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Determination of analyte concentration using two labelling markers

a labeling marker and analyte technology, applied in the direction of measuring devices, instruments, material testing goods, etc., can solve the problem of negligible contribution

Inactive Publication Date: 2003-05-01
EKINS ROGER PHILIP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This approach provides a reliable and accurate measurement of analyte concentration, minimizing errors and eliminating the need for precise receptor standardization, enabling precise analyte concentration determination in various ranges, including picomolar to micromolar concentrations.

Problems solved by technology

Generally speaking, such errors customarily amount (in total) to 10% or less, and the binding of 5% or less of the total analyte in test samples would therefore be likely to cause inter-sample measurement errors arising from this particular source to contribute negligibly to the total.

Method used

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  • Determination of analyte concentration using two labelling markers
  • Determination of analyte concentration using two labelling markers

Examples

Experimental program
Comparison scheme
Effect test

example 2

[0047] Establishment of a dual fluorescence T4 assay method.

[0048] Example 1 illustrates the route taken to establish a valid "dual label ratio" competitive immunoassay method. Substitution of a fluorescein label for .sup.125I for labelling T4, and of a rhodamine label for .sup.131I to label anti-T4 antibody have enabled establishment of an analogous dual-fluorescence ratio immunoassay method. The labelling of antibody and T4 with these fluorescent labels was effected by conventional methods "Immunochemistry in Practice", A Johnston and R Thorpe, Blackwell Scientific Pub (1982). However, in order to assist in selection of the correct reagent concentrations, certain experiments were conducted using dual-labelled antibody (i.e. labelled with .sup.133I and rhodamine), and dual labelled T4 (i.e. labelled with .sup.125I and fluorescein). Furthermore, antibody was coated (by adsroption) onto small polystyrene discs, rather than covalently linked to microcellulose as described in Example 1...

example 3

[0049] Establishment of a dual fluorescence T4 assay method involving time-resolved pulse fluorescence.

[0050] Instead of labelling the T4 and anti-T4 antibody with fluorescein and rhodamine as in Example 2, they were labelled respectively with terbium and europium chelates with EDTA (ethylene diamine tetraacetic acid) coupled onto the antibody in a known manner. The signal ratio *was measured by known pulsed-light fluorescence techniques using a known time-resolving fluorimeter, and the results obtained with the unknown sample compared with the calibration curve obtained with standard solutions. Again satisfactory agreement was obtained with results obtained by other methods.

example 4

[0051] A kit for use in the estimation of RSH (thyrotrophin) according to the invention is composed of the following components:

[0052] (a) A monoclonal anti-TSH antibody commercially available from the Department of Endocrinology, the Middlesex Hospital Medical School, Mortimer Street, London, is immobilised on a solid plate and labelled with fluorescein.

[0053] (b) Standard solutions contain 0.2, 1.0, 5.0, 20.0 and 100 micro-international units of TSH per mol.

[0054] (c) The back-titration reagent is likewise a commercially available anti-TSH monoclonal antibody, this time labelled with a europium (III) chelate with cupric tri-fluoroacetylacetone and formaldehyde in a manner similar to that proposed in published International Patent Application WO 86 / 01604. The first antibody is permanently fluorescent and the second is capable of estimation by time-resolved pulse fluorescence.

[0055] Such a kit can be used for the estimation of TSH by a similar procedure to that described in Examples...

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Abstract

Methods for measuring the concentration of an analyte in a sample are provided. The sample is contacted with a receptor molecule having binding sites for the analyte which is labeled with a first marker under conditions whereby only a small fraction of the binding sites on the receptor become occupied by the analyte. The receptor having fractionally occupied binding sites is then back-titrated via a back titration technique which includes a second marker different from the first, and the relative strengths of the two signals produced by the markers are measured thereby providing a value representative of the fractional occupancy of the binding sites on the receptor molecule by the analyte. This value is compared with one or more corresponding values obtained in the same way using one or more standard liquid samples of known analyte concentration.

Description

[0001] The present invention relates to a method of measuring the concentration of analytes in liquids using two different labelling markers by immunoassay or immunometric techniques, also to an analytical device and kit.[0002] It is known to measure the concentration of an analyte such as a drug or hormone in a liquid by exposing the liquid to a receptor having binding sites on its molecule for the analyte, separating the receptor containing bound analyte from the liquid, measuring a value, representative of the proportion of the available binding sites on the receptor molecule that have been occupied by analyte molecules (referred to as the fractional occupancy) and comparing that value with a corresponding measured value obtained with a solution of known concentration of the analyte.[0003] The measurement of the value in question can be achieved by a back-titration technique involving contacting the receptor molecule containing bound analyte with a labelled version of the analyte...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): G01N33/543G01N33/58G01N33/78
CPCG01N33/54386G01N33/582Y10S436/808Y10S436/80G01N33/78
Inventor EKINS, ROGER PHILIP
Owner EKINS ROGER PHILIP
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