Before describing how we define the limit of detection (LOD) and limit of quantification (LOQ), we need to define what these terms mean. LOD is the confirmation of a signal whereas LOQ is a quantitative measure of how much above 0(zero) you have.
One method to determine LOD and LOQ of an assay is to get a rough estimation of LOD by measuring a negative control. Or by measuring a blank sample many times and then calculating the mean and the standard deviation of the negative control. By going 3.3 standard deviations away from the mean, you can calculate LOD. If you go 10 standard deviations away from the mean you can calculate the limit of quantification (LOQ). This will tell you the strength of a signal measured in absorbency from our assay which is significantly distinguished from the noise of the negative control. The limit of detection in terms of absorbency for GlycoSpot protease assay is 0.015 AU and the limit of quantification is 0.03 AU.
Another way of determining LOD and LOQ is by creating a standard curve for a protease of interest like Alcalase. You start by making a dilution series of the enzyme and follow it with a linear regression. That will show a dose-response curve, in which an increasing enzyme gives an increased signal. By calculating the standard deviation of the Y intercept and the slope of the linear regression line, you can calculate the LOD and LOQ.
In which σ is the standard deviation of the Y intercept and S being the slope of the dose-response curve. By calculating LOD and LOQ this way, you get a rough estimation of where you can expect to be able to distinguish the signal from the negative control (sample without enzyme) and where you can start quantifying how many enzyme units (e.g., in ppm or ppb) you have in your tested sample.
A third method to determine LOQ and LOD is by testing it experimentally. Based on the mathematical estimations described above, you get a rough estimation of the LOD. By testing several samples or a dilution series with concentration values near the predicted LOD, it is possible to measure which samples are statistically different from the negative control (sample without enzyme), giving you a value for LOD.
GlycoSpot has developed a portable test solution consisting of the SIRIUS reader and a test-kit. With this in hand, the effective removal of proteases can be validated down to single-digit ppm levels of the enzyme raw material. Our test method can also be used to optimize your overall CIP and washing processes by assessing where and when your proteases are most active. The test method works with commonly used serine-proteases such as Alcalase™️ and Savinase™️