This method allows a more effective way to quantify chitinase activity in biological samples. Chitinase activity has been shown to serve as a good marker for diagnosis and therapeutic efficacy in numerous conditions and diseases. Earlier methods for the measurement of chitinase activity were often time consuming and difficult to perform, both problems that have been solved by the assay shown here.
This technique will help fulfill the potential chitinases have as a diagnostic and therapeutic marker by making it easier to measure chitinase activity;for example, it can be used to monitor the treatment of Gaucher disease because chitinase activity has been shown to decrease during effective treatment. Begin by preparing substrates, standards, and solutions for the assay. Prepare 1x McIlvain buffer according to the manuscript directions and use it to dilute chitin substrates to 500 micromolar each.
Dissolve 4-methylumbelliferone, the standard, to a concentration of 0.1 millimolar in stop buffer to create a standard curve stock solution. For every 10 samples, mix 2.17 milliliters of 1x McIlvain buffer with 100 microliters of the chitin substrate to create a working solution, then pipette 95 microliters of the working solution into each well of a 96-well plate. Add five microliters of the test sample to each well and mix it into the working solution.
Cover the plate and shake it for five seconds, then incubate the plate at 37 degrees Celsius for 15 minutes to allow the enzymatic reaction to take place. Meanwhile, dilute the stock solution of 4-methylumbelliferone to make a five-micromolar working standard solution and create a standard curve by preparing a series of dilutions according to the manuscript. Start with the five-micromolar concentration and mix each dilution well.
After the incubation, add 200 microliters of stop buffer to each well to stop the reaction. Add 300-microliter duplicates of each standard to the same plate, then read the plate at an excitation of 360 nanometers and an emission of 455 nanometers. This protocol was successfully used to measure chitinase activity in bronchoalveolar lavage and serum fluid of wild type and chitotriosidase over-expressing transgenic mice.
The assay confirms that overexpression of the CHIT-1 gene in mice results in increased chitinase activity. A standard curve was used to determine chitinase based on fluorescence of the samples. The most important thing to remember when first attempting the protocol is to add the samples as quickly and efficiently as possible while still mixing thoroughly.
When you first start out, it may be beneficial to do fewer samples per run to ensure accuracy in measurements, due to a shorter incubation period.
