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GAD-AB
ENG
(May 2016 – Model 14)
1. NAME AND INTENDED USE
GAD-AB is a radioimmunossay kit for the measurement of the Glutamic Acid Decarboxylase (GAD) autoantibodies in human serum or
in EDTA plasma.
2. INTRODUCTION
Insulin-dependent diabetes mellitus (=IDDM = type I) is an autoimmune disease resulting from an insidious destruction of pancreatic
islet  cells. In this disease several auto-Antibodies could be found, including : Insulin Antibodies (IAA), cytoplasmic Islet Cells
Antibodies (ICA), and Glutamic Acid Decarboxylase Antibodies (GAD-AB). As a result, GAD-AB measurement is proposed (either
alone or in conjunction with ICA and/or IAA) in the case of type I diabetes (IDDM).
The non Insulin Dependent Diabetes Mellitus (NIDDM) is a heterogeneous and multiform disease due to peripheral resistance of
insulin.
However some patients with NIDDM gradually develop insulin deficiency, a condition referred to slowly progressive IDDM (especially
in non obese patients). The  cell destruction seen in this case is thought to be induced by pancreatic autoimmunity. The presence of
autoantibodies to glutamic acid decarboxylase (GAD-AB) may be useful to in distinguishing patients with type I versus those with type
II disease in adults.
GAD exits in 2 majors isoforms : 65 and 67 kD. But it is now established that antibodies against the 65 kD form are more specific in
the above purposes. For this reason, this kit specifically measures GAD65-autoantibodies.
3. PRINCIPLE
In the GAD auto-antibody assay, test serum samples are incubated, first with 125I-labelled human GAD. This is followed by addition of
solid phase protein A to precipitate the labelled GAD-GAD antibody complexes. After centrifugation, the precipitates are counted for
125I and the amount of radioactivity in the precipitate is proportional to the concentration of GAD antibody in the test sample.
4. REAGENTS
Each kit contains enough reagents for 50 tubes. The expiry date is printed on the external label.
REAGENTS
PROTEIN A suspension: freeze dried.
Protein A, buffer and sodium azide.
125I-GAD:
freeze dried. 125I labelled human recombinant
GAD65, buffer, bovine albumin, red dye and sodium azide. 
50 kBq (  1.4 µCi ).
STANDARDS 0 to 6: ready to use. Human serum, sodium
azide and rabbit antibodies anti human GAD at the following
concentrations :
0 - 1 - 3 - 10 - 30 - 120 and 300 U/mL (*).
CONTROLS I and II: ready to use. Human serum, sodium
azide and rabbit antibodies anti human GAD at the following
concentrations :4 and 25 U/mL (*)
BUFFER: ready to use. Buffer, bovine albumin and sodium
azide.
QUANTITY
1 vial
qs 2.6 mL
of buffer
1 vial
qs 2.6 mL
buffer
7
0.15 mL
vials
2
0.15 mL
vials
1
120 mL
vial
STORAGE
2-8°C until the expiry date.
2-8°C 2 weeks after reconstitution
2-8°C until the expiry date.
2-8°C 2 weeks after reconstitution
2-8°C until the expiry date.
2-8°C until the expiry date.
2-8°C until the expiry date.
(*) The values shown above are only target values : the exact values of the standards are indicated on the labels in U/mL.
Units are currently RSR arbitrary units. 1U.RSR = 25 U WHO 97/550.
5. PRECAUTIONS FOR USE
5.1. Safety measures
Raw materials of human origin contained in the reagents of this kit have been tested with licensed kits and found negative for the antiHIV 1, anti-HIV 2, anti-HCV antibodies and the HBs antigen. However as it is impossible to strictly guarantee that such products will
not transmit hepatitis, the HIV virus, or any other viral infection, all raw materials of human origin including the samples to be assayed
must be treated as potentially infectious.
Do not pipette by mouth.
Do not smoke, eat or drink in areas in which specimens or kit reagents are handled.
