Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1996-02-14
2000-04-25
Saunders, David
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 78, 435 793, 436501, 436503, 436504, 436514, 436518, G01N 33558, G01N 33566, G01N 33567
Patent
active
060542829
DESCRIPTION:
BRIEF SUMMARY
This application claims benefit of international application PCT/GB94/01799, filed Aug. 17, 1994.
The present invention relates to improved methods of detecting biological interactions especially in receptor binding assays. In particular the invention involves the use of radial partition.
Conventional radioligand binding assays are involved, ie. they employ vacuum filtration and other complex procedures. Such procedures are outlined for example in Receptor-Ligand Interactions, E. D. Hulme (Ed), 1992, IRL Press. A need therefore exists for further improved techniques which are less cumbersome to perform and more suitable, for example, for the identification of new drug leads by high throughput screening.
Radial partition was first described as long ago as 1982 (J. L. Giegel et al, Clin. Chem., 28, 1894-1898(1982)). Since then it has become well established as the basis for assays in the immunodiagnostics field. A typical method employs an antibody immobilised on a paper filter with sequential application of analyte and second antibody conjugate. A wash (optionally containing substrate for enzyme immunoassays) is then applied such that unbound conjugate is washed radially away from the centre leaving bound conjugate as a central dot. Quantitation according to the signal used gives, by reference to a standard curve, the concentration of the analyte.
We have now surprisingly found that the principle of radial partition may be successfully applied in novel assays for the identification of compounds which bind to a biological receptor of interest. Such novel partition assays do not require vacuum filtration or other complex procedures. These may be readily applied in rapid or high throughput screening procedures, and used for the identification of novel active compounds in, for example, the pharmaceutical and agrochemical industries.
Therefore according to a first aspect of the present invention we provide a method for the detection of a compound which modulates binding of a ligand to a biological receptor, which method comprises contacting the ligand, biological receptor and test compound at a locus on a solid phase matrix, the matrix allowing movement of fluids therein by capillary action, under conditions which permit binding of the ligand to the biological receptor and partition of any unbound ligand on the solid phase matrix, and detecting any modulation of binding by the test compound by reference to any such partition.
Components of the above assay may be added to the matrix in any order. optionally three, preferably two of the components may be mixed prior to application to the matrix. Preferably, the order should be such that the ligand does not contact the biological receptor before the test compounds, since this would require any active compound to displace the ligand and this might be kinetically slow and reduce the sensitivity of the assay. Preferably the first addition should contain the biological receptor. Preferably the biological receptor is immobilised on the solid phase matrix.
Alternatively the method of the invention may be used to determine interactions between a biological receptor of interest and a test compound. Therefore in a further aspect of the present invention we provide a method for the detection of a compound which binds to a biological receptor, which method comprises contacting the biological receptor with a test compound at a locus on a solid phase matrix, the matrix allowing movement of fluids therein by capillary action, under conditions which permit binding of the test compound to the biological receptor and partition of any unbound test compound on the solid phase matrix, and detecting binding of the test compound to the biological receptor by reference to any such partition.
Conveniently the test compound is immobilised on the solid phase matrix prior to application of the other assay component.
By "immobilised" we mean covalent or non-covalent attachment to the matrix, or attachment to a substance which is unable to migrate through the matrix by capillary action. In the
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Giegel, et al: "Radial partition immunoassay", Clinical Chemistry, vol. 28, No. 9, Sep. 1982, pp. 1894-1898.
Search Report for application No. GB 9416583.4, UK Patent Office (citing above documents not already of record.).
Saunders David
Zeneca Limited
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