ICA screen ELISA Kit

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.