Determination of analyte concentration using two labelling markers

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...

AI Technical Summary

Benefits of technology

[0014] When I refer to an insignificant fraction of the analyte I mean a fraction sufficiently small that the errors introduced by permitting a change in the initial analyte concentration are as small as, or smaller than, the errors unavoidably introduced into the measuring procedure elsewhere by limitations in the accuracy of sample and reagent manipulation, signal measurement, standardisation, temperature variation and the like. 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. Nevertheless this limit can sometimes be exceeded without detriment. Ideally, however, it is preferable to minimise measurement error by reducing the amount of analyte bound to the receptor to 1-2% (or less) of the total.

[0026] The receptor used will be one having binding sites on its molecule for the analyte to be estimated. These binding sites should be essentially constant in number per molecule and should thus be chemical binding sites rather than physical adsorption sites. They should also be capable of occupation solely by the analyte as compared to any other ingredient of the samples undergoing estimation and will thus preferably be specific to the analyte. Antibodies, e.g. monoclonal antibodies, are particularly preferred receptor molecules, but enzymes specific to individual analytes are other examples of receptor molecules that can be used. Antibodies to a wide variety of analytes are known or described in the literature or are commercially available and other antibodies specific to other hormones etc. can be manufactured by known techniques forming no part of this invention. To maximise the precision of measurement the receptor is preferably chosen with an affinity constant for the analyte such that between 25% and 75% of the binding sites on the receptor molecule are occupied by the analyte at its expected concentration in the unknown sample, i.e. it has an affinity constant from one third to 3 times the reciprocal of the expected analyte concentration. Affinity constants for receptors may be determined by a standard Scatchard analysis (Ann. N.Y. Sci., 51, (1949, 660).

[0031] In a further related invention it is possible to bind a variety of different labelled receptor molecules at spaced-apart locations on a single extended solid substrate such as a plate or rod, e.g. of inert plastics material such as polystyrene, each of the different locations being provided with receptor molecules having binding sites for one particular analyte, so that different locations bind different analytes. Such a multi-spot device can then be used to estimate concentrations of a plurality of analytes in a single liquid sample, separate labelled back-titration reagents being provided for each different analyte such that each pair of labels is capable of being differentiated and the ratio of the strengths of the signals determined. This is of particular advantage where a liquid such as a body fluid contains or may contain several different constituents of interest and the concentration of each needs to be known. (Although the use of labelled receptor molecules facilitates the production of such a multi-spot device because the amount of receptor molecule need not then be constant from spot to spot and device to device, it would also be possible to produce such a plate by using unlabelled receptor molecules provided that the amount of receptor molecule in each spot could be accurately known by other means and / or accurately controlled so as to be constant from device to device.)

[0033] In use of such a device having labelled microbeads the microbeads 22 on the plate 20 are contacted with the sample containing analyte(s) to be estimated and then with appropriate back-titration reagents bearing appropriate labels followed by estimation of the relative signal strengths of the two markers. Different markers will be chosen for different beads and back-titration reagents to enable the readings for one bead to be separated from those for another or alternatively each bead will be scanned separately, the latter alternative being facilitated by the use of fluorescent or fluorogenic markers.

[0037] A further advantage is that, when using fluorescent markers and scanning with a light beam, the light beam need not encompass the entire spot containing the receptor molecules but can merely sample a portion of the spot because the total amounts of bound analyte and receptor molecule need not be studied.

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.

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