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220 Su Min Namkung, et al. Competitive Enzyme-Linked Immunosorbent Assay for Detection of Dopamine and Serotonin ORIGINAL ARTICLE Korean J Clin Lab Sci. 2017;49(3):220-226 https://doi.org/10.15324/kjcls.2017.49.3.220 pissn 1738-3544 eissn 2288-1662 Detection of Dopamine and Serotonin by Competitive Enzyme-Linked Immunosorbent Assay Su Min Namkung 1, Jeong Su Choi 1, Ji Hyang Park 1, Man Gil Yang 2, Min Woo Lee 1, Suhng Wook Kim 1 1 Department of Integrated Biomedical and Life Sciences, Graduate School, Korea University, Seoul, Korea 2 Biomedical Research Institute, Seoul National University, Seoul, Korea 경쟁적 ELISA 를이용한도파민과세로토닌의검출 남궁수민 1, 최정수 1, 박지향 1, 양만길 2, 이민우 1, 김성욱 1 1 고려대학교대학원의생명융합과학과, 2 서울대학교병원의생명연구원 Dopamine (DA) and serotonin (5-Hydroxytryptamine, 5-HT) are neurotransmitters and hormones that exist in small amounts but have important role in the body. Serum and 24-hour urine are used as specimens, and are usually examined by HPLC-MS. In this study, we tried to detect DA and 5-HT by competitive ELISA using antigen-antibody (Ab) reaction. After immobilizing 5 g/ml BSA conjugate on a 96-well surface, hormone and primary Ab, which are respectively diluted to different concentrations, were treated. Then, HRP-conjugated secondary Ab and TMB were added to measure absorbance. The regression equation and R 2 value were calculated based on absorbance, and sensitivity of Ab to hormone as well as the correlation between hormone concentration and absorbance were determined. In DA ELISA, R 2, the correlation between the concentration of hormone and absorbance, was the highest by 0.91 when anti-dopamine Ab was diluted 6,000 times and 7,000 times. In 5-HT ELISA, R 2 was bigger than 0.90 in every concentration except 3,000 times and 6,000 times. Both DA and 5-HT were not effectively detected at low concentrations (less than 1.0 10 7 M); and because reference value of serum DA is lower than this, HPLC-MS was required to detect serum DA. However, competitive ELISA may be effective in detecting 24-hour urine DA, serum, and 24-hour 5-HT. Further studies are needed to detect hormones more accurately at lower concentrations. Key words: Competitive ELISA, Dopamine, Serotonin Corresponding author: Suhng Wook Kim Department of Integrated Biomedical and Life Sciences, Graduate School, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul 02841, Korea Tel: 82-2-3290-5686 Fax: 82-2-940-2829 E-mail: swkimkorea@korea.ac.kr This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Copyright 2017 The Korean Society for Clinical Laboratory Science. All rights reserved. Received: July 31, 2017 Revised: August 21, 2017 Accepted: August 26, 2017 서론도파민 (dopamine, DA) 과세로토닌 (serotonin, 5-Hydroxytryptamine, 5-HT) 은신경전달물질및호르몬으로서체내에미량존재하지만그범위를벗어날경우심각한질병상태를초래할수있다. 도파민은모노아민에속하는카테콜아민계열신경전달물질중하나로전구체는 L-DOPA 이고, 인간뇌에서노르에피네프린으로생합성된다. 혈액뇌장벽 (Blood-Brain Barrier, BBB) 을통과하지못하기때문에뇌와말초에서의합성과기능은독립적이다 [1]. 혈관확장및심박수와혈압증가, 배뇨량증가, 인슐린생성감소, 운동조절, 동기부여, 보상기전등의기능을하며 [2] 운동조절중에서도자발적인움직임에중요한기능을하기때문에부족할경우파킨슨병을유발한다 [3]. 도파민의혈청농도는 30 ng/l (0.195 10 9 M) 미만이며, 24시간뇨에서의농도는 26 480 g/l (170 3,319 10 9 M) 이다 [4]. 혈청농도가매우낮기때문에검사시혈청보다는 24시간뇨검체

Korean J Clin Lab Sci. Vol. 49, No. 3, September 2017 221 를주로이용한다. 세로토닌은모노아민신경전달물질로생화학적으로트립토판에서유도된다 [5]. 인간을포함한동물의위장관, 혈소판, 중추신경계에서발견되며그중대다수는위장관에분포하면서장운동을조절한다 [6]. 또한뇌의광범위한영역에걸쳐분포하며기분, 기억, 수면등을담당한다 [7]. 최종대사산물은 5-Hydroxyindoleacetic acid (5-HIAA) 이다. 세로토닌의혈청농도는 101 283 g/l (0.57 1.61 10 6 M) 이며 [8], 24시간뇨에서의농도는 178 g/l (1.01 10 6 M) 이하이다 [9]. 세로토닌도검사시 24시간뇨검체를주로이용한다. 도파민은주로 high-performance liquid chromatography-mass spectrometry (HPLC-MS) 를이용해검출하며, limit of detection (LOD) 은 6.5 g/l 이다. 세로토닌은 HPLC-MS, liquid chromatography-tandem mass spectrometry (LC- MS/MS) 등을이용해검출하며, LOD는 0.78 g/l이다 [10]. 저농도도검출할수있기때문에민감도가높은정확한측정방법이라할수있다. 하지만 HPLC 는장비가매우비싸고검체세척시간이필요하며경우에따라특이도가높지않을수있다. 때문에보다간단하고정확한검사를위해전기화학적방법등대안을찾는연구들이존재한다 [11 14]. ELISA 는간단하고항원-항체반응을이용하기때문에특이도가높다는장점이있다. 따라서본연구에서는 ELISA를이용해도파민과세로토닌을검출하고그효과를보고자하였다. 재료및방법 1. 시약 Dopamine hydrochloride와 serotonin hydrochloride는 Sigma-Aldrich (St. Louis, MO, USA) 에서구입하였다. Bovine serum albumin (BSA) 은 Merck Millipore (Kankakee, Illinois, USA) 에서구입하였고, Pierce SnakeSkin pleated dialysis tubing 7,000 Da은 Thermo Fisher Scientific (Waltham, MA, USA) 에서구입하였다. 토끼의도파민에대한 polyclonal antibody (pab) (ab8888), 세로토닌에대한 pab (ab8882), horseradish peroxidase (HRP) 가결합된당나귀의토끼 IgG에대한 pab (ab6802) 는 Abcam (Cambridge, UK) 에서구입하였다. 또한도파민과세로토닌에대한토끼의 pab를 AbClon (Seoul, Korea) 에의뢰하여제작하였다. 3,3,5,5 -tetramethylbenzidine (TMB) substrate reagent set는 BD Biosciences (San Diego, CA, USA) 에서구입하였다. 2. 측정기기 96 well immunoplate (SPL, Pocheon, Korea) 에서 HRP와 TMB 반응까지완료한후 ChroMate Microplate Reader (Awareness Technology, Palm City, FL, USA) 로 450 nm에서흡광도를측정하였다. 