Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving oxidoreductase
Reexamination Certificate
2001-01-26
2003-08-05
Gitomer, Ralph (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving oxidoreductase
C435S025000, C435S188000
Reexamination Certificate
active
06602679
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to chemiluminescent compounds and methods of use therefor. More particularly, the present invention concerns novel stabilized chemiluminescent formulations containing all the necessary components for chemiluminescence light generation. Even more particularly, the present invention relates to a single or one-part reagent for detecting and quantifying various biological molecules through chemiluminescence as well as detecting DNA fragments in DNA sequencing applications and methods of use therefore.
2. Description of Related Art
Radioimmunoassay has provided the most practical approach to achieve detection of antigens and antibodies in picomolar concentrations and is, thereforee, widely used in both clinical and pharmacological laboratories. However, the very success and widespread use of radioimmunoassays has raised several problems which include: (1) shelf-life and stability of radiolabeled compounds, (2) high cost of radioactive waste disposal, and (3) health hazards as a result of exposure to the use of not only radioactive materials but to the solvent necessary for liquid-scintillation counting, as well.
Chemiluminescence, i.e. the production of light by chemical reaction, and bioluminescence, i.e. the light produced by some living organisms, have been tested as potential replacements for radioactive labels, not only in competitive and sandwich-type immunoassays, but also, DNA sequencing and other related research. Chemiluminescence provides a major advantage over radioactive labeling because it generates cold light i.e. its generated light is not caused by vibrations of atoms and/or molecules involved in the reaction but by direct transformation of chemicals into electronic energy. Thus, research on the chemiluminescence of organic compounds is an on-going area of major emphasis. Parenthetically, chemiluminescence is also advantageous in detecting and measuring trace elements and pollutants for environmental control. The best known chemiluminescent reactions are those which employ either stabilized enzmye triggerable 1,2-dioxetanes, acridanes, acridinium esters, luminol, isoluminol and derivatives thereof or lucigenin, as the chemical agent, reactant or substrate.
Enzymatic triggerable 1,2-dioxetanes such as those described by A. P. Schaap, R. S. Handley and B. P. Giri.
Tetrahedron Lett.,
935 (1987); A. P. Schaap, T. S. Chen, R. S. Handley, R. DeSilva, and B. P. Giri,
Tetrahedron Lett.,
1159 (1987) as well as in U.S. Pat. No. 5,707,550 and PCT/US99/20590 are superior in immunoassays and other related applications compared to presently known peroxidase substrates such as luminol and the like, because these 1,2-dioxetane substrates are highly sensitive and detect an enzyme concentration up to 10
−21
M in solution, as well as on a membrane. Futher, stabilized 1,2-dioxetane substrates provide high signal, low background, wide dynamic range, rapid results and excellent reproducibility.
On the other hand, peroxidase is widely distributed in higher plants and in especially high concentrations in fig sap and horseradish. It is also found in some animal tissues and in microorganisms. Because of its wide availability, horseradish peroxidase(HRP) is widely used in labeling haptens, antibodies, protein A/G, avidin, streptavidin and DNA for enzyme immunoassays, immunocytochemistry, immunoblot and DNA detection.
In an assay procedure, HRP is used in lieu of the enzymatic triggerable dioxetane because detection of enzyme activity takes advantage of the reactive cooperation between the enzyme and a highly sensitive chemiluminescent agent. Peroxidase-based chemiluminescent assays, while demonstrating improved detection sensitivity, suffer from the lack of reproducibility such that the obtained data is not always reliable.
