Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1987-08-19
1992-03-24
Ceperley, Mary E.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 8, 536 41, 536 174, 548114, 548178, G01N 3358, C07F 938, C07D41704
Patent
active
050988280
DESCRIPTION:
BRIEF SUMMARY
The invention concerns D-luciferin derivatives, their application and processes for the detection of ligands marked with an enzyme in the determination of substances, in particular biochemically active substances.
The determination of substances which play a role in biological processes, are biologically active, occur in biological processes, etc., constitutes a significant problem.
Thus, for example, in the field of immunology the determination of antigens, haptens or antibodies is extremely important.
The development of the first radio immuno assays suitable for use in the determination of antigens or antibodies of this type was originated more than 20 years ago. Since then, various immunological methods were developed, in which marked reagents (tracers) are used for the determination of antigens or antibodies.
Enzyme immuno assays, in which enzymes serve as markers, are of particular importance. Enzyme immuno assays of this type are being employed increasingly compared to radio-immuno assays, as there is no radioactive waste, the reagents may be stored longer and the tracer is not destroyed.
Enzyme immuno assays are divided into homogeneous (homogeneous enzyme assays; EMIT) and heterogeneous (heterogeneous enzyme immuno assays; ELISA).
A review of the most important methods of such enzyme immuno assays may be found in:
J. Clin. Chem. Clin. Biochem., Vol. 18, 1980, pages 197-208, "Enzyme immuno assays in clinical chemistry: present status and trends" by M Oellerich and
"Principles of Enzyme Immuno Assays" by M. Oellerich in "Methods of Enzymatic Analysis", Vol. 1, H. U. Bergmeyer, ed., p. 233-260, Verlag Chemie, Weinheim/Bergstrasse (1983).
A summary article concerning the possible applications of enzymes in immuno assay methods is found in:
Analyst, May 1984, Vol. 109, pages 533-547, "Use of Enzymes in Immuno Assay Techniques, a Review" by Christopher Blake and Barry J. Gould.
In the case of immuno assays of this type enzymes serve as markers. In the process, the enzymes combine with a ligand into a ligand-enzyme conjugate. The ligand may be an antibody, an antigen or a hapten. The ligand marked by an enzyme (for example an antibody marked with an enzyme) is then reacted with its biochemical "counter part" (for example an antigen), wherein the ligand is added to its biochemical counter part.
For the detection of ligand-enzyme conjugates, they are reacted with a substrate. The enzymes release a reaction product from the substrate, which may be detected analytically, for example by photometry, potentiometry, thermometry or by scintillation spectrometry.
In these enzyme immuno assays therefore the amplification effect by way of the substrate reaction of the enzymes per unit time is utilized. It is possible in this manner to determine quantitatively the amount of the reaction product formed and thus also the quantity of bound ligands marked by an enzyme (antibody, antigen, hapten).
The quality of an enzyme immuno assay thus depends among others on the amplification effect and the limit of detection relative to the reaction product.
Frequently, however, such enzyme immuno assays are not sensitive enough. This may be the result of the fact that the amplification effect is not sufficiently large in the substrate reaction or that the limit of detection for the reaction product is too high.
It has now been discovered surprisingly that luciferin derivatives may be used as substrates for the detection of ligands marked by enzymes. The enzyme releases the luciferin compound from the derivatives, which in turn may be again reacted with an enzyme, i.e. the luciferase of the fire-fly Photinus pyralis with the emittance of light. From the amount of light emitted in the process the amount of the ligand-enzyme conjugate may be determined quantitatively, from which in turn the quantity of the substance to be determined may be calculated.
It is therefore the object of the present invention to provide compounds used in the process.
This object is attained by the formation of D-luciferin derivatives of the following general
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Geiger Reinhard
Miska Werner
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