Chemistry: analytical and immunological testing – Involving producing or treating antigen or hapten – Producing labeled antigens
Patent
1986-04-17
1989-08-15
Warden, Robert J.
Chemistry: analytical and immunological testing
Involving producing or treating antigen or hapten
Producing labeled antigens
436500, 436518, 436537, 436547, 436805, 436808, 530390, 530391, 530405, 530802, 534 16, 546245, 546249, 546321, G01N 33533
Patent
active
048574759
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
This invention relates to the assaying of organic substances found in human and animal body fluids and the like. More particularly it relates to a fluorometric immunoassay and substances for use as labelled reagents in such an assay.
BACKGROUND ART
There are now a number of applications in which it is required to label antibodies (or other organic substances, for example macromolecules such as proteins, and haptens) with metal ions, either radioactive metal ions for use in radioimmunoassay and other nuclear medicine studies or lanthanide metal ions for fluorometric immunoassay and other studies involving fluorescence. For these purposes the organic substances are conventionally labelled with metal ions through the agency of chelates. Hitherto the chelates have been modified with bridging reagents which convert them into bifunctional reagents so that they retain their chelation function whilst being readily attachable by covalent bonding to the molecule to be labelled.
One class of chelating reagents which has been used for this purpose is the class of .beta.-diketones, such as trifluoroacetylacetone and benzoyl and .alpha.- and .beta.-naphthoyl trifluoroacetone, and chelates of lanthanide metal ions with such reagents have been coupled to antibodies using EDTA-analogues (see European patent application No. 0.064,484). It has also been proposed in GB Patent Specification No. 1,560,402 to modify a .beta.-diketone ligand such as thenoyl-trifluoroacetylacetone by the attachment of an aminomethyl substituent and to use one molecule of the modified ligand and two molecules of unmodified ligand to form a lanthanide metal ion complex which readily couples to an antibody after conversion of the amino group to an isothiocyanate group.
However, bifunctional-chelating agents are difficult to synthesize and many of the reactions by which covalent bonding of the ligand-metal ion complex to the molecule to be labelled is achieved have only a low yield of the desired labelled product and may also confer undesirable properties on the labelled molecule.
DISCLOSURE OF INVENTION
The present invention seeks to provide an alternative method of achieving satisfactory lining of a metal ion complex to an organic molecule and thereby conferring specialized functionality to the labelled molecule.
According to the present invention a method of potentiating an NH.sub.2 -bearing macromolecule for labelling with a metal ion comprises reacting the NH.sub.2 -bearing macromolecule with a .beta.-diketone in the presence of an aldehyde to form a cyclic condensation product.
The invention also provides a method of potentiating an organic macromolecule for labelling with a metal ion, comprising reacting an NH.sub.2 -bearing compound having an active group capable of reaction with the organic macromole with a .beta.-diketone in the presence of an aldehyde to form a cyclic condensation product and linking the resulting product to the organic macromolecule via the active group.
Further it provides a substance capable of being labelled with a metal ion for use in a fluorometric assay technique, comprising a cyclic condensation product of an NH.sub.2 -bearing macromolecule, a .beta.-diketone and an aldehyde.
It also provides a labelled reagent for use in fluorometric assay comprising a cyclic condensation product of an aldehyde, a .beta.-diketone and an organic substance having an NH.sub.2 group, the condensation product being chelated to a lanthanide metal ion to form a complex capable of being estimated fluorometrically, and a process for the fluorometric assay of an organic substance in which such a labelled reagent is used.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention is illustrated by the accompanying drawings which are graphs relating to assays of alphafaeto-protein (selected as using a typical NH.sub.2 -containing antibody), as described in the Examples below.
FIG. 1 is a calibration curve using rabbit and anti-alpha-faeto-protein (AFP) directly labelled with trifluoroacetylacetone.
FIG. 2 is a calibration curve
REFERENCES:
patent: 3956341 (1976-05-01), Loev
patent: 4374120 (1983-02-01), Soini et al.
Jerry March, Advanced Organic Chemistry, 2nd Ed., pp. 816 & 722 (1980), McGraw-Hill, N.Y.
Chemical Abstracts, vol. 100, No. 188, 162q (1984), Ilkka et al-Europium as a Label in Time-Resolved Immunofluorometric Assays.
Clinical Chemistry-vol. 25, No. 3, pp. 353-361, Fluoroimmunoassay: Present Status and Key Problems-Soini et al.
Benson Robert
Ekins Roger Philip
Warden Robert J.
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