3. BSA conjugation BSA와신경전달물질을결합시키기위해신경전달물질, BSA, 5% glutaraldehyde 를혼합하고색이약간노란빛을띨때까지 10분정도기다린후 20 mm BH 4 를혼합하여실온에서 60 분간반응시킨다. BSA는 carrier protein 역할을하고, glutaraldehyde 가 linker 역할을하여 hapten인신경전달물질과 BSA를 crosslinking시킨다. BH 4 는 crosslinking 과정에서생성되는 Schiff base를환원시켜서 link를안정되게한다. 결합하지않은신경전달물질과 glutaraldehyde 를제거하기위해 dialysis tubing을이용하여 24시간동안 7,000 Da molecular weight cut-off (MWCO) dialysis 를한후 20 C에서보관한다. BSA-DA 는도파민 20 mg/ml stock solution 1 ml, BSA 30 mg/ml 1 ml, 5% glutaraldehyde 50 L, 20 mm BH 4 1 ml를혼합하여만들고, BSA-serotonin 은세로토닌 10 mg/ml stock solution 100 L, DW 900 L, BSA 30 mg/ml 1 ml, 5% glutaraldehyde 100 L, 20 mm BH 4 1 ml를혼합하여만든다 [15]. 4. Competitive ELISA 96 well plate에서진행하였다. 5 g/ml BSA conjugate 를 coating buffer (50mM bicarbonate buffer, ph 9.6) 로 1,000 배희석하여 100 L 분주한후실온에서 2시간동안 shaking incubation 하여 well 표면에고정되도록한다. 고정되지않은 BSA conjugate는 wash buffer (10 mm PBS, ph 7.2, 0.05% Tween-20) 200 L를 3회처리하여제거한다. 그후비특이적인결합을막기위해 blocking buffer (3%, w/v, 10 mm PBS에 BSA, ph 7.2) 200 L를넣고실온에서 30분동안 shaking incubation 시킨다. 신경전달물질을 assay buffer (10 mm PBS, ph 7.2, 0.01% Tween-20) 로 10배씩연속희석하여 1.0 10 2 M부터 1.0 10 9 M 농도로준비한후 well에 50 L 분주한다. 1차항체로사용할신경전달물질에대한토끼의 pab 를 assay buffer로희석하여 50 L 분주한다. Abcam 제품은 6,000배로희석하였고, 제작한항체는적절한농도를찾기위해희석배수를다르게하였다. 도파민항체는 1,000, 3,000,

222 Su Min Namkung, et al. Competitive Enzyme-Linked Immunosorbent Assay for Detection of Dopamine and Serotonin 5,000, 6,000, 7,000, 8,000배희석하였고, 세로토닌항체는 1,000, 3,000, 4,000, 5,000, 6,000, 7,000배희석하였다. 실온에서 1시간 shaking incubation 시킨후 wash buffer 200 L를 3회처리한다. 2차항체로토끼 IgG에대한당나귀의 pab를사용하는데, 이항체는 HRP와결합되어있다. Assay buffer로 5,000배희석하여 50 L 분주한후실온에서 30분동안 shaking incubation시키고 wash buffer 200 L를 3회처리한다. TMB substrate reagent 를 100 L 분주한후 10분간반응시킨다. HRP와 TMB가반응하면색이파래지는데, 이를멈추기위해 2 M H 2SO 4 를 50 L 첨가하면노래진다. 색이완전히노래지면 450 nm에서흡광도를측정한다 [16]. 5. 결과처리항체희석배수별로 10회씩반복실험한후신경전달물질의농도에따라흡광도값을정리하였다. 농도별로데이터의상위 10% 와하위 10% 를제외한나머지값으로평균을냈고, 회귀선과 R 2 (coefficient of determination, 결정계수 ) 을통해농도와흡광도의연관성을확인하였다. R 2 0.65 인경우에만연관성이있는것으로고려하였다. 결과이실험은경쟁적 ELISA로, well 표면에부착된항원과 buffer에희석된항원이경쟁적으로 1차항체와결합한다. 그다음단계에서 wash buffer를처리할때 well 부착항원과이에결합한항체만남기고나머지는제거하기때문에앞서희석했던신경전달물질의농도에따라남아있는항체의양이달라지게되며, HRP가결합된 2차항체를처리하여발색기질인 TMB 와반응시킨후흡광도를측정하면그정도를수치화하여확인할수있다. 실험은농도를아는표준시료를항원으로하여진행하였으며, 실험결과로 standard curve를나타냈다. 