1. Field of the Invention
This invention relates to nonradioisotopically-labeled proteins and polypeptides useful as labeled conjugates in specific binding assays for determining such proteins and polypeptides, or specific binding partners thereof, in liquid media such as body fluids, particularly serum. In particular, the present invention relates to flavin adenine dinucleotide-labeled proteins (e.g., immunoglobulins) and polypeptides useful in nonradioisotopic immunoassays.
2. Description of the Prior Art
Specific binding assay methods have undergone a technological evolution from the original competitive binding radioimmunoassay (RIA) in which a radioisotope-labeled antigen is made to compete with antigen from a test sample for binding to specific antibody. In the RIA technique, sample antigen is quantitated by measuring the proportion of radioactivity which becomes associated with the antibody by binding of the radiolabeled antigen (the bound-species of the labeled antigen) to the radioactivity that remains unassociated from antibody (the free-species) and then comparing that proportion to a standard curve. A comprehensive review of the RIA technique is provided by Skelly et al, Clin. Chem. 19: 146(1973). While by definition RIA is based on the binding of specific antibody with an antigen or hapten, radiolabeled binding assays have been developed based on other specific binding interactions, such as between hormones and their binding proteins. All radiolabeled specific binding assays are by necessity heterogeneous, that is, the bound- and free-species of the labeled conjugate must be physically separated and the label (i.e., radioactivity) measured in one of the separated species.
From the radiolabeled binding assays have evolved nonradioisotopic binding assays employing labeling substances such as enzymes as described in U.S. Pat. Nos. 3,654,090 and 3,817,837. Recently, further improved nonradioisotopic binding assays have been developed as described in German Offenlegungschriften Nos. 2,618,419 and 2,618,511, based on U.S. Ser. Nos. 667,982 and 667,996, filed on Mar. 18, 1976 and assigned to the present assignee, employing particularly unique labeling substances, including coenzymes, cyclic reactants, cleavable enzyme substrates, and chemiluminescent molecules. Flavin adenin dinucleotide (FAD) is mentioned as being useful as a coenzyme label since FAD functions as a coenzyme in reactions which can be used to monitor specific binding reactions. The majority of the recently developed nonradioisotopic specific binding assays can be performed in a homogeneous format, that is, without separating the bound- and free-species of the labeled conjugate, due to the fact that the label expresses a different activity in the bound-species compared to the free-species.
In addition to its use as a coenzyme label, FAD has also been found to be useful as a prosthetic group label as described in U.S. Patent Applications Ser. Nos. 917,961 and 45,423, filed June 22, 1978 and June 4, 1979, respectively, both assigned to the present assignee. Various FAD-labeled ligand conjugates are described in the aforesaid applications. It is highly desirable to prepare FAD-labeled conjugates for proteins and polypeptides of clinical significance so as to enable the homogeneous, nonradioisotopic specific binding assay determination of such proteins and polypeptides, and their binding partners. Preparation of such labeled protein and polypeptide conjugates is complicated by the complex structure and heterogeneity of proteins and polypeptides; the molecular size, fragility, and susceptibility to denaturation of such ligands; the need to maintain water solubility in the labeled conjugates; the need to maintain proper configuration in the conjugated protein or polypeptide; and the expected instability of chemically modified proteins and polypeptides over long storage periods.
Flavin adenine dinucleotide (FAD) has the following chemical structure [The Merck Index, 9th ed. (1976) p. 532]: ##STR2## which hereinafter is abbreviated as: ##STR3## wherein Riboflavin--Phos.sub.2) Ribose represents the riboflavin-pyrophosphate-ribose residue in FAD.
The numerous conventional methods for modifying proteins and polypeptides and for coupling such ligands to solid supports and other materials are described in the following: for general reviews see Methods in Enzymology, vol. XLIV "Immobilized Enzymes", ed. Mosbach, Academic Press (New York 1976), Affinity Chromatography, Lowe and Dean, John Wiley and Sons (New York 1974), and Clin. Chem. 22:726(1976); and for specific references see Science 144:1344(1967) [the carbodiimide reaction], Erlanger et al, Methods in Immunology and Immunochemistry, ed. Williams and Chase, Academic Press (New York 1967), p. 149 [the mixed anhydride reaction], Peptides and Amino Acids, Kopple, W. A. Benjamin, Inc. (New York 1966) [the acid azide and active ester reactions], and Proc. Nat. Acad. Sci. USA 66:651(1970) [the bis-imidate reaction].
USPTO Backgrounds
USPTO Backgrounds