According to the prior art, two problems arise in the detection of peroxidase activity. First, a HRP assay is employed as either a two component reaction system or a two step reaction process. See, inter alia U.S. Pat. Nos. 4,598,044; 5,171,668; 5,206,149; 5,552,298; 5,601,977; 5,593,845; 5,670,644; 5,723,295. The two component system provides two reagents, one reagent containing an organic compound for light production such as luminol and the second an oxidizing agent such as hydrogen peroxide. When the two are mixed together, peroxidase enzyme detection occurs. However, the resulting solution, after mixing, is not stable for a long period of time and due to the instability of the mixed reagent the background is very high. Indeed, the chemiluminescence of most of the prior art systems is like a flash or a glow for a short period of time. Further, in a two-step chemiluminescence reaction process, the first step, i.e. the peroxidase catalyzed reaction, is done at a lower pH than the second step of the reaction. This causes accumulation of an intermediate compound which is subsequently induced to produce a burst of light by raising the pH of the solution. Thus, the multistep nature of peroxidase-catalyzed luminescent reaction, with rate limiting steps and competing side reactions, creates several problems in efficiency and reproducibility when used in the immunological field. The second problem is the lost activity of the peroxidase enzyme in buffers. The stock solutions of peroxidase below 10 ng/ml are not stable, even in the presence of bovine serum albumin. For example, at 10 pg/ml, activity is lost within one minute, and even at 1 ng/ml, the solution is stable for not more than two minutes. See K. Pugnet, A. M. Michelson, and S. Avrameas, Anal. Biochem. 79 477-456 (1977). The same results are observed when peroxidase is assayed using luminol-hydrogen peroxidase as a substrate in the presence of N-methylphenothiazine and phenolindophenol as an enhancer as shown in
FIG. 2
of U.S. Pat. No. 5,171,668. Furthermore, this reference shows the non-linear relationship between the concentration of a peroxidase-labeled AFP antibody and light emission.
In spite of its known deficiencies horseradish peroxidase, as noted above, is still widely used for assays because it is widely available and inexpensive to use. Horseradish peroxidase catalyzes the luminescent oxidation of a wide range of substrates including cyclic hydrazide, phenol derivatives, acridane derivatives and components of bioluminescent systems. Other suitable substrates, also, include: (a) luminol and related compounds, as taught by [L. Ewetz, and A. Thore.
Anal. Biochem.,
71, 564 (1976), A. D. Pronovost and A. Baumgarten,
Experientia,
38, 304 (1982), H. Arakawa, M. Maeda and A. Tsuji,
Anal. Biochem.,
97, 248 (1979), L. S. Hersh, W. P. Vann, and S. A. Wilhelm,
Methods in Enzymology,
73, 608 (1981)]; (b) pyrogallol, and purpurogallin as taught by [B. Velan and M. Halmann,
Immunochemistry,
15, 331 (1978), M. Halmann, B. Velan, T. Sery and H. Schupper,
Photochem. Photobiochem.,
30,165 (1979), G. Ahnstrom and R. Nilsson, Acta.
Chim. Scand.,
19, 313 (1965), (c) acridanecarboxylic acid derivatives as taught by [H. Akhavan-Tafti, R. DeSilva, Z. Arghavani, R. A. Eickholt, R. S. Handley, B. A. Schoenfelner, K. Sugioka, Y. Sugioka and A. P. Schaap,
J. Org. Chem.,
63, 930 (1998)]; and (d) luciferins isolated from
Pholas dactlus,
and the firefly
Photinus pyralis
or Cypridina as disclosed by [K. Puget, A. M. Michelson and S. Avrameas,
Anal. Biochem.,
79, 447 (1977), D. Slawikska, J. Siwinski, W. Pukacki and K. Polewski, in “
Analytical Application of Bioluminescence and Chemiluminescence”
(E. Schram and P. Stanley, eds.), p. 239,
State Publishing and Printing,
Westlake Village, Calif., 1979. T. Kobayashi, K. Saga, S. Shimizu and T. Goto,
Agric. Biol. Chem.,
46, 1403 (1981)]. These light producing reactions differ widely in their detection limits, specificity, reagent availability and magnitude and kinetics of light emission. This, of course, restricts their applicability.
As noted above, peroxidase catalyzed luminescence suffers from two problems, namely, (1) peroxidase enzyme instabi
Gitomer Ralph
Plunkett & Cooney, P.C.
Weintraub, Esq. Arnold S.
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