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Demeditec:: Anti-GAD ELISA

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Diabetes
Cat.-No.: DEGDE96

Product: Anti-GAD ELISA

TEST SPECIFICATIONS

Anti-GAD ELISA

Enzyme immunoassay for the quantitative determination of GAD autoantibodies in human serum.

Technology

: ELISA

Kit size

: 96 wells

Sample material

: serum

Sample preparation

: -

Sample volume

: 25 µl

Standard range

: 5 - 2000 U/ml

Incubation

: 1 h, 1 h, 20 min, 20 min

Measuring system

: TMB at 450 nm

Sensitivity

: 0.57 U/ml


Special remarks:

INTENDED USE 

The GAD 65  autoantibody (GADAb) ELISA kit is intended for use by professional persons only, for the quantitative determination of GAD autoantibodies in human serum. 
Autoantibodies to pancreatic beta cell antigens are important serological markers of type 1 diabetes mellitus (type 1 DM). The antigens recognised by these antibodies include insulin, glutamic acid decarboxylase (GAD 65  kDa isoform) and the islet cell antigen IA-2 or ICA-512. 

ASSAY PRINCIPLE 
In DEMEDITEC’s GADAb  ELISA, GAD  autoantibodies in patients’ sera, calibrators and controls are allowed to interact with GAD 65  coated onto ELISA plate wells. After an 1 hour incubation, the samples are discarded leaving GAD autoantibodies bound to the immobilised GAD 65  on the plate. GAD 65 -Biotin is added in a 2 nd incubation step where, through the ability of GAD autoantibodies in the samples to act  divalently,  a  bridge  is  formed  between  GAD 65   immobilised  on  the  plate  and  GAD 65 -Biotin.  The amount of GAD 65 -Biotin bound is then determined in a 3 rd incubation step by addition of Streptavidin Peroxidase,  which  binds  specifically  to  Biotin.  Excess,  unbound  Streptavidin  Peroxidase  is  then washed away and addition of 3,3’,5,5’ – tetramethylbenzidine (TMB) results in formation of a blue colour. This reaction is stopped by addition of stop solution causing the well contents to turn yellow. The absorbance of the yellow reaction mixture at 450 nm and 405 nm is then read using an ELISA plate reader. A higher absorbance indicates the presence of GAD autoantibody in the test sample. Reading at  405  nm  allows quantitation  of  high  absorbances  (and  should  be  used  for  concentrations  of 200 U/mL or more). Low values (less than 10 U/mL) should be read off the 450 nm calibration curve. The measuring interval is 5 – 2000 U/mL.