대한의생명과학회지 (Biomedical Science Letters)
- 제2권1호
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- Pages.121-126
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- 1996
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- 1738-3226(pISSN)
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- 2288-7415(eISSN)
ELISA Inhibition Assay에 의한 Vinblastine의 측정
Vinblastine Determination Measured by a Sensitive ELISA Inhibition Assay
- Jae Wha kim (Molecular and Cellular Biology Research Group, Korea Research Institute of Bioscience and Biotechnology, KIST) ;
- Mi Young Han (Molecular and Cellular Biology Research Group, Korea Research Institute of Bioscience and Biotechnology, KIST) ;
- Hee Gu Lee (Molecular and Cellular Biology Research Group, Korea Research Institute of Bioscience and Biotechnology, KIST) ;
- Eun Young Song (Molecular and Cellular Biology Research Group, Korea Research Institute of Bioscience and Biotechnology, KIST) ;
- Tai Wha Chung (Molecular and Cellular Biology Research Group, Korea Research Institute of Bioscience and Biotechnology, KIST) ;
- Kyung Soo Nam (Department of Pharmacology, College of Medicine, Dongguk University) ;
- In Seong Choe (Molecular and Cellular Biology Research Group, Korea Research Institute of Bioscience and Biotechnology, KIST)
- 발행 : 1996.06.01
초록
Vinblastine을 포함하는 bis-indole alkaloids에 대한 단일클론 항체를 생산하여 Vinca alkaloids의 양을 측정할 수 있는 간편한 immunoassay체계를 확립하였다. Vinca alkaloids는 periwinkle식물체의 배양된 세포로부터 추출하여 BSA와 접합한 후 Balb/c생쥐에 면역시켜 얻은 비장세포와 골수종양세포의 융합을 유도하여 VBL-BSA에 반응하는 클론을 ELISA 방법으로 분석하였으며 이들 클론 중 bis-in-dole alkaloids와 특이적으로 반응하는 항체는 inhibition assay를 통하여 분리할 수 있었고 그 결과 두개의 단일클론 항체를 형성하는 세포주(KN-1과 KN-2)를 확립하였다. KN-1의 경우 dimeric bis-indole alkaloids 와는 상당한 교차반응을 나타낸 반면 monomeric bis-indole alkaloids 와는 교차반응을 나타내지 않았으며 이 클론의 항체를 이용하여 배양된 세포 추출물에 포함된 Vinca alkaloids의 양을 측정한 결과 0.05 nM정도의 dimeric Vinca alkoloids까지도 측정할 수 있었다.
Specific monoclonal antibodies(mAbs) against bis-indole alkaloids related to vinblastine were established to develop a simple and specific immunoassay system for the quantitation of Vinca alkaloids. Vinca alkaloids were extracted from tissue cultured cells of periwinkle plant (Vinca rosea L.). Spleen cells from Balb/c mice immunized with vinblastine-bovine serum albumin(VBL-BSA) conjugate as immunogen were fused with myeloma cells(Sp2/0-Ag.14) in the presence of polyethylene glycol. In the preliminary experiments, 32 clones which highly reacted with VBL-BSA conjugate were selected by ELISA(Enzyme-linked immunosorbent assay).These clones were further analyzed by inhibition assay of ELISA. The results obtained with two typical monoclonal antibodies, KN-1 and KN-2, were described. KN-1 exhibited considerable reactivities with soluble dimeric bis-indole alkaloids, whereas no cross reacted with monomeric bis-indole alkaloids. However KN-2 showed cross reactivity with mono- and di-meric bis-indole alkaloids. Furthermore, KN-1 was applied to the immunoassay system for determining the VBL amounts of in vitro cultured cell extracts. This assay system could detect dimeric vinca alkaloid as low as 0.05 nM.