Background/Purpose: Anti-carbamylated protein antibody (anti-CarP) is seen in 45% of
rheumatoid arthritis (RA) patients [1]. The precise target antigen (Ag) of
anti-CarP had not been elucidated, since the authors of the first report used
fetal calf serum (FCS) as target Ag which contains various kind of proteins. Although
several proteins have been reported to be target Ags of anti-CarP, the repertoire
of anti-CarP has not been fully understood. In addition, all reported carbamylated
Ags are also known as citrullinated Ags, and carbamylation-specific Ag has not been
reported. Since the largest fraction of FCS is albumin, we hypothesized that human
albumin (ALB) is one of target Ags of anti-CarP. The purpose of this study is to
demonstrate whether anti-carbamylated ALB antibody (anti-car-ALB) is detected
in RA and to evaluate its clinical importance.

Methods: ALB and FCS were carbamylated by potassium cyanate. ALB was also
citrullinated by peptidyl arginine deiminase. We established in house ELISA
systems of anti-carbamylated FCS antibody (anti-car-FCS), anti-car-ALB, and anti-citrullinated
ALB antibody (anti-cit-ALB) as previously described [1]. Sera and clinical data
of 455 RA patients were obtained from KURAMA database which was established to
store clinical information and specimens from RA patients in Kyoto University. Inhibition
ELISA was done by 8 representative sera, in which they were pre-incubated with
increasing concentration of unmodified FCS, car-FCS, car-ALB, or cit-ALB and
tested for reactivity against car-FCS.

Results: The sensitivity of anti-car-FCS, anti-car-ALB, and anti-cit-ALB were
27.0%, 44.2%, and 7.3%, respectively (Table 1). Positive result of anti-car-ALB
was related to bone damage and smoking (Table 2). Inhibition ELISA showed
anti-car-FCS was inhibited by car-ALB, but not by cit-ALB (Figure 1A). Strength
of inhibition by car-ALB was different among sera (Figure 1B). The titer of
anti-car-FCS and anti-car-ALB showed good correlation (rho=0.63), but anti-CCP
did not correlate with anti-car-ALB (rho=0.28) or anti-cit-ALB (rho=0.16).

Conclusion: Anti-car-ALB was more sensitive than anti-car-FCS for diagnosing RA and
correlated with bone damage and smoking. Car-ALB is one of the target Ags of
anti-CarP. This is the first report of carbamylation-specific Ag.

Reference: [1] Shi J. Proc Natl Acad Sci U S A. 2011; 108: 17372.