대한임상병리사회지 : 제 23 권 1 호 1991 증강화학발광면역법을이용한잡상선홀몬측정평가 한국의학연구소분석부 김순기 김용남 허순자 1. 서론 지금까지내분비홀몬검사는주로방사면역측정 법 (radioimmunoassay : RIA) 을이용하고있다. 그 러나방사성동위원소사용

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1 대한임상병리사회지 : 제 23 권 1 호 1991 증강화학발광면역법을이용한잡상선홀몬측정평가 한국의학연구소분석부 김순기 김용남 허순자 1. 서론 지금까지내분비홀몬검사는주로방사면역측정 법 (radioimmunoassay : RIA) 을이용하고있다. 그 러나방사성동위원소사용에는고가의장비, 짧은 반감기로인한시약의소모와관리의번거로움으로 인한검사원가의누증, 별도의시설기준, 허가의복 잡성, 검사자의방사선취급면허취득, 폐기물처 리등의문제를안고있어서, 비핵물질을이용하는검사법이꾸준히개발되고있다 2, 3, 4, 7, 8) 예를들면효 소면역측정법 (enzyme immunoassay: EIA) 형광 면역측정법 (fluoroimmunoassay: FIA) 1,7,8,11), 화 학발광면역측정법 (chemiluminescence immunoassay: CIA) 에기초한증강화학발광면역검사법 (enhanced luminescence immunoassary: ELIA) 등이 다. 본연구에서는 ELIA 검사방법을이용하여잡상선 호몬트리요도터로닌 (triiodo thyronine : T 3), 터 록신 (thyroxine : T 4 ) 갑상선자극홀몬 (thyroid stimulating hormone : TSH) 을측정하여 RIA 및 EIA 와그결과를비교하여보았다. 1. 원리 II. 재료및방법 ELIA 의원리는고상 (s 이 id phase) 에말이나양, 당나귀, 흰쥐항체에 horseradish peroxidase 를피 복시쳐, 단일클론항체인 conjugate 를넣고 37 0 C 에 서반응시킨다음반응되지않은물질을세척과정에 서제거하고, signal 시약에포함된화학발팡 (1 uminescence) 물질파촉매제를넣어줌으로서 H 2 O 2 나 KSFe(CN)6 등의산화제와반응할때 aminophthalic acid 가되며, 이것이기저상태로될 때일어냐는발광을측정하게된다 (Fig. 1). N "-./ I ~q Perox 뺀 se (En 피 Fig 1. Principle of enhanced luminescence assays -102-

2 2. 검사방법 1) T 3 경쟁법 (competitive method) 으로서나귀양항체 (donkey anti-sheep) 를피복시킨기벽 (we ll) 에표 준혈청 (standard serum) 과대조혈청 (control serum), 검체 25μl 를각각넣고, conjugate (horseradish peroxidase) 와측정시약 (sheep anti - T 3 antibody) 을넣어반응시킨다음, 결합되지않 은항체를세척과정에서제거하고, 효소와기질반응 을유도하기위하여면역증강제 (immuno enhancer) 및 hydrogen peroxidase 가혼합된 signal 시약올 250μl 씩분주하여생성되는빛의강도가유지되는 2-20 분안에 Amerlite reader 에넣어농도를구한 다 (Fig.2). Assemble strip holder 25μ1 standard (0, 0.4, 1. 0, 2.0, 40, 80) ng/ml control, sample 100μ1 conjugate 100μ1 assay reagent Incubation at 3ic /hr Aspirate and wash using ELIA analyzer 250μ1 signal reagent Read coated wells in ELIA washer between 2-20 min. after addition of signal reagent Results are printed out Fig 2. Flow chart for enhanced luminescence assay for T 3 2) T 4 T 4 는 T 3 와같이경쟁법으로서 conjugate 에 sheep anti-t 3 대신 sheep anti-t 4 와측정시약에 sheep anti-t 4 antibody 를사용한것으로검사방법은동일 하다. 3) TSH TSH 는 T 3, T 4 와는달리 immunometric 방법으 로검정되며, TSH 의 β'-subunit 에대한흰쥐단일 클론항체 (mouse monoclonal antibody) 를피복시 킨기벽에표준혈청, 대조혈청, 환자의혈청올각각 100μl 씩넣고, horseradish peroxidase 에단일클론 항체가용해되어있는 conjugate 용액을 100μl 씩넣 어 3iC 에서 1 시간반응시킨다음세정기 (washer) 로 세척하고 250μl 의 signal 시약을넣어판독한다 (Fig. 4)

