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OBJECTIVE: Studies suggest that anti-52 kDa Ro antibodies are more sensitive and specific than anti-60 kDa Ro antibodies for neonatal lupus. However, these studies mainly used immunoblot or ELISA using recombinant protein, which have poor sensitivity for anti-60 kDa Ro antibodies. In addition, the control patients were not disease matched. We reassessed the sensitivity and specificity of anti-52 kDa Ro, anti-60 kDa Ro, and anti-La, addressing these limitations. METHODS AND RESULTS: To assess sensitivity, 125 mothers of children with neonatal lupus (NLM) were recruited. All maternal sera were assessed using a commercial line immunoassay that uses natural 60 kDa Ro protein (Inno-LiaTM ANA Update, Innogenetics NV, Gent, Belgium). By this method, 96% of the sera had antibodies to 60 kDa Ro, 86% to 52 kDa Ro, and 78% to 48 kDa La. Immunoblot of 65 NLM showed significantly fewer positive results for anti-60 kDa Ro (p < 0.001) and anti-52 kDa Ro (p < 0.05). Sensitivity of the 3 antibodies was assessed in the symptomatic mothers of children with congenital heart block (CHB) (78 women) and disease matched controls with unaffected children (65 women) using Inno-LiaTM ANA Update. The sensitivity of each antibody was compared by multiple logistic regression to adjust for maternal disease. There was no significant difference between the groups for 60 kDa Ro or for anti-52 kDa Ro antibody. However, there was a significant difference for the anti-La antibody (p = 0.001), with an odds ratio of 3.59. This translates to an increase in risk from a published 2% for CHB in an anti-Ro-positive mother to 3.1% if the woman is also anti-La antibody-positive, and to a decrease in risk to 0.9% if anti-La-negative. CONCLUSION: Contrary to previous reports, 52 kDa Ro as detected by Inno-LiaTM ANA Update is not more specific for or frequent in CHB than 60 kDa Ro. However, the presence of anti-La antibodies significantly increases the risk for CHB.