CPC G01N 1/405 (2013.01) [B01D 15/3809 (2013.01); B01D 15/3823 (2013.01); G01N 33/54306 (2013.01); G01N 33/56966 (2013.01); G01N 33/56972 (2013.01); G01N 2333/7051 (2013.01); G01N 2333/70517 (2013.01)] | 13 Claims |
1. A method of isolating a target cell, wherein the target cell is a CD4+ lymphocyte, the method comprising:
(a) contacting a stationary phase chromatographic matrix suitable for cell separation with a receptor binding reagent, wherein:
the stationary phase chromatographic matrix is a nonmagnetic or non-magnetizable material and comprising a streptavidin mutein affinity reagent immobilized thereon,
the receptor binding reagent comprises (1) a Fab monomer fragment that binds CD4 on the surface of lymphocytes with (i) a dissociation constant (KD) of 10−3 to 10−7 M and/or (ii) a koff rate of 3×10−5 sec−1 or greater, wherein the Fab fragment comprises a heavy chain comprising the sequence set forth in SEQ ID NO: 1 and a light chain comprising the sequence set forth in SEQ ID NO: 2, and the receptor binding reagent further comprises (2) a binding partner C that comprises a streptavidin-binding peptide fused to the C-terminus of the Fab monomer fragment, wherein the binding partner C is capable of reversibly binding to a binding site Z of the streptavidin mutein affinity reagent that is immobilized on the stationary phase, the binding site Z having a lower dissociation constant (KD) for biotin or a biotin analog than for the binding partner C, thereby immobilizing the receptor binding reagent on the stationary phase chromatographic matrix;
(b) loading a sample comprising one or more CD4+ lymphocyte target cells onto a selection affinity chromatography column to form a complex in the column, wherein the selection affinity chromatography column has been formed by loading the chromatographic matrix comprising the receptor binding reagent immobilized thereof of (a) into a chromatography column, and wherein the complex comprises multimers of the monomeric Fab fragment fusion bound to the target cells, thereby reversibly immobilizing the one or more CD4+ lymphocyte target cells on the chromatographic matrix;
(c) washing the selection affinity chromatography column to remove unbound cells; and
(d) eluting the one or more CD4+ lymphocyte target cells bound on the stationary phase, comprising contacting the selection affinity chromatography column with a competition reagent selected from the group consisting of a biotin or biotin analog capable to disrupt the binding between the streptavidin-binding peptide of the receptor binding reagent and the streptavidin mutein, thereby displacing the receptor binding reagent and releasing the one or more CD4+ lymphocyte from the monomeric Fab fragment fusion of (b) in an eluate, wherein the eluate comprises the one or more CD4+ lymphocytes target cells, the Fab monomers and biotin or biotin analog,
wherein the streptavidin-binding peptide comprises the amino acid sequence of SEQ ID NO. 13 or SEQ ID NO. 3.
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