Insulin Autoantibodies ELISA Kit

$475.00

The Insulin Autoantibodies ELISA Kit is an enzyme immunoassay for the quantitative determination of IgG autoantibodies and antibodies to insulin in human serum. The Eagle Biosciences IAA ELISA is for research use only and not for use in diagnostic procedures.

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Insulin Autoantibodies ELISA Kit

Insulin Autoantibodies ELISA Kit Developed and Manufactured by Medipan

Size: 1×96 wells
Sensitivity: 0.08 U/ml
Dynamic Range: 0.1 – 20 U/mL
Incubation Time: 90 minutes
Sample Type: Serum
Sample Size: 5 µL
Alternative Names: IAA ELISA, Insulin Auto Antibody ELISA, Human IAA ELISA
For Research Use Only


Calibration
The Insulin Autoantibodies ELISA Kit is artificially calibrated and concentrations of IAA are therefore expressed in U / ml.

Linearity
On the basic of the heterogeneous nature of the autoantibody population and in view of epitope specificity and affinity of the autoantibodies the theoretical values expected by dilution with IAA free human serum do not correspond with the measured concentrations in some cases.


Scientific 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 in all ages.

The presence of Insulin autoantibodies (IAA) in samples never treated with insulin, as opposed to insulin antibodies (IAb), is an evidence of an ongoing destruction process of pancreatic beta cells in type 1 diabetes. IAA are particularly important when determining type 1 diabetes risk since their prevalence is significantly elevated in subjects developing the disease in childhood and moreover, they are often the first autoantibodies to be detected before onset of the disease. The prevalence of IAA is inversely correlated with the age of diagnosis.

In type 1 diabetics with recent onset of the disease in the age < 5 years IAA can be determined in > 90 % of the samples, whereas in type 1 diabetics in the age > 20 years the prevalence of IAA is < 20 %.  The IAA measurement, together with that of antibodies to glutamic acid decarboxylase (GAD65 Ab), protein tyrosine phosphatase-like antigen IA2 and Islet cells antigens (ICA) forms the basis of current strategies for predicting future onset of type 1 diabetes.  In addition to autoantibodies to human insulin, antibodies induced by insulin treatment can be frequently found. Such antibodies occur at much higher concentration and are mainly directed against de-natured (incorrectly folded) fraction of applied insulin. This may lead to formation of antigen-antibody-complexes and by this reducing of the activity of the injected insulin. Measurements are necessary to obtain an optimal disease management when applying insulin.


Products Related to Insulin Autoantibodies ELISA Kit

Insulin Ultrasensitive ELISA Kit
Insulin ELISA Assay Kit
ICA screen ELISA Kit

Additional Information

Assay Principle


IAA ELISA ASSAY KIT is an enzyme immunoassay for the quantitative determination of IgG autoantibodies and antibodies to insulin in human serum.  In the first step Insulin AAb from the diluted sample bind to human recombinant insulin coated on the microtiter plate. After an incubation of 60 minutes at 37 °C unbound components are removed by washing. In a next step bound antibodies reacts with the added anti-human-IgG horseradish peroxidase (HRP) complex. Excessive conjugate is removed after 15 minutes at 37 °C by another washing step. HRP converts the colorless substrate TMB added into a blue product. The enzyme reaction is stopped by adding an acid solution after 15 minutes at 37 °C. The absorbance of the resulting yellow product is measured at 450 / 620 nm within 30 minutes. The obtained OD is direct proportional to the amount of bound antibodies.

Assay Procedure


  1. Pipette into the corresponding wells according to assay scheme:  100 μl calibrators (1 – 5), 100 μl diluted sample and control serum (C).
  2. Cover the plate and incubate for 60 min at 37 °C.
  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 anti-human IgG – HRP (D) to each well.
  5. Cover the plate and incubate for 15 min at 37 °C.
  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 substrate solution (E) to each well and shake shortly.
  8. Incubate for 15 min in the dark at 37 °C.
  9. Add 100 μl stop solution (F) to each well.  Avoid any time shift during pipetting the samples and reagents.
  10. Read the optical density at 450 nm versus 620 or 690 nm within 30 min after adding the stop solution.

Manual

Product Manual


Publications

Citations


  • Hirata Y, H Ishizu, N Ouchi, S Motomura, M Abe, Y Hara, H Wakasugi, I Takahashi, H Sakano, M Tanaka, H Kawaao & T Kanesaki: Insulin autoimmunity in a case with spontaneous hypoglycemia; Japan J Diabet 1970, 13: 312-319
  • Palmer JP, CM Asplin, P Clemens, K Lyen, O Tatpati, PK Raghu & TL Paquette: Insulin antibodies in insulin-dependent diabetes before insulin treatment; Science 1983, 222:1337-1339
  • Palmer JP, CM Asplin, PK Raghu, P Clemens, K Lyen, O Tatpati, B McKnight, TL Paquette, M Sperling, L Baker & R Guthrie: Anti-insulin antibodies in insulin-dependent diabetes before insulin treatment – a new marker for autoimmune beta cell damage?; Pediatr Adolesc Endocrinol 1986, 15:111-116
  • Ziegler, AG, R Ziegler, P Vardi, RA Jackson, JS Soeldner & GS Eisenbarth: Life-table Analysis of Progression to Diabetes od Anti-Insulin Autoantibody-positive Relatives of Individuals with Type 1 Diabetes; Diabetes 1989, 38:1320-1325
  • Williams AJK, PJ Bingley, E Bonifacio, JP Palmer & Eam Gale: A novel Micro-assay for Insulin Autoantibodies; J Autoimmunity 1997; 10:473-478
  • Lindberg B, SA Ivarsson, M Landin-Olsson, G Sundkvist, L Svanberg & A Lernmark: Islet autoantibodies in cord blood from Children who developed Type I (insulin-dependent) diabetes mellitus before 15 years of age; Diabetologia 1999; 42:181-187
  • Potter KN & T J Wilkins: The molecular specificity of insulin autoantibodies; Diabetes Metab Res Rev 2000; 16:338-353