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United States Patent  [li] Patent Number: 5,464,741
Hendrix  Date of Patent: Nov. 7,1995
 PALLADIUM (II) OCTAETHYLPORPHINE
ALPHA-ISOTHIOCYANATE AS A
PHOSPHORESCENT LABEL FOR
 Inventor: John L. Hendrix, Palm Harbor, Fla.
 Assignee: Henwell, Inc., Palm Harbor, Ha.
 Appl. No.: 134,133
 Filed: Oct. 8,1993
 Int. CI.6 C12Q 1/70
 U.S. CI 435/5; 435/6; 435/7.1;
435/7.2; 435/7.3; 435/7.4; 435/7.5; 435/7.6; 435/968; 436/546; 436/548; 436/816; 436/817;
 Field of Search 435/5, 6, 7, 7.6,
435/968; 540/145; 424/1.1, 85.8, 88, 89, 1.49; 436/546, 548, 816-818
 References Cited
U.S. PATENT DOCUMENTS
4,058,732 11/1977 Wieder 250/461 B
4,234,455 11/1980 Homeier 252/430
4,578,491 3/1986 Amundsen 556/137
4,614,723 9/1986 Schmidt et al 436/536
4,622,174 11/1986 McKoy 252/582
4,659,676 1/1987 Rhyne 436/56
4,751,068 6/1988 Bickai 423/437
4,783,529 11/1988 Lavallee et al 540/145
4,863,875 9/1989 Bailey 436/518
4,885,114 12/1989 Gordon 252/589
4,898,985 2/1990 Ito et al 568/344
4,935,166 6/1990 Lee 252/582
5,082,642 1/1992 Bickar 423/402
5,102,213 4/1992 Lee 359/890
5,102,625 4/1992 Milo 422/82.07
5,120,882 7/1992 Ellis, Jr. 568/910
5,120,886 7/1992 Lyons 568/909.8
5,155,149 10/1992 Atwater 524/88
5,156,840 10/1992 Goers et al 424/85.91
5,158,922 10/1992 Hinney et al 502/175
5,162,231 11/1992 Cole et al 436/64
5,268,371 12/1993 Mauclaire et al 514/185
FOREIGN PATENT DOCUMENTS
2171656 12/1988 Japan 436/546
2086042 5/1982 United Kingdom 436/545
8002076 10/1980 WIPO .
Biological Abstract 88(7):74113, Savitskii et al, "Phosphorescence immunoassay: Metalloporphyrins as an Alternative of rare earth fluorescent labels", abstract published 01 Oct. 1989.
Tietz (ed), Textbook of Clinical Chemistry, (W. B. Sanders Company 1986) pp. 64 and 1590.
Primary Examiner—Carol A. Spiegel
Attorney, Agent, or Firm—Joseph C. Mason, Jr.; Ronald E.
Smith; Louise A. Foutch
A phosphorescence assay system. The phosphorescent label for immunoassays is palladium (II) octaethylporphine alphaisothiocyanate. The labeling agent has a Stokes shift of not less than 150 nanometers. Method for preparing the palladium (II) phosphorescent label is also shown.
7 Claims, 1 Drawing Sheet
PALLADIUM (II) OCTAETHYLPORPHINE ALPHA-ISOTfflOCYANATE AS A PHOSPHORESCENT LABEL FOR IMMUNOASSAYS
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a novel phosphorescent 1Q compound and to a method of use of such phosphorescent compound as a label or marker for binding with immunoglobulins or chemical analytes. It specifically relates to palladium (II) octaethylporphine alpha-isofhiocyanate as a novel phosphorescent compound.
2. Description of the related art
Generally, the assays of the art have included radioimmunoassays for insulin and other hormones. The radioisotopes attach to hormones which have been chemically modified and then mixed with non-radioisotope labeled 20 hormone. This admixed solution is contacted with a limited amount of antibody which is specific for the hormone. After separating the bound antibody from the unbound a determination can be made as to the concentration of the hormone
in the patient's blood by comparing the radioactivity level of 25 the patient's hormone to the radioactivities of known concentrations of hormones.
In other areas of the art, biological assays have provided a valuable tool in the evaluation of the effect of pharmacologically active compounds including biologically active 30 proteins. Procedures developed for the bioassay of these types are usually biphasic. One procedure incubates the substrate cell population with the substance to be tested. The art has developed widely divergent procedures to carry out the quantitative analysis of the cellular response to the 35 sample compound.
To obtain the quantitative analysis the art includes immunomediated assays which are quantitative only for the binding of the molecule to the cell. Another type of assay requires cell staining and visual or microscopic observation of a change in cellular morphology. Such assays are dependent on a subjective evaluation of the results.
