Neuraminidase - Assay

Prepare a 37°C water bath. Adjust the spectrophotometer to 549 nm. Into numbered tubes pipette as follows:

Mucin	0.4 ml
0.1 M acetic acid	0.5 ml

At timed intervals, add 0.1 ml of the respective enzyme dilution. Include a blank of 0.1 ml of water in place of the enzyme. Incubate for 30 minutes in the 37°C water bath. Stop the reaction at timed intervals by adding 1 ml of 5% phosphotungstic acid to each tube. Centrifuge for 10 minutes. Remove a 0.5 ml aliquot of each supernatant to dry clean test tubes. To each, add 0.1 ml of 0.2 M sodium metaperiodate. Allow to stand for 20 minutes at room temperature, add 1.0 ml of 0.755 M sodium arsenite. Shake the tubes until the brown color disappears, then add 3.0 ml of the 0.6% thiobarbituric acid. Heat for 15 minutes in a boiling water bath, cool in an ice bath for 5 minutes and add 4.6 ml of cyclohexanone to each tube. Extract the color in cyclohexanone layer by vortexing. Centrifuge at high speed for 15 minutes. Remove colored cyclohexanone layer and read A₅₄₉ of the colored cyclohexanone layer versus a reagent grade water blank.

Note: The final A₅₄₉ of sample should not exceed 0.5 absorption unit.