Correlation Between Different Anti-Ro52 Antibody Subtypes And Clinical Manifestations

Objective1.To determine if there is a difference in the subtype of anti-Ro52 antibodies in SLE/SS/IIM patients by detecting the subtype of anti-Ro52 antibodies(eg,IgA,IgG,IgM)in different patient sera from SLE/SS/ⅡM.2.To determine whether different clinical manifestations of SLE/SS/IIM and related experimental indicators are related to different anti-Ro52 antibody subtypes by SLE/SS/IIM patient case statistics;and if there is a difference in clinical manifestations and related laboratory parameters between anti-Ro52 antibody positive patients and anti-Ro52-negative patients.3.Through the above studies,to determine whether different subtype of anti-Ro52 antibodies can predict different diseases target organ damage,prognosis and clinical outcomes.Method1.Case Collection:Randomly collecting 50/30/30 cases of patients with SLE/SS/IIM and immunological detection of anti-Ro52 antibody positive/negative from Department of Rheumatology in the Second Affiliated Hospital of Kunming Medical University and to statistics the relevant clinical manifestations,laboratory indicators and activity score.2.Serum collection:randomly collecting a clear diagnosis of SLE/SS/ⅡM and immunological detection of anti-Ro52 antibody positive serum including 50/30/30 copies respectively from Department of Rheumatologyin the Second Affiliated Hospital of Kunming Medical University.3.Recombinant protein:Ro52 protein was recombined by molecular cloning method and expressed and purified.The recombinant Ro52 protein was identified by Western-blot.4.Detection:Indirect ELISA were used to determine the anti-Ro52 antibodies in different anti-Ro52 antibodies in patients with subtype of anti-Ro52,the OD value obtained by calculating the samples of different anti-Ro52 antibody subtypes relative concentration,and thus statistics SLE/SS/ⅡM patients is what anti-Ro52 antibody subtype based.5.Statistical Analysis:The GraphPad Prism 5 statistical software will be used to analysis the statistical data.Normal distributions are expressed as mean ± standard error.Paired T-test was used to compare the two groups;ANOVA was used to compare the multiple groups.P values<0.05 considered statistically significant.Result1.Protein recombination results:(1)Identification of recombinant plasmids:The Ro52 protein gene was synthesized by a bioengineering company and the gene sequence was optimized.The Ro52 gene has a total of 1440 bp and its protein size is about 50 kDa,which is about 66 kDa in size when ligated to the pHisA vector.The recombinant plasmid(pHisA-Ro52)EcoR I/Sac I restriction enzyme digestion site identified by the size of about 50kDa about the purpose of the band,and the band and PCR identified by the purpose of the band and biological,and the target genes synthesized by the company are at the same level.All of them confirm that the target gene is correctly inserted into the expression vector,and the target gene can be expressed and purified.(2)Identification of the target protein:Western Blot was performed on the protein initially expressed at a position of 66 kDa.One group was blocked by patient sera and the other was blocked by an anti-Ro52 antibody purchased from Abcam manufacturer.The results showed that only the location of the size of the band of 66kDa significant color,suggesting that the target protein.The purified target protein was identified by Western-Blot,and was first blocked with primary antibody Ro52 and secondary IgG,and the result of exposure showed that the band was specifically colored and distinct.It is confirmed as a target protein,which can be used for subsequent ELISA experiments.2.Indirect ELISA results(using t-test):(1)In 110 serum samples(including 50 SLE,30 SS,30 ⅡM),the level of anti-Ro52 antibody subtype IgG(1.772±0.01287)was significantly higher than that of IgM(1.592±0.02076)(P<0.0001)and IgA(1.469±0.02816)(P<0.0001)levels;and IgM levels were also significantly higher than IgA levels(P<0.0001).(2)In 50 serum samples of SLE,30 serum samples of SS,and 30 serum samples of IIM.① There was no significant difference in the level of anti-Ro52 antibody IgG between SLE(1.776 ± 0.01816)and SS(1.814 ± 0.01965)(P = 0.1793),SLE and IIM(1.724 ± 0.02877)(P=0.1112),but SS was higher than in IIM(P=0.0124).