Anti IA2 ELISA Assay Kit

$710.00

The Eagle Biosciences anti-IA2 ELISA Assay Kit is an enzyme immunoassay for the quantitative determination of autoantibodies to Protein Tyrosine Phosphatase (IA2 Abs) in human serum. This anti-IA2 ELISA is for research use only and not for use in diagnostic procedures.

SKU: IA231-K01 Categories: , ,

Anti IA2 ELISA Assay Kit

For Research Use Only

Size: 1×96 wells
Sensitivity: Cut-Off Control
Dynamic Range: 1 – 400 IU/ml
Incubation Time: 3 hours
Sample Type: Serum
Sample Size: 50 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 an environmental agent. 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.

During the preclinical phase, this autoimmune process is marked by circulating autoantibodies to beta cell antigens. These autoantibodies are present years before the onset of type 1 diabetes and prior to clinical symptoms. Early studies utilized the immunofluorescence test for islet-cell antibodies (ICA), which has been difficult to standardize and is now replaced by a combination of several radioimmunoassays for antibodies against specific beta cell antigens, such is insulin (IAA), glutamic acid decarboxylase (GAD) and tyrosine phosphatase ICA 512 (IA2).

IA2, a member of the protein tyrosine phosphatases family is localized in the dense granules of pancreatic beta cells and the second defined recombinant islet cell antigen. IA2 shares sequence identity with the islet cell antigen 512. The higher frequency of antibodies to IA2 is explained by the presence of autoantibodies directed to the COOH terminus of IA2 which is lacking in the ICA512 molecule.  IA2 autoantibodies are present in the majority of individuals with new-onset type 1 diabetes and in individuals in the pre-diabetic phase of the disease. The appearance of autoantibodies to IA2 seems to be correlated with the rapid progression to overt type 1 diabetes.

Assay Principle

Anti-IA2 ELISA Assay Kit is an enzyme immunoassay for the quantitative determination of autoantibodies to Protein Tyrosine Phosphatase (IA2 Abs) in human serum.  Anti-IA2 ELISA Assay Kit uses the ability of IA2 Abs acting divalently and forming a bridge between immobilized IA2 and liquid-phase IA2-Biotin. In the first step IA2 Ab from the sample bind to IA2 coated on the microtiter plate. In a second step IA2-Biotin binds to this complex. The bound IA2-Biotin correlates with the amount of IA2 Abs in patient’s serum. Unbound IA2-Biotin is removed by washing.  The bound IA2-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: 50 μl negative control (C I) and calibrators (1 – 4), 50 μl patient’s samples and control serum (C II).
  2. Pipette 25 μl Enhancer (K) into each well.
  3. Cover the plate, shake for 5 seconds > 500 rpm and incubate over night (at least 16 h) at 2 – 8 °C.
  4. Prepare sufficient amount of reagents (B, D / G, E, H/J) and allow the covered plate to reach room temperature (at least 30 minutes preferable while shaking – possible precipitates will disappear).
  5. 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.
  6. Add 100 μl of reconstituted IA2-Biotin solution (prepared from H and J) to each well.
  7. Cover the plate and incubate for 60 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 diluted SA-POD (prepared from D and G) to each well.
  10. Cover the plate and incubate for 20 min at room temperature (18 – 25 °C) while shaking > 500 rpm.
  11. 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.
  12. Add 100 μl substrate solution (E) to each well and shake for 5 seconds.
  13. Incubate for 20 min in the dark at room temperature.
  14. Add 100 μl stop solution (F) after exact 20 min for each well. Shake the plates for 5 seconds > 200 rpm.
  15. Read the optical density at 450 nm versus 620 nm (690 nm) within 5 minutes after adding the stop solution.

Manual

Product Manual


Publications

References

  • Lan MS, Wasserfall C, Maclaren NK & Notkins AL: IA-2, a transmembrane protein of the protein tyrosine phosphatase family, is a major autoantigen in insulin-dependent diabetes mellitus; Proc. Natl. Acad. Sci. USA 1996, 93: 6367-6370
  • Pietropaolo M, Hutton JC & Eisenbarth GS: Protein tyrosine phosphatase-like proteins: Link with IDDM; Diabetes Care 1997, 20: 208-214
  • Batstra M, HJ Anstoot & P Herbrink: Prediction and diagnosis of type 1 diabetes using ß-cell autoantibodies; Clin Lab 2001, 47: 497-507
  • Seissler J, E.Hatziagelaki & WA Scherbaum: Modern concepts for the prediction of type 1 diabetes; Exp Clin Endocrinol Diabetes 2001, 109 Suppl 2: S304-S316
  • Pozzilli P, S Manfrini & L Monetini: Biochemical markers of type 1 diabetes; clinival use, Scand J Clin Lab Invest 2001, 61: 38-44
  • Winter WE, N Harris & D Schatz: Immunological markers in the diagnosis and prediction of autoimmune Type 1a diabetes

Recent 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).