Binding assay using binding agents with tail groups

Details Binding assay using binding agents with tail groups Binding assay using binding agents with tail groups

1996-10-23

2001-11-13

Woodward, Michael P. (Department: 1631)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S007100, C436S518000, C436S532000, C530S391100, C530S387300

Reexamination Certificate

active

06316186

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to binding assays using binding agents with tail groups, and in particular binding agents having oligonucleotide tail groups. These binding assays are useful in determining the concentration of analytes in liquid samples.
BACKGROUND OF 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 sample with a binding agent immobilised on a solid support, the 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 fraction of the binding sites of the binding agent that are occupied by the analyte. Typically, the concentration of the analyte in the liquid sample can then be determined by comparing the value representative of the fraction of the binding sites occupied by analyte against values obtained from a series of standard solutions containing known concentrations of analyte.
In the past, the measurement of the fraction of the binding sites occupied 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 or an anti-idiotypic antibody capable of recognising empty binding sites of the binding agent. 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 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 to the occupied binding sites on the binding agent. The fraction of the binding sites occupied by analyte 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 to allow the developing agent to be detected. 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 assays 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 111-116. Berson and Yalow proposed that in the performance of competitive immunoassays, maximum sensitivity is achieved when an amount of binding agent is used to bind approximately 30-50% 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 WO 84/01031, 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 “ambient analyte” 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 fraction of the binding sites on the binding agent occupied by the analyte is effectively independent of the volume of the sample.
This approach is further refined in EP 304,202 which discloses that the sensitivity and ease of development in the assays in WO 84/01031 are improved by using an amount of binding agent less than 0.1V/K moles located on a small area (or “microspot”) on a solid support, where V is the violume of the sample and K is the affinity constant of the binding agent for the analyte. In both of these references, the fraction of the binding sites occupied by the analyte is measured using either a competitive or non-competitive technique as described above.
SUMMARY OF THE INVENTION
There is continuing need to develop binding assays which have enhanced kinetics to allow assays to be carried out more quickly and easily. In addition, it would be desirable to provide a binding assay which the user of the assay can easily customise for the detection of different groups of analytes.
Accordingly, in a first aspect, the present invention provides a method of determining the concentrations of analytes in a liquid sample comprising:
(a) immobilising one or more capture agents on a solid support, each capture agent being capable of specifically binding a given binding agent;
(b) contacting the liquid sample with one or more binding agents, each binding agent having binding sites specific for a given analyte so that a fraction of the binding sites become occupied by the analyte, and a tail group adapted to bind to a corresponding capture agent;
(c) contacting the liquid sample, either simultaneously or sequentially with the step (b), with the immobilised capture agents so that the binding agents become bound to their respective capture agents; and
(d) determining the fraction of the binding sites of a binding agent occupied by analyte to determine the concentration of the analyte in the liquid samples.
Accordingly, the present invention provides an assay in which the binding of the analytes takes place in the liquid phase, rather than at a surface of a solid substrate. This enhances the kinetics of the reaction between analyte and binding agent.
Thus, in one embodiment, contacting the liquid sample with the binding and capture agents simultaneously allows the assay to be carried out in a single step, eg using a single reaction vessel. Alternatively, sequential contact of the binding agent(s), and capture agent(s) may be preferred, especially where the liquid is serum or blood, and non-specific binding is an important source of error. In these cases, the binding agent can be first contacted with the liquid sample in a first vessel and then the sample transferred to a second vessel to allow the capture agent to bind the binding agent to the solid support.
In a second aspect, the present invention provides a method of immobilising one or more binding agents on a support, each binding agent having binding sites specific for a given analyte and a tail group adapted to bind to a capture agent, comprising:
(a) immobilising one or more capture agents on a support each capture agent being capable of binding to the tail group of a given binding agent and,
(b) contacting the binding agents with the support having the capture agents immobilised thereon so that the binding agents become specifically bound to their respective capture agents through their tail groups.
The above method can additionally comprise the step of attaching the tail groups to the binding agents prior to exposing them to the capture agents immobilised on the support.
Thus, it is possible for the user of the assay to customise binding agents for use in determining the concentration of different groups of analytes and using the customised binding agents in conjunction with a universal support having capture agents immobilised on it, to which the binding agents can individually bind by virtue of their tail groups.
In this aspect of the invention, the assay is carried out by exposing the support to a liquid sample after the binding agent(s) has or have become bound to the capture agent(s).
In either aspect, the present invention

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