类风湿因子检测试剂盒的研发
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摘要
类风湿性关节炎(rheumatoid arthritis,RA)是一种常见的风湿性疾病,患病率约为0.4%-1.0%,该病如不能及时诊断和治疗,常在起病后1-3年出现关节畸形,严重影响日常工作和生活,提高其早期诊断率是医务工作者所关注的问题。类风湿因子(rheumatoid factor,RF)是诊断类风湿性关节炎的重要标准之一,快速高效准确地检测RF对RA的诊断及预后具有非常重要的意义。目前RF的检测方法有:胶乳凝集法、ELISA(enzyme linked immunosorbent assay)法和免疫比浊法(透射比浊法,散射比浊法,胶乳增强比浊法)。胶乳凝集法是较早的测定RF的方法,但仅能对RF进行半定量测定,且只能测定IgM-RF。后两种方法可对RF进行定量测定,ELISA法可测定RF及其亚型。目前,随着全自动生化分析仪的普及,散射比浊法和透射比浊法是近年来使用较多的RF测定方法,在这两种检测方法的基础上又建立了灵敏度更高乳胶增强比浊法,但目前的检测试剂为国外进口,费用昂贵,开发低价质优的国产RF检测试剂盒对于减轻患者负担具有重要的意义。
本课题的旨在根据胶乳比浊法的基本原理,开发具有自我知识产权国产RF定量检测试剂盒,为临床诊断提供价格低廉、临床诊断价值高的试剂。
将大小均一粒径为100nm的聚苯乙烯胶乳颗粒进行透析处理,制备工作微球。应用硫酸铵沉淀及亲和层析的方法,从健康人血清中提取纯化人免疫球蛋白IgG,并将提取的IgG交联到工作微球上,得到免疫微球。以不同浓度的PEG6000为加速剂,观察免疫微球与RF的凝聚情况。选择PEG6000的最适浓度、RF最佳反应时间、反应温度、加样量、检测波长,并绘制RF标准曲线,确定RF检测的线性范围。通过重复性实验测定批间批内变异系数,回收实验确定系统误差,确定正常人群RF参考值范围。测定通过金标准确认RA患者血清RF,计算敏感性、特异性。
实验结果表明胶乳比浊法RF定量检测的最佳反应条件为:PEG的最佳浓度为3.8%,胶乳的最佳浓度为0.30%,加样量为10μL,检测波长500nm,反应时间共15分钟。批内、批间变异系数分别为1.80～4.33%和7.5 3～14.14%;RF检测试剂的评价结果为:检测敏感度为81.5%,特异性为69.0%,跃顿指数50.5%;样品中加入已知量RF测定,回收率为94.2～1 06.5%,对含高浓度RF的血清样品稀释测定,结果表明方法的健全性良好。最低测定灵敏度为3IU/ml。稳定实验试剂保质期为12月以上(4℃)。
成功地制备了胶乳比浊法RF定量检测试剂盒,为临床实验室RF的检测提供了新的选择。
Rheumatoid Arthritis (RA) is a common rheumatic disease, the prevalence rate of it is about 0.4% to 1.0%, if the disease can not be diagnosed or treated in time, it often cause joint deformities, which will seriously affect the ability to work and daily life, after one to three years. Increasing its rate of early diagnosis is the doctors concern. Rheumatoid factor (RF) is one of the most important bases for the diagnosis of rheumatoid arthritis, the fast and accurate detection of RF is very valuable to the diagnosis and prognosis of RA. There are kinds of methods to measure RF: latex agglutination, ELISA (enzyme linked immunosorbent assay), imnunoturbidimetry(nephelometryand and tronsmisson turbidimetry and immunolatex turbimetry). Latex agglutination of RF is a classical method, but it's only a semi-quantitative determination, and can only through IgM-RF. The last two methods can carry out quantitative determination of the RF, ELISA method can determine the RF and its subtypes. According to the recent development of automation equipment, scattering turbidity and transmission method has been used a lot in clinical diagnosis. Most of the current reagents are imported, they are very expensive.
