Anti-IA2 ELISA Assay Kit

Additional Information

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.