Chemistry: analytical and immunological testing – Biospecific ligand binding assay
Patent
1997-05-13
1999-04-06
Saunders, David
Chemistry: analytical and immunological testing
Biospecific ligand binding assay
435 6, 435 71, 435973, 436523, 436533, 436534, 436800, 436805, G01N 33533, G01N 33546, G01N 33566
Patent
active
058917381
DESCRIPTION:
BRIEF SUMMARY
This invention relates to a biospecific multiparameter assay method. Immunoassays are a well established group of biospecific assays and now widely used in routine diagnostics and research laboratories. Another group of biospecific assays, is DNA hybridization assays. Biospecific assays generally employ one or several biospecific reactants (e.g. antibody, DNA probe) and normally one of these reactants is labelled. The labels currently used are radioisotopic, enzymatic, luminescent and fluorescent labels.
In routine diagnostics there is a growing need for multiparameter (multianalyte) analysis. Unfortunately, the current methodology does not allow the use of more than two or three simultaneous labels because the spectrometric signals from different labels cannot be sufficiently separated. The emission spectra of different radioisotopic labels and photoluminescent labels overlap significantly and as a consequence they provide inadequate separation of different analytes over a required concentration range.
The purpose of this invention is to present a better method for multiparameter biospecific assays. The method according to this invention is based on methods that are generally known within the field of immunology and DNA hybridization. Normally, they are performed as follows. The method uses two biospecific probes that recognize the analyte molecule k. In this text, these probes are referred to as the primary probe Ab(k,1) and the secondary probe Ab(k,2). When the secondary probe is labeled, for example, with a photoluminescent label F, it is denoted with the symbol Ab.sup.F (k,2). In the reaction solution, there is an excess of primary and secondary probes compared to the number of analyte molecules M.sub.k. When the analyte molecule, which is either a polypeptide or a macromolecule, has separate epitopes i.e. molecule structures that bind specifically to the probes, they form together a complex Ab(k,1)+M.sub.k +Ab.sup.F (k,2). In principle, the amount of complex formed is directly proportional to the amount of the analyte, and the excess of primary and secondary probes remain in the solution. The complexes are separated from the free probes using a commonly known technique, for example, in which the primary probe is bound to a solid carrier and the free probes are washed away from the sample. Finally, the signal of bound label F in the complexes is measured in a traditional way which depends on the label chosen. The intensity of the signal obtained is directly proportional to the amount of label in the solution, and the response of the system is linear.
If the analyte to be measured is a small molecule without two or more epitopes which specifically bind to the probes, one can use a secondary probe that reacts specifically with the complex formed by the analyte and the primary probe (C. H. Self et al., Clin. Chem. 40 (1994) 2035-2041).
BACKGROUND OF THE INVENTION
Certain multiparameter biospecific assay methods have been introduced earlier. It has been common practice to use multiple labels to label biospecific reagents and to perform the separation of the signals on the basis of their different emission spectra. In most cases, however, the known multiparameter methods are based on the use of a solid support where the biospecific reagents can be immobilized at separate and optically distinguishable areas, or that are based on the use of artificial microparticles as a solid support. Some of the methods are reviewed below:
1. A method, in which various biospecific probes are attached to a matrix, which is formed by small areas on a planar solid support, is described in the patent PCT WO 84/01031. In this method, after the reaction and the wash, the signals from the photoluminescent labels in each area are measured separately, for example, using a laser scanning microscope.
2. A method, in which the identification of the analyte category is based on the color of the microparticles, which are used as a solid support and which is achieved by optically measuring the light absorption of the particle to be an
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Hanninen Pekka
Soini Erkki
Soini Juhani
Saunders David
Soini Erkki
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