus allow more substrate to be available for reaction. This would increase the linearity.
Decreasing the sample volume while keeping the monitoring parameters constant might decrease the assay’s precision, especially at lower levels of enzyme activity. The smaller the sample volume, the lower the final activity in, the reaction mixture, the smaller the $\Delta$A/min. The smaller the $\Delta$A, the greater the error of analysis. The laboratory would have to evaluate the effect of changing the sample volume on the assay’s precision and decide whether the decrease, if any, in precision was acceptable.
$$\text{Enzyme activity,} \frac{U}{L} = \frac{\Delta A/ minute \times \text{total reaction volume (mL)} \times \text{sample volume}}{\text{Micromolar extinction coefficient}\; (\epsilon) \times \text{optical pathlength}}$$See Chapter 54.
An automated instrument calculates the final result for an enzyme measurement by multiplying the measured change of absorbance ($\Delta$A /min) by a “factor.” This factor consists of all the constants of the equation in answer #6, that is $$\frac{\text{Total Volume} \times \text{Sample Volume}}{\text{extinction coefficient}\; (\epsilon) \times \text{path length}} \ldotp$$See calculation example for ALP (See Alkaline Phosphatase method in CD-ROM).