흡광도가신경전달물질의농도에의존적으로분포하는것이가장이상적인결과이며, 시판되는 Abcam 제품 (ab8888, ab8882) 을기준으로삼아제작한항체의성능을보고자하였다. 먼저각항원에대한항체의특이성을보기위해교차반응실험을 3회진행하였다. 항원은같은모노아민신경전달물질인히스타민, 도파민, 세로토닌, 에피네프린을사용하였고, 항체는도파민과세로토닌에대한것을사용하였다. 흡광도측정결과목표항원을제외한나머지항원은농도에따른차등적인변화를보이지않았으며, 항체희석배수에따른변화또한보이지않 Table 1. Absorbance of cross-reactivity test using anti-dopamine Ab Concentration of Ag Histamine Dopamine Serotonin Epinephrine 1.0 10 2 M 0.67 0.73 0.50 0.39 1.0 10 3 M 0.70 0.88 0.58 0.36 1.0 10 4 M 0.59 1.05 0.53 0.37 1.0 10 5 M 0.59 1.11 0.53 0.38 1.0 10 6 M 0.73 1.32 0.46 0.37 1.0 10 7 M 0.61 1.30 0.53 0.39 1.0 10 8 M 0.69 1.40 0.56 0.35 1.0 10 9 M 0.50 1.38 0.46 0.33 Absorbance of dopamine is result of 6,000 times diluted Ab. Other neurotransmitters had similar range of absorbance in every dilution factor. Table 2. Absorbance of cross-reactivity test using anti-serotonin Ab Concentration of Ag Histamine Dopamine Serotonin Epinephrine 1.0 10 2 M 0.80 0.65 1.25 0.67 1.0 10 3 M 0.85 0.71 1.41 0.63 1.0 10 4 M 0.80 0.71 1.65 0.64 1.0 10 5 M 0.65 0.79 1.75 0.59 1.0 10 6 M 0.73 0.57 1.95 0.66 1.0 10 7 M 0.76 0.72 1.92 0.66 1.0 10 8 M 0.80 0.75 2.21 0.61 1.0 10 9 M 0.71 0.65 2.15 0.58 Absorbance of serotonin is result of 1,000 times diluted Ab. Other neurotransmitters had similar range of absorbance in every dilution factor.

Korean J Clin Lab Sci. Vol. 49, No. 3, September 2017 223 았다 (Table 1, 2). 따라서교차반응실험결과항체가각항원에대한특이도가있다고판단하고연구를진행하였다. 도파민의경우시판되는제품을 6,000배희석하여사용했을때 R 2 =0.96 으로항원농도와흡광도의상관관계가높은것을확인할수있었다. 제작한항체는농도별로희석하여사용했는데, 1,000배희석한경우 R 2 =0.61로매우낮은것을확인하였다. 회귀식과 R 2 값은 Table 3에정리하였고, 흡광도그래프는 Figure 1으로정리하였다. 6,000배와 7,000배에서높은수준의상관관계를확인할수있었다. 또한유의한 R 2 값을갖는모든희석배수에서제품보다회귀선의기울기의절대값이크게나왔으므로제작한항체의도파민에대한민감도가제품보다더크다고판단된다. 세로토닌의경우시판되는제품을 6,000배희석하여사용했을때 R 2 =0.73으로그다지크지않았다. 제작한항체는농도별로희석하여사용하였으며, 회귀식과 R 2 값은 Table 4에정리하 였고, 흡광도그래프는 Figure 2로정리하였다. 3,000배와 6,000배를제외한모든희석배수에서높은수준의상관관계를확인할수있었다. 또한회귀선의기울기의절대값이제품에비해매우크게나왔으므로제작한항체의세로토닌에대한민감도가더크다고판단된다. Table 3. Results of ELISA for dopamine detection: regression equation, coefficient of determination Dilution rate of primary antibody Regression equation R 2 Abcam product (1:6,000) y= 0.0622x+0.2872 0.96 1:1,000 y= 0.0644x+1.2048 0.61 1:3,000 y= 0.135x+0.5788 0.86 1:5,000 y= 0.1029x+0.6277 0.89 1:6,000 y= 0.0968x+0.6127 0.91 1:7,000 y= 0.0878x+0.6347 0.91 1:8,000 y= 0.0773x+0.667 0.83 Figure 1. Absorbance graphs of ELISA for dopamine detection.

224 Su Min Namkung, et al. Competitive Enzyme-Linked Immunosorbent Assay for Detection of Dopamine and Serotonin 고찰 경쟁적 ELISA 를통해신경전달물질의농도변화에따른흡광도차이를확인할수있었다. 항체희석배수에따라연관성의정도는다르게나타났지만도파민과세로토닌모두전반적으로 Table 4. Results of ELISA for serotonin detection: regression equation, coefficient of determination Dilution rate of primary antibody Regression equation R 2 Abcam product (1:6,000) y= 0.0145x+0.359 0.73 1:1,000 y= 0.1359x+1.0387 0.95 1:3,000 y= 0.1277x+0.6456 0.82 1:4,000 y= 0.1054x+0.7178 0.91 1:5,000 y= 0.0814x+0.6227 0.90 1:6,000 y= 0.0721x+0.7252 0.71 1:7,000 y= 0.0745x+0.6299 0.90 0.8 0.9 범위의높은결정계수를나타냈으며, 회귀선을통해항원에대한민감도를확인하고교차반응실험을통해특이도를확인하였다. 도파민의경우 1차항체를 1,000배희석했을때 R 2 =0.61이었으므로신경전달물질농도와흡광도간에연관성이없다고판단하였다. 항원-항체반응에있어서 prozone에해당하는경우로생각된다. 1차항체를 6,000배와 7,000배로희석했을때 R 2 =0.91로신경전달물질농도와흡광도의상관관계가가장컸다. 또한회귀선의기울기의절댓값이시판제품은 0.06인것에반해제작항체는최대 0.1029 로훨씬큰값이나왔으므로시판제품보다제작항체가항원인도파민에대해더높은민감도를갖고있다고판단하였다. 그러나전반적으로 1.0 10 7 M 이하의저농도에서유의한정도로농도의존적인흡광도가나오지않았기때문에검출에한계가있는것으로보인다. 저농도의항원을효과적으로검출하지못하는것으로인해모든항체희석 Figure 2. Absorbance graphs of ELISA for serotonin detection.

Korean J Clin Lab Sci. Vol. 49, No. 3, September 2017 225 배수에서시판제품 (R 2 =0.96) 보다낮은결정계수를나타냈으며, 1.0 10 7 M 이하농도에서의흡광도를제외하면민감도가증가하며 R 2 0.98 로매우높은상관관계를나타낸다. 따라서항원-항체반응을이용한 ELISA는도파민이 0.195 10 9 M 미만으로존재하는혈청보다는 170 3,319 10 9 M의농도로존재하는 24시간뇨에서의도파민검출에적합할것으로사료된다. 세로토닌의경우시판제품은 R 2 =0.73 으로낮았던것에반해제작항체는 6,000배를제외한모든희석배수에서 R 2 0.8로상관관계가높은것을확인할수있었다. 또한세로토닌에서도회귀선의기울기의절댓값이큰차이를보였는데, 시판제품은 0.0145로매우작았고제작항체는 0.0721 0.1359 범위로나왔으므로, 시판제품보다제작항체가항원인세로토닌에대해훨씬높은민감도를갖고있다고판단하였다. 도파민과마찬가지로 1.0 10 7 M 이하의저농도에서는세로토닌을효과적으로검출하지못하였고, 해당농도에서의흡광도를제외하면민감도가증가하며 0.95 R 2 0.99 로매우높은상관관계를나타낸다. 그러나세로토닌은혈청에서 1.305 10 6 M 이하, 24 시간뇨에서 1.191 10 6 M 이하의농도로존재하기때문에혈청과 24시간뇨검체모두에서검출이가능할것으로사료된다. 도파민과세로토닌모두 ELISA보다 HPLC의 LOD가훨씬낮다. 다른모노아민신경전달물질인히스타민을대상으로하는경우에도같은결과를확인하였다 [17]. 하지만시간과비용면에서 ELISA의장점이명확하며 24시간뇨검체의참고치보다낮은 LOD를갖기때문에검사에이용할수있으므로, 현재신경전달물질의검사에사용되는 HPLC-MS 의장점인정확성을갖춘다면 ELISA 를이용한검사방법을이용하는것이효과적일것이라사료된다. 이를위해보다낮은농도를검출하기위한방법은추후연구가필요하다. 요약도파민과세로토닌은신경전달물질및호르몬으로서미량이지만체내에서중요한기능을한다. 검체는혈청과 24시간뇨를쓰며, 주로 HPLC-MS 를이용하여검사한다. 본연구에서는항원-항체반응을이용한경쟁적 ELISA를통해도파민과세로토닌을검출하고자하였다. 5 g/ml BSA conjugate를 96 well 표면에고정시킨뒤각각농도를다르게한신경전달물질과 1차항체를넣어반응시키고 HRP가결합된 2차항체와 TMB를처리하여흡광도를측정하였다. 