NRH activity assay

  (Neuraminidase) 

  GCB1226 

1.0 Principle: neuraminidase

 Sialyllactose + H2O N-Acetylneuraminate + Lactose 

 N-acetylneuraminic acid aldolase 

N-Acetylneuraminate N-Acetyl-D-mannosamine +Pyruvate 

  Lactate dehydrogenase 

Pyruvate +NADH+H+ L-Lactate +NAD+ 

The disappearance of NADH can be detected through light absorption at 340nm.

2.0 Reagents:

A. 0.1M PIPES-NaOH buffer solution, pH 6.5 B. 8.0 mM Sialyllactose

C .50mMCaCl2

D.100u/ml NAL

E.100u/mlLDH F .2.5mM NADH

G. Enzyme Diluent: 0.1M PIPES + 0.125% BSA, pH 6.5

3.0 Operating procedures:

3.1 Instrument parameter setting

If there are no saved parameters in the instrument, set the parameters according to the following instructions. If relevant parameters already exist, retrieve them and confirm.

Detection method: Kinetic scanning

Measurement wavelength: 340nm, reaction time: 600S

Delay time: 420S Integration time: 180S

Coefficient/Factor: 4.02 Measured Temperature: 37±0.5℃

3.2 Sample preparation

If the sample to be tested is a solid, it can be dissolved at a ratio of 10mg sample to 1000ul ultrapure water. After dissolution, it should be placed at 2-8 degrees for 30 minutes.

3.3 Detection method

3.3.1 Add 1.2ml of Reagent B, 0.5ml of Reagent C, 0.25ml of Reagent D, 0.25ml of Reagent E, and 0.2ml of Reagent F to a quartz cuvette, and incubate at 37 degrees for 5 minutes.

3.3.2 Add 100ul of sample, gently mix, and then proceed with the measurement.

3.3.3 After the measurement is completed, record the corresponding values: initial reading, △A/min, and activity value (U/ml).

3.3.1 The range of activity value (U/ml) is 0.05-0.20U/ml. If it exceeds the range, the sample to be tested needs to be diluted with reagent G and tested again.

3.3.5 Calculation formula Activity (U/ml) = △A340nm×4.02×df (dilution factor)