A direct binding assay for the vascular cell adhesion molecule-1 (VCAM1) interaction with alpha 4 integrins
A direct binding assay for the vascular cell adhesion molecule-1 (VCAM1) interaction with alpha 4 integrins
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DOI：
10.3109/15419069509081293
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发表时间：
1995-01-01
期刊：
影响因子：
--
通讯作者：
Whitty, A
中科院分区：
文献类型：
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作者：
Lobb, RR;Antognetti, G;Whitty, A
血管细胞粘附分子-1（VCAM 1）是免疫球蛋白（IG）超家族的成员，与α 4整联蛋白α 4 β 1（极晚期抗原4：VLA 4）和α 4 β 7相互作用，这些蛋白在许多白细胞亚群上组成性表达，并在细胞运输和激活中发挥关键作用。使用重组VCAM-IgG融合蛋白（VCAM-Ig）作为α 4 β 1的可溶性配体，我们通过荧光分析直接证明α 4 β 1受体可以以不同的亲和力状态存在于细胞表面上，并且高亲和力状态由锰离子或某些活化抗β 1单克隆抗体诱导（Jakubowski et al.，1995年b）。在这里，我们通过开发一种快速和可重复的检测方法来扩展这些观察结果，该方法使用碱性磷酸酶（AP）偶联的VCAM-Ig（VCAM-Ig-AP），以微量滴定板的形式测量VCAM 1和α 4整联蛋白之间的相互作用。该试验使我们能够直接评估金属离子、抗β 1单克隆抗体以及不同细胞类型和种属对VCAM 1/α 4整联蛋白相互作用的影响。最重要的是，该测定系统提供了一种快速评价α 4整联蛋白定向抑制剂的方法，而没有粘附测定中固有的后配体结合事件的复杂性。
Vascular cell adhesion molecule-1 (VCAM1) is a member of the immunoglobulin (Ig) superfamily which interacts with the alpha 4 integrins alpha 4 beta 1 (very late antigen 4: VLA4) and alpha 4 beta 7, which are constitutively expressed on many leukocyte subsets and play a key role in cell trafficking and activation. Using a recombinant VCAM-IgG fusion protein (VCAM-Ig) as a soluble ligand for alpha 4 beta 1 we directly demonstrated by fluorescence analysis that the alpha 4 beta 1 receptor can exist in different affinity states on the cell surface, and that a high affinity state is induced by manganese ions or certain activating anti-beta 1 monoclonal antibodies (Jakubowski et al., 1995b). Here we have extended these observations by developing a rapid and reproducible assay using alkaline phosphatase (AP)-coupled VCAM-Ig (VCAM-Ig-AP) which measures the interaction between VCAM1 and alpha 4 integrins in a microtiter plate format. This assay has allowed us to evaluate directly the effects of metal ions, anti-beta 1 mAbs, and different cell types and species on the VCAM1/alpha 4 integrin interaction. Most importantly, the assay system provides a means to rapidly evaluate alpha 4 integrin-directed inhibitors without the complication of post-ligand binding events inherent in adhesion assays.