Neuraminidase Assay Kit, BioAssay(TM)

Neuraminidase (also known as Sialidase) is an enzyme that hydrolyzes terminal sialic acid residues on poly-saccharide chains. It is predominantly expressed in microorganisms such as bacteria and viruses. Cleavage of sialic acid residues by neuraminidase is believed to play several roles in infection by influenza viruses. It is thought to assist in the penetration of mucosal linings, the invasion of target cells, the elution of progeny viruses from infected cells, and the prevention of self-aggregation. Thus, neuraminidase is an important target for influenza drug development and simple, direct and automation-ready procedures for measuring neuraminidase activity find wide applications in research and drug discovery. Neuraminidase assay measures the sialic acid released by neuraminidase in one step. The change in color intensity of the reaction product at 570nm or fluorescence intensity at lem/ex=585/530nm is directly proportional to neuraminidase activity in the sample.\n\nKey Features:\nSensitive and accurate. Linear detection range at 37 degrees C in 96-well plate: 0.1 to 10 U/L for Colorimetric Assay:s and 0.01 to 2 U/L for Fluorimetric Assay:s.\nSimple and convenient. Homogeneous assay requiring only two absorbance measurements. Assay can be completed in 60 min.\nHigh-throughput. Can be readily automated as a high-throughput 96-well plate assay to screen thousands of samples per day.\n\nApplications:\nDirect Assays: neuraminidase activity in biological samples.\nDrug Discovery: evaluation of neuraminidase inhibitors.\n\nKit Contents: (100 tests in 96-well plates)\nAssay Buffer: 6ml Substrate: 6ml Cofactors: 120ul\nDye Reagent: 60ul Enzyme: 120ul Standard: 500ul\nStorage conditions. The kit is shipped on ice. Store all reagents at -20 degrees C. Shelf life of three months after receipt.\nPrecautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.\n\nProcedures:\nColorimetric Procedure:\nNote: SH-group containing reagents (e.g. mercaptoethanol, DTT) may interfere with this assay and should be avoided in sample preparation.\n1. Equilibrate all components to desired reaction temperature (i.e 37 degrees C). Prepare a 400uM Standard Premix by mixing 20ul of the 10mM Standard and 480ul dH2O. Dilute Standard in distilled water as follows.\nNo Premix+H2O Sialic Acid (uM)\n1 50ul+0ul 400\n2 30ul+20ul 240\n3 15ul+35ul 120\n4 0ul+50ul 0\nTransfer 20ul standards into separate wells of a clear flat-bottom 96-well plate.\n2. Transfer 20ul of each sample into two separate wells of the same plate. One well will be used for the sample activity and one for the sample blank.\n3. Immediately prior to starting the reaction, prepare enough Working Reagent (WR) for all sample and standard wells by mixing per reaction tube: 30ul Assay Buffer, 55ul Substrate, 1ul Cofactors, 1ul Enzyme and 0.5ul Dye Reagent. For the sample blank wells, substitute 55ul Assay Buffer for the 55ul Substrate. Add 80ul of the appropriate WR to each well.\n4. Incubate the reaction plate protected from light at 37 degrees C (or desired temperature) for 20 min. Measure the OD at 570 nm (OD20min). Incubate reaction plate for a further 30 min, again protected from light and at 37 degrees C (or desired temperature). Measure the OD (OD50min).\n\nFluorimetric Procedure:\n1. Dilute the Standards prepared in Colorimetric Procedure: 1:5 in H2O. Transfer 20ul standards into separate wells of a black 96-well plate.\n2. Transfer 20ul of each sample into two separate wells of the same plate. One well will be used for the sample activity and one for the sample blank.\n3. Add 80ul of appropriate Working Reagent (see Colorimetric Procedure:) to each well. Tap plate to mix.\n4. Incubate the reaction plate protected from light at 37 degrees C (or desired temperature) for 20 min. Measure the F (lex/em=530/570 nm) (F20min). Incubate reaction plate for a further 30 min, again protected from light and at 37 degrees C (or desired temperature). Measure the F (F50min).\n\nCalculation:\nPlot the OD or F measured at 50 min for each standard against the standard concentrations. Determine the slope using linear regression fitting. Subtract the optical density or fluorescence values for the 20 min time point from the values of the 50 min time point for the sample, sample blank and H2O (water, #4) reactions. The neuraminidase activity of a Sample is calculated as\nNeuraminidase Activity =\nDRSAMPLE

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SPECIFICATIONS

Catalog Number

N2100-15

Size

1Kit

References

1. Sugahara, K. et al. (1980). Enzymatic Assay of Serum Sialic Acid. Clinica Chimica Acta 108: 493-8.\n2. Simpson, H. et al. (1993). Serum sialic acid enzymatic assay based on microtitre plates: application for measuring capillary serum sialic acid concentrations. Br J Biomed Sci. 50: 164-7.