3 Assemble strip holder 10μ1 standard (0, 25, 50, 100, 150, 200)μg/dl control, sample 10μl conjugate Assay reagent loo.u1 loo.u1 Incubation at 37 0 C /lhr Aspirate and wash using ELIA washer 250μ 1 signal reagent Read coated wells in ELIA analyzer between 2-20 min. after addition of signal reagent Result are printed out Fig 3. Flow for enhanced luminescence assay for T 4 Assemble strip holder 100,ul standard (0, 0.2, 5, 40, 200) control, sample 100μl conjugate 100μ l Incubation at 37"C /lhr Aspirate and wash using ELIA washer 250μ1 si 맹 al reagent j Read coated wells in ELIA analyzer between 2-20 min. after addition of signal reagent Result are printed out Fig 4. Flow chart for enhanced luminescence assay for TSH -104-

4 high 애III. 결과 1.37, 9.2, 5.65, 고농도 3.3, 15.0, 29.4 로표시된 1. 정밀도 기대범위에포함될뿐만아니라평균값에근접되었 다 (Table 2, Fig. 5) 측정항목마다저, 중, 고농도의검체를각각 20 회 씩반복측정하여, T 3, T 4, TSH 의변이계수 (C.V) 를구한바각각저농도 5. 6, 1. 6, 3. 0% 중간농도 3.2, 2. 1, 1. 5%, 고농도 5.0, 3.0, 1. 5% 로양호하 였다 (Table 1). Table 1. Within assay precision of ELIA for T3, T4, TSH in serum(n=25) 앓섭띈 T3 T4 TSH (ng/ml) (μg/d l) (μiu/m l) X Low SD CV X Medium SD CV X πωmω25 r / / // / medium 서 Low Expected range i/ 1이 이1 // ~ 1ll / ~ J T3 ng/dl T4 μg/dl TSH μiu/ml Fig 5. Accuracy of ELIA for. T3, T 4, TSH in serum / High SD CV Table 2. Accuracy of ELIA for T3, T4, 꾀선띈 Low expected TSH in serum (n = 20) T3 T4 TSH (ng/ml) (μg/d l) (μiu/m l) result range Medium expected result range High expected 2. 정확도 result range 상품화된대조혈청저, 중, 고농도를각각 20 회 반복측정하여그평균치를비교하여. T 3, T 4, TSH 에서각각저농도 0.45, 3. O. 0.16, 중간농도 3. 밤사면역측정법과의비교 서로다른검체 T 3 27, T 4 31, TSH 27 개를방사 면역측정법의결과와비교하여, 기울기를 T 3, y= 0.99x 十 0.6. T 4 y=0.98x+0.27, TSH, y= 1. 0 x+0.04(y=elia, x=ria) 로얻었고, 상관계수 (r) 는 T T TSH 로서양호하 였고, t 값은각각 0.185, 0.318, , p<0.05 로 유의한차는없었다 (Table 3. Fig. 6, 7, 8) Table 3. Comparison of serum T3, T4. 꾀침 TSH concentrations determined by RIA and ELIA methods T3 T4 TSH Unit ng/ml μg/dl μiu/ml n 31 r V 0.99x 十 x x P<0.01 P<O.Ol P<0.05 m 씨

5 {니띠Fig 7. 1'4 of correlation with RIA vm m3 4. 자동효소면역측정법과비교 톤 2 b.o I 멀 1. 5 μj 2.5 y = 0. 99x r=0.986 n=27 p<0.01 결과가다른검체를각각 T 3 25, T 4 36, TSH 33 개를 IMX (Abbott Co. USA) 에서측정한결과기 울기가 T 3 y=0.83x+0.170, T 4 O. 77x+0.182, TSH y= O. 75x (y=ELIA, x= IMX) 였고, 상관계수 (r) 는 T , T , TSH 으 로 RIA 와의비교보다는저조하나양호한상관을보 였다 (Table 4, Fig. 9, 10, 11). 0.5 () o x IMx (ng/ml), Table 4. Comparison of serum T 3, T 4, ~ I~;i Tish i r~ ncid h TSH cocentrations determined by IMX and ELIA methods n~r1 rl- n T3 T4 TSH y Fig 6. 1'3 of correlation with RIA Unit ng/ml μg/dl μiu/ml n r y 0.83x+0.17 O. 77x x+0.l0 L { ~야 럭그)( 1i ng 7 F y=0.98x 十 0.27 r=0.988 n=31 p< x lmx{μg/dl) Y ~ 야 E 0.8 걷 려 y = 0. 83x r=0.93 n=25 니띠EX).Ig~Pl-o ; O X lmx{ng/md Fig 9. T3 of correlation with IMx ωm때 m M 8 ω6 4 2 (y= 1x r=0.999 n=27 p<0.05 o x IMx{μIU/m}) Fig 8. TSH correlation with RIA 력니띠영~뼈륙)y i {0. y=0.77x r=0.86 n= x IMx{μg/dD Fig 10. T4 of correlation with IMx -106-