Wear disposable gloves while handling kit reagents or specimens and wash hands thoroughly afterwards.
Avoid splashing.
Decontaminate and dispose of specimens and all potentially contaminated materials as if they contained infectious agents. The
recommended method of doing this is autoclaving for a minimum of one hour at 121.5°C.
Sodium azide may react with lead or copper piping to form highly explosive metal azides. During waste disposal, flush the drains
thoroughly to prevent a build-up of these products.
5.2. Basic radioprotection rules
This radioactive product may only be received, purchased, stored or used by persons so authorized, and by laboratories covered by
such authorization. The solution should under no circumstances be administered to humans or to animals.
The purchase, storage, use or exchange of radioactive products are subject to the laws in force in the user's country.
6
GAD-AB
ENG
(May 2016 – Model 14)
The enforcement of the basic rules for handling radioactive products ensures adequate security.
A summary of these is given below :
Radioactive products must be stored in their original containers in a suitable area.
A record of the receipt and storage of radioactive products must be kept up to date.
Handling of radioactive products should take place in a suitably-equipped area with
restricted access (controlled zone).
Do not eat, drink, smoke or apply cosmetics in a controlled zone. Do not mouth-pipette radioactive solutions.
Avoid any direct contact with all radioactive products by using laboratory coats and protective gloves.
Contaminated laboratory equipment and glassware must be disposed of immediately after contamination to prevent
cross-contamination of different isotopes.
Any contamination or radioactive substance loss should be dealt with in accordance with the established procedures.
All radioactive waste disposal must be carried out according to the regulations in force.
5.3. Handling precautions
Do not use kit components beyond their expiry date.
Do not mix reagents from different batches.
Avoid microbial contamination of the reagents.
Fully respect the incubation times and the temperature during the assay.
6. SPECIMEN COLLECTION AND PREPARATION OF SERUM SAMPLES
The assay is performed on serum or on EDTA plasma.
If the assay is to be performed within 24 hours of sample collection, the serum samples may be stored at 2-8°C. Otherwise, it is best
to divide them into aliquots and store deep-frozen (-20°C)
Specimen should be thawed before using at room temperature then mixed (vortex).
Do not refreeze serum samples for later use.
Samples which show turbidity, haemolysis, hyperlipemia or contain fibrin may give misleading results.
7. ASSAY PROCEDURE
7.1. Material required
Precision micropipettes or similar with disposable tips, capable of dispensing 20 µL, 50 µL and 1 mL (or 1 mL multipipette type).
Their calibration should be checked regularly.
Distilled water. Coniform plastic tubes (CONICAL-TUBE: 50 / plastic bag). Vortex-type mixer. Refrigerated multitube centrifuge at 28°C (1500 g minimum).
Test tube rack with inversion possibility. Gamma scintillation counter calibrated for 125 iodine measurement.
7.2. Reconstitution of the tracer and the Protein A suspension
Reconstitute the tracer and the Protein A suspension with 2.6 mL of Buffer.
Recap the vial.
Mix gently by inversion to assure complete dissolution of the freeze-dried material. A clear solution is formed.
Do not freeze the reconstituted preparation.
NB.: Protein A suspension should be shaken just before use.
7.3. Protocol
The assay requires the following groups of tubes :
T group for the determination of total activity,
Standard group,
Control group,
Sx group for the samples to be assayed.
It is recommended that the assay is performed in duplicate for the standards, controls and samples.
Observe the order in which reagents are to be added :
Dispense 20 µL of standards, controls and serum samples into the correspondingly labelled tubes.
Add 50 µL of 125I-GAD to each tube (including T group).
Mix each tube gently with a vortex-type mixer. Cover the tubes.
Incubate 2 hours at room temperature (18-25°C). Just before the end of the incubation, mix the Protein A suspension with a vortex
type mixer.
Add 50 µL of Protein A suspension to each tube (except the T tubes).
Mix each tube gently with a vortex-type mixer. Cover the tubes.
