Enzymatic Assay of Neuraminidase

1. Objective

To standardize a procedure for the enzymatic determination of neuraminidase.

2. Scope

This procedure applies to products that have a specification for neuraminidase content by enzymatic determination.

Do NOT use this assay for neuraminidase from Newcastle disease virus (Product No. N5146) or neuraminidase from Streptococcus sp. (Product No. N5271).

3. Definitions

Unit Definition - One unit will liberate 1.0 μmol of N-acetylneuaminic acid per minute at pH 5.0 at 37 ^oC using NANA-Lactose as substrate.

Purified Water - water from a deionizing system, resistivity > or = 18MΩ•cm @ 25 ºC

NANA - N-Acetylneuraminic Acid

4. Discussion

N-Acetylneuramin-Lactose + H₂O ^{Neuraminidase} > NANA + Lactose

5. Responsibilities

Analytical services personnel should follow this protocol as written.

6. Safety

Refer to the Safety Data Sheet (SDS) for hazards and appropriate handling precautions.

7. Procedure

7.1 Conditions

37ºC, pH 5.0, A_550nm, Light path = 1 cm

7.2 Method: Colorimetric

7.3. Reagents

7.3.1 100 mM Sodium acetate buffer with 2 mM calcium chloride, pH 5.0 at 37^oC (Buffer)

7.3.1.1 Prepare 100 mL in purified water using sodium acetate, anhydrous, (Product No. S8750) and dalcium dhloride, dihydrate, (Product No. C3881).

7.3.1.2 Adjust to pH 5.0 at 37^oC with 1 M HCl.

7.3.1.3 Only neuraminidase from Vibrio cholera requires calcium ions; however, the presence of calcium ions does not interfere with the activity of neuraminidase from other sources.

7.3.2 0.085% (w/v) N-Acetylneuramin-lactose (2→3) isomer solution (NAN-Lactose). Prepare a 0.85 mg/mL solution in Reagent 7.3.1 using N-acetylneuramin-lactose (Product No. A3307).

This assay system is operating at less than Km value for the substrate. It is critical that the concentration of the 2→3 isomer is 0.085% (w/v). Please correct for water and isomer content.

7.3.3 0.2% (w/v) Bovine serum albumin solution (BSA)
Prepare a 2 mg/mL solution in purified water using albumin, bovine, (Product No. A6003).

7.3.4 5% (w/v) Phosphotungstic Acid Solution (PT)
Prepare 50 mg/mL in 2.5 M HCl using Phosphotungstic Acid, free acid, (Product No. P4006).

7.3.5 Neuraminidase Enzyme Solution (Enzyme)
Immediately before use, prepare a solution containing approximately 0.03 – 0.09 unit/mL of neuraminidase in cold Reagent 7.3.3.

7.3.6 9 M Phosphoric Acid Solution (PAS)
Prepare 20 mL in purified water using phosphoric acid (Product No. 215104).

7.3.7 200 mM Sodium m-periodate Solution (Per)
Prepare 10 mL in Reagent 7.3.6 using sodium m-periodate (Product No. S1878).

7.3.8 10% (w/v) Sodium m-arsenite with 50 mM sulfuric acid and 500 mM sodium sulfate solution (Ars)
Prepare 50 mL in purified water using sodium m-arsenite (Product No. S7400), sulfuric acid (Product No. 258105), and sodium sulfate, anhydrous, (Product No. S9627).

7.3.9 0.6% (w/v) Thiobarbituric acid and 500 mM sodium sulfate solution (TBA)
Prepare 200 mL in purified water using 2-thiobarbituric Acid, (Product No. T5500), and sodium sulfate, anhydrous, (Product No. S9627).

7.3.10 5% (v/v) HCl in Butanol solution (But)
Prepare by combining 5 mL of concentrated HCl, (Product No. 258148) and 95 mL of n-butanol (Product No. B0878).