② The levels of anti-Ro52 antibody IgM were:1.774±0.03353,1.578±0.03693,1.635±0.03631.The t test was used to compare the three groups.The P values were:0.9458,0.2429,and 0.2737.Therefore,there was no significant difference in the levels of anti-Ro52 antibody IgM between the three groups.③The level of anti-Ro52 antibody IgA was not significantly different between SLE(1.501 ± 0.03664)and SS(1.543 ±0.0461)(P = 0.4802),but higher in SLE than in IUM(1.343 ± 0.06504)(P = 0.0250),SS is higher than ⅡM(P=0.0150).(3)① In 50 SLE serum,the anti-Ro52 antibody subtype IgG level(1.77610.01816)was higher than IgM(1.57410.03353)(P<0.0001),IgA(1.501±0.03664)(P<0.0001);And there was no significant difference between IgM and IgA(P=0.1400).②In 30 SS sera,the anti-Ro52 antibody IgG subtype(1.81410.01965)was higher than IgM(1.578±0.03693)(P<0.0001),IgA(1.54310.04610)(P<0.0001);There was no significant difference between IgM and IgA(P=0.5513)·③ In 30 IIM sera the anti-Ro52 antibody subtype IgA level(1.34310.06504)was lower than IgG(1.724±0.02877)(P=0.0002),IgM(1.635±0.03631)(P<0.0001);whereas,There was no significant difference between IgG and IgM(P=0.0610).3.Statistical results of the cases:(1)①In 50 SLE patients tested positive for anti-Ro52 antibodies:The systemic lupus erythematosus activity indexes(SLEDAI)scores of patients with anti-Ro52 antibodies were in IgG type and non-IgG type(That is IgM and IgA)was no significant difference(using t test,P=0.3376).Lupus nephritis(LN)(P=0.5602),blood platelet PLT(P=0.2520),and incidence of skin rash(P=0.5683)were not significantly different between IgG and non-IgG types.The incidence of arthritis was lower in IgG patients than in non-IgG patients(P=0.0451)(using X2test).② In 50 patients with positive anti-Ro52 antibodies and 50 patients with negative anti-Ro52 antibodies:there was no significant difference in SLEDAI scores(t test,P=0.2197).There was no significant difference in LN(P=0.8369),PLT decrease(P=0.6893),incidence of skin rash(P>0.9999),and the incidence of arthritis in patients of anti-Ro52 antibody-positive lower than negative(P=0.0018)(useing X2 test).(2)Among the 30 SS patients with positive anti-Ro52 antibodies tested,the EULAR primary Sjogren’s syndrome disease activity indexes(ESSDAI)score was not significant differences between IgG and non-IgG anti-Ro52 antibody patients.(using t test,P=0.1447).The incidence of elevated IgG(P=0.4189)and interstitial lung disease(ILD)(P=0.6707)was not significantly different between IgG and non-IgG patients(using X2 test).(3)① In 30 IIM patients with positive anti-Ro52 antibodies tested:Myositis activity indexes(MDI)(P=0.1703),Cutaneous dermatomyositis disease area and severity index,CDASI(activity)(P=0.6678)and CDASI(injury)(P=0.4464)were not significantly different between IgG and non-IgG anti-Ro52 antibodies(using t-test);however,ILD occurred is higher in IgG patients(using X2 test,P=0.0261).② Among 30 patients with positive anti-Ro52 antibodies and 20 patients with negative anti-Ro52 antibodies:MDI was higher in patients with anti-Ro52 antibodies than in negative patients(P = 0.0077),CDASI(activity)(P = 0.0551),and CDASI(injury)(P=0.5408)There was no significant difference between the two(using the t-test);the incidence of ILD was higher in patients with anti-Ro52 antibody(X2 test,P=0.0221).Conclusion1.The subtype of anti-Ro52 antibody in serum of SLE/SS/IIM patients is mainly IgG,followed by IgM.2.In patients with SLE,patients with positive anti-Ro52 antibody have a higher probability of joint involvement,and patients with anti-Ro52 antibody IgG subtype have a smaller probability of joint involvement;while SLEDAI scores and LN,rash,PLT decrease incidence was not related to anti-Ro52 antibody.3.In SS patients,there was no association between ESSDAI score,IgG elevation,and incidence of ILD with anti-Ro52 antibody subtypes.4.In IIM patients,the incidence of ILD was higher in patients with positive anti-Ro52 antibodies,and the incidence of ILD was higher in patients with anti-Ro52 antibody IgG subtypes;CDASI(activity),CDASI(injury)is not related to anti-Ro52 antibodies and subtypes.Patients with anti-Ro52 antibodies had higher MDI scores,but were not associated with this antibody subtype.