Apparatus, compositions and methods for rapid competitive homogeneous assay

A method for detecting the presence, absence or concentration of a target molecule in a sample comprising:

• (i) providing a sample containing a target molecule;
• (ii) exposing the sample to a traceable binding agent wherein the traceable binding agent specifically binds to the target molecule to form a target molecule-traceable binding agent complex;
• (iii) mixing the target molecule-traceable binding agent complex with unbound target molecule to provide a sample mixture;
• (iv) adding the mixture to a vertical stacked electrophoresis matrix, wherein the matrix consists of two or more phases increasing in density or viscosity from top to bottom, and one or more phases of the matrix have an uncharged polymer (p) capable of associating with a binding agent electrophoresed through the polymer containing phase(s);
• wherein
• (a) the vertical stacked electrophoresis matrix is provided in a vertically arranged vessel with a hydrodynamic radius of at least 0.5 mm;
• (b) at least one of said phases comprises a gradient of increasing density from 1.0 to 1.1 g/ml within the vertically arranged vessel;
• (c) the gradient comprises
• (1) a sample mixture layer of density 1.0 to 1.02 g/ml;
• (2) a capture layer of density from 1.002 to 1.05 g/ml;
• and
• (3) a stacking layer of density from 1.01 to 1.1 g/ml,
• wherein the stacking layer has a higher density than the capture layer, and the capture layer has a higher density than the sample layer;
• and
• detecting and measuring the presence, absence, or concentration of the target molecule-traceable binding agent complex by measuring or detecting relative amounts of the traceable binding agent in relation to the unbound target molecule to determine the presence, absence, or concentration of the target molecule in the sample.