Incubate 1 hour at room temperature ( 18-25°C).
Add 1 mL of cold (2-8°C) buffer solution to each tube (except the T tubes).
Mix the contents of the tubes.
Centrifuge all the tubes (except the T tubes) at 1500 g for 30 mn at 2-8°C.
Eliminate the liquid by aspiration or inversion (except the T tubes), taking care not to disturb the pellets.
Measure the radioactivity bound to the tubes with a gamma scintillation counter.
8. QUALITY CONTROL
Good laboratory practices require that control samples be used in each series of assays to check the quality of the results obtained.
These samples must be treated in exactly the same way as the samples to be assayed, and it is recommended that the results be
analyzed with appropriate statistical methods.
7
GAD-AB
ENG
(May 2016 – Model 14)
9. RESULTS
Typical results (example only) : these data must not be substituted for results obtained in the laboratory.
Group of tubes
T
Standard 0
Standard 1
Standard 3
Standard 10
Standard 30
Standard 120
Standard 300
Control I
Control II
CPM Mean
44318
975
2747
6071
14935
27167
38379
40595
8021
25615
B/T x 100
2.2
6.2
13.7
33.7
61.3
86.6
91.6
18.1
57.8
Concentration (U/mL)
0
1
3
10
30
120
300
4.1
25.5
10. EXPECTED VALUES
Each laboratory must establish its own range of normal values. The values given below are only indicative.
Cut-off: Values below or equal to 1 U/mL are considered as negative. Values higher than 1 U/mL are considered as positive.
GAD Ab Performance Evaluation
Clinical Specificity : Assay of sera from 100 individual healthy blood donors were assayed in GAD-AB.100 % were identified as
being negative for GAD autoantibodies..
In a study of GADAb in different patient groups using GADAb kit, GADAb were not detected in patients with Hashimoto’s thyroiditis
(n= 12), myasthenia gravis (n= 19) nor in patients with Addison’s disease (n= 20). However, 1 of 27 (4%) of patients with Grave’s
disease was GADAb positive.
Clinical Sensitivity : Samples from 93 patients with Type I DM of differing ages and disease duration were tested using GAD Ab kit.
66 (71%) were identified as being identified as being positive for GAD Ab.
In the 2005 DASP study, GAD-AB kit showed 95% specificity (n=100) and 84% sensitivity (n=50).
11. SPECIFIC CHARACTERISTICS OF THE ASSAY
11.1. Imprecision
This was evaluated with 2 samples assayed 25 times in the same series or in 25 different series.
Intra-run
Inter-run
U/mL
CV %
U/mL
CV %
6.4
3.6
6.1
4.9
43
3.7
43
7
11.2. Recovery test
Known quantities of anti-GAD were added into different serum pools. The recovery percentage obtained were between 84% and
120%.
11.3. Specificity
The GAD-AB assay is specific for glutamic acid decarboxylase autoantibodies. There is no interference with other autoantibodies such
as IAA (Insulin auto-antibodies as well as polyimmunopathys with TPOAB, TRAB and 21-OH-AB).
11.4. Detection limit
The detection limit is defined as being the smallest detectable concentration different from 0 with a probability of 95%. It has been
assessed as being 0.11 U/mL.
11.5. Interference
No interference was observed when samples were spiked with the following materials: haemoglobin up to 5 mg/mL or Intralipid up to
3000 mg/dL
ASSAY FLOW-CHART – GAD-AB
Tubes
Standards
Controls
Samples
µL
T
125I-GAD
µL
Mix gently
Protein A
µL
-
50
----
-
----
-
Standards
20
50
50
20
50
Incubate
1 h at
18-25 °C
1000
Controls
Incubate
2 h at
18-25 °C
Samples
20
50
50
50
8
Mix gently
Buffer
µL
1000
1000
Mix gently
---Centrifuge
30 min
at 2-8°C
and 1500 g
---Eliminate
the liquid
---Count