Objective To investigate the roles of anti-U1-ribonucleoprotein(RNP)antibody and anti-RNPA/RNP68 antibodies in the diagnosis of autoimmune diseases(AID).Methods From October 2021 to March 2024,136 AID patients from Shanghai Fourth People's Hospital of Tongji University and Renji Hospital of Shanghai Jiao Tong University School of Medicine were enrolled[AID group,including 23 cases of mixed connective tissue disease(MCTD),49 cases of systemic lupus erythematosus(SLE),14 cases of Sjögren's syndrome(SS),14 cases of lupus nephritis,17 cases of autoimmune hepatitis(AIH),19 cases of rheumatoid arthritis(RA)],and 136 non-AID patients(non-AID group)and 100 healthy subjects(healthy control group)were enrolled as well.The anti-RNPA/RNP68 antibodies were determined by flow dot matrix immunoluminescence,and anti-U1-RNP antibodies were determined by western blotting.Kappa test was used to evaluate the consistency of different methods.Receiver operating characteristic(ROC)curve was used to evaluate the efficacy of each index in diagnosing AID.Results The positive rates of anti-U1-RNP antibody and anti-RNPA/RNP68 antibody in AID group were higher than those in non-AID group and healthy control group(P<0.016 7),and the positive rates of the 2 antibodies in non-AID group were also higher than those in healthy control group(P<0.0167).There was no statistical significance in the positive rate of anti-U1-RNP antibody and anti-RNPA/RNP68 antibody among all the groups(P>0.05).The consistency of the 2 antibodies was better in healthy control group(Kappa=0.92),and it was poor in AID group(Kappa=0.10).The area under curve(AUC)of the combined determination of anti-U1-RNP antibody and anti-RNPA/RNP68 antibody(both positive)was 0.80,which was higher than those of single determinations(0.70,0.73)(P<0.01).For MCTD and SLE,the AUC of anti-U1-RNP antibody and anti-RNPA/RNP68 antibody combined determination were 0.81 and 0.75,respectively,which were higher than those of single determinations of anti-U1-RNP antibody(AUC were 0.73 and 0.70,respectively)(P<0.01).The AUC of the combined determination of the 2 antibodies for SLE was higher than that of the single determination of anti-RNPA/RNP68 antibody(0.67)(P<0.01).For lupus nephritis,the AUC of the combined determination of anti-U1-RNP antibody and anti-RNPA/RNP68 antibody was 0.52,which was lower than those of the single determinations of the 2 antibodies(0.62,0.69)(P<0.05).For SS,the AUC of anti-RNPA/RNP68 antibody single determination was 0.72,which was higher than that of anti-U1-RNP antibody single determination(0.51)(P<0.05).There was no statistical significance between the AUC of anti-U1-RNP antibody,anti-RNPA/RNP68 antibody and combined determination in diagnosing other AID(P>0.05).Conclusions Anti-U1-RNP antibody and anti-RNPA/RNP68 antibody have relatively high efficacy in the diagnosis of lupus nephritis and SS,respectively.The combined determination of 2 antibodies can improve the diagnostic ability of SLE and MCTD,and has certain value in clinical diagnosis.