US 2003/0170734 A1 · Multiplexed assays using electrophoretically separated molecular tags

Abstract

The invention provides methods and algorithms for measuring one or more analytes in a sample by using a plurality of releasable molecular tags attached to binding compounds specific for the analytes of interest. After binding compounds specifically bind to their respective analytes to form complexes, the molecular tags of the binding compounds forming such complexes are cleaved and released, while the molecular tags of those binding compounds not forming such complexes are not released. The released molecular tags are then electrophoretically separated along with one or more electrophoretic standards to generate electropherogram data, and the identity of each molecular tag is determined by the location of its corresponding peak in such data relative to the one or more of the electrophoretic standards. In this way, distortions in the electropherogram data due to factors such as instrumentation differences, assay conditions, reagent variability, or the like, can be taken into account and experimental results from different electrophoresis systems, different assays, or the like, may be compared.

Claims16

Dependent claims

A method of determining a plurality of analytes in a sample, the method comprising the steps of:

generating for each analyte present in an amount in the sample a quantity of a molecular tag, the quantity being monotonically related to the amount of the analyte and each molecular tag having a different electrophoretic mobility such that molecular tags associated with different analytes form distinct peaks upon electrophoretic separation;
combining the sample with one or more electrophoretic standards, each electrophoretic standard having a known quantity and a different electrophoretic mobility such that upon electrophoretic separation each electrophoretic standard forms a peak distinct from the peaks of other electrophoretic standards and the molecular tags;
electrophoretically separating the molecular tags and the one or more electrophoretic standards to generate electropherogram data;
determining a peak location in the electropherogram data of at least one electrophoretic standard;
determining for each molecular tag a peak location relative to a peak location of at least one electrophoretic standard and correlating the peak location of each molecular tag to the identity of an analyte in the sample.

2 more elements

A method of determining locations of peaks of a plurality of molecular tags in electropherogram data, each molecular tag having a different electrophoretic mobility such that upon electrophoretic separation different molecular tags form distinct peaks in the electropherogram data, the method comprising the steps of:

(a) electrophoretically separating the molecular tags and one or more electrophoretic standards to generate electropherogram data, each electrophoretic standard having a different electrophoretic mobility such that upon electrophoretic separation each electrophoretic standard forms a peak distinct from the peaks of the molecular tags and the other electrophoretic standards, and each peak of such one or more electrophoretic standards and molecular tags having a migration interval and each migration interval having a mean;
(b) determining a peak location of at least one electrophoretic standard within a migration interval in the electropherogram data;
(c) determining peak locations of peaks within a migration interval in the electropherogram data closest to an electrophoretic standard or a qualified peak in a qualified peak set, the peak locations being relative to the location of the closest electrophoretic standard or qualified peak, and correlating with a molecular tag or an electrophoretic standard a peak within the migration interval whose location is closest to the mean of the migration interval;
(d) determining a peak signal-to-noise ratio of the peak and adding the peak to the qualified peak set if the peak signal-to-noise ratio is greater than or equal to 1.5;
(e) repeating steps (c) and (d) until a peak location is correlated to every molecular tag having a peak in the electropherogram data.

2 more elements

A computer-readable product embodying a program for execution by a computer to identify a plurality of molecular tags by determining locations of peaks in electropherogram data correlated with such molecular tags, the program comprising instructions for:

(a) reading electropherogram data from a storage medium, the electropherogram data obtained by electrophoretic separation of the plurality of molecular tags and one or more electrophoretic standards, each electrophoretic standard and molecular tag having a different electrophoretic mobility such that upon electrophoretic separation each electrophoretic standard and molecular tag forms a distinct peak in the electropherogram data;
(b) determining a peak location of at least one electrophoretic standard in the electropherogram data;
(c) determining peak location of a peak in the electropherogram data, the peak location being relative to the location of an electrophoretic standard and correlating the peak location with a molecular tag or an electrophoretic standard;
(d) repeating steps (c) until a peak location is correlated to every molecular tag having a peak in the electropherogram data.

1 more element

Dependencies are inferred from the claim text.

Multiplexed assays using electrophoretically separated molecular tags

Life of this patent

Multiplexed assays using electrophoretically separated molecular tags

Life of this patent

Abstract

Claims16