Korean J Lab Med 21;3:675-84 DOI 1.3343/kjlm.21.3.6.675 Original Article Diagnostic Immunology Evaluation of Anti-dsDNA Antibody Tests: Crithidia luciliae Immunofluorescence Test, Immunoblot, Enzyme-linked Immunosorbent Assay, Chemiluminescence Immunoassay Jin-young Yang, M.D., Eun-Jee Oh, M.D., Yonggoo Kim, M.D., and Yeon-Joon Park, M.D. Department of Laboratory Medicine, The Catholic University of Korea School of Medicine, Seoul, Korea Background : Anti-double stranded DNA antibody (anti-dsdna) test is useful for the diagnosis and monitoring of systemic lupus erythematosus (SLE). Although several methods are available, none of them is completely satisfactory and differences among them have been reported. We evaluated the diagnostic performance of 6 commercial kits for anti-dsdna detection. Methods : A total of 142 sera (SLE [N=74], other systemic rheumatic diseases [N=], other diseases [N=18]) were tested by 6 different assay kits using different antigenic sources of DNA: Crithidia luciliae immunofluorescence test (CLIFT), salmon testes (immunoblot, IB), human (ELISA I), salmon testes with nucleosome linker (ELISA II), plasmid (ELISA III), and synthetic oligonucleotides (chemiluminescence immunoassay, CLIA). Results : With manufacturers cut-off values, 6 test kits showed sensitivities of 55.4-91.9%. ELISA I had a greater sensitivity than the other five assays (P<.1). The specificities of ELISA II, ELISA III, CLIA, and CLIFT were higher than those of ELISA I and IB (P<.5). In ROC curve analysis, 3 ELISA kits and CLIA showed AUC values of.845-.893, and revealed no significant differences among them (P>.5). With cut-off values set at 95% of specificity, ELISA II had a higher sensitivity than ELISA III (63.5% vs. 41.9%, P<.5). IB had poor concordance rates with other assays (42.- 65.%). Pearson correlation coefficients among 4 quantitative assays were.667-.798. Conclusions : Six different assays showed various performances depending on the methods and cut-off values used. Except IB, the other five assays can be used for the detection of anti-dsdna. (Korean J Lab Med 21;3:675-84) Key Words : Anti-dsDNA, Crithidia luciliae immunofluorescence test, ELISA, Chemiluminescence immunoassay, Immunoblot assay 서 전신홍반루푸스 (systemic lupus erythematosus, SLE) 는염증성자가면역질환으로다양한자가항체를특성으로하고있 Received : May 31, 21 Manuscript No : KJLM1-12 Revision received : August 18, 21 Accepted : October 9, 21 Corresponding author : Eun-Jee Oh, M.D. Department of Laboratory Medicine, Seoul St. Mary s Hospital, 5 Banpo-dong, Seocho-gu, Seoul 137-71, Korea Tel : +82-2-2258-1641, Fax : +82-2-2258-1719 E-mail : ejoh@catholic.ac.kr *This work was supported by a grant of the Korea Healthcare technology R & D project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (A92258). ISSN 1598-6535 론 The Korean Society for Laboratory Medicine 다. 항 double-stranded DNA (dsdna) 항체의검출은 SLE의진단에중요하며 [1], American College of Rheumatologists 의 SLE 진단기준에포함되어있다 [2]. 또한 SLE 질병의활성도와항 dsdna항체수준의상관성이보고되어있으므로항 dsdna항체정량검사는 SLE의치료전후추적에매우유용하다 [1, 3-5]. 항 dsdna항체의검출을위한검사방법으로방사선면역측정법 (radioimmunoassay, RIA) 을이용한 Farr assay, Crithidia luciliae를이용한면역형광측정법 (immunofluorescence test) (CLIFT), 효소면역측정법 (enzyme-linked immunosorbent assay, ELISA), 화학발광면역측정법 (chemiluminescence immunoassay, CLIA), 면역블롯법 (immunoblot, IB) 등다양한검사가소개되어왔다. 이중 Farr assay는높은총 675
676 Korean J Lab Med 21;3:675-84 결합력 (high avidity) 을가진항 dsdna항체만을검출하므로 SLE 진단에특이도가높으나 [6], 동위원소를사용하고, 항체의동형감별이어려우며, 일반적으로자동화검사가아니므로이용이급격히감소되고있다 [7, 8]. 또한경도의 SLE에서는낮은친화력 (low affinity) 을가진항체만검출되므로 Farr assay만을이용한선별검사에서는 SLE 환자에서위음성결과를보일수도있다 [9]. CLIFT는중간정도에서높은정도의총결합력을가진항체를검출하므로특이도가높은검사로알려져있으나, Crithidia luciliae의 kinetoplast는 dsdna뿐만아니라 histones을발현하므로, 항 histone항체에의한위양성이있을수있고 [1], 정량검사가아니므로 SLE 환자의추적검사에이용할수없어유용성이떨어진다 [5]. ELISA는검사방법이간단하고재현성이높으며자동화가가능하여다양한시약이소개되어있고, 그사용이증가하고있다. 그러나높은친화력을가진항체뿐아니라낮은친화력을가진항체도검출하므로민감도는높으나특이도가낮다고알려져있으며 [5, 11-14], single stranded DNA (ssdna) 에의한위양성결과를초래할수있고 [14], 검사에이용되는항원과검출방법등이시약마다달라검사의표준화가어려운단점이있다 [15]. 최근이러한 ELISA 의단점을보완하여재조합또는합성된항원이나 plasmid 등보다정제된항원을이용하고, 항원결합방법과항체검출을개선한차세대 ELISA 키트와 CLIA가소개되었다. IB는다양한항 extracted nuclear antigen (ENA) 항체검출이가능하여자가면역질환의선별검사로이용되고있는데, dsdna 항원이포함된경우항 dsdna항체의검출도가능하다. 국내에서는다양한 ELISA, CLIA, CLIFT, IB가소개되어사용되고있으나, 최근소개된차세대 ELISA 시약들의비교평가는보고된바없으므로, 저자들은정량검사인세가지 ELISA 키트와 CLIA, 정성검사인 CLIFT, IB를시행하고각검사의임상적유용성을비교평가하였다. 대상및방법 1. 대상본원에항 dsdna항체검사가의뢰된총 142명의검체를대상으로하였다. 대상군은남자 16명, 여자 126명이었으며평균연령은 43.8세 (43.8±15.) 였다. 대상군의임상진단은항 ds- DNA항체검사가의뢰된시점의의무기록을기준으로하였으며, 질환의활성도가확인된 SLE 환자 74명과 SLE를제외한전신자가면역질환환자 (other systemic rheumatic disease, ORD) 명, 그외다른질환환자 ( 이하 others) 18명을포함하였다. SLE 환자 74명은모두 SLE 진단기준 [2] 11개항목중 4 개이상을만족하여 SLE로진단받았으며, 항 dsdna항체이외에항인지질항체또는항 Sm 항체양성이거나항 dsdna항체결과와상관없이 SLE 진단이가능한환자였다. 또한보체감소, 치료후추적검사양성등 ECLAM scoring system [16] 에의해질환의활성도를확인하였다. 환자중 3명 (4.%) 은검사의뢰당시 SLE가의심되어추후확진되었고, 62명 (83.8%) 은본원과타병원에서 SLE로진단받고치료중이었으며, 나머지 9명 (12.2%) 은질환이악화되어입원한환자들이었다. 진단당시사용한항 dsdna항체검사키트는 ds-dna IgG antibodies ELISA kit (Genesis Diagnostics, Cambridgeshire, UK) 였다. ORD 예에는쇼그렌증후군 22예, 미분화결체조직질환 8예, 류마티스성관절염 5예, 염증성근병증 3예, 레이노드증후군 2예, 전신성경화증 2예, 성인발병 Still병 2예, 베체씨병 2예, 항인산증후군 2예, 강직성척추염 1예, 자가면역성간염 1예가포함되었다. 그외다른질환은관절염 9예, 뇌경색 2예, 경부림프절병증, 만성신장부전, 결절성홍반, 과민성폐렴, 폐색전증, 척수암, 위암각각 1예씩이었다. 정상인으로부터얻은 pooled sera 3가지를음성대조액으로사용하였다. CLIFT, ELISA, CLIA, IB는시약회사의지침에따라시행하였으며, 정량검사 (ELISA, CLIA) 는 WHO reference preparation (Wo/8) 에의한 calibration에의해농도를산출하였고단위는 IU/mL 로표시하였다. 각검사에사용된 dsdna 항원, 2차항체, 검사방법, 제조사에서제시하는경계치, 검출농도범위, 민감도, 특이도등은 Table 1에요약하였다. 2. 방법 1) Crithidia luciliae를이용한면역형광측정법 (CLIFT) CLIFT는 FLUORO ndna TEST (Medical & biological laboratories Co., Nagoya, Japan, 이하 CLIFT) 시약을이용하여시약회사의지침에따라검사하였다. 1:5로희석된혈청 4 ml를 Crithidia luciliae의항원이부착된슬라이드위에떨어뜨리고습윤상자에넣어실온에서 2분간반응시켰다. 세척후 fluorescein isothiocyanate를결합시킨 goat anti-human IgG를슬라이드의각 well에한방울씩떨어뜨리고, 실온에서 2분간반응시킨후세척하였다. Mounting medium을얹고형광현미경으로 2명의연구자가관찰하여 kinetoplast에형광이발현되는것을양성으로판독하였다.
