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.