6 4 η섭띠 ζ~ {륙)력Y Jqιy= O. 75x+0.10 r=0.98 n=33 문제점이지적되었다 년영국 Birmingham 의 Walfson 연구소에서 enhancer 를첨가시킴으로서상당시간까지빛올지 속시킬수있는방법을개발하였고, 1986 년 Whitehead 17) 등에의해임상검사의이용이가능해 짐에따라서갑상선흘몬은물론암의표지 (cancer marker), 바이러스, 간염등의검사까지도가능하게 되었다. ELIA 는정확성을기하기위하여반복측정을원칙 x IMx(μIU/m l) Fig 11. TSH of correlation with IMx 으로하고있으나, 절약방법으로명명되어있는 EOS (economy of standard) 방법이프로그랩되어 있다. 이 3) 등은반복측정과 1 회측정언 EOS 법과의 비교에서기울기 y = x (y = EOS 법, x= 반복측정법 ) 와상관계수 (r) 은보고하였 IV. 고찰 면역학적정량측정법의획기적인전기는 1959 년 Yallow 와 Beron l8 ) 이당뇨병환자의체내에서 insulin 항체를측정하는데방사면역측정법 (Radio immunoassay) 을사용한것이라고하겠다. 오늘날 훌몬은물론바이러스종양관련각종항원이나항체 약물까지 RIA 로검사하는범위는넓다. 그러나방 사성물질을취급함에는쉽게사용하기어려운여러 가지문제가따르므로, 효소면역측정법, 형광면역측정법 (fluoro immunoassay) 등을개발하게되었 다 년 Schroder 6 ) 은 luminal 이나 isoluminal 등 파같은화학물질의존재하에서 H20 2, K3Fe(CN)6 으며, 본연구에서도 y=0.86x+0.15, r=0.963 을 얻어양호한결과를보였다 (Table 5). Table 5. Correlation of between E.O.S and duplicateds 싫 grjt Test name n I y-intercept Slop r Lee s etc TSH T3 Auther T TSH 등의산화제와반응할때생기는 aminophthalic RIA 와의비교는국내보다는국외에서더많은보 acid 가기저상태로될때발광을형성하는데, 이빛고가있었다 씨 ) 의강도를측정함으로서물질의농도를알수있는이 2) 등은 TSH 를 RIA 와비교해서 y=o.648x+0. 분석법을개발하였으나, 발광을지속시킬수없다는 170, r=0.978 을, 최 4) 등은 Luteinizing hormone Table 6. Comparison of serum thyroid hormone by RIA and ELIA method 꾀삶냄느 Test name n y-intercept Slop r Lee's etc TSH Lee s etc TSH Choi's etc LH Rhys John s etc TSH a-fp T Auther T \).988 TSH