Yang J-Y, et al., Evaluation of 6 Anti-dsDNA Kits 677 Table 1. Characteristics of assay kits used for the detection of anti-dsdna Methods Assay kits Antigen Conjugated Cut-off AMR (IU/mL) Sensitivity Specificity ELISA I Genesis Human DNA Rabbit anti-human IgG-HRP IU/mL -4 NA* NA* ELISA II Euroimmun Salmon testes Rabbit anti-human IgG-HRP IU/mL 2.6-757 6. 99. ELISA III Scimedx Plasmid Goat anti-human IgG-HRP 35 IU/mL 5-99.2 CLIA LIAISON Synthetic oligonucleotides Mouse MoAb- isoluminol 25 IU/mL.5-24 84.9 98. IB Immunoblot Salmon testes Goat anti-human IgG-ALP 1+ NA 85. Euroimmun CLIFT FLUORO ndna Crithidia luciliae, kinetoplast Goat anti-human IgG-FITC 1+ NA 87.5 98.5 Cut-off values, AMR, sensitivity, and specificity are those provided by the manufacturers. *Not shown in manufacturer s instruction. Abbreviations: AMR, analytical measurable range; ELISA, enzyme-linked immunosorbent assay; HRP, horseradish peroxide; NA, not available; CLIA, chemiluminescence immunoassay; IB, immunoblot assay; ALP, alkaline phosphatase; CLIFT, Crithidia luciliae immunofluorescence test. 2) 효소면역측정법 (ELISA) (1) ELISA I (ds-dna IgG antibodies) Human DNA를항원으로이용하는 ds-dna IgG antibodies ELISA kit (Genesis Diagnostics, 이하 ELISA I) 를사용하였다. 1:으로희석된혈청을분주한후실온에서 3분간반응시켰다. 3회세척후 horseradish peroxidase (HRP) 를결합시킨 rabbit anti-human IgG ml를넣고실온에서 3 분반응시킨후세척하였다. TMB substrate ml를넣고실온에서 1분간반응시키고정지액 ml를넣은후 4 nm 에서흡광도를측정하였다. (2) ELISA II (anti-dsdna-ncx ELISA [IgG]) Salmon testes에서추출한 dsdna를 nucleosome을 linker로이용하여 microplate에부착한 anti-dsdna-ncx ELISA (IgG) (Euroimmun, Lu_beck, Germany, 이하 ELISA II) 를사용하였다. 1:으로희석된혈청을반응시킨후, rabbit anti-human IgG-peroxidase를첨가하였다. 다른검사과정은 ELISA I과동일하였다. (3) ELISA III (EIA kit for anti-dsdna antibody) Plasmid DNA를항원으로사용하는 EIA kit for anti-dsdna antibody (Scimedx corporation, NJ, USA, 이하 ELISA III) 를사용하였다. 1:으로희석된혈청을반응시켰고, conjugate로 goat anti-human IgG-HRP를사용하였다. 다른검사과정은 ELISA I과동일하였다 3) 화학발광면역측정법 (CLIA) LIAISON dsdna (DiaSorin, Vercelli, Italy) 시약으로자동화장비 (LIAISON, DiaSorin) 를이용하여검사하였다. Synthetic dsdna가 streptavidin-biotin 결합에의해 magnetic particles에부착되어있고, isoluminol derivative가표지자로붙은 mouse monoclonal antibody를사용하여혈청내항 ds- DNA 항체 (IgG) 를검출하였다. 4) 면역블롯법 (IB) SLE-Profil EUROASSAY (Euroimmun) 시약과 Euroblot Master (Euroimmun) 스캐너를사용하여검사하였다. Salmon testes에서추출한 dsdna 항원이부착된스트립에 1:으로희석된혈청 ml 를분주하고실온에서 3분간 shaking (3 rpm) 하면서반응시켰다. 세척후 alkaline phosphatase를결합시킨 goat anti-human IgG ml를분주하여실온에서 3분반응시키고, 세척후기질액 ml를넣어실온에서 1분간발색반응시켰다. 증류수로세척후건조시키고, 발색강도를판독하여 1+ 이상을양성으로판독하였다. 3. 통계적분석 6가지항 dsdna항체검사의 SLE 환자검출을위한민감도와특이도를구하였다. 진단명을바탕으로 SLE 환자에서의항 dsdna항체양성률을민감도로정하고, SLE를제외한전신자가면역질환자와그외다른질환자에서의항 dsdna항체음성률을특이도로정하였다. 정량검사는 1) 제조회사가제시한경계치, 2) ROC 곡선을구하여민감도와특이도의합이최대가되도록정한경계치, 3) ROC 곡선분석에서특이도가 95% 일때의경계치로구분하여분석하였다. 각검사간일치율은 interrater agreement 방법을이용하였으며, Kappa value.2 이하를 poor,.21-.6을 fair to moderate,.61 이상을 good agreement로판정하였다. 정량검사들의상관성분석은 Pear-
