Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Patent
1996-06-14
1998-11-17
Spiegel, Carol A.
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
435 6, 435 71, 435 792, 435962, 435970, 435973, 435975, 436518, 436809, G01N 3353
Patent
active
058375511
DESCRIPTION:
BRIEF SUMMARY
This application is the U.S. national stage of PCT/GB94/02814, filed Dec. 23, 1994.
FIELD OF THE INVENTION
The present invention relates to binding assays, e.g. for determining the concentration of analytes in liquid samples.
BACKGROUND TO THE INVENTION
It is known to measure the concentration of an analyte, such as a drug or hormone, in a liquid sample by contacting the liquid with a binding agent having binding sites specific for the analyte, separating the binding agent having analyte bound to it and measuring a value representative of the proportion of the binding sites on the binding agent that are occupied by analyte (referred to as the fractional occupancy). Typically, the concentration of the analyte in the liquid sample can then be determined by comparing the fractional occupancy against values obtained from a series of standard solutions containing known concentrations of analyte.
In the past, the measurement of fractional occupancy has usually been carried out by back-titration with a labelled developing reagent using either so-called competitive or non-competitive methods.
In the competitive method, the binding agent having analyte bound to it is back-titrated, either simultaneously or sequentially, with a labelled developing agent, which is typically a labelled version of the analyte. The developing agent can be said to compete for the binding sites on the binding agent with the analyte whose concentration is being measured. The fraction of the binding sites which become occupied with the labelled analyte can then be related to the concentration of the analyte in the liquid sample as described above.
In the non-competitive method, the binding agent having analyte bound to it is back-titrated with a labelled developing agent capable of binding to either the bound analyte or the occupied binding sites on the binding agent. The fractional occupancy of the binding sites can then be measured by detecting the presence of the labelled developing agent and, just as with competitive assays, related to the concentration of the analyte in the liquid sample as described above.
In both competitive and non-competitive methods, the developing agent is labelled with a marker. A variety of markers have been used in the past, for example radioactive isotopes, enzymes, chemiluminescent markers and fluorescent markers.
In the field of immunoassay, competitive immunoassays have in general been carried out in accordance with design principles enunciated by Berson and Yalow, for instance in "Methods in Investigative and Diagnostic Endocrinology" (1973), pages 111 to 116. Berson and Yalow proposed that in the performance of competitive immunoassays, maximum sensitivity is achieved if an amount of binding agent is used to bind approximately 30 to 50% of a low concentration of the analyte to be detected. In non-competitive immunoassays, maximum sensitivity is generally thought to be achieved by using sufficient binding agent to bind close to 100% of the analyte in the liquid sample. However, in both cases immunoassays designed in accordance with these widely accepted precepts require the volume of the sample to be known and the amount of binding agent used to be accurately known or known to be constant.
In International Patent Application WO84/01031, I disclosed that the concentration of an analyte in a liquid sample can be measured by contacting the liquid sample with a small amount of binding agent having binding sites specific for the analyte. In this method, provided the amount of binding agent is small enough to have only an insignificant effect on the concentration of the analyte in the liquid sample, it is found that the fractional occupancy of the binding sites on the binding agent by the analyte is effectively independent of the volume of the sample.
This approach is further refined in EP304,202 which discloses that the sensitivity and ease of development of the assays in WO84/01031 is improved by using an amount of binding agent less than 0.1V/K moles located on a small area (or "microspot") of a solid
REFERENCES:
patent: 5096807 (1992-03-01), Leaback
patent: 5324633 (1994-06-01), Fodor et al.
patent: 5432099 (1995-07-01), Ekins
patent: 5508200 (1996-04-01), Tiffany et al.
patent: 5552272 (1996-09-01), Bogart
patent: 5599668 (1997-02-01), Stimpson et al.
Ekins et al., "Multianalyte Microspot Immunoassay--Microanalytical `Compact Disk` of the Future," Clinical Chemistry, 37 (11):1955-67, 1991.
Berson and Yalow, "Methods in Investigative and Diagnostic Endocrinology", pp. 111-116 (1973).
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