Additional Information

Assay Principle

Medizym® anti-CCP Ref is an enzyme immunoassay for the quantitative determination of IgG autoantibodies to cyclic citrullinated peptides (CCP) in human serum.

In the first step CCP AAb from the diluted sample (as well as from the calibrators and control) bind to cyclic citrullinated peptides coated on the microtiter plate. After an incubation of 60 minutes at room temperature (RT) unbound components are removed by washing step.

In a next step bound antibodies reacts with the added anti-human-IgG horseradish peroxidase (HRP) complex. Excessive conjugate is removed after 30 minutes at RT 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 RT. The color changes from blue to yellow. The absorbance of the resulting 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
2. 100 µl calibrators (1 – 5)
3. 100 µl diluted patient sample and control serum (C).
4. Cover the plate and incubate for 60 min at RT (18 – 25 °C).
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 anti-human IgG – HRP (D) to each well.
7. Cover the plate and incubate for 30 min at RT.
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 substrate solution (E) to each well and shake shortly.
10. Incubate for 15 min in the dark at RT.
11. Add 100 µl stop solution (F) to each well.
12. Avoid any time shift during pipetting the samples and reagents.
13. Read the optical density (OD) at 450 nm versus 620 or 690 nm within 30 min after adding the stop solution.