678 Korean J Lab Med 21;3:675-84 son 상관계수를이용하였다. 통계분석은 SPSS 12. (SPSS Inc., Chicago, IL, USA) 과 MedCalc version 11.3 (MedCalc Software, Mariakerke, Belgium) 을사용하여 P<.5인경우를통계적으로유의하다고판정하였다. 결 1. 항 dsdna항체검사방법들의 SLE 환자검출을위한민감도와특이도분석 각검사방법의민감도와특이도는 Table 2-4에제시하였으며, 정량검사는 3가지의경계치를기준으로분석되었다. 항 dsdna항체의정량결과를환자군 (SLE, ORD, Others) 에따라구분하여 Fig. 1에나타냈다. 제조회사에서제시하는경계치 Table 2. Analytical performance of various anti-dsdna antibody assays based on the manufacturers cut-off values Assay kits Cut-off (IU/mL) Abbreviations: SLE, systemic lupus erythematosus; ORD, other systemic rheumatic diseases; CLIFT, Crithidia luciliae immunofluorescence test; IB, immunoblot assay; ELISA, enzyme-linked immunosorbent assay; CLIA, chemiluminescence immunoassay. 과 N of patients with positive results SLE (N=74) ORD (N=) Others (N=18) Sensitivity Specificity CLIFT 51 (68.9) 7 (14.) (.) 68.9 89.7 IB 41 (55.4) 28 (56.) 9 (.) 55.4 45.6 ELISA I 68 (91.9) 17 (34.) 3 (16.7) 91.9 7.6 ELISA II 53 (71.6) 4 (8.) 1 (5.6) 71.6 92.6 ELISA III 35 46 (62.2) 5 (1.) (.) 62.2 92.6 CLIA 25 41 (55.4) 3 (6.) (.) 55.4 95.6 를기준으로분석한결과, 본연구에포함된 6가지검사의민감도는 55.4-91.9%, 특이도는 45.6-95.6% 였다. 민감도는 ELISA I이 91.9% 로가장높았으며나머지 5가지검사의민감도보다유의하게높았고 (P<.1), 특이도는 ELISA II, ELISA III, CLIA, CLIFT가 ELISA I (7.6%) 과 IB (45.6%) 에비해유의하게높았다 (P<.5). 정량검사들의 ROC 곡선분석을통한민감도는 71.6-83.8% 였고, 특이도는 82.4-89.7% 였으며, ELISA II 의특이도가 89.7% 로가장높았다. 특이도를 95.% 로설정할경우, 4가지정량검사의민감도는 41.9-63.5% 로분포되었으며, ELISA II의민감도 (63.5%) 가 ELISA III의민감도 (41.9%) 에비해유의하게높았다 (P<.5). 2. ROC 곡선분석 3가지 ELISA와 CLIA의 SLE 환자검출을위한 ROC 곡선분석결과, AUC 값은 ELISA I.893, ELISA II.892, ELISA III.845, CLIA.849로서 ELISA I의 AUC 값이가장높았으나, 검사들간 AUC 값의차이는통계적으로유의하지않았다 (P>.5) (Fig. 2). 3. 항 dsdna항체검사결과의일치율분석본연구에포함된 6가지검사의 3가지경계치에따른일치율을 Table 5에나타냈다. 각검사들간의일치율은 42.-88.8% 였으며, Kappa 계수에따라 good agreement 이상 (Kappa value.61) 인경우일치율에밑줄을그어표시하였다. CLIFT 는 IB 를제외한 ELISA 및 CLIA와모두양호한일치율을나타냈고, ELISA II와 CLIA가 CLIFT 결과와가장높은일치율 Table 3. Analytical performance of various anti-dsdna antibody assays based on the cut-off values determined by ROC curve analysis Table 4. Analytical performance of various anti-dsdna antibody assays based on the cut-off values set at 95% specificity by ROC curve analysis Assay kits Cut-off (IU/mL) N of patients with positive results SLE (N=74) ORD (N=) Others (N=18) Sensitivity Specificity Assay kits Cut-off (IU/mL) N of patients with positive results SLE (N=74) ORD (N=) Others (N=18) Sensitivity Specificity ELISA I 76.4 62 (83.8) 11 (22.) 1 (5.6) 83.8 82.4 ELISA II 59.8 61 (82.4) 6 (12.) 1 (5.6) 82.4 89.7 ELISA III 16.9 58 (78.4) 1 (2.) 2 (11.1) 78.4 82.4 CLIA 9.61 53 (71.6) 8 (16.) (.) 71.6 88.2 Abbreviations: SLE, systemic lupus erythematosus; ORD, other systemic rheumatic diseases; CLIFT, Crithidia luciliae immunofluorescence test; IB, immunoblot assay; ELISA, enzyme-linked immunosorbent assay; CLIA, chemiluminescence immunoassay. ELISA I 224.2 39 (52.7) 3 (6.) (.) 52.7 95. ELISA II 182.4 47 (63.5) 2 (4.) 1 (5.6) 63.5 95. ELISA III. 31 (41.9) 4 (8.) (.) 41.9 95. CLIA 16. 43 (58.1) 3 (6.) (.) 58.1 95. Abbreviations: SLE, systemic lupus erythematosus; ORD, other systemic rheumatic diseases; CLIFT, Crithidia luciliae immunofluorescence test; IB, immunoblot assay; ELISA, enzyme-linked immunosorbent assay; CLIA, chemiluminescence immunoassay.
