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High-throughput high-information content label-free cell biology screening methods

  • US 8,846,575 B2
  • Filed: 09/29/2009
  • Issued: 09/30/2014
  • Est. Priority Date: 03/05/2008
  • Status: Expired due to Fees
First Claim
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1. A method for hit compound identification in a high throughput, label-free biosensor cellular assay, the method comprising:

  • (a) generating at least one signaling pathway-specific kinetic profile for a target cell membrane receptor in a live-cell, wherein the at least one signaling pathway-specific kinetic profile for the target cell membrane receptor is obtained by;

    (i) providing an optical biosensor having a single type of live-cell immobilized on a surface of the biosensor, the biosensor being situated in a well of a microtiter plate, and the live-cell having a first and second target, wherein the first target is a cell membrane receptor, wherein the second target is a cellular protein, wherein the cellular protein is a member of the signaling pathway triggered by activation of the cell membrane receptor,(ii) separately stimulating the live-cell expressing target cell membrane receptor with a plurality of candidate compounds after the live-cell is attached to the surface of the biosensor;

    (iii) contacting the ligand candidate-treated live-cell with a mixture containing two markers; and

    (iv) determining the effect of the ligand candidate on the marker mixture-induced biosensor output;

    (b) generating a relational table, which table incorporates each signaling pathway-specific kinetic profile for the plurality of candidate compounds;

    (c) determining a delta response based on the relational table for each of the plurality of candidate compounds screened against the target cell membrane receptor; and

    (d) identifying hit compounds from the plurality of candidate compounds based upon the separation of the delta response from a threshold.

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