ICA screen ELISA Kit

$705.00

The ICA (Islet Cell Antibody) screen ELISA Assay Kit (Enzyme immunoassay) is for screening of Islet cell autoantibodies in human serum.  The Eagle Biosciences ICA (Islet Cell Antibody) screen ELISA Assay Kit is for research use only and not to be used in diagnostic procedures.

SKU: ICA31-K01 Categories: , ,

ICA screen ELISA Kit

ICA screen ELISA Kit Developed and Manufactured by Medipan

Size: 1×96 wells
Dynamic Range: Cut-off Control
Incubation Time: Overnight
Sample Type: Serum
Sample Size: 50 µL
Alternative Names: Islet Cell Antibody screen ELISA, Human ICA screen ELISA
For Research Use Only


Assay Background

Type 1 diabetes, also known as insulin-dependent diabetes mellitus (IDDM), results from a chronic autoimmune process which destructs the insulin-secreting pancreatic beta cells. This is caused by simultaneous action of specific auto-reactive CD4+ and CD8+ T lymphocytes. Even before the onset of type 1 diabetes autoantibodies against different antigens of the islet cells can be detected in the serum of those patients. This process may take years to complete and may occur at any time at all ages.

These autoantibodies represent the most important markers to identify persons with increased risk to develop diabetes at a time when all metabolic tests available still show normal results.  Islet cell autoantibodies (ICA) have been the first serological markers described for type 1 diabetes mellitus. These antibodies belong to the IgG subtype and are directed against a variety of islet cell antigens. They are detected by means of immunohistochemical methods using slides from the pancreas.

Although there have been several international workshops since the first description in 1974 these methods still reveal some methodological problems. Especially the standardization of this technique is very difficult. In spite of this it is considered as the “gold standard” and used in many routine laboratories. After discovery of the two main protein antigens of ICA – the enzyme glutamic acid decarboxylase (GAD65) and the islet cell protein IA2 which belongs to the family of tyrosine phosphatases – it becomes possible to measure these specific autoantibodies by means of sensitive radio ligand assays. Recently also ELISA (anti-GAD ELISA and Anti-IA2 ELISA) tests became available.


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Additional Information

Assay Principle


ICA Screen ELISA Assay Kit is an enzyme immunoassay for the semi-quantitative determination of islet cell autoantibodies (ICA) in human serum.  The ICA Screen ELISA Assay Kit system uses the ability of IC autoantibodies to act di-valently, that is, to form a bridge between autoantigens (highly purified material from primate source) coated onto the plate and liquid phase autoantigen-Biotin. In the first step IC autoantibodies from the sample bind to autoantigens coated on the microtiter plate. In a second step IC-autoantigen-Biotin binds to this complex which correlates with the amount of IC autoantibodies in patient’s serum. Unbound IC-autoantigen-Biotin is removed by washing.  The bound IC-autoantigen-Biotin then is quantified by addition of Streptavidin- peroxidase and a colorogenic substrate (TMB) followed by reading the optical density (OD) at 450 nm.

Assay Procedure


  1. Pipette into the corresponding wells according to assay scheme: 50 μl controls (CI, CC, CII), 50 μl patient’s samples
  2. Pipette 25 μl Enhancer (K) into each well.
    Cover the plate, shake for 5 seconds > 400 rpm and incubate over night (at least 16 h) at 2 – 8 °C.
  3. 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).
  4. 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 sec. soaking time each.
  5. Add 100 μl of reconstituted IC-autoantigen-Biotin solution (prepared from H and J) to each well.
  6. Cover the plate and incubate for 60 min at room temperature (18 – 25 °C) while shaking > 400 rpm.
  7. 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 sec. soaking time each.
  8. Add 100 μl diluted 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 > 400 rpm.
  9. 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 sec. soaking time each.
  10. Add 100 μl substrate solution (E) to each well and shake for 5 seconds.
  11. Incubate for 20 min in the dark at room temperature.
  12. Add 100 μl stop solution (F) after exact 20 min to each well. Shake the plates for 5 seconds > 400 rpm.
  13. Read the optical density at 450 nm versus 620 nm (690 nm) within 5 minutes after adding the stop solution.

Documents


Publications

Citations


Harms RZ, Ostlund KR, Cabrera M, et al. Frequencies of CD8 and DN MAIT Cells Among Children Diagnosed With Type 1 Diabetes Are Similar to Age-Matched Controls. Front Immunol. 2021;12:604157.

Harms, R.Z., Ostlund, K.R., Cabrera, M.S., et al. Confirmation and Indentification of Biomarkers Implicatingn Environmental Triggers in the Pathogenesis of Type 1 Diabetes. Frontiers in Immunology (2020) https://dx.doi.org/10.3389%2Ffimmu.2020.01922

Bottazzo, GF, A Florin-Christensen and D Doniach: Islet cell antibodies in diabetes mellitus with polyendocrine deficiencies; Lancet II 1974; 1273-1283

Soeldner JS, M Tuttleman, S Skrikanta, OP Ganda and GS Eisen-Barth: Insulin-dependent diabetes and initiation of autoimmunity: Islet cell autoantibodies, insulin autoantibodies and beta cell function; New Engl J Med 1985; 313: 839-900

Boitard C, E Bonifacio, GF Botazzo, H Gleichmann and J MolenaarImmunology and diabetes workshop: Report on the third internatiional (stage 3) workshop on standardization of cytoplasmatic islet cellantibodies; Diabetologia 1988; 31:451-452

Batstra MR, HJ Aanstoot and P Herbring: Prediction and diagnosis of Type 1 Diabetes using ß-cell autoantibodies; Clin Lab 2001; 9+10, 497-507