Yang J-Y, et al., Evaluation of 6 Anti-dsDNA Kits 679 4 8 3 7 ELISA I (Genesis) 3 SLE ORD Others Cut-off III Cut-off II Cut-off I A ELISA II (EUROIMMUN) 6 4 3 SLE ORD Others Cut-off III Cut-off I Cut-off II B ELISA III (Scimedx) Cut-off III Cut-off I CLIA (LIAISON) Cut-off I Cut-off II Cut-off III SLE ORD Others Cut-off II C SLE ORD Others D Fig. 1. Anti-dsDNA levels measured by ELISA I (A), II (B), III (C), and CLIA (D) in 74 SLE patients, patients with other systemic rheumatic diseases (ORD) and 18 patients with other diseases (Others) (Cut-off I provided by manufactures, cut-off II determined by ROC curve, and cut-off III set at 95% specificity are indicated.). Abbreviations: SLE, systemic lupus erythematosus; ORD, other systemic rheumatic diseases; IB, immunoblot assay; ELISA, enzyme-linked immunosorbent assay; CLIA, chemiluminescence immunoassay. 1. (83.2%) 을보였다. 제조사에서제시한경계치를적용한경우, ELISA II 와 ELISA.8 III만 86.7% 의양호한일치율을나타냈고, 나머지검사들사이에서는 Kappa 계수.5-.6의 moderate agreement 를보 Sensitivity.6.4.2 ELISA I (AUC=.893) ELISA II (AUC=.892) ELISA III (AUC=.845) CLIA (AUC=.849) 였다. ROC 곡선분석에의한경계치나특이도 95% 를기준으로한경계치를적용한경우에는 ELISA I과 ELISA II의일치율이 88.8% 로가장높았으나, 나머지일치율도모두.58 이상의 Kappa 계수를나타내었다. IB는나머지모든검사와 42.- 65.% 의낮은일치율을보였다. 4. 정량검사결과의상관성분석...2.4.6.8 1. 1-specificity Fig. 2. ROC curve of the 3 ELISA assay kits and one CLIA. Abbreviations: ELISA, enzyme-linked immunosorbent assay; CLIA, chemiluminescence immunoassay. 4가지정량검사들간의상관성을 Fig. 3에나타냈다. Pearson 상관계수는.667-.798로 ELISA I과 CLIA의상관계수가.667로가장낮았고, ELISA I과 ELISA III 사이의상관계수가.798로가장높았다.
68 Korean J Lab Med 21;3:675-84 Table 5. Concordance rates of anti-dsdna detection results among different assay kits based on various cut-off values Method CLIA ELISA III ELISA II ELISA I IB CLIFT 83.2* 81.1 83.2 75.5 53.8 88.1 8.4 83.2 8.4 84.6 78.3 8.4 8.4 IB 65. 57.3 56.6 42. 55.9 53.8 53.8 46.9 63.6 61.5 59.4 62.2 ELISA I 64.3 72. 75.5 81.1 79. 88.8 83.2 86.7 86. ELISA II 81.8 86.7 83.9 83.2 83.2 82.5 ELISA III 81.1 83.9 85.3 *Agreement using the manufacturers cut-off values; agreement using the cut-off values determined by ROC curve analysis; agreement using the cut-off values set at 95% specificity by ROC curve analysis, underlined values showed good agreement (kappa value.61). Abbreviations: CLIA, chemiluminescence immunoassay; ELISA, enzyme-linked immunosorbent assay; IB, immunoblot assay; CLIFT, Crithidia luciliae immunofluorescence test. 고 6가지항 dsdna항체검사결과를제조사의경계치를기준으로분석한경우, 민감도는 55.4-91.9%, 특이도는 45.6-95.6% 로분포하였으며검사방법에따라다양한결과를나타냈다. CLIFT는일반적으로 ELISA에비해특이도가높고민감도가낮다고알려져있으나 [17, 18], 본연구에서는 CLIFT의특이도가 89.7% 로 ELISA II, III나 CLIA의특이도 92.6-95.6% ( 제조회사의경계치적용시 ) 보다낮은결과를보였다. 또한 ROC 곡선분석에의한경계치적용시, CLIFT의특이도는정량검사들과비슷하였으나민감도는 3가지 ELISA보다낮은결과를나타냈다. 이는 Antico 등 [19] 의연구에서 ELISA의특이도가 CLIFT보다높고, 최근소개된차세대 ELISA의특이도가향상되었다고한보고와일치한다. CLIA는 DiaSorin사에서개발한 LIAISON dsdna 시약을사용하였는데, histone과다른핵단백에대한항체의위양성반응을배제할수있는장점이있다. 본연구에서도제조회사에서제시한경계치적용시비교검사들중가장높은특이도 (95.6%) 를나타냈다. 4가지정량검사의분석에서는제조사의경계치를적용할경우, 민감도 55.4-91.9%, 특이도 7.6-95.6% 의결과를보였고, 민감도는 ELISA I이가장높았으나, 특이도는 CLIA가가장높았다. 