측정결과를토대로회귀식과 R 2 값을구하여신경전달물질에대한항체의민감도와신경전달물 질농도와흡광도사이의상관관계를판단하였다. 도파민은 1차항체를 6,000배, 7,000배로희석했을때 R 2 =0.91로신경전달물질농도와흡광도의상관관계가가장높게나타났고, 세로토닌은 3,000배와 6,000배를제외한모든희석배수에서 R 2 0.90의높은상관관계를나타냈다. 도파민과세로토닌모두 1.0 10 7 M 이하의저농도에서는효과적으로검출되지않았기때문에참고치가이보다낮은혈청도파민검출은 HPLC-MS 이용이필요해보이지만, 24시간뇨도파민과혈청및 24시간뇨세로토닌의검출에는 competitive ELISA가효과적일것이라사료된다. 보다낮은농도의신경전달물질도정확하게검출하기위해서는추가연구가필요하다. Acknowledgements: None Funding: None Conflict of interest: None REFERENCES 1. The National Collaborating Centre for Chronic Conditions. Parkinson's Disease. 1st ed. London: Royal College of Physicians; 2006. p59-100. 2. Goldberg L. Cardiovascular and renal actions of dopamine: Potential clinical applications. Pharmacol Rev. 1972;24(1):1 29. 3. Bernheimer H, Birkmayer W, Hornykiewicz O, Jellinger K, Seitelberger F. Brain dopamine and the syndromes of Parkinson and Huntington clinical, morphological and neurochemical correlations. J Neurol Sci. 1973;20(4):415 455. 4. Gardner D, Shoback D, et al. Greenspan s Basic & clinical endocrinology. 9th ed. New York City: The McGraw-Hill Companies; 2011. Appendix. 5. González-Flores D, Velardo B, Garrido M, González- Gómez D, Lozano M, Ayuso M, et al. Ingestion of Japanese plums (Prunus salicina Lindl. cv. Crimson Globe) increases the urinary 6-sulfatoxymelatonin and total antioxidant capacity levels in young, middle-aged and elderly humans: Nutritional and functional characterization of their content. J Food Nutr Res. 2011; 50(4):229-236. 6. Berger M, Gray JA, Roth BL. The expanded biology of serotonin. Annu Rev Med. 2009;60:355-366. 7. Neumeister A, Young T, Stastny J. Implications of genetic research on the role of the serotonin in depression: emphasis on the serotonin type 1A receptor and the serotonin transporter. Psychopharmacology. 2004;174(4):512 524. 8. Siddiqi HA, Salwen MJ, et al. Laboratory diagnosis of gastrointestinal and pancreatic disorders. In: McPherson RA, Pincus MR, editors. Henry's Clinical diagnosis and management by laboratory methods. 23rd ed. St Louis, MO: Elsevier; 2017. Chap 22. 9. Pussard E, Guigueno N, Adam O, Giudicelli JF. Validation of

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