QuantiChrom™ Hyaluronidase Inhibitor Screening Assay Kit

Protocol SDS

Application

• For evaluation and high-throughput screen (HTS) of hyaluronidase modulators.

Key Features

• Rapid and reliable. The entire assay can be completed in 30 min.
• Simple and Convenient. Simple procedure with an enzymatic reaction and addition of stop reagent. No wash or reagent transfer steps are involved.
• Robust and amenable to HTS. Can be readily automated on HTS liquid handling systems for processing thousands of samples per day.

Method

• Turbidimetric (OD 600nm)

Samples

• Compounds that affect hyaluronidase activity

Species

• All

Procedure

• 30 min

Size

• 100 tests

Shelf Life

• 12 months

More Details

HYALURONIDASES are a family of enzymes that catalyze the degradation of the glycosaminoglycan, hyaluronic acid. Hyaluronic acid is one of the major constituents of the extracellular matrix in organisms where it contributes to both cell proliferation and migration. The role of hyaluronidases in breaking down this key factor in cell proliferation makes them a possible target for cancer treatment. One hypothesis is that increased hyaluronidase may help prevent tumor invasion by breaking down the extracellular matrix needed for tumor expansion. Conversely, decreasing hyaluronidase activity might prevent metastasis by stopping cancer cells from escaping primary tumor masses. The study to determine hyaluronidases’ exact role in cancer pathology is still ongoing. BioAssay Systems’ Hyaluronidase Inhibitor Screening Assay Kit uses a two-step turbidimetric reaction to measure hyaluronidase activity by the amount of hyaluronic acid that is hydrolyzed. A stop reagent halts the enzymatic reaction and forms turbidity with any residual hyaluronic acid in the well. The decrease in turbidity at 600 nm is directly proportional to hyaluronidase activity in the sample.

How do I calculate the concentration of test compound in the reaction?

The final concentration of the test compound in the 100 µL reaction is 5× lower than the 20 µL of test compound. So if 20 µL of 100 µM test compound was added to the well, the final concentration of test compound in the 100 µL enzymatic reaction would be 20 µM. The final concentration after adding 160 µL Stop Reagent is irrelevant because the reaction is stopped after its addition.

My percent inhibition readout is greater than 100%?

Inhibition readouts slightly over 100% (e.g. 100% to 110%) are not an issue and can be assumed to just be 100% inhibition. Inhibition readouts significantly over 100% may mean there is a substance interfering with the assay.

My percent inhibition readout is negative?

Inhibition readouts that are slightly negative (e.g. -1% to -10%) are not an issue and can be assumed to be 0% inhibition. Inhibition readouts that are significantly negative may mean there is a substance interfering with the assay or that the test compound is acting as an activator to improve the enzyme activity rather than inhibit it.

I am using a different species or a different brand of hyaluronidase, how do I determine the optimal hyaluronidase concentration for inhibitor screening?

How to Determine Optimal Hyaluronidase Concentration for Inhibitor Screening:
1. Prepare stock Hyaluronidase solution in Enzyme Buffer (start at a high concentration, you can always dilute down). In eppendorf tubes, serially dilute 50 µL of stock Hyaluronidase in Enzyme Buffer.
2. Transfer 40 µL of each Hyaluronidase dilution into separate wells of a clear, flat bottom 96-well plate
3. Transfer 40 µL of dH2O into two separate wells for the No Enzyme Control (NEC) and No Substrate Control (NSC).
4. Prepare enough Working Reagent for each well by combining 10 µL Substrate and 35 µL of Assay Buffer. Add 40 µL Working Reagent to sample wells and the NEC. Add 40 µL Assay Buffer to the NSC well.
6. Tap plate briefly to mix and incubate the plate for 20 minutes at room temperature.
7. Add 160 µL Stop Reagent to each well. Tap plate to mix briefly and thoroughly.
8. Incubate for 10 minutes at room temperature and read optical density at 600 nm.

Select a concentration of enzyme that yields an OD600nm reading about half way between the NEC and NSC OD600nm readings.

Note: the low concentrations of enzyme may produce OD600nm readings slightly higher than the NEC, this is normal.

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If you or your labs do not have the equipment or scientists necessary to run this assay, BioAssay Systems can perform the service for you.

– Fast turnaround
– Quality data
– Low cost

Please email or call 1-510-782-9988 x 2 to discuss your projects.