그러나 ROC 곡선분석에서는 4가지정량검사의 AUC값 찰 차이에통계적유의성이없었으며 (P>.5), 민감도 71.6-83.8%, 특이도 82.4-89.7% 로서검사들의수행능차이가감소되었다. ELISA I은제조사의경계치를적용할경우, 특이도는 7.6% 로낮았고민감도는 91.9% 로서우수하였는데, 이는 ELISA I의경계치가다른정량검사에비해민감도가우수하도록설정되었기때문으로생각된다. 높은민감도를보인 ELISA I은검출항원으로 human DNA를사용하는데, Janyapoon 등 [2] 의연구에서사람혈액에서추출된 DNA를사용한 ELISA가박테리아에서추출된 DNA를사용한검사에비해높은민감도를보였다는보고와일치한다. 그러나 SLE 환자의진단에이용되는항 dsdna항체검사는높은민감도보다는높은특이도가요구되므로 ELISA I 시약을사용하는검사실에서는임상의와상의후경계치의상향조정이필요할것으로생각한다. 치료중인 SLE 환자에서질환의활성도는항 dsdna항체검사키트의민감도에영향을줄수있는데, Hora@k 등 [21] 의연구에의하면, 활성도가감소된환자에서는항 dsdna항체가음성으로전환될수있다. 따라서본연구에포함된치료중인 SLE 환자 62명에서는의무기록조회를통해보체감소, 치료후추적검사양성등 ECLAM scoring system [16] 에의해질환의활성도가있음을확인하였다. 또한기존에사용중이던 ELISA I 시약을사용하여 SLE 환자로진단되었을경우대상군선정에오류가있을수있으므로, 의무기록을검토하여 dsdna항체이외에항인지질항체또는항 Sm 항체양성이거나항 dsdna항체결과와상관없이 SLE 진단이가능한환자만을포함하였다. 항 dsdna항체는다양한질환에서검출될수있고건강한사람에서도양성일수있으므로 SLE 진단을위한검사는특이도가높아야한다 [22]. 이에본연구에서는제조사의경계치에따라각검사키트의민감도와특이도를분석하고, Antico 등 [19] 의연구에서와같이 ROC 곡선분석을통해특이도 95% 를나타내는경계치값으로각검사키트의민감도와특이도를분석하였다. 본연구에서제조사의경계치값과 ROC 곡선분석에의한경계치값에차이가큰이유는항 dsdna항체검사의특이도를높이기위해경계치를높게설정했거나, 제조사에서자체적으로검사키트의수행능평가시포함한대상군과본연구에포함된대상군의분포가다르기때문으로생각된다. 특히제조사에서는자체평가시정상대조군으로헌혈자의검체를많이포함하였으나 (ELISA II, 41.3%; ELISA III, 46.6%; CLIA, 38.3%), 일반적으로정상인을대상으로한선별검사에항 dsdna항체검사가포함되지않으므로본연구에서정상인은대상군에포함하지않았다. 이러한이유로제조사와 ROC 곡선분석에의한
Yang J-Y, et al., Evaluation of 6 Anti-dsDNA Kits 681 8 ELISA II (IU/mL) 7 6 4 3 r=.761, P<.1 ELISA III (IU/mL) r=.798, P<.1 3 4 ELISA I (IU/mL) 3 4 ELISA I (IU/mL) CLIA (IU/mL) r=.667, P<.1 ELISA III (IU/mL) r=.733, P<.1 3 4 ELISA I (IU/mL) 4 6 8 ELISA II (IU/mL) CLIA (IU/mL) r=.713, P<.1 CLIA (IU/mL) r=.735, P<.1 4 6 8 ELISA II (IU/mL) ELISA III (IU/mL) Fig. 3. The correlation among three commercial ELISA and one CLIA kits for anti-dsdna detection in 142 serum samples. Abbreviations: ELISA, enzyme-linked immunosorbent assay; CLIA, chemiluminescence immunoassay. 경계치값에차이를보인것으로해석된다. 본연구에서사용한검사결과들의일치율분석에서는 ELISA II와 ELISA III의일치율이 86.7% ( 제조사의경계치적용시 ) 로가장높았고, 동일한 ELISA 간의일치율도 72.-88.8% 로차이가있었다. 기존의여러연구들에서도각검사키트마다다양 한민감도와특이도를보고하였는데 [23-25], 항 dsdna항체검출을위한 ELISA 키트들사이에결과의일치율이낮은이유로는사용하는항원의특이성정도, 항체의결합력, 면역글로불린 class 등이다양하여사용하는검사방법에의해영향을받을수있고, 동일한 ELISA 방법을사용하더라도그항원의원
682 Korean J Lab Med 21;3:675-84 천이 human DNA, salmon testes DNA, plasmid DNA, recombinant DNA, calf thymus DNA 등으로차이가있기때문이다. 그외에피토프의크기와 3차원적인구조의차이 (conformational difference), 고체상 (solid phase) 에부착시킨 DNA 조각들의항원성의변이, DNA의구조적인재배열 [26], 항체반응의제한성, dsdna coating linker, 항 ssdna 항체에의한위양성, 검사시약마다다른경계치를사용하는점등도차이를유발하는원인이될수있다 [15]. Coating linker 로사용되는 protamine poly-l-lysine, protamine sulphate, methylated bovine serum albumin 등은혈청내면역복합체나면역글로불린에의해위양성을유발할수있는데, ELISA II는최근 dsdna와결합력이높은고순도의 nucleosome을 coating linker로교체하였으며, 본연구에서도이키트를평가에이용하였다. 고순도의 nucleosome은순수한 dsdna와결합력이높아검사의민감도와특이도를향상시킬수있는데, 본연구에서 ELISA II의민감도와특이도가 ROC 곡선분석에서 82.4% 와 89.7% 로서양호한결과를보였고, 특이도를 95.% 로설정한경우에는 ELISA II가비교한정량검사들중가장높은민감도 (63.5%) 를보였다. Kim 등 [17] 은 4가지 ELISA의비교에서 Euroimmun 항 dsdna항체검사의민감도가 14.7% 로가장낮았다고보고하였는데, 이는 Kim 등이사용한키트의항원이 salmon testes로본연구와동일하나 coating linker가교체되기이전의시약으로평가하였기때문으로해석된다. Nitrocellulose 막에여러항원을부착하여 IB로다양한자가항체검출이가능한 line immunoassay는민감도가 ELISA와비교할만하다고보고되어있으며, 비용면에서효율적이므로항 ENA항체검출을위한선별검사와함께항 dsdna항체검출에사용되고있다 [27]. 