Adenosine Deaminase Assay Kit

Product Name:	Adenosine Deaminase Assay Kit (BA0209)
SKU:	BA0209
Detection Method:	Fluorimetric (lambda ex/em = 415/475 nm)
Detection Range:	0.3 - 30 U/L adenosine deaminase
Sample Type:	Serum, plasma, saliva and other biological samples
Species Reactivity:	All
Assay Time:	Within 50 minutes
Kit Size:	100 Assays
Equipment Required:	Not applicable
Storage:	Shipped at room temperature. Store all components at -20 degrees C upon receipt.
Shelf Life:	6 months after receipt
Shipping:	Room Temperature

Adenosine deaminase (EC 3.5.4.4) is a key enzyme in purine metabolism and plays an important role in the normal immune function of humans. ADA deficiency is one cause of Severe Combined Immunodeficiency (SCID), and elevated levels of ADA have been observed in patients with tuberculosis and immune-related diseases. This kit provides a convenient fluorimetric method to measure adenosine deaminase activity in biological samples. In this assay, liberated ammonia reacts with the reagents where the increase in fluorescence at lambda ex/em = 415/475 nm is directly proportional to enzyme activity.

Fast and safe. Assay can be completed within 50 minutes. Non-radioactive assay.
Sensitive and accurate. Linear detection range is 0.3 to 30 U/L adenosine deaminase in a 96-well plate assay.
Convenient and high-throughput. Homogeneous mix-incubate-measure type assay. Can be readily automated to assay thousands of samples per day.

For quantitative determination of adenosine deaminase enzyme activity in 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	Prior to the assay, equilibrate all components to room temperature and briefly centrifuge the tubes before opening; the Working Reagent (WR) should be prepared fresh for each assay run.
2	Prepare the enzyme in an enzyme buffer, e.g. 0.05% BSA in 200 mM potassium phosphate, pH 7.4; the protocol is optimised for adenosine deaminase from calf intestine and the optimal amount of a different enzyme should be determined experimentally.
3	Dilute serum and plasma samples at least 1:20 and saliva at least 1:50 in water prior to the assay run.
4	Prepare a 1 mM Standard Premix by combining 10 uL of 20 mM Standard and 190 uL of water, then prepare all standards according to the standard dilution table (1000, 600, 300 and 0 uM).
5	Transfer 10 uL of each standard into separate wells of a black 96-well plate.
6	Transfer 10 uL of each sample into separate wells of the plate.
7	Prepare enough WR for all assay wells by mixing 2 uL of 5 mM Substrate and 40 uL of Assay Buffer for each well.
8	Initiate the reaction by adding 40 uL of WR to all wells, tap the plate to mix and incubate for 30 minutes at room temperature.
9	Add 50 uL of Detection Reagent to all wells, tap the plate to mix and incubate for 20 minutes.
10	Measure fluorescence intensity at lambda ex/em = 415/475 nm.

Subtract the blank value (Standard #4) from the standard values and plot delta-F against the standard concentrations. Determine the slope (per uM) and calculate the ADA activity in each sample as: ADA Activity = [(F_Sample - F_Blank) / (Slope (per uM) x t (min))] x n (U/L), where F_Sample and F_Blank are the measured fluorescence values of the sample and blank, t is the reaction time (30 min) and n is the sample dilution factor. Unit definition: 1 Unit (U) of ADA will catalyse the deamination of 1 micromole of adenosine per minute at room temperature and pH 7.4. If sample ADA activity exceeds 30 U/L, dilute samples in enzyme buffer and repeat the assay.

Component	Quantity	Storage
Assay Buffer	4 mL	-20 degrees C
20 mM Standard	50 uL	-20 degrees C
5 mM Substrate	200 uL	-20 degrees C
Detection Reagent	5 mL	-20 degrees C