Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-01-12
2002-01-01
Le, Long V. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S002000, C435S004000, C435S007200, C435S007230, C435S007240, C435S007720, C435S007900, C435S040510, C435S040520, C435S173300, C436S546000, C436S063000, C436S064000, C436S800000, C436S805000, C436S808000, C436S813000
Reexamination Certificate
active
06335173
ABSTRACT:
The present invention relates to methods of using tyramide coated live cells for flow cytometry, preferably using catalyzed reporter deposition and amplification staining.
BACKGROUND OF THE INVENTION
The following information is presented solely to assist the understanding of the reader, and none of the information is admitted to describe or constitute prior art to the claims of the present invention.
Flow cytometry is a sensitive and quantitative method for measuring the fluorescence or light scatter of particles or cells. This method has been widely used to study cellular physiology, especially as it relates to the immune system and control of the cell cycle. Nolan et al, “The Emergence of Flow Cytometry for Sensitive, Real-time Measurements of Molecular Interactions”,
Nature Biotechnology,
Vol. 16, (1998), which is incorporated by reference herein in its entirety including any drawings, describe recent flow cytometry developments for fields as diverse as ligand binding and enzyme kