Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals – Carrier is inorganic
Patent
1993-04-29
1997-11-25
Housel, James C.
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
Carrier is inorganic
435 79, 435 792, 435 793, 435 794, G01N 33553
Patent
active
056912072
DESCRIPTION:
BRIEF SUMMARY
This invention relates to a method for the qualitative or quantitative determination of the presence of an analyte in an aqueous medium.
The detection and/or assay of analytes using immunoassay techniques is well established, particularly in relation to proteins such as antigens and antibodies, as well as sugars, lectins and nucleic acids. However, many current techniques, while being of great sensitivity, are often laborious in requiring a number of steps each of which may be of long duration. It has proved possible to simplify some of such assays, however, by immobilising one of the components of the assay system on a solid support, since this facilitates removal of excess reagents. Such assays will normally involve the use of a labelled macromolecule, which may be the analyte itself or a binding partner for the analyte, carrying a suitable label such as a radioisotope, a fluorophore or an enzyme producing a characteristic reaction.
One simplification which has been proposed is to use a coloured substance attached to one of the immunoassay reactants as a visible marker. However, very few coloured substances are able to produce a sufficiently intense signal. U.S. Pat. No. 4,313,734 of Akzona Inc. describes the use of, inter alia, colloidal gold as such a coloured material, specifying that the gold particles should have a particle size of at least 5 nanometers, preferably 10 to 100 nm.
An improved immunoassay system is described in WO89/06801 in which at least 75% of the gold particles have a mean diameter of less than 5 nanometers. This is said to give more rapid reaction of the gold reagent with the immobilised reactant together with an increase in colour intensity. We have now found that a yet further increase in colour intensity may be obtained when the very small gold particles of W089/06801 are formed into larger particles (superaggregated gold-protein colloids) by a novel aggregation process. The superaggregated particles are very different from the monolithic gold particles used in immunoassays to date and allow for analysis of substances at even lower concentrations than the already impressively low levels made possible by the systems of W089/06801.
Small gold particles are also used as markers in a blotting system, as described in U.S. Pat. No. 4,775,636 of Janssen Pharmaceutica N.V. However, there is no suggestion that the particles are aggregated, rather they are simply bound to the component which it is desired to visualise.
According to the present invention we provide a method for the qualitative or quantitative determination of an analyte in a test sample wherein a labelled reagent comprising a gold sol bound to a substance capable of specifically binding to said analyte or to a specific binding partner therefor, is caused to be immobilised in bound form on a solid phase to provide an indication of the presence or quantity of the analyte in the sample, characterized in that the labelled reagent comprises a superaggregated complex of said substance or specific binding partner therefor and a gold sol wherein at least 75% by weight of the gold particles of the gold sol have a mean diameter of less than 20 nanometers.
In many types of solid phase assay it is advantageous to couple an analyte analogue or a specific binding partner for said analyte to a solid support to provide the solid phase onto which the labelled reagent is immobilised. As a further aspect of the invention therefore, we provide a method for the qualitative or quantitative determination of an analyte in a liquid sample, wherein said sample is contacted in an aqueous assay medium with (i) an analyte analogue or a specific binding partner for said analyte immobilised on a solid support and (ii) a labelled reagent comprising a gold sol attached to a molecule capable of specifically binding said analyte or a specific binding partner therefor, and optionally an enzyme capable of generating a characteristic reaction, whereby a quantity of said labelled reagent is immobilised on said support, inspection or determination of the colour
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Gogstad Geir Olav
Holtlund Jostein
Housel James C.
Nycomed Pharma AS
Tanigawa Gary
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