그러나본연구에서 IB와다른검사들의일치율은 42.-65.% (Kappa 계수.2 이하 ) 로서낮은일치율을보였다. 이는 IB와 ELISA 결과를비교하여 67.9% 의낮은일치율을보고한이전연구보고와일치하는데 [28], 앞에서언급한대로항원부착기질과부착방법및검출방법의차이에의한것으로생각된다. 불일치를보이는검사에서는 IB 양성결과가 IB 음성결과보다많았으므로, IB의위양성이의심되며, 본연구결과 IB를 SLE 환자의진단과추적검사에이용할경우반드시특이도가높은추가검사가동반되어야할것으로생각한다. 항 dsdna항체의농도는 SLE 환자의질병활성도와관련이있으므로치료전후추적검사로서이용된다 [1, 3-5]. 따라서항 dsdna항체의정성적인검출과동시에정량적인농도측정 이중요하고, 정량검사는 WHO international reference serum Wo/8을사용하여 calibration을시행하고 international units으로보고하여야한다 [29]. 본연구에서사용된정량검사들사이의상관성비교에서 ELISA I과 CLIA의상관계수가.667로가장낮았고, ELISA I과 ELISA III 사이의상관계수가.798로가장높았으나정확한평가를위해서는추후 SLE 환자의질병활성도와항 dsdna항체농도의상관성평가가필요하다. 결론적으로본연구에서항 dsdna항체검출을위한 6가지검사의비교결과, 검사시약의항원종류, 부착방법, 검출방법, 사용된경계치등에따라다양한민감도, 특이도, 일치율등을보였으며, 95% 특이도설정시검사의민감도는 ELISA II에서가장높았다. IB를제외한 3가지 ELISA와 CLIA, CLIFT는항 dsdna항체검출에유용하나, 정량검사는검사실내평가를통한적절한경계치수치의설정이필요하다고판단된다. 요약배경 : 항 double stranded DNA (dsdna) 항체는전신홍반루푸스 (systemic lupus erythematosus, SLE) 의진단과질환의활성을추적하는데유용한진단도구이다. 여러방법들이사용되고있지만그어느방법도완전히만족할만한것은아니며각방법들사이에차이가보고되어왔다. 본연구에서는항 ds- DNA항체검출을위해 6가지검사키트의진단적수행능과임상적유용성을평가하였다. 방법 : 142개의혈청검체 (SLE 환자 74개, SLE 이외의다른전신성류마티스환자 개, 그외다른질환을가진환자 18개 ) 를모아 6가지검사키트로항 dsdna항체를측정하였다. 각검사에서사용된 dsdna 항원은 Crithidia luciliae immunofluorescence test (CLIFT), salmon testes (immunoblot, IB), human DNA (ELISA I), salmon testes with nucleosome linker (ELISA II), plasmid DNA (ELISA III), synthetic oligonucleotides (chemiluminescence immunoassay, CLIA) 였다. 결과 : 6가지검사키트의제조사양성기준에따른민감도는 55.4-91.9% 로서 ELISA I의민감도가나머지 5가지검사키트에비해유의하게높았다 (P<.1). 특이도는 ELISA II, ELISA III, CLIA, CLIFT가 ELISA I과 IB에비해유의하게높았다 (P<.5). ROC 곡선분석에서 3가지 ELISA 키트와 CLIA의 AUC 값은.845-.893이었고, 각검사들의 AUC 값차이에통계적유의성은없었다 (P>.5). 특이도를 95% 로정했을때
Yang J-Y, et al., Evaluation of 6 Anti-dsDNA Kits 683 는 ELISA II의민감도 (63.5%) 가 ELISA III (41.9%) 에비해유의하게높았다 (P<.5). IB는다른검사들과의일치율이 42.- 65.% 로낮았다. 4가지정량검사들간의상관성분석결과, Pearson 상관계수는.667-.798이었다. 결론 : 6가지항 dsdna항체검사키트는검출방법과사용된경계치에따라다양한검사수행능을나타냈으며, IB를제외한나머지검사들은항 dsdna항체의검출을위해유용하게사용할수있을것이다. 참고문헌 1. Kavanaugh AF and Solomon DH; American College of Rheumatology Ad Hoc Committee on Immunologic Testing Guidelines. Guidelines for immunologic laboratory testing in the rheumatic diseases: anti-dna antibody tests. Arthritis Rheum 2;47:546-55. 2. Tan EM, Cohen AS, Fries JF, Masi AT, McShane DJ, Rothfield NF, et al. The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 1982;25:1271-7. 3. Reveille JD. Predictive value of autoantibodies for activity of systemic lupus erythematosus. Lupus 4;13:29-7. 4. Isenberg D and Smeenk R. Clinical laboratory assays for measuring anti-dsdna antibodies. Where are we now? Lupus 2;11: 797-8. 5. Sinico RA, Bollini B, Sabadini E, Di Toma L, Radice A. The use of laboratory tests in diagnosis and monitoring of systemic lupus erythematosus. J Nephrol 2;15(S6):S2-7. 6. Tozzoli R, Bizzaro N, Tonutti E, Villalta D, Bassetti D, Manoni F, et al. Guidelines for the laboratory use of autoantibody tests in the diagnosis and monitoring of autoimmune rheumatic diseases. Am J Clin Pathol 2;117:316-24. 