Anticitrulline antibodies can be caused by homocitrulline-containing proteins in rabbits

To clarify the possible roles of protein-bound homocitrulline in causing an antibody response to citrulline and as a confounding factor in citrulline detection assays.

Native, carbamoylated, and citrullinated albumins were used for testing the specificity of commercial antibodies against modified citrulline by Western blot. Rabbits were immunized with human albumin and/or bone type I collagen, both of which were treated with cyanate to produce homocitrulline, or with citrullinated synthetic telopeptide of type I collagen. These antisera were tested for binding to cyclic citrullinated peptide 2 (CCP-2), mutated citrullinated vimentin, and type I or II collagen telopeptides containing either citrulline or homocitrulline.

Commercial antibodies that had been considered to be specific for chemically modified citrulline were found to recognize both homocitrulline- and citrulline-containing albumins. The rabbits immunized with proteins containing homocitrulline produced high-affinity antibodies against CCP-2 and, to a lesser extent, against mutated citrullinated vimentin. The antisera also bound homocitrulline-containing collagen telopeptides and, less strongly, citrulline-containing telopeptides.

Our findings demonstrate that homocitrulline, which is a structural analog of citrulline (longer by 1 carbon, and readily formed in the body), can be involved in raising citrulline-recognizing antibodies in experimental animals and can cause false-positive reactions in assays for citrulline.