Description
Hyaluronidase Inhibitor Screening Assay Kit (BA0220) (BA0220)
The Hyaluronidase Inhibitor Screening Assay Kit (SKU: BA0220) provides a rapid turbidimetric method for screening hyaluronidase inhibitors. Hyaluronidases catalyse the degradation of hyaluronic acid, a major constituent of the extracellular matrix that contributes to cell proliferation and migration, making these enzymes a possible target for cancer treatment. The kit uses a two-step turbidimetric reaction in which a stop reagent halts the enzymatic reaction and forms turbidity with any residual hyaluronic acid; the decrease in turbidity at 600 nm is directly proportional to hyaluronidase activity. The entire assay can be completed in 30 minutes with a simple procedure and no wash or reagent-transfer steps. It is robust and amenable to high-throughput screening on automated liquid handling systems. Note that neither the enzyme hyaluronidase nor a control inhibitor is included in the kit.
| Product Name: | Hyaluronidase Inhibitor Screening Assay Kit (BA0220) |
| SKU: | BA0220 |
| Detection Method: | Turbidimetric; read at 600 nm |
| Detection Range: | Not specified |
| Sample Type: | Test compounds (inhibitors); purified hyaluronidase enzyme |
| Species Reactivity: | All |
| Assay Time: | Approximately 30 minutes |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | Store kit components at -20 degrees C upon receipt. |
| Shelf Life: | 12 months after receipt |
| Shipping: | Room Temperature |
Hyaluronidases are a family of enzymes that catalyse the degradation of the glycosaminoglycan hyaluronic acid, one of the major constituents of the extracellular matrix, where it contributes to both cell proliferation and migration. The role of hyaluronidases in breaking down this key factor makes them a possible target for cancer treatment. This kit uses a two-step turbidimetric reaction to measure hyaluronidase activity by the amount of hyaluronic acid hydrolysed: a stop reagent halts the enzymatic reaction and forms turbidity with residual hyaluronic acid, and the decrease in turbidity at 600 nm is directly proportional to hyaluronidase activity in the sample.
- Rapid and reliable: the entire assay can be completed in 30 minutes.
- Simple and convenient: a simple procedure with an enzymatic reaction and addition of a stop reagent, with no wash or reagent transfer steps involved.
- Robust and amenable to HTS: can be readily automated on HTS liquid handling systems for processing thousands of samples per day.
- High-throughput screening for evaluation of hyaluronidase inhibitors.
Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1 | This assay is based on an enzyme-catalysed kinetic reaction, so addition of Working Reagent should be quick and mixing brief but thorough; use of a multi-channel pipettor is recommended. Note that neither the enzyme hyaluronidase nor a control inhibitor is included in the kit. |
| 2 | Prior to assay, equilibrate all components to room temperature; the Working Reagent should be prepared fresh and used within two hours. |
| 3 | Prepare hyaluronidase in Enzyme Buffer and use it fresh; albumin and other proteins interfere with the assay and should not be included in the Enzyme Buffer. The protocol is optimised for human hyaluronidase (Acro Biosystems Cat # PH0-H5225) or bovine hyaluronidase (Calzyme Cat # 091A0300); dilute human hyaluronidase to 20 U/mL, or bovine hyaluronidase to 10 U/mL, in Enzyme Buffer. |
| 4 | Dissolve the test compounds (inhibitors) in a solvent of choice; DMSO at concentrations of 1 v/v% or less in the 100 microlitre enzymatic reaction will not interfere (the 20 microlitres of test compound may be in 5% DMSO). |
| 5 | For each inhibitor and inhibitor concentration being tested, transfer 40 microlitres of hyaluronidase into separate wells of a 96-well plate. |
| 6 | Transfer an additional 40 microlitres of hyaluronidase and 40 microlitres of Enzyme Buffer into separate wells for the No Inhibitor Control (NIC) and No Enzyme Control (NEC) respectively. |
| 7 | To the NIC and NEC wells, add 20 microlitres of the solvent in use with the test compounds. |
| 8 | To the sample wells, add 20 microlitres of each respective test compound; the concentration of test compound in the 20 microlitres should be 5x the desired final concentration in the 100 microlitre enzymatic reaction. |
| 9 | Incubate wells with test compounds for 15 minutes at room temperature. |
| 10 | Prepare enough Working Reagent for each well by combining 10 microlitres of Substrate and 35 microlitres of Assay Buffer, add 40 microlitres of Working Reagent to every well and tap the plate to mix immediately. |
| 11 | Incubate the plate for 20 minutes at room temperature. |
| 12 | Add 160 microlitres of Stop Reagent to each well and tap the plate to mix briefly and thoroughly. |
| 13 | Incubate for 10 minutes at room temperature and read optical density at 600 nm. This assay can be run in 384-well plates by using one quarter of the 96-well volumes. |
Calculate the % inhibition as: % Inhibition = [1 - ((ODNo Enzyme - ODNo Inhibitor) / (ODNo Enzyme - ODTest Cpd))] x 100%, where ODNo Enzyme, ODNo Inhibitor and ODTest Cpd are the optical density values of the No Enzyme Control, No Inhibitor Control and Test Compound respectively.
| Component | Quantity | Storage |
| Substrate | 1.5 mL | -20 degrees C |
| Stop Reagent | 20 mL | -20 degrees C |
| Assay Buffer | 5 mL | -20 degrees C |
| Enzyme Buffer | 5 mL | -20 degrees C |