7.3.11 0.20 mM N-Acetylneuraminic acid standard solution (NANA)
Prepare 100 mL in purified water using N-acetylneuraminic acid, (Product No. A2388)

7.4 Enzyme Incubation Test Method

7.4.1 Pipette (in milliliters) the following reagents into suitable containers:

		Test
	Test	Blank
Reagent 7.3.1 (Buffer)	0.20	0.20
Reagent 7.3.2 (NAN-Lactose)	0.20	0.20

7.4.2 Mix by inversion and equilibrate to 37 ^oC. Then add:

Reagent 7.3.5 (Enzyme)	0.10	------
Reagent 7.3.3 (BSA)	------	0.10

7.4.3 Immediately mix by inversion and incubate at 37 ^oC for exactly 10 minutes. Then add:

Reagent 7.3.4 (PT)

0.50

0.50

7.4.4 Immediately, mix by inversion and centrifuge for 3 minutes at low speed.

7.5 Standard Curve

7.5.1 Pipette (in milliliters) the following reagents into suitable containers:

							Std
	Std 1	Std 2	Std 3	Std 4	Std 5	Std 6	Blank
Reagent 7.3.11 (NANA)	0.05	0.10	0.20	0.30	0.40	0.50	-----
Purified water	0.45	0.40	0.30	0.20	0.10	-----	0.50
Reagent 7.3.4 (PT)	0.50	0.50	0.50	0.50	0.50	0.50	0.50

7.5.2 Immediately, mix by inversion and centrifuge for 3 minutes at low speed.

7.6 Color Development

7.6.1 Pipette (in milliliters) the following reagents into suitable containers:

		Test							Std
	Test	Blank	Std 1	Std 2	Std 3	Std 4	Std 5	Std 6	Blank
Test supernatant	0.50	-------	-------	-------	-------	-------	-------	------	------
Test Blank supernatant	-------	0.50	-------	-------	-------	-------	-------	------	------
Std 1 supernatant	-------	-------	0.50	-------	-------	-------	-------	------	------
Std 2 supernatant	-------	-------	-------	0.50	-------	-------	-------	------	------
Std 3 supernatant	-------	-------	-------	-------	0.50	-------	-------	------	------
Std 4 supernatant	-------	-------	-------	-------	-------	0.50	-------	------	------
Std 5 supernatant	-------	-------	-------	-------	-------	-------	0.50	------	------
Std 6 supernatant	-------	-------	-------	-------	-------	-------	-------	0.50	------
Std Blank supernatant	-------	-------	-------	-------	-------	-------	-------	------	0.50
Reagent 7.3.7 (Per)	0.10	0.10	0.10	0.10	0.10	0.10	0.10	0.10	0.10

7.6.2 Mix by inversion and incubate for 20 minutes at 25 °C. Then add:

Reagent 7.3.8 (Ars)

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

7.6.3 Vortex until the yellow-brown color disappears. Then add:

Reagent 7.3.9 (TBA)

3.00

3.00

3.00

3.00

3.00

3.00

3.00

3.00

3.00

7.6.4 Transfer to a boiling water bath and incubate for 15 minutes. Allow to cool to room temperature. Then add:

Reagent 7.3.10 (But)

4.60

4.60

4.60

4.60

4.60

4.60

4.60

4.60

4.60

7.6.5 Shake or vortex the solution until most of the pink color has been extracted from the lower layer.

7.6.6 Centrifuge for 2-3 minutes at low speed, ~1000 rpm.

7.6.7 Transfer the upper layer to suitable cuvettes and immediately record the A_550nm for Test, Test Blank, Standard, and Standard Blank.

7.7 Calculations

7.7.1 Standard Curve:

ΔA550nm Standard = A_550nm Standard – A_550nm Standard Blank

Plot the ΔA_550nm Standard vs. micromoles N-acetylneuraminic acid. Obtain the slope, y-intercept and R2 for the standard curve.

7.7.2 Sample Determination: Determine the micromoles of N-acetylneuraminic acid liberated using the Standard Curve.

	Units/mL enzyme =	(μmol of NANA liberated)(df)
		(0.10)(10)

df = Dilution factor of enzyme
0.10 = volume (in milliliters) of enzyme used
10 = time (in minutes) of the assay per the unit definition

7.8 Final Assay Concentration
In a 0.50 mL reaction, the final concentrations are 80 mM sodium acetate, 1.6 mM calcium chloride, 0.034% (w/v) n-acetylneuramin-lactose (2→3 isomer), 0.04% (w/v) bovine serum albumin, and 0.003 – 0.009 unit neuraminidase.

References

1. Schneir, M.L. and Rafelson, M.E., Jr. (1996) Biochim. Biophys. Acta. 130, 1-11.

2. Cassidy, J.T., Jourdia, G.W., and Roseman, S. (1965) J. Biol. Chem. 240, 3501-3506.

3. Warren, L. (1959) J Biol. Chem. 234, 1971-1975.