The theme of this research is to study the latex Turbidimetry volume of RF, and develop the domestic intellectual property rights RF kit for clinical diagnosis of low-cost, high value clinical diagnostic reagents
The modest size of PEG, uniform particles of polystyrene microspheres,were chosen and pre-treated for the preparation of working microsphere. The human immunoglobulin IgG was exstracted and purified by ammonium sulfate precipitation and affinity chromatography. The immuno-mirospheres were obtained by cross linking the IgG to working microspheres. The serials of concentration of PEG6000 were added into the antigen-antibody reaction system, and the optimal concentration of accelerating agent(PEG6000) was determined. The optimal reaction time, reaction temperature,the quantity of application of sample and test wave length were determined by orthogonal experiment. the standard curve was made and the linearity range was determined. The coefficient of variability of inter-batch and intra-batch were checked by repeatability test, and the systematic error was checked by recovery test. The reference range was established. The sensitivity and specificity of the reagents were test by testing of clinical samples.
Highly purified human immunoglobulin IgG was obtained successfully. The desired immuno-microspheres were successfully prepared. The optimal concentration of PEG6000 was 3.8%, and the optimal concentration of latex was 0.3%; the optimized quantity of application of sample was 10μl,the detection wave length was 500nm, the total reaction time was 15 minutes. The coefficient of variability of intra-batches and inter-batches was 1.80-4.33% and 7.53-14.14%, respectively. The recovery rate was 94.2-106.5%.Detection sensitivity was 81.5% and specificity was 69.0%, youden index was 50.5%. The lowest detective sensitivity was 3IU/ml,and the assured stable experiment reagent time was 12 monthes at 4℃refrigerator.
The latex Turbidimetry rheumatoid factor detecting kits was successfully developed, which providing an alternative selection for the dection of rheumatoidfactor in clinical laboratory.
本课题的旨在根据胶乳比浊法的基本原理,开发具有自我知识产权国产RF定量检测试剂盒,为临床诊断提供价格低廉、临床诊断价值高的试剂。
将大小均一粒径为100nm的聚苯乙烯胶乳颗粒进行透析处理,制备工作微球。应用硫酸铵沉淀及亲和层析的方法,从健康人血清中提取纯化人免疫球蛋白IgG,并将提取的IgG交联到工作微球上,得到免疫微球。以不同浓度的PEG6000为加速剂,观察免疫微球与RF的凝聚情况。选择PEG6000的最适浓度、RF最佳反应时间、反应温度、加样量、检测波长,并绘制RF标准曲线,确定RF检测的线性范围。通过重复性实验测定批间批内变异系数,回收实验确定系统误差,确定正常人群RF参考值范围。测定通过金标准确认RA患者血清RF,计算敏感性、特异性。
实验结果表明胶乳比浊法RF定量检测的最佳反应条件为:PEG的最佳浓度为3.8%,胶乳的最佳浓度为0.30%,加样量为10μL,检测波长500nm,反应时间共15分钟。批内、批间变异系数分别为1.80～4.33%和7.5 3～14.14%;RF检测试剂的评价结果为:检测敏感度为81.5%,特异性为69.0%,跃顿指数50.5%;样品中加入已知量RF测定,回收率为94.2～1 06.5%,对含高浓度RF的血清样品稀释测定,结果表明方法的健全性良好。最低测定灵敏度为3IU/ml。稳定实验试剂保质期为12月以上(4℃)。
成功地制备了胶乳比浊法RF定量检测试剂盒,为临床实验室RF的检测提供了新的选择。
Rheumatoid Arthritis (RA) is a common rheumatic disease, the prevalence rate of it is about 0.4% to 1.0%, if the disease can not be diagnosed or treated in time, it often cause joint deformities, which will seriously affect the ability to work and daily life, after one to three years. Increasing its rate of early diagnosis is the doctors concern. Rheumatoid factor (RF) is one of the most important bases for the diagnosis of rheumatoid arthritis, the fast and accurate detection of RF is very valuable to the diagnosis and prognosis of RA. There are kinds of methods to measure RF: latex agglutination, ELISA (enzyme linked immunosorbent assay), imnunoturbidimetry(nephelometryand and tronsmisson turbidimetry and immunolatex turbimetry). Latex agglutination of RF is a classical method, but it's only a semi-quantitative determination, and can only through IgM-RF. The last two methods can carry out quantitative determination of the RF, ELISA method can determine the RF and its subtypes. According to the recent development of automation equipment, scattering turbidity and transmission method has been used a lot in clinical diagnosis. Most of the current reagents are imported, they are very expensive.
