Anti-GAD (Glutamic Acid Decarboxylase) ELISA Assay Kit

$710.00

The Eagle Biosciences Anti-GAD (Glutamic Acid Decarboxylase) ELISA Assay Kit is an enzyme immunoassay for the determination of autoantibodies to Glutamic Acid Decarboxylase (GAD65) in human serum. The Anti-GAD ELISA Assay Kit is for research use only and not for use in diagnostic procedures.

SKU: GAD31-K01 Categories: , ,

Anti-GAD (Glutamic Acid Decarboxylase) ELISA Assay Kit

For Research Use Only

Size: 1×96 wells
Sensitivity: Cut-Off Control
Dynamic Range: 1-250 IU/mL
Incubation Time: 3 hours
Sample Type: Serum
Sample Size: 25 µL

Additional Information

Assay Background

Type 1 diabetes, also known as insulin-dependent diabetes mellitus (IDDM), results from a chronic autoimmune destruction of the insulin-secreting pancreatic beta cells, probably initiated by exposure of genetically susceptible host to environmental agents. Autoimmune destruction of beta cells is thought to be completely asymptomatic until 80-90% of the cells are lost. This process may take years to complete and may occur at any time in all ages.

During the preclinical phase, this autoimmune process is marked by circulating autoantibodies to beta cell antigens. These autoantibodies, such as anti-insulin (IAA), anti-glutamic acid decarboxylase (GAD) and anti-tyrosine phosphatase ICA 512 (IA2), are present years before the onset of type 1 diabetes and prior to clinical symptoms. GAD, the enzyme that catalyzes the conversion of glutamate to GABA, has been identified in two isoforms, molecular weight 65.000 (GAD65) and 67.000 (GAD67). Although GAD autoantibodies are found in type 1 diabetes and in the rare neurological disorder Stiff-man syndrome (SMS), the GAD autoantibodies profile in the two diseases differs. Autoantibodies of SMS patients recognize a combination of linear and conformational epitopes of GAD while GAD65 autoantibodies in patients with type 1 diabetes are predominantly directed to the conformational epitopes. GAD65 autoantibodies (GAD65 Abs) are present in 70-80% of newly diagnosed patients with type 1 diabetes.

Assay Principle

Anti-GAD ELISA Assay Kit is an enzyme immunoassay for the quantitative determination of autoantibodies to glutamic acid decarboxylase (GAD65 Abs) in human serum.  The Anti-GAD ELISA Assay Kit uses the ability of GAD65 Abs acting divalently and forming a bridge between immobilized GAD65 and liquid-phase GAD65-Biotin. In the first step GAD65 Ab from the sample bind to GAD65 coated on the microtiter plate. In a second step GAD65-Biotin binds to this complex. The bound GAD65-Biotin correlates with the amount of GAD65 Abs in patient’s serum. Unbound GAD65-Biotin is removed by washing.  The bound GAD65-Biotin could be quantified by addition of Streptavidin- peroxidase and a colorogenic substrate (TMB) and reading the optical density (OD) at 450 nm.

Assay Procedure

  1. Pipette into the corresponding wells according to assay scheme : 25 μl negative control (C I) and calibrators (1 – 5), 25 μl patient’s samples and control serum (C II).
  2. Cover the plate and incubate for 60 min at room temperature (18 – 25 °C) while shaking > 500 rpm.
  3. Aspirate or “flick out” by striking the wells sharply onto absorbent paper to remove any residual droplets. Wash 3 times with 300 μl washing solution (diluted from B) with 5 seconds soaking time each.
  4. Add 100 μl of reconstituted GAD65-Biotin solution (prepared from H and J) to each well.
  5. Cover the plate and incubate for 60 min at room temperature (18 – 25 °C) while shaking > 500 rpm.
  6. Aspirate or “flick out” by striking the wells sharply onto absorbent paper to remove any residual droplets. Wash 3 times with 300 μl washing solution (diluted from B) with 5 seconds soaking time each.
  7. Add 100 μl reconstituted SA-POD (prepared from D and G) to each well.
    Cover the plate and incubate for 20 min at room temperature (18 – 25 °C) while shaking > 500 rpm.
  8. Aspirate or “flick out” by striking the wells sharply onto absorbent paper to remove any residual droplets. Wash 3 times with 300 μl washing solution (diluted from B) with 5 seconds soaking time each.
  9. Add 100 μl substrate solution (E) to each well and shake shortly.
    Incubate for 20 min in the dark at room temperature.
  10. Add 100 μl stop solution (F) after exact 20 min to each well. Shake the plates for 5 seconds > 200 rpm.
  11. Add 100 μl stop solution (F) after exact 20 min to each well. Shake the plates for 5 seconds > 200 rpm.
  12. Read the optical density at 450 nm versus.

Manual

Product Manual


Publications

Citations

Arroyo-Jousse et. al. “La metilación global del ADN y los niveles de homocisteína en plasma se encuentran disminuidos en pacientes con diabetes mellitus tipo 1.” Revista Medica Chile. 2015:14(5).