SIALIC ACID is a general name for nine carbon acidic sugars with N- or O-substituted derivatives. The most common member of these sugars is N-acetylneuraminic acid (NANA). Sialic acid is widely distributed throughout mammalian tissues and fluids including serum. Sialylated oligosaccharides have been shown to exhibit antiviral properties and are also known to influence blood coagulation and cholesterol levels. The sialic acid level in body fluids is also an important marker for diagnosing cancer. Simple and direct procedures for measuring sialic acid concentrations find wide applications in research and drug discovery. BioAssay Systems' sialic acid assay uses an improved Warren method, in which sialic acid is oxidized to formylpyruvic acid which reacts with thiobarbituric acid to form a pink colored product. The color intensity at 549 nm or fluorescence intensity at lem/ex = 585/555 nm is directly proportional to sialic acid concentration in the sample.

10% TCA: 5 mL Hydrolysis Reagent: 10 mL

DMSO: 12 mL Standard: 500 μL 10 mM Sialic Acid

Storage conditions. The kit is shipped at ambient temperature. Store the Standard at -20°C, all others at room temperature. Shelf life of three months after receipt.

Precautions: 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.

1. Standards. Equilibrate all components to room temperature. Prepare a 1000 μM sialic acid standard Premix by mixing 25 μL of the 10 mM Standard and 225 μL distilled water dH2O. Dilute Standard as follows.

Transfer 20 μL standards into four labeled Eppendorf tubes, add 5 μL 10% TCA.

2. Samples treatment. To determine total sialic acid (TSA), samples need to be hydrolyzed to release bound sialic acid as follows. In an Eppendorf tube, mix 20 μL sample, 40 μL dH2O and 40 μL Hydrolysis

Reagent. Heat at 80°C for 60 min, let cool and briefly centrifuge. Add 25 μL 10% TCA, vortex and centrifuge at 14,000 rpm for 10 min. Transfer 25 μL supernatant into a clean tube and label it “TSA”. To determine free sialic acid (FSA), directly precipitate protein by mixing 40 μL sample and 10 μL 10% TCA. Vortex and centrifuge at 14,000 rpm for 10 min. Transfer 25 μL supernatant into a clean tube and label it “FSA”.

3. Oxidation. Prepare working reagent for each tube by mixing 15 μL Hydrolysis Reagent, 50 μL dH2O and 65 μL Oxidation Reagent. Add 125 μL working reagent to each tube and let stand for 60 min at room temperature.

4. Color Reaction. Add 50 μL Dye Reagent to each tube. Mix and heat for 10 min at 100°C. Let cool for another 5-10 min. Add 100 μL DMSO to each tube. Mix and centrifuge for 5 min at 14,000 rpm. Transfer 250 μL supernatant into separate wells of a clear, flat-bottom 96-well plate.

5. Read optical density at 549 nm (540-555nm).

FLUORIMETRIC PROCEDURE

The fluorimetric assay is 10-fold more sensitive than the colorimetric assay. Prepare standards at 0, 30, 60 and 100 μM sialic acid in dH2O. The sample treatment, oxidation and color reaction steps are the same, except that the final reaction mixture is transferred into wells of a black, flat-bottom 96-well plate. Read fluorescence intensity at lex = 555 nm and lem = 585 nm.

Subtract blank value (#4) from the standard values and plot the DOD or DF against standard concentrations. Determine the slope and calculate the sialic acid concentration of Sample,

RSAMPLE and RBLANK are optical density or fluorescence intensity readings of the Sample and dH2O Blank (#4), respectively. n is the sample dilution factor, n = 5 for TSA assays and n = 1 for FSA assays.

Note: if the Sample OD value is higher than that for the 1000 μM Standard, or sample fluorescence intensity higher than that for the 100 μM Standard, dilute sample in water and repeat the assay. Multiply result by the fold of dilution.

Conversions: 1000 μM NANA equals 30.9 mg/dL or 309 ppm.

MATERIALS REQUIRED, BUT NOT PROVIDED

Pipeting devices, centrifuge tubes, centrifuge, heat block, clear flatbottom 96-well plates, black 96-well plates (e.g. Corning Costar) and plate readers.

1. Warren, L. (1959). The Thiobarbituric Acid Assay of Sialic Acids J. Biol. Chem. 234: 1971-1975.

2. Stefenelli, N. et al (1985). Serum sialic acid in malignant tumors, bacterial infections, and chronic liver diseases. J Cancer Res Clin Oncol. 109(1):55-59.

EnzyChromTM Sialic Acid Assay Kit (Cat# ESLA-100)

Quantitative Colorimetric/Fluorimetric Sialic Acid Determination

Sensitive and accurate. Use as little as 10 μL samples. Linear detection range in 96-well plate: 0.02 to 1 mM sialic acid for colorimetric assays and 2 to 100 μM for fluorimetric assays. Simple and convenient. Can detect free sialic acid by addition of a single working reagent and incubation for 60 min at room temperature or total sialic acid by pre-treating samples with a 60 min hydrolysis step.