Korean Journal of Clinical Laboratory Science (대한임상검사과학회지)

Korean Society for Clinical Laboratory Science (대한임상검사과학회)
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Detection of Dopamine and Serotonin by Competitive Enzyme-Linked Immunosorbent Assay

경쟁적 ELISA를 이용한 도파민과 세로토닌의 검출

  • Namkung, Su Min (Department of Integrated Biomedical and Life Sciences, Graduate School, Korea University) ;
  • Choi, Jeong Su (Department of Integrated Biomedical and Life Sciences, Graduate School, Korea University) ;
  • Park, Ji Hyang (Department of Integrated Biomedical and Life Sciences, Graduate School, Korea University) ;
  • Yang, Man Gil (Biomedical Research Institute, Seoul National University) ;
  • Lee, Min Woo (Department of Integrated Biomedical and Life Sciences, Graduate School, Korea University) ;
  • Kim, Suhng Wook (Department of Integrated Biomedical and Life Sciences, Graduate School, Korea University)
  • 남궁수민 (고려대학교 대학원 의생명융합과학과) ;
  • 최정수 (고려대학교 대학원 의생명융합과학과) ;
  • 박지향 (고려대학교 대학원 의생명융합과학과) ;
  • 양만길 (서울대학교병원 의생명연구원) ;
  • 이민우 (고려대학교 대학원 의생명융합과학과) ;
  • 김성욱 (고려대학교 대학원 의생명융합과학과)
  • Received : 2017.07.31
  • Accepted : 2017.08.26
  • Published : 2017.09.30
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Abstract

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 $5{\mu}g/mL$ 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 $1.0{\times}10^{-7}M$); 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.

도파민과 세로토닌은 신경전달물질 및 호르몬으로서 미량이지만 체내에서 중요한 기능을 한다. 검체는 혈청과 24시간 뇨를 쓰며, 주로 HPLC-MS를 이용하여 검사한다. 본 연구에서는 항원-항체반응을 이용한 경쟁적 ELISA를 통해 도파민과 세로토닌을 검출하고자 하였다. $5{\mu}g/mL$ BSA conjugate를 96 well 표면에 고정시킨 뒤 각각 농도를 다르게 한 신경전달물질과 1차 항체를 넣어 반응시키고 HRP가 결합된 2차 항체와 TMB를 처리하여 흡광도를 측정하였다. 측정 결과를 토대로 회귀식과 $R^2$값을 구하여 신경전달물질에 대한 항체의 민감도와 신경전달물질 농도와 흡광도 사이의 상관관계를 판단하였다. 도파민은 1차 항체를 6,000배, 7,000배로 희석했을 때 $R^2=0.91$로 신경전달물질 농도와 흡광도의 상관관계가 가장 높게 나타났고, 세로토닌은 3,000배와 6,000배를 제외한 모든 희석배수에서 $R^2{\geq}0.90$의 높은 상관관계를 나타냈다. 도파민과 세로토닌 모두 $1.0{\times}10^{-7}M$ 이하의 저농도에서는 효과적으로 검출되지 않았기 때문에 참고치가 이보다 낮은 혈청 도파민 검출은 HPLC-MS 이용이 필요해 보이지만, 24시간 뇨 도파민과 혈청 및 24시간 뇨 세로토닌의 검출에는 competitive ELISA가 효과적일 것이라 사료된다. 보다 낮은 농도의 신경전달물질도 정확하게 검출하기 위해서는 추가 연구가 필요하다.

Keywords

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