Background/Purpose:

Anti-citrullinated protein antibodies (ACPA) play an important role in the diagnosis and prognosis of Rheumatoid Arthritis (RA). The Anti-Modified Citrulline (AMC) (‘Senshu’) method is the most frequently used method to detect citrullinated proteins. Recently, we identified antibodies against carbamylated proteins (anti-CarP) and wished to know whether the ‘AMC-Senshu’ method could discriminate citrullinated and carbamylated proteins. More importantly, we also wished to know to what degree human autoantibodies can discriminate between these two modifications.

Methods:

We analyzed the reactivity of the ‘AMC-Senshu’ method and selected sera of RA patients on western blots targeting citrullinated, carbamylated or non-modified Fetal Calf Serum (FCS) and Fibrinogen (Fib). The levels of ACPA and anti-CarP antibodies in sera before and after ACPA depletion were compared and potential cross-reactivity analysed.

Results:

The ‘AMC-Senshu’ method strongly stained both citrullinated and carbamylated FCS and as well as citrullinated and carbamylated Fib but not the non-modified counterparts. This indicates that this method can not discriminate between these two modifications. Using patient sera we demonstrate that autoantibodies present in human serum can be specific for either carbamylated Fib or citrullinated Fib. In addition many RA patients can be double positive for both reactivities. To analyze if these patients are double positive because of cross-reactivity or because they contain two antibody families we used ACPA depletion columns to deplete all ACPA followed by analysis of ACPA levels and anti-CarP levels. After ACPA depletion, more than 98% of ACPA in the sera were depleted while more than half of anti-CarP antibodies remained in the flow through in 5 out of 7 samples tested. These data indicate that part, but not all anti-CarP antibodies are cross-reactive to citrullinated epitopes.

Conclusion:

In conclusion, the ‘AMC-Senshu’ method identifies both citrullinated and carbamylated proteins. In contrast human autoantibodies can discriminate between these two modifications indicating the presence of two separate families of autoantibodies, one directed against citrullinated protein and one directed against carbamylated proteins with a limited degree of cross-reactivity.

Disclosure: These studies were financially supported by Janssen Biologics BV, (Johnson & Johnson)

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/recognition-of-citrullinated-and-carbamylated-proteins-by-human-antibodies-specificity-cross-reactivity-and-the-amc-senshu-method/