7 (LH),:u} l:ll.iil.~»a1 y=l.336x -2.8, r=0.933%, op> %.g. TSHOllAl y=0.959x , r=0.996% Rhy John's 13 > % ::_ TSHOllA1 y=o. 752x-0.092, r = ~.:L t;.l.jl a-f etoprotein Oil Al y = x+2.9, r=0.960% ~.Jlc5}.Jl ~~o:l ~ <t!-t-olla1.5:. T 3 y=0.99x+0.06 r=0.986, T4 y=0.98x+o. 27 r=0.988, TSH y=x+0.04, r=0.999~ t+s}4j o1 RIA.Q} ~ ~41 c5}e:l s.. 'i"~ t:-}.jl ~~ 7 --t~ ~ e-t(table 6). 41*el -t-~~a.c oj~s... ~o1s..,?8ojs_, xa~.5:.7} -E-.g. ~ ~.. ~~ {! l:lj-a}pj ~ 4:AJ ~% ~If-* A%t}JL ~~t+. l:lj-a~ %~1-'{!±~ ~1-B-<St.C~-11 u:t ~-c ~J\117} ~ t:-}. ~ C{!-T-OllA1.C T 3, T 4, TSH~ f5-7j-&}~ ~::i!j- oj ~ ~?8~% ol- -c5}e:l ~?8c5}.Jl 71~ A- -t}.c RIA, EIA.Q} l:ll.iil. ~ 7}~» ~ ~,:u} t:-}%,:u} 7 J~lt:-}. 1. if-.5:.7} e-t~ x1, 'F.Jlif-.5:..9-l ~ ~~a% 7--t 20 1 ~ l:lj* ~?8 <S}e:l Al * s.. Oil Al ttl 0 17-ll 4- T , T 4 1.6, TSH 3.0%, '3'-7Jif-S.. ttl 0 l7-ll4- T 3 3.2, T 4 2.1, TSH 1.5%,.Jli;-.5:..9-l ttl 0 17-ll 4- T3 5.0%, T %, TSH 1. 5% t}~ e-}. 2. ~,:i1}5:l~ ~.Jl ~.C xl, '3'-,.Jli;-.5:..9-l ~ ~ ~a% zt 2o&1~ ej*~?8<n ~jj} T30llA1 o.45(o ), 1. 37( ), 3. 3ng/ml ( ng/ml), T 40llA1 3.0( ), 9.2( ), 15. O,ug/dl ( ,ug/dl), TSHOll Al 0. 16( ), 5.65( ), 29.4,ulu/dl ( l.O,ulu/dl).. ~~ 0 JOll 11--q~ "5-1%~~1011 ~ o}yt;.} ~it~l0l1 XJ-2-~ol ~~E.... Ul.iil.~?8~ ~o1 ~~t:-}. 3. A1.. t:-}~ ~~41 T 3 27, T 4 31, TSH 277»~ ~ Xa<S}e:l RIA l:ll.iil.<t}- ~jj} T3 y=0.99x+0.06, r=o. 986, T 4 y=0.98x+0.27, r=0.958, TSH y=x r=o. 990(y=ELIA, x= RIA).. 0 J=~c5}~~ o:l. ~7-ll~ %.9-l<t}- 5:}.C ~~t:-}(p<0.01). 4. EIA ~ oj IMX (Abotte Co. USA) ~ o 1% <tll:ll.iil..c T 3 25, T4 36, TSH 337».9-l ~~1 ~ ~?8<5} e:l T3 y=0.83x+0.17, r=0.938, T4 y=0.77x+ 1.82, r=0.869, TSH, y=o. 75x+0.10, r=o. 986(y=ELIA, X=IMX) ~ 0 J=~c5}~t:-}. ~1.2} ~ol &}~ ~::i!j- tct~ *~~~ ::_ RIA~ ~~J ~ 4- ~% ~ ~...iijtj~ o:j ~ C2.l=~ oj EOS l:lj-~.g. ~ J\11 ~ o l.jl t'-t.g. 0 J=.g. ~J 4fc5}rll *1 t;.l ~ 4- ~% ~ ~...iijtj~ ~ t:-}. Evaluation of Enhanced Chemiluminescence Enzyme Immuno Assay (ELlA) in the Determination of Thyroid Hormone (T 3, T 4, TSH) using Amerlite System Kim, S.K., Kim, Y.N., Her, S.Z. Dept. of Korea Medical Institute Analytical Dpt. ABSTRACT Whereas radioimmunoassay technique which is very sensitive as well as accurate has been widely used for the assay of most endocrinological hormones, numbers of related problems launched the search for many lab. techniques that can replace the use of radioisotopes. Authors employed "Enhanced Luminescence Immunoassay" (ELlA) technique as the one to assay and evaluate thyroid hormone(t3, T 4) and thyroid stimulating hormone(tsh). The results are as follows : 1. Precision rate was calculated by assaying T 3, T 4. TSH and 25 times repetitively at 3 diffeerent concentration for each-low, medium, high -. and so was judged excellent as the coefficient of variation(cv) was 5.6% 1.6% 3% (Low), 3.2%, 2.1%, 1.5% (medium). 5.0%, 3%. 1.5% (High). 2. Control serum sample with known assay value labelled as Low, Medium, High was tested for 20 times for each and the results are 0. 45ng/ml ( ), 1.37ng/ml(l ), 3.3ng/ml ( ) for T 4 : 3.0,ug/dl( ), 9.2,ug/ -108-

