Uric Acid/Uricase Assay Kit

1. Uric Acid Standard : One 100 mg tube of powder.
2. Fluorescence Probe : One 200 μL amber tube of a 10 mM solution in DMSO.
3. HRP : One 100 μL tube of 100 U/mL solution in glycerol.
4. 10X Assay Buffer : One 25 mL bottle.

Box 2 (shipped on blue ice packs)

1. 1N NaOH and deionized water
2. 100 mM Tris, pH 7.5
3. 1X PBS for sample dilutions and controls as necessary
4. 10 μL to 1000 μL adjustable single channel micropipettes with disposable tips
5. 50 μL to 300 μL adjustable multichannel micropipette with disposable tips
6. Standard 96-well fluorescence black microtiter plates
7. Multichannel micropipette reservoir
8. Fluorescence microplate reader capable of reading excitation in the 530-570 nm range and emission in the 590-600 nm range.

• Detection sensitivity limit of 0.5 μM of uric acid or 1 mU/mL uricase
• Measures uric acid concentrations in biological samples such as serum, plasma, and urine without any need for pretreatment

Note: All reagents must be brought to room temperature prior to use.

• 1X Assay Buffer: Warm the 10X Assay Buffer to room temperature prior to using. Prepare a 1X Assay Buffer by diluting the 10X Assay Buffer with deionized water. Add 25 mL 10X Assay Buffer to 225 mL deionized water for 250 mL total. Mix to homogeneity. Store the 1X Assay Buffer at 4 °C up to 12 months.
• Uric Acid Working Reagent (Uric Acid Assay): If measuring uric acid, prepare a Uric Acid Working Reagent by diluting the Fluorescence Probe 1:100, HRP 1:250, and Uricase 1:250 in 1X 4 Assay Buffer (e.g. For 100 assays, combine 50 μL of Fluorescence Probe, 20 μL HRP, and 20 μL Uricase with 1X Assay Buffer to a 5 mL total volume). Mix thoroughly and protect the solution from light. For best results, use the Uric Acid Working Reagent within 30 minutes of preparation. Prepare only enough for immediate use. Do not store the Working Reagent solution.
• Uricase Working Reagent (Uricase Assay): Prior to measuring uricase activity, weigh out the Uric Acid Standard powder for a 10 mg/mL solution in 1N NaOH. This 10 mg/mL is equivalent to a concentration of 60 mM. Once dissolved, dilute the 60 mM uric acid solution to a concentration of 10 mM in 100 mM Tris, pH 7.5. Vortex thoroughly. Next, prepare the Uricase Working Reagent by diluting the Fluorescence Probe 1:100, HRP 1:250, and uric acid solution 1:10 in 1X Assay Buffer (e.g. for 100 assays, combine 50 μL of Fluorescence Probe, 20 μL HRP, and 500 μL Uric Acid with 1X Assay Buffer to a 5 mL total volume). Mix thoroughly and protect the solution from light. For best results, use the Uricase Working Reagent within 30 minutes of preparation. Prepare only enough for immediate use. Do not store the Working Reagent solution. Note: If uric acid solution has visible precipitation, or does not readily go into solution, warm the solution at 37 °C and vortex thoroughly to resuspend and dissolve.

• Cell Culture Supernatant: To remove insoluble particles, centrifuge at 10,000 rpm for 5 min. The supernatant can be assayed directly or diluted as necessary. Prepare the standard curve in the same non-conditioned media. Note: Maintain pH between 7 and 8 for optimal working conditions as the Fluorescent Probe is unstable at high pH (>8.5).
• Cell Lysate: Resuspend cells at 1-2 x 106 cells/mL in 1X Assay Buffer or PBS. Homogenize or sonicate the cells on ice. Centrifuge to remove debris. Cell lysates can be assayed undiluted or titrated as necessary.
• Serum: Collect blood without using an anticoagulant and allow to clot for 30 minutes at 25 °C. Centrifuge at 2000 x g and 4 °C for 15 minutes. Remove the serum layer and store on ice. Avoid disturbing the white buffy layer. Aliquot samples for testing and store at -80 °C. Perform dilutions in 1X Assay Buffer or PBS.
• Plasma: Collect blood with heparin or citrate and centrifuge at 500-1000 x g and 4 °C for 10 minutes. Remove the plasma layer and store on ice. Avoid disturbing the white buffy layer. Aliquot samples for testing and store at -80 °C. Perform dilutions in 1X Assay Buffer or PBS.
• Urine: To remove insoluble particles, centrifuge at 10,000 rpm for 5 min. The supernatant can be assayed directly or diluted as necessary. Dilute in 1X Assay Buffer or PBS. Aliquot samples for testing and store at -80 °C. Notes:
• All samples should be assayed immediately or stored at -80 °C for up to 1-2 months. Run proper controls as necessary. Optimal dilution conditions for samples must be determined by the investigator. Always run a standard curve with samples.
• Samples with NADH concentrations above 10 μM and glutathione concentrations above 50 μM will oxidize the probe and could result in erroneous readings. To minimize this interference, it is recommended that superoxide dismutase (SOD) be added to the reaction at a final concentration of 40 U/mL (Votyakova and Reynolds, Ref. 2). 5
• Avoid samples containing DTT or β-mercaptoethanol since the probe is not stable in the presense of thiols (above 10 μM).
• Uric acid values from urine can be normalized to creatinine levels.

I. Uric Acid

1. Prepare and mix all reagents thoroughly before use. Each sample, including unknowns and standards, should be assayed in duplicate or triplicate.
2. Add 50 μL of each sample (uric acid standards, hydrogen peroxide controls or unknowns) into an individual microtiter plate well.
3. Add 50 μL of Uric Acid Working Reagent to each well. Mix the well contents thoroughly and incubate for 20 minutes at 37 °C and protected from light. Note: This assay is continuous (not terminated) and therefore may be measured at multiple time points to follow the kinetics of the reactions.
4. Read the plate with a fluorescence microplate reader equipped for excitation in the 530-570 nm range and for emission in the 590-600 nm range.
5. Calculate the concentration of uric acid within samples by comparing the sample RFUs to the uric acid standard curve. Subtract the value from the zero uric acid control well.

II. Uricase

1. Prepare and mix all reagents thoroughly before use. Each sample, including unknowns and standards, should be assayed in duplicate or triplicate.
2. Add 50 μL of each sample (uricase standards, hydrogen peroxide controls or unknowns) into an individual microtiter plate well.
3. Add 50 μL of Uricase Working Reagent to each well. Mix the well contents thoroughly and incubate for 60 minutes at 37 °C and protected from light. Note: This assay is continuous (not terminated) and therefore may be measured at multiple time points to follow the kinetics of the reactions.
4. Read the plate with a fluorescence microplate reader equipped for excitation in the 530-570 nm range and for emission in the 590-600 nm range.
5. Calculate the concentration of uricase within samples by comparing the sample RFUs to the uricase standard curve. Subtract the value from the zero uricase control well. 7