The theme of this research is to study the latex Turbidimetry volume of RF, and develop the domestic intellectual property rights RF kit for clinical diagnosis of low-cost, high value clinical diagnostic reagents
The modest size of PEG, uniform particles of polystyrene microspheres,were chosen and pre-treated for the preparation of working microsphere. The human immunoglobulin IgG was exstracted and purified by ammonium sulfate precipitation and affinity chromatography. The immuno-mirospheres were obtained by cross linking the IgG to working microspheres. The serials of concentration of PEG6000 were added into the antigen-antibody reaction system, and the optimal concentration of accelerating agent(PEG6000) was determined. The optimal reaction time, reaction temperature,the quantity of application of sample and test wave length were determined by orthogonal experiment. the standard curve was made and the linearity range was determined. The coefficient of variability of inter-batch and intra-batch were checked by repeatability test, and the systematic error was checked by recovery test. The reference range was established. The sensitivity and specificity of the reagents were test by testing of clinical samples.
Highly purified human immunoglobulin IgG was obtained successfully. The desired immuno-microspheres were successfully prepared. The optimal concentration of PEG6000 was 3.8%, and the optimal concentration of latex was 0.3%; the optimized quantity of application of sample was 10μl,the detection wave length was 500nm, the total reaction time was 15 minutes. The coefficient of variability of intra-batches and inter-batches was 1.80-4.33% and 7.53-14.14%, respectively. The recovery rate was 94.2-106.5%.Detection sensitivity was 81.5% and specificity was 69.0%, youden index was 50.5%. The lowest detective sensitivity was 3IU/ml,and the assured stable experiment reagent time was 12 monthes at 4℃refrigerator.
The latex Turbidimetry rheumatoid factor detecting kits was successfully developed, which providing an alternative selection for the dection of rheumatoidfactor in clinical laboratory.
引文
1.Walter E.,On the occurrence of a factor in human serum activating the specific agglutination of sheep blood corpuscles,Acta Pathol Microbiol Scand.,1940,17:172-188
2.Rose H.M.,et al.,Differential agglutination of normal andsensitized sheep erythrocytes by sera of patient with rheumatoid arthritis,Proc.Soc.Exp.Biol.Med.,1949,68:1
3.Terry L Moore,Rheumatoid factors,Clinical Immunology,1998,18(9):89-104
4.Roitt I.M.,Essential Immunology,Oxford,Eng.:Black well Scientific Publications,1977
5.Spector T.D.,Rheumatoid arthritis,Rheum.Dis.North.Am.,1990,16:513
6.Hochberg M.C.,Epideminology of the rheumatic diseases in Schumacher HR (eds),Primer onrheumatic diseases Atlanta GA,hrthritis Found,1988:48-51
7.滕庆,刘永革,何晓琥.抗环瓜氨酸肽抗体和隐匿性类风湿因子免疫球蛋白M 型在诊断早期幼年类风湿关节炎中的意义[J].实用儿科临床杂志,2004,19(10):845 847.
8.Zeng X,AiM,Tian X,et al.Diagnostic value of anticyclic citrulli-nated peptide antibody in patients with rheumatoid arthritis [J].J Rheumatol,2003,30:1451-1455
9.Bas S,Perneger TY.Diagnostic tests for rherumatoid arthritis:comparison of anticyclic cirt rullinatedpetide antibodies,antikerat in antibodies and IgM rheumatoid factors[J].Rheumatology,2002;41(7):809
10.van VenrooijWJ,Zendman AJ,Pruijn GJ.Autoantibodies to citrulli-nated antigens in(early)rheumatoid arthritis[J].Autoimm un Rev,2006,6(1):37-41.
11.穆荣,孙晓云,栗占国.类风湿因子和抗环瓜氨酸多肽抗体联合检测在类风湿关节炎诊断中的意义.Journal of Peking University(Health Sciences)Vol.37 No.5 Oct.2005 498-500
12.Aho K,Palusuo T,Kurki P.Marker antibodies of rheumatoid arthritis:diagnostic and pathogeneticimp lications[J].Semin Arthritis Rheum,1994,23:379-387
13.何菁,贾汝琳,韩蕾,等.隐性类风湿因子在类风湿关节炎诊断中的意义.中华风湿病学杂志,2001,5:24-26
14.Verpoort KN,Jol-van der Zijde CM,Papendrecht-van der Voort EA,et al.Isotype distribution of anti-cyclic citrullinated pep tide anti2bodies in undifferentiated arthritis and rheumatoid arthritis reflects anongoing immune response[J].Arthritis Rheum,2006,54(12):3799-3808.
