Description
Sialic Acid 1-Step Assay Kit (BA0148) (BA0148)
The Sialic Acid 1-Step Assay Kit (SKU: BA0148) provides a simple, direct and automation-ready procedure for measuring sialic acid concentration. Sialic acid is a general name for nine-carbon acidic sugars, the most common of which is N-acetylneuraminic acid (NANA); it is widely distributed throughout mammalian tissues and fluids and its level in body fluids is an important marker in cancer diagnosis. This assay uses a single working reagent that combines NANA aldolase, pyruvate oxidase and hydrogen-peroxide determination in one step. The colour intensity of the product at 570 nm, or the fluorescence intensity at 585/530 nm, is directly proportional to the sialic acid concentration in the sample. Free sialic acid can be measured directly, while total sialic acid is measured following a hydrolysis step, using as little as 10 uL of sample.
| Product Name: | Sialic Acid 1-Step Assay Kit (BA0148) |
| SKU: | BA0148 |
| Detection Method: | Colorimetric / Fluorometric |
| Detection Range: | Colorimetric 0.02 - 1 mM; fluorometric 2 - 100 uM sialic acid |
| Sample Type: | Serum and other biological samples |
| Species Reactivity: | All |
| Assay Time: | 60 minutes |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | Store all kit components at -20 C. |
| Shelf Life: | 6 months after receipt |
| Shipping: | Gel Pack |
A single-reagent enzymatic assay for the quantitative colorimetric or fluorometric determination of sialic acid. NANA aldolase and pyruvate oxidase generate hydrogen peroxide, which reacts with a dye to yield a coloured product at 570 nm or a fluorescent product at 585/530 nm. Free sialic acid is measured directly and total sialic acid following a 60 min hydrolysis step.
- Sensitive and accurate, using as little as 10 uL of sample
- Linear detection range: colorimetric 0.02 - 1 mM, fluorometric 2 - 100 uM sialic acid
- Measures both free and total sialic acid
- Simple single-working-reagent format
- Direct measurement of sialic acid in serum and other biological samples
Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1 | For total sialic acid, hydrolyse bound sialic acid: combine 20 uL of sample with 80 uL Hydrolysis Reagent, incubate at 80 C for 60 min, cool, centrifuge and add 20 uL Neutralisation Reagent. Skip this step for free sialic acid. |
| 2 | Equilibrate all components to room temperature. Prepare a 1 mM standard premix (50 uL of 10 mM Standard in 450 uL water) and dilute to 1.0, 0.6, 0.3 and 0 mM. Transfer 10 uL of each standard and 10 uL of each sample into wells of a clear flat-bottom 96-well plate. |
| 3 | Prepare Working Reagent per well by mixing 93 uL Assay Buffer, 1 uL Dye Reagent and 1 uL Enzyme. Transfer 90 uL to each well and tap to mix. |
| 4 | Incubate for 60 min at room temperature and read optical density at 570 nm (550-585 nm). |
| 5 | For the fluorometric assay, dilute the standards 1:10, use a black plate and read fluorescence at 530/585 nm after 60 min. A 384-well format is also supported using 5 uL standards and 45 uL Working Reagent. |
Subtract the water blank from the standard values and plot against sialic acid concentration to determine the slope. Calculate [Sialic Acid] = ((R_SAMPLE - R_WATER) / Slope) x n, where n is 6 for hydrolysed samples and 1 for free sialic acid samples. One mM NANA equals 30.9 mg/dL or 309 ppm.
| Component | Quantity | Storage |
| Assay Buffer | 10 mL | -20 C |
| Hydrolysis Reagent | 10 mL | -20 C |
| Enzyme | 120 uL | -20 C |
| Neutralisation Reagent | 5 mL | -20 C |
| Dye Reagent | 120 uL | -20 C |
| Standard (10 mM Sialic Acid) | 500 uL | -20 C |
