Cell proliferation-based amplified detection of analytes
OwnerM. Boris Rotmaninventor
Life of this patent
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Abstract
A test kit and method for the highly sensitive detection of specific analytes in a sample is provided. The presence of the analyte in the sample results in a decrease in the concentration of a growth inhibiting substance leading to proliferation of cells in the region of the analyte. The presence or absence of the analyte is determined by detecting the presence of increased numbers of cells. Assay sensitivity is accounted for by the exponential amplification of cell number that occurs during cell proliferation in the presence of analyte.
Claims17
A method for determining an analyte in a sample comprising the steps of:
- (a) providing the sample;
- (b) contacting the sample with a probe comprising an enzyme conjugated to a specific binder which specifically binds to the analyte to form a probe-analyte complex if said analyte is present, wherein said enzyme is capable of degrading a preselected bacterial growth inhibitory substance;
- (c) removing unbound probe from the sample comprising said probe-analyte complex;
- (d) contacting the complex of step (c) with a homogeneous reaction layer comprising a matrix comprising (i) a sample of bacterial cells whose growth is inhibited by said growth inhibitory substance, (ii) a complete growth medium in an amount sufficient to promote proliferative growth of said bacterial cells, and (iii) said growth inhibitory substance in an amount sufficient to reduce said proliferative growth of said bacterial cells, to produce an assay system;
- (e) incubating said assay system of step (d) for a time sufficient for said enzyme to degrade said growth inhibitory substance, thereby providing enhanced growth of said bacterial cells in regions where said growth inhibitory substance has been degraded;
- and,
- (f) determining said analyte by correlating the presence or amount of said enhanced growth regions, as compared to a background region where said growth inhibitory substance has not been degraded, to the presence or amount of said analyte in said sample.
A kit for the detection of an analyte in a sample said kit comprising:
- (a) a probe comprising an enzyme conjugated to a specific binder which specifically binds to said analyte to form a probe-analyte complex, wherein said enzyme is capable of degrading a preselected bacterial growth inhibitory substance;
- (b) a sample holding means capable of receiving said sample and said probe;
- (c) a homogeneous reaction matrix comprising (i) a hydrogel, (ii) a sample of bacterial cells whose growth is inhibited by said growth inhibitory substance, (iii) a complete growth medium in an amount sufficient to promote proliferative growth of said bacterial cells, and (iv) said growth inhibitory substance in an amount sufficient to reduce said proliferative growth of said bacterial cells, wherein said reaction matrix is fixed to a backing material.
Dependencies are inferred from the claim text.