15.Pai S,Pai L,Birkenfeldt R.Correlation of serum IgA rheumatoid factor levels with disease severity in rheumatoid arthritis[J].Scand J Rheumatol,1998,27(4):252-256.
16.Bizzaro N,Mazzanti G,Tonutti E,et al.Diagnostic accuracy of the anti-citrulline antibody assay for rheumatoid arthritis[J].Olin them,2001,47(6)1089-1093.
17.Jonson T.Is measurement of rheumatoid factor isotypes clinically useful[J]?Ann Rheum Dis,1993,52(3):161
18.William aon MeColl GJ.Early rheumatoid arthritis:can weprediot its outcome[J].IntMed J,2001,31(3):168-180.
19.辛华,牟洪香,郑强等.免疫比浊法和胶乳凝集法测定类风湿因子的比较.Hei Long Jiang Medicine And Pharmacy Jun.2007,Vol.30 No.3:27-28
20.徐胜前,高松,张锐等.免役比浊法和胶乳凝集法测定类风湿因子的比较.中华风湿学杂志,2001,5(3):201-202
21.陈丽华.早期类风湿关节炎病人中类风湿因子的临床意义.中华内科杂志,1995,34(7):449-451
22.Josson T,SteinssonK,JossonH,eta l.C ombinede levation of IgM and IgA Rheumatoid factor has high diagnostic specificity for rheumatoida rthritis.Rheumatol Int,1998,18(3):119-122.
23.John C.Thompson,Alan R.Craig,Carol L.Davey,David J.Kinetics and proposed mechanism of the reaction of an immunoinhibition,particle-enhanced immunoassayClin.Chem.,Dec 1997;43:2384-2389.
24.P.Englebienne,A.Van Hoonacker,and J.Valsamis Rapid Homogeneous Immunoassay for Human Ferritin in the Cobas Mira Using Colloidal Gold as the Reporter ReagentClin.Chem.,December 1,2000;46(12):2000-2003.
1.Jonsson T,Steinsson K,Jonsson H,et al.Combined elevation of IgM and Igh rheumatoid factor has high diagnostic specificity for rheumatoid arthritis[J].Rheumatol Int,1998,18(3):119-122
2.Aho K,Heliovaara M,Maatela J,et al.Rheumatoid factors antedating clinical rheumatoid arthritis.J Rheumatol,1991,18:1282-1284.
3.Visser H,Gelinck LB,Kampfaath AN,et al.Diagnostic and prognostic characteristics of the enzyme linked immunosorbent rheumatoid factor assays in rheumatoid arthritis.Ann Rheum Dis,1996,55(3):157-161.
4.Josson T,Thorsteinsson J,Kolbeinsson A,et al.Population study of the importance of rheumatoid factor isotypes in adults.Ann Rheum Dis,1992,51(7):863-868.
5.Josson T,Steinsson K,Josson H,et al.Combined elevation of IgM and IgA rheumatoid factor has high diagnostic specificity for rheumatoid arthritis.Rheumatol Int,1998,18(3):119-122.
6.Zeben D,Hazes JM,Zwinderman AH,et al.Clinical significance of rheumatoid factors in early rheumatoid arthritis:results of a follow-up study.Ann Rheum Dis,1992,51:1029.
7.陈丽华.早期类风湿关节炎病人中类风湿因子的临床意义.中华内科杂志,1995,34(7):449-451.
8.Pai S,Pai L,Birkenfeldt R.Correlation of serum IgA rheumatoid factor levels with disease severity in rheumatoid arthritis.Scand J Rheumatol,1998,27(4):252-256.
9.Houssien DA,Josson T,Davies E,et al.Clinical significance of IgA rheumatoid factor subclasses in rheumatoid arthritis.J Rheumatol,1997,24(11):2119-2122.
10.杨嘉林,于彦,史丽等.斑点免疫吸附法侧定IgE型类风湿因子.中华医学杂志,1992,72(10):587-589.
11.OliverJE,Worthington J,SilmanAJ,et al.Genetic epidemiologyof rheumatoid arthritis[J].CurrOpin Rheumatol,2006,18(2):141-146.
12.SircarD,GhoshB,Ghoshh,et al.Juvenile idiopathic arthritis[J].Indian Pediatr,2006,43(5):429-433.