7. Emlen W, Jarusiripipat P, Burdick G. A new ELISA for the detection of double-stranded DNA antibodies. J Immunol Methods 199;132: 91-11. 8. Radice A and Sinico RA. A new oligonucleotide-based ELISA for the detection of anti-double-stranded DNA antibodies. Autoimmunity 6;39:113-9. 9. Nossent JC, Huysen V, Smeenk RJ, Swaak AJ. Low avidity antibodies to dsdna as a diagnostic tool. Ann Rheum Dis 1989;48:748-52. 1. Deng JS, Sontheimer RD, Lipscomb MF, Gilliam JN. The binding of antihistone antibodies to Crithidia luciliae kinetoplasts is growth cycledependent. Arthritis Rheum 1985;28:163-8. 11. Haugbro K, Nossent JC, Winkler T, Figenschau Y, Rekvig OP. AntidsDNA antibodies and disease classification in antinuclear antibody positive patients: the role of analytical diversity. Ann Rheum Dis 4;63:386-94. 12. Sinico RA. Interdisciplinary forum on autoimmune disease research: guidelines. G Ital Nefrol 2;19:212-24. 13. Smeenk RJ. Methodological update detection of antibodies to ds- DNA: current insights into its relevance. Clin Exp Rheumatol 2; 2:294-3. 14. Hylkema MN, Huygen H, Kramers C, vd Wal TJ, de Jong J, van Bruggen MC, et al. Clinical evaluation of a modified ELISA, using photobiotinylated DNA, for the detection of anti-dna antibodies. J Immunol Methods 1994;17:93-12. 15. Rouquette AM and Desgruelles C. Detection of antibodies to dsdna: an overview of laboratory assays. Lupus 6;15:43-7. 16. Bencivelli W, Vitali C, Isenberg DA, Smolen JS, Snaith ML, Sciuto M, et al. Disease activity in systemic lupus erythematosus: report of the consensus study group of the European workshop for rheumatology research. III. Development of a computerised clinical chart and its application to the comparison of different indices of disease activity. The European Consensus Study Group for Disease Activity in SLE. Clin Exp Rheumatol 1992;1:549-54. 17. Kim KH, Han JY, Kim JM, Lee SW, Chung WT. Clinical significance of ELISA positive and immunofluorescence negative anti-dsdna antibody. Clin Chim Acta 7;38:182-5. 18. Villalta D, Bizzaro N, Corazza D, Tozzoli R, Tonutti E. Evaluation of a new automated enzyme fluoroimmunoassay using recombinant plasmid dsdna for the detection of anti-dsdna antibodies in SLE. J Clin Lab Anal 2;16:227-32. 19. Antico A, Platzgummer S, Bassetti D, Bizzaro N, Tozzoli R, Villalta D. Diagnosing systemic lupus erythematosus: new-generation immunoassays for measurement of anti-dsdna antibodies are an effective alternative to the Farr technique and the Crithidia luciliae immunofluorescence test. Lupus 21;19:96-12. 2. Janyapoon K, Jivakanont P, Surbrsing R, Siriprapapan W, Tachawuttiwat T, Korbsrisate S. Detection of anti-dsdna by ELISA using different sources of antigens. Pathology 5;37:63-8. 21. Horák P, S&c&udla V, Her&manová Z, Pospís&il Z, Falty@nek L, Budíková M, et al. Clinical utility of selected disease activity markers in patients with systemic lupus erythematosus. Clin Rheumatol 1;2:337-44. 22. Wasmuth JC, Gru_n B, Terjung B, Homrighausen A, Spengler U.
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