Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Reexamination Certificate
1996-08-07
2002-06-18
Chin, Christopher L. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
C435S007920, C435S007940, C436S524000, C436S528000, C436S536000, C436S807000, C436S810000
Reexamination Certificate
active
06406920
ABSTRACT:
This invention relates to processes for carrying out specific binding assays, e.g. immunoassays, and to apparatus, e.g. test kits, for carrying out these processes.
In particular embodiments the invention is applicable to enzyme-linked and fluorescent-marker-linked specific binding assays, including immunoassays.
A wide variety of immunoassays and other specific binding assays is already known.
Examples of such assays and the materials used for them are given in S. Spector, Ann.Rev.Pharm. (1973) 13, 359-70 (radioimmunoassays), L. E. M. Miles and C. N. Hales, Nature (1968) 219, 186-189 (assays using radioactive, enzyme and other markers), E. Habermann, z.klin.Chem.u.klin.Biochem. (1970) 8, 51-55 (radioactively-labelled and enzyme-labelled assay procedures), and in G.B. patent specification No. 1,363,565 (enzyme-labelled immunoassays) and U.S. Pat. Nos. 4,150,949 and 4,160,818 (fluorescence-labelled immunoassay).
An important group of such assays comprises those in which a ternary complex is formed between a specific adsorbent, the material under assay, and a marker-conjugated material with specific binding capacity for the material under assay (“conjugate”). Certain assays of this kind have been called “sandwich” or “antiglobulin” assays. They have the property that the quantity of marker becoming fixed to the specific adsorbent is directly rather than inversely related to the quantity of the material under assay that participates in the ternary complex, and this can be simply measured after separation of the immobilised material from the remaining free marked conjugate, in whatever manner is appropriate to the marker in use.
However, as appears for example from the above-mentioned Habermann (1970) publication and G.B. specification No. 1,363,565, the performance of these assays is not without difficulty: they demand either a number of successive manipulation steps to carry out the assay reactions, or else suffer from low sensitivity, which careful choice of reagents has not so far been able to overcome.
Commercial test kits are available comprising essentially:
(a) a plate consisting of an array of tubes or pre-formed wells which are coated with an antibody or antigen as the case may be;
(b) an enzyme linked to an appropriate antibody (a so-called conjugate) against a substance to be detected if present in a test sample and
(c) a substrate for the determination of the activity of the enzyme.
One standard procedure for conducting an assay for antigen or antibody involves:
(1) determining the working dilution for the test sample;
(2) removing any excess of antibody or antigen used to sensitise the wells;
(3) washing the wells;
(4) introducing a proportion of the suitably diluted test sample;
(5) incubating for about two hours to allow the substance to be detected in the test sample to bind to the sensitising substance;
(6) washing the wells to remove unreacted material;
(7) introducing the suitably diluted conjugate (incubate for about 2 hours);
(8) washing the wells to remove unreacted material;
(9) adding a solution of the substrate;
(10) incubating until a suitable intensity of colour develops as a result of the reaction of the substrate with the enzyme;
(11) stopping the reaction, e.g. with a strong alkali and
(12) measuring the optical density of the reacted substrate solution.
This procedure is also time-consuming since each of the several antibody-antigen reactions requires several hours to reach equilibrium. In practice, shorter incubation times are used but only at the expense of sensitivity and/or economy.
According to the results of the present work, it is believed that an obstacle to the use of fewer assay steps is an unwanted interference with the formation of the desired immobilised complex which can originate in interfering reactions between two of the components. By using a specific binding agent of selected narrow specificity, or in slow-release form, such interference can be avoided, and high-sensitivity assays carried out using fewer manipulation steps.
According to this invention there is provided a process for carrying out a specific binding assay (for example an immunoassay) in which (a) a sample under assay, possibly containing a substance being tested for, is reacted with (b) a specific binding partner for the substance being tested for, immobilised on a solid support, and (c) a specific binding partner for the substance being tested for which is conjugated to a detectable marker, thereby to form a complex by reaction between whatever quantities are present of the substance being tested for with reagents (b) and (c), in which the marker is immobilised to the support via the substance being tested for, and is detected or assayed as an index of the quantity present in the sample (a) of any of the substance being tested for; characterised in that reaction ingredients (a), (b) and (c) are all mixed in a single step for reaction in a single reaction liquid, and competitive interference between the binding reactions of the substance being tested for and reagents (b) and (c) is avoided either by use of an antibody of narrow specificity, such as a monoclonal antibody, to avoid the interference, or by use of a slow-release form of reagent (c).
The narrow specificity required of the antibody is a capacity to bind specifically with the substance under test but without preventing the binding reaction between the substance under test and its other specific binding partner. Such an antibody can be selected out of a number of antibodies with an affinity for the substance under test, by using normal methods to verify the progress of a binding reaction between the other specific binding partner and a complex previously formed between the substance to be tested in the assay and the narrow-specificity antibody to be selected.
According to a preferred embodiment of the present invention, the conjugate between antibody and the enzyme or other marker, and/or the antibody (if any) which is coupled to the solid surface, comprises a monoclonal antibody or other antibody of sufficiently narrow specificity to ensure that the desired assay reaction or reactions are not impeded by competition between the conjugate and the immunosorbent in their reactions with whatever quantities are present of the substance being tested for in the sample under assay. Monoclonal antibody of sufficiently narrow specificity can, for example, be produced as antibody derived from a line of antibody-producing cells, derived from a single antibody-producing progenitor cell or cells. Such a line can, for example, be produced by known cell fusion, culture and isolation techniques using very pure antigens as comparative material.
Alternatively, in many cases antibody of sufficiently narrow specificity can be obtained in the (polyclonal) immunoglobulins of antisera raised against discrete chemical or physical molecular fragments of the material under test, for example, antibody against Fc fragments (or against smaller peptide fragments) of immunoglobulins to be tested for, or against sub-units or peptides of protein antigens to be tested for. The object in each case is to ensure substantial freedom from interference which can arise particularly, for example, in carrying out immunoassays of the “sandwich” or “antiglobulin” test configurations.
In a “sandwich” test configuration, antigen under test can be specifically adsorbed to a first antibody bound to a solid surface, and a second antibody carrying an enzymic or other (e.g. fluorescent or radioactive) marker is specifically bound to the adsorbed antigen under test. Marker specifically so bound is used for measurement and determination of the antigen under test, e.g. by direct measurement, such as radiometry or fluorimetry, or exposure of enzyme marker to substrate followed by product measurement. Thus, in preferred sandwich tests, the two antibodies used can have different, non-interfering specificity with respect to the same antigen under test.
In an “antiglobulin” test configuration, sometimes also referred to as a “sandwich” test configuration, the positi
Davis Paul James
Porter Philip
Chin Christopher L.
Inverness Medical Switzerland GmbH
Oppedahl & Larson LLP
LandOfFree
Processes and apparatus for carrying out specific binding... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Processes and apparatus for carrying out specific binding..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Processes and apparatus for carrying out specific binding... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2906860