13.Phelan JD,Thompson SD.Genomicprogress in pediatric arthritis:Recent work and future goals.Pediatric and heritable disorders [J].CurrOpin Rheumatol,2006,18(5):482-489.
14.滕庆,刘永革,何晓琥.抗环瓜氨酸肽抗体和隐匿性类风湿因子免疫球蛋白M型在诊断早期幼年类风湿关节炎中的意义[J].实用儿科临床杂志,2004,19(10):845 847.
15.van VenrooijWJ,Zendman AJ,Pruijn GJ.hutoantibodies to citrullinated antigens in(early)rheumatoid arthritis[J].Autoimmun Rev,2006,6(1):37-41.
16.BrunnerJK,Sitzmann FC.Anticyclic citrullinated peptide antibodies injuvenile idiopathic arthritis[J].Mod Rheumatol,2006,16(6):372-375.
17.van RossumM,van Soesbergen R,de Kort S,et al.Anti-cyclic citrullinated peptide(anti-CCP)antibodies in children with juvenile idiopathic arthritis[J].J Rheumatol,2003,30(4):825-828.2
18.Verpoort KN,Jol-van der Zijde CM,Papendrecht-van der Voort EA, et al. Isotype distribution of anti- cyclic citrullinated peptide antibodies in undifferentiated arthritis and rheumatoid arthritis reflects anongoing immune response [J]. Arthritis Rheum, 2006, 54 (12): 3799- 3808.
19. Robert HS, Thomas L. The rheumatoid factor: an analysis of clinical utility. Am,J Med, 1991,91:528-533.
20. Tutuncu Z, Reed G, Kreruz J, et al. Do patientswith older onset rheumatoid arthritis receive less aggressive treatment [J]. Ann Rheum Dis, 2006, 65 (9): 1 226 - 1 229.
21. Olivieri I, Palazzic, Peruz G, et al. Management issues with elderly - onset rheumatoid arthritis: anupdate[J]. Drug Aging, 2005, 22(10): 809 - 822.
22. Htllier J P, Eliaou J F, Daures J P, et al. HLA-DRB1 genes and patients with late onset rheumatoid arthritis [J]. Ann Rheum Dis, 2001, 60(5): 531 - 533.
23. Forslin K, Vincent C, Serre G, etal. Antifilaggrin antibodies in early rheumatoid arthritis may predict radiological progression [J]. Scand J Rheumatol, 2001,30:221 - 224
24. Forslind K, AhlmenM, EberhardtK, etal. Prediction of radiological outcome in early rheumatoid arthritis in clinical practice: role ofantibodies to citrullinated peptides [J]. Ann Rheum Dis, 2004,63:1,090-1095
2.Rose H.M.,et al.,Differential agglutination of normal andsensitized sheep erythrocytes by sera of patient with rheumatoid arthritis,Proc.Soc.Exp.Biol.Med.,1949,68:1
3.Terry L Moore,Rheumatoid factors,Clinical Immunology,1998,18(9):89-104
4.Roitt I.M.,Essential Immunology,Oxford,Eng.:Black well Scientific Publications,1977
5.Spector T.D.,Rheumatoid arthritis,Rheum.Dis.North.Am.,1990,16:513
6.Hochberg M.C.,Epideminology of the rheumatic diseases in Schumacher HR (eds),Primer onrheumatic diseases Atlanta GA,hrthritis Found,1988:48-51
7.滕庆,刘永革,何晓琥.抗环瓜氨酸肽抗体和隐匿性类风湿因子免疫球蛋白M 型在诊断早期幼年类风湿关节炎中的意义[J].实用儿科临床杂志,2004,19(10):845 847.
8.Zeng X,AiM,Tian X,et al.Diagnostic value of anticyclic citrulli-nated peptide antibody in patients with rheumatoid arthritis [J].J Rheumatol,2003,30:1451-1455
9.Bas S,Perneger TY.Diagnostic tests for rherumatoid arthritis:comparison of anticyclic cirt rullinatedpetide antibodies,antikerat in antibodies and IgM rheumatoid factors[J].Rheumatology,2002;41(7):809
10.van VenrooijWJ,Zendman AJ,Pruijn GJ.Autoantibodies to citrulli-nated antigens in(early)rheumatoid arthritis[J].Autoimm un Rev,2006,6(1):37-41.