The use of radioactive metabolites to determine viability of an antibody is less subjective than the colorimetric 45 systems but the processing of numerous samples is time consuming, labor intensive and expensive. Further, counting of assay cell with the aid of an electronic particle counter to determine cell growth requires large multiples of each sample for accurate results and does not truly reflect the 5Q viability of the assay cells.
Even more recently, the art has developed colorimetric analysis of cell growth either by direct staining of the cell monolayer or by cellular dye uptake. For example, dye molecules have been used as labels for proteins (e.g. anti- 55 bodies) to make reagents which have been associated with fluorescence readings. However, it is known in the art that labeling an antibody with a dye molecule and measuring the absorbance value does not provide a level of sensitivity sufficient to enable an accurate quantitative measurement of go ligands in immunoassay systems.
Thus, the present state of the art indicates that although radioimmunoassays have been the cornerstone of modern immunoassay procedures, non-radioisotopic methods such as enzyme-linked immunosorbent assays and fluorescent 65 immunoassays have become predominant in clinical laboratories due to aforementioned shortcomings of the radio
immunoassays and due to the licensing, special skills, expensive equipment, shipping and disposal problems associated with radioisotopes.
However, the present state of the art still has objections and has lack of sufficient sensitivity for immunoassay procedures to achieve full acceptability in the art. The nonradioisotope immunoassays generally lack the ability to detect molecular concentrations to the extent that radioimmunoassays can measure in large part due to the light scatter and other types of interference inherent in the testing procedure. Enzyme-linked immunosorbent assays require bulky, labile enzymes, are susceptible to inhibition and denaturation, and an additional incubation step is needed with a substrate to monitor the enzyme's activity.
It is recognized that fluorescence immunoassays achieve greater levels of sensitivity than enzyme-linked immunosorbent assays due to fluorescence molecules, i.e., the fluorophore labels, absorbing energy at one wavelength, i.e., excitation, and radiating energy at another wavelength, i.e., emission. This difference in wavelengths, known as the Stokes shift, can be used advantageously to gain sensitivity by having the excitation light project at 90° to the emission light being detected by a photomultiplier tube or other known detection devices. It is also recognized that each of the materials used as a fluorescent label has its own characteristics of required wavelength of excitation and resulting wavelength of fluorescent emission, i.e., has its own Stokes shift. If the label has a low Stokes shift, it is difficult and expensive to design light sensors which will respond to the wavelength of fluorescence and be relatively insensitive to the wavelength of the excitation light. Such sensors, as mentioned, use expensive detraction gratings inserted between the sample containing the fluorescent labels and the optical sensor. These are placed so that they are orthogonal to the direction of a beam of light at the excitation wavelength. There is still some background scatter and noise to contend with due to soluble molecules, small colloidal particles, or the presence of solid-phase material.
Thus, there is a need for a better luminescent compound for use in immunoassays, but the prior art considered as a whole, neither teaches nor suggests how the prior art compounds, if any, might be improved.
SUMMARY OF THE INVENTION
Accordingly, it is an object of this invention to provide a luminescent compound which has high selectivity as used in immunoassays.
It is a further object of this invention to provide an improved immunoassay system utilizing a novel luminescent compound having a relatively long Stokes shift.
It is a still further object of this invention to provide a immunoassay method utilizing a novel luminescent compound as a label or marker for binding with immunoglobulins or chemical analytes.
It is another object of the invention to provide a method for preparation of such novel luminescent compound.
It is therefore an object of the present invention to provide a sensitive phosphorescent label for immunoassays.
It is still another object of the present invention to provide a phosphorescent antibody comprising an antibody specific to an antigen to be detected, and conjugated to said antibody, a novel phosphorescent compound as a label.
Additional objects, advantages and novel features of the invention will be set forth in the description which follows,
Another and particular embodiment of the invention is a method of preparing a phosphorescent compound which comprises the following protocol in sequence:
(a) preparing and recovering as a first sub-product of the formula
an example of which is palladium (II) octaethylporphine alpha-isothiocyanate.
In accordance with another aspect of the invention a 30 phosphorescent labeled reagent is provided comprising antibodies, antigens, hormones, virus particles, haptens, bacterial components, drugs, monoclonal antibodies, anti-antibodies, immunoglobulins or proteins.
Another and further embodiment of this invention is a 35 method for the preparation of a reagent useful for the detection of a ligand in a biological fluid, said reagent comprising an antibody to said ligand bound to the compound palladium (II) octaethylporphine alpha-isothiocyanate, comprising adding palladium (II) octaethylporphine 40 alpha-isothiocyanate to a serum solution of immunoglobulin specific for the analyte of interest and incubating the admixture under conditions sufficient to enable said antibody to bind to the palladium (II) octaemylporphine alpha-isothiocyanate thereby forming said reagent. 45
A still further aspect of this invention is a phosphorescent antibody comprising an antibody specific to an antigen to be detected, and conjugated to said antibody, a phosphorescent compound of the formula