8 dl( ), 15Jlg/dl( ) for T4: and 0.16JLIU/ml ( ), 5. 65JLIU/ml ( ), 29.4JLIU/ml( l.O)for TSH. They are all in permissable range shown in the parenthesis and close the mean value, and so was judged relatively accurate. 3. Comparison with other techniques. 1) For 27 samples of T 3, 31 T4 samples, 27 TSH samples which show different values from the assay values by radioimmunoassay technique, the slope was Y=0.99x+0.06, Y=0.98x+0.27, Y = x (Y =ELlA, x =RIA) and the correlation coefficient was , , in good correlation, and so did not show any statistically appreciable difference (p < 0. 01, p < 0. 05). 2) For comparison with IMx which is also a enzyme immunoassay technique, 25 samples of T3, 36 T4 samples, 33 TSH samples were tested. The resultant slope was Y = 0. 83x , Y = x , Y = 0. 75x+0.10(Y =ELlA, x = IMx) and the correlation coefficient was , , in good correlation for each. Accroding to the above results Amerlite, a "Enhanced Luminescence Immunoassay" technique, may be regarded as a substitute for RIA technique and the E. 0. S method (Economy of Standard) was not only economic but also able to manipulate a large quantity speedily. REFERENCES 1. ~-'tiif, 7cl~Tf, 01.X..-!-: A17J-~t ~~J-Pj_ ~~-'8 ~% o1%~ Human alpha feto protein(ha FP) 9-1 ~78' tl1 ~<?J AJ-l:IJ t:-1"& 1 Al, Ai19-T-! Aill..., o 11;-.:t!-, o 1 ~ ~ : Enhanced Luminescence~ o1%~ 7J-AJ--'ti?;}~~-?:- ~78oJ1.:t!-~.iiJ7} x~ ~' ~ ILJ--tJ., ~~it : }'&l:lj".j5l-~±t;! ~ ~ 78 ~-% o1 %itl- Human thyrotropin (TSH) 9-1 ~78 4. ~ ~ 4;-' s.. qj_ Ail, ~.!f-... : ~ F LH ~ AtoJ1 tl1 itl- Enhanced Luminescence Immunoassay (ELlA).2}.!f- 7}A1 -ifo1 ~ RIA.2}9-1 l:l1.iil ~ ELIA ~.e.. -F 1-Jl ~ At. <?J AJ-l:IJ t:-1.q} xa.x. ~t:-1-o:f )J;], Ai]11-T:J. Ai]l..., Bergs trand CG, Czar B : Demonstration of a new protein fraction in serum from the human fetus, and J clinical lab. Invest 8, 174, Barnard GIR and Collins WP: The development of Luminescence Immuno Accays, Medical Laboratory sciences 44, , Daterson N., Biggart EM, Champman RS Best all GH ; Evaluation of a time resolved immunoflurometric assay for serum thyroid stimulating hormone, Am clinical biochem 22, 606, Hemmila Iikka : Flu oro immuno assay and Immuno flurometric assays, Clinical chem, 31/3, , Ian \V, \Vood head JS : Chemiluminescence Immuno assay, J. Clinical Immuno assay 7, 152, John R. Henly R. Sharrkland D. : Evaluation of an enhanced luminescence assay for alpha feto protein., Clinical chem 32, 2066, Lawson N. Mike N. Wilson R. Raudom H. : Assesment of a time resolved fluoro immuno assay for thyrotropin in routine clinical practice, Clinical chem, 32/4, , Miles LEM., Hales CN : Labelled antibodys and immunological assay systems., Nature 219, July 13, Rhy John, Robert Henley, David Shank and Alen M, McGregor: Enhanced luminescence lmmuno assay evqluation of a New, More sensitive thyrotropin assays. 14. Rhys John, Robert Henley and David Shank laud : Evaluation of an enhanced luminescence assay for Alpha feto protein, Clinical chem. 32/11, , Simpson JSA, Campbell AK, Ryall MET. Woodhead JS. : A stable chemiluminescent assays., Nature , Vol 14, June, Week I. Woodhead JS. : Chemiluminesceuce immunoassays, J. Clinical Immunoassay, 7 : -109-

9 19, Whitehead, T. P., Thorpe GHG, Carter T JN, Grocuttc and Kricha LJ : Enhanced luminescence procedure for sensitive determination of peroxidase labelld conjugates in Immuno assay., Nature 305, , Y allow RS. Berson SA : Immuno assay of endogenous plasma insulin in man J, Clinical Invest 39, 1157,

유해중금속안정동위원소의 분석정밀 / 정확도향상연구 (I) 환경기반연구부환경측정분석센터,,,,,,,, 2012

11-1480523-001163-01 유해중금속안정동위원소의 분석정밀 / 정확도향상연구 (I) 환경기반연구부환경측정분석센터,,,,,,,, 2012 목 차 ⅰ ⅲ ⅳ Abstract ⅵ Ⅰ Ⅱ Ⅲ i 목 차 Ⅳ ii 목 차 iii 목 차 iv 목 차 v Abstract vi Abstract σ ε vii Abstract viii Ⅰ. 서론 Ⅰ. 1 Ⅰ. 서론.