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CCR 4 expressing cells cultured adherently on a capillary wall and formaldehyde fixed as the stationary phase for ligand screening by ACE
Author(s) -
Yakufu Pazilaiti,
Qi Hui,
Li Meina,
Ling Xiaomei,
Chen Wenjing,
Wang Ying
Publication year - 2013
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201200376
Subject(s) - chromatography , ligand (biochemistry) , chemistry , membrane , receptor , chemokine receptor , stationary phase , cell culture , biophysics , chemokine , biochemistry , biology , genetics
We have developed an on‐line screening method for CC chemokine receptor 4 ( CCR 4) ligands, in which the whole cells expressed with CCR 4 were cultured adherently and immobilized on the inner wall of the capillary as the stationary phase for the first time. Moreover, in this method it is unnecessary to isolate and purify the target receptors from cell membranes. Therefore, it is possible to almost completely preserve the native conformation of the target receptors. The binding activities of the immobilized CCR 4 did not change. A known antagonist of CCR 4, compound A , was employed to validate the bioactivity of the cell layer and stability of this method. The intraday, interday, and batch‐to‐batch reproducibilities were investigated ( RSD ≤ 13.9%). Nonlinear chromatography was used to calculate the binding constant between the compound A and CCR 4 (6.4 × 10 4 /M, RSD = 4.96%). Using this method, the qualitative and quantitative characterizations of 23 computer‐aided drug design compounds were achieved and the kinetic parameters ( K , k a , k d , and k ′) were obtained by nonlinear chromatography. Three active compounds were screened out, which also showed activity in chemotaxis inhibition assay. The experimental results show that this method is simple, sensitive, and efficient for drug screening. Moreover, it offered a novel way to detect the nonspecific interactions between ligands and cell membrane.