ADA- Adenosine Deaminase

Colorimetric- Kinetic
1X30 mL

Quantitative determination of ADA by means of photometric method. Reagent deterioration:

Presence of particles and turbidity.
Only for in vitro use in clinical laboratory (IVD).
Materials required but not provided
Clinical Significance - Thermostatic bath at 37ºC.
ADA is an enzyme catalyzing the deamination reaction from adenosine to inosine. - Spectrophotometer or photometer thermostable at 37ºC with a 546 nm filter.
The enzyme is widely distributed in human tissues, especially high in T
lymphocytes. Elevated serum ADA activity has been observed in patients with Specimen and Stability
acute hepatitis, alcoholic hepatic fibrosis, chronic active hepatitis, liver cirrhosis,
viral hepatitis and hepatoma. Increased ADA activity was also observed in patients Fresh serum and non-hemolyzed serum or plasma,Pleural,Pericardial,Ascitic fluid
with tuberculous effusions. Determination of ADA activity in patient serum may add and CSF can be used.
Ideally, venous blood should be collected and handled anaerobically. Do not use
unique values to the diagnosis of liver diseases in combination with ALT or γ-GT citrate or oxalate as anticoagulant.
(GGT) tests. ADA assay may also be useful in the diagnostics of tuberculous
pleuritis. Stability: 7 days at 2-4ºC.

Principle Assay Procedure

The ADA assay is based on the enzymatic deamination of adenosine to inosine 1. Assay Conditions
which is converted to hypoxanthine by purine nucleoside phosphorylase (PNP). - Wavelength ............................................................. 546 (540-550)nm
Hypoxanthine is then converted to uric acid and hydrogen peroxide (H2O2) by - Cuvette .............................................................. 1 cm light path
- Temperature ....................................................................... 37C
xanthine oxidase (XOD). H2O2 is further reacted with TOOS and 4-
2. Pipette into a Cuvette:
aminoantipyrine (4-AA) in the presence of peroxidase (POD) to generate quinone
dye which is monitored in a kinetic manner. The entire enzymatic reaction scheme
is shown below. Calibrator Sample
ADA R 1 (µl) 360 360
Adenosine + H2O Inosine + NH3 Calibrator (µl) 10 -
Sample (µl) - 10
PNP
Inosine + Pi Hypoxanthine + Ribose 1-phosphate 3. Mix and incubate for 5 min at 37C.
4. Add:
XOD
Calibrator Sample
Hypoxanthine + 2H2O + 2O2 Uric acid + 2H2O2
R 2 (µl) 180 180
POD
5. Read the absorbance after 180 seconds and read again
2H2O2+4-AA+ EHSPT 4H2O+Quinone dye (max 556nm)
after 1 and 2 minutes.
6. Calculate absorbance change per minute ( A/min).
One unit of ADA is defined as the amount of ADA that generates one µmole of inosine
from adenosine per min at 37ºC
Application sheets for automated systems are available on request

Reagents Calculations
Asample /min
Contents Concentration ADA (U/L) = × Calibrator value
R1 Acalibrator /min
Tris-HCl pH 8.0 25 mmol/L
4-AA 4 mmol/L Quality Control
PNP 0.3 U/mL Commercially available normal and pathological control sera are recommended
XO 0.4 U/mL to monitor the performance of the procedure.
Peroxidase 0.1 U/mL If control values are found outside the defined range, check the instrument,
Stabilizer reagents and calibrator for problems.
R2
Tris-HCl pH 4.0 25 mmol/L
Serum controls are recommended for internal quality control. Each laboratory should
Adenosine 11 mmol/L establish its own Quality Control scheme and corrective actions.
EHSPT 4 mmol/L
Precautions
Solution R1 and CAL contain Sodium Azide. Avoid ingestion or contact with skin or Reference Values
mucous membranes. In case of skin contact, flush affected area with copious
amounts of water. In case of contact with eyes or if ingested, seek immediate
medical attention. Serum/Plasma : 4-24 U/L
All specimens used in this test should be considered potentially infectious. CSF:-
Normal : <10 U/L
Calibration Positive : >10 U/L
Pleural, Pericardial & Ascitic Fluids:-
Recommend that this assay should be calibrated using the ADA CAL (liquid
stable) included in the kit. Normal : <40 U/L
Suspect: >40 U/L to <60 U/L
Positive: >60 U/L
Storage and Stability
All the components of the kit are stable until the expiration date on the label when
stored tightly closed at 2-8ºC, (These values are for orientation purpose).
Protected from light and contaminations prevented during their
use. Do not use reagents over the expiration date.
It is suggested that each laboratory establish its own reference range.
Performance Characteristics Literatures

Interference 1. Kobayashi F, Ikeda T, Marumo F, Sato C: Adenosine deaminase

isoenzymes in liver disease. Am. J. Gastroenterol. 88: 266-271 (1993)
The following analytes were tested up to the levels indicated and found not to
interfere: 2. Kallkan A., Bult V., Erel O., Avci S., and Bingol N. K. : Adenosine
Hemoglobin: up to 800 mg/dl deaminase and guanosine deaminase activities in sera of patients with viral
hepatitis. Mem Inst. Oswaldo Cruz 94(3) 383-386 (1999)
Intralipid: up to 1000 mg/dl
Ascorbic acid: up to 50 mg/dl 3. Burgess LJ, Maritz FJ, Le Roux I, et al. Use of adenosine deaminase as a
diagnositic tool for tuberculous pleurisy. Thorax 50: 672-674 (1995)
Precision
The CV of the test should be less than 5%.

Intra assay precision Manufactured by:Asritha Diatech India Pvt.Ltd.

N=20 Level1 Level 2
Mean 29.0 140.4
SD 0.17 0.29
Marketed By: Peerless Biotech Pvt. Ltd.,
CV 0.59% 0.20% # 99 & 100, Nehru Nagar Industrial Estate, 2nd Link Street,
Inter assay precision Kottivakkam, Chennai -600041 Phone:+91-44-24541688Email:
N=5 Level1 Level 2 admin@peerlessbio.com, www.peerlessbi o.com
Mean 28.6 135.7
SD 0.12 1.10
CV 0.42% 0.81%

Linearity
The method is linear up to 176 U/L. Sample above this concentration should
be diluted with 0.9% NaCl and reassay. Multiply the result by dilution factor.

Sensitivity
The minimum detectable concentration of ADA with an acceptable level of
precision was determined as 4 U/L.

Correlation
This method (Y) was compared with another commercially available method
(X) and the following linear regression equation obtained:
Y=0.9766X+0.0206, and a correlation coefficient of 0.9989, 50 patient
samples were analyzed.

Test Parameters

Mode Kinetic
Wavelength(nm) 546 nm
Sample Volume (l) 10 µL
Working Reagent Volume 540 µL
Delay time 180 sec
Read time 120 sec
Calibrator Con. As on vial
Reaction Temperature ( C) 37
Reaction Direction Increasing
Blank with Distilled Water
Units U/L
Linearity Limit 176 U/L