11.穆荣,孙晓云,栗占国.类风湿因子和抗环瓜氨酸多肽抗体联合检测在类风湿关节炎诊断中的意义.Journal of Peking University(Health Sciences)Vol.37 No.5 Oct.2005 498-500
12.Aho K,Palusuo T,Kurki P.Marker antibodies of rheumatoid arthritis:diagnostic and pathogeneticimp lications[J].Semin Arthritis Rheum,1994,23:379-387
13.何菁,贾汝琳,韩蕾,等.隐性类风湿因子在类风湿关节炎诊断中的意义.中华风湿病学杂志,2001,5:24-26
14.Verpoort KN,Jol-van der Zijde CM,Papendrecht-van der Voort EA,et al.Isotype distribution of anti-cyclic citrullinated pep tide anti2bodies in undifferentiated arthritis and rheumatoid arthritis reflects anongoing immune response[J].Arthritis Rheum,2006,54(12):3799-3808.
15.Pai S,Pai L,Birkenfeldt R.Correlation of serum IgA rheumatoid factor levels with disease severity in rheumatoid arthritis[J].Scand J Rheumatol,1998,27(4):252-256.
16.Bizzaro N,Mazzanti G,Tonutti E,et al.Diagnostic accuracy of the anti-citrulline antibody assay for rheumatoid arthritis[J].Olin them,2001,47(6)1089-1093.
17.Jonson T.Is measurement of rheumatoid factor isotypes clinically useful[J]?Ann Rheum Dis,1993,52(3):161
18.William aon MeColl GJ.Early rheumatoid arthritis:can weprediot its outcome[J].IntMed J,2001,31(3):168-180.
19.辛华,牟洪香,郑强等.免疫比浊法和胶乳凝集法测定类风湿因子的比较.Hei Long Jiang Medicine And Pharmacy Jun.2007,Vol.30 No.3:27-28
20.徐胜前,高松,张锐等.免役比浊法和胶乳凝集法测定类风湿因子的比较.中华风湿学杂志,2001,5(3):201-202
21.陈丽华.早期类风湿关节炎病人中类风湿因子的临床意义.中华内科杂志,1995,34(7):449-451
22.Josson T,SteinssonK,JossonH,eta l.C ombinede levation of IgM and IgA Rheumatoid factor has high diagnostic specificity for rheumatoida rthritis.Rheumatol Int,1998,18(3):119-122.
23.John C.Thompson,Alan R.Craig,Carol L.Davey,David J.Kinetics and proposed mechanism of the reaction of an immunoinhibition,particle-enhanced immunoassayClin.Chem.,Dec 1997;43:2384-2389.
24.P.Englebienne,A.Van Hoonacker,and J.Valsamis Rapid Homogeneous Immunoassay for Human Ferritin in the Cobas Mira Using Colloidal Gold as the Reporter ReagentClin.Chem.,December 1,2000;46(12):2000-2003.
1.Jonsson T,Steinsson K,Jonsson H,et al.Combined elevation of IgM and Igh rheumatoid factor has high diagnostic specificity for rheumatoid arthritis[J].Rheumatol Int,1998,18(3):119-122
2.Aho K,Heliovaara M,Maatela J,et al.Rheumatoid factors antedating clinical rheumatoid arthritis.J Rheumatol,1991,18:1282-1284.
3.Visser H,Gelinck LB,Kampfaath AN,et al.Diagnostic and prognostic characteristics of the enzyme linked immunosorbent rheumatoid factor assays in rheumatoid arthritis.Ann Rheum Dis,1996,55(3):157-161.
4.Josson T,Thorsteinsson J,Kolbeinsson A,et al.Population study of the importance of rheumatoid factor isotypes in adults.Ann Rheum Dis,1992,51(7):863-868.
5.Josson T,Steinsson K,Josson H,et al.Combined elevation of IgM and IgA rheumatoid factor has high diagnostic specificity for rheumatoid arthritis.Rheumatol Int,1998,18(3):119-122.
6.Zeben D,Hazes JM,Zwinderman AH,et al.Clinical significance of rheumatoid factors in early rheumatoid arthritis:results of a follow-up study.Ann Rheum Dis,1992,51:1029.
7.陈丽华.早期类风湿关节炎病人中类风湿因子的临床意义.中华内科杂志,1995,34(7):449-451.
8.Pai S,Pai L,Birkenfeldt R.Correlation of serum IgA rheumatoid factor levels with disease severity in rheumatoid arthritis.Scand J Rheumatol,1998,27(4):252-256.
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