Description
Neuraminidase Activity Assay Kit (Colorimetric or Fluorometric) (BA0135) (BA0135)
The Neuraminidase Activity Assay Kit (Colorimetric or Fluorometric) (SKU: BA0135) enables quantitative determination of neuraminidase (sialidase) activity in biological samples. Neuraminidase is an enzyme that hydrolyses terminal sialic acid residues on polysaccharide chains and is predominantly expressed in microorganisms such as bacteria and viruses. It is believed to play several roles in infection by influenza viruses and is therefore an important target for influenza drug development. This assay measures the N-acetylneuraminic acid (NANA) released by neuraminidase in one step, with the change in colour intensity at 570 nm or fluorescence at 585/530 nm being directly proportional to neuraminidase activity in the sample. The homogeneous assay requires only two absorbance measurements and can be completed in 60 minutes. The linear detection range at 37 degrees C is 0.1 to 10 U/L for colorimetric assays and 0.01 to 2 U/L for fluorometric assays.
| Product Name: | Neuraminidase Activity Assay Kit (Colorimetric or Fluorometric) (BA0135) |
| SKU: | BA0135 |
| Detection Method: | Colorimetric or Fluorometric |
| Detection Range: | 0.1 to 10 U/L (colorimetric); 0.01 to 2 U/L (fluorometric) at 37 degrees C |
| Sample Type: | Biological samples |
| Species Reactivity: | All |
| Assay Time: | 60 minutes |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | -20 degrees C |
| Shelf Life: | 6 months after receipt |
| Shipping: | Gel Pack |
This kit measures the N-acetylneuraminic acid (NANA) released by neuraminidase in one step. The change in colour intensity of the reaction product at 570 nm or fluorescence intensity at 585/530 nm is directly proportional to neuraminidase activity in the sample.
- Sensitive and accurate with a linear detection range at 37 degrees C in 96-well plate of 0.1 to 10 U/L for colorimetric assays and 0.01 to 2 U/L for fluorometric assays
- Simple and convenient homogeneous assay requiring only two absorbance measurements and completed in 60 minutes
- High-throughput assay that can be readily automated as a 96-well plate assay to screen thousands of samples per day
- Direct measurement of neuraminidase activity in biological samples
- Drug discovery evaluation of neuraminidase 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 | Equilibrate all components to the desired reaction temperature (i.e. 37 degrees C) and prepare a 400 uM Standard Premix by mixing 20 uL of the 10 mM Standard and 480 uL distilled water, then dilute the standard as described in the dilution table. |
| 2 | Transfer 20 uL standards into separate wells of a clear flat-bottom 96-well plate, and transfer 20 uL of each sample into two separate wells, one for sample activity and one for the sample blank. |
| 3 | Immediately prior to starting the reaction, prepare enough Working Reagent for all sample and standard wells by mixing per reaction tube 30 uL Assay Buffer, 55 uL Substrate, 1 uL Cofactors, 1 uL Enzyme and 0.5 uL Dye Reagent; for the sample blank wells substitute 55 uL Assay Buffer for the 55 uL Substrate. |
| 4 | Add 80 uL of the appropriate Working Reagent to each well. |
| 5 | Incubate the reaction plate protected from light at 37 degrees C for 20 minutes and measure the OD at 570 nm, then incubate for a further 30 minutes protected from light and measure the OD again; for the fluorometric procedure use a black 96-well plate and measure fluorescence at 530/570 nm. |
Plot the OD or F measured at 50 minutes for each standard against the standard concentrations and determine the slope by linear regression. Subtract the 20 minute values from the 50 minute values for the sample, sample blank and H2O reactions, then Neuraminidase Activity = (delta RSAMPLE - delta RBLANK - delta RH2O) / Slope x (1/t) (U/L), where Slope is the slope of the standard curve in uM-1 and t is the reaction time between readings (30 minutes). One Unit (U) of neuraminidase will catalyse the release of 1 umole of NANA per minute under assay conditions.
| Component | Quantity | Storage |
| Assay Buffer | 6 mL | -20 degrees C |
| Substrate | 6 mL | -20 degrees C |
| Cofactors | 120 uL | -20 degrees C |
| Dye Reagent | 60 uL | -20 degrees C |
| Enzyme | 120 uL | -20 degrees C |
| Standard | 500 uL | -20 degrees C |
