Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals – Carrier is particulate and the particles are of...
Patent
1996-09-06
1998-04-14
Chin, Christopher L.
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
Carrier is particulate and the particles are of...
422 61, 422 8208, 435 71, 435 793, 435 794, 435 795, 435962, 436518, 436523, 436538, 436824, G01N 33543, G01N 33537
Patent
active
057390428
DESCRIPTION:
BRIEF SUMMARY
This invention relates to a method of assaying an analyte and to kits useful in such a method.
Assay techniques for determining the presence and desirably also the concentration of an analyte using a binding partner having specificity for that analyte are frequently encountered, e.g. in the fields of biochemistry and clinical chemistry. Thus, for example, a wide range of immunological and related techniques has been proposed for determining materials such as antigens in serum, using an appropriate binding partner for the analyte, such as a specific antibody (e.g. a monoclonal antibody) for a particular antigen.
One such technique comprises competitive binding assays, in which a known amount of a labelled version of an analyte to be determined (e.g. carrying a radioactive label) and a relatively small known amount of a binding partner therefor are incubated with the analyte to be determined, whereby the labelled and the naturally-occurring analyte compete for the binding partner. The amount of labelled analyte bound to the binding partner is thereafter determined and the concentration of the naturally-occurring analyte, which will bear an inverse relationship to this amount, is assessed from a previously established standard curve.
Another useful technique comprises sandwich assays. These employ an excess of binding partner, the analyte which binds thereto being labelled by treatment with a labelled ligand also having affinity for the analyte. The amount of bound and labelled analyte is then determined and permits the analyte concentration to be assessed by reference to a standard calibration curve.
The binding partner and the labelled ligand in such sandwich assays preferably have affinities for different binding sites (e.g. epitopes) on the analyte. The ligand may, for example, be labelled for reading on the basis of radioactivity, light absorption or fluorescence.
Sandwich assays tend to exhibit greater sensitivity than competitive binding assays and are therefore usually preferred. It will be appreciated that high sensitivity is essential in, for example, immunoassays in clinical laboratories, where it may be required to quantify e.g. antigens present in serum at concentrations in the nmol/l to pmol/l range or even lower.
The binding partner in both the above-described types of assay is commonly coupled to a solid support in order to facilitate isolation of the bound analyte and competing or analyte-bound label. Thus, for example, the binding partner may be coupled to the surface of a reaction vessel, e.g. to the surfaces of the wells of a microtitre plate made from a suitable plastics material, so as to facilitate washing to remove unbound excess labelled ligand.
Alternatively the binding partner may be coupled to the surfaces of an array of particles, for example made of a suitable plastics material such as polystyrene or polyacrylate. Separation of the bound analyte/label from free label may then be effected by, for example, filtration or, in the event that superparamagnetic particles are employed, by application of a magnetic field. The particles are advantageously of microscopic size in order to present a large total surface area coated with the binding partner. The use of monosized microparticles is preferred since it ensures that the particles exhibit standard binding properties.
A disadvantage of the above-described basic assay techniques is that separation of the bound analyte and label and associated washing steps to remove unbound label are inherently time-consuming and labour-intensive. It is known, however, that this problem may in principle be avoided in the case of particle-based assays if the particles are analysed by means of flow cytometry. This typically involves passage of a suspension of particles through the measurement region of a photometer in such a way that successive individual particles are irradiated with excitation light, causing emission of a pulse of scattered light related to the size of the particle and a further signal, e.g. a pulse of fluorescent light, related to the
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Chin Christopher L.
Nguyen Bao-Thuy L.
Sinvent AS
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