Vinblastine Determination Measured by a Sensitive ELISA Inhibition Assay

ELISA Inhibition Assay에 의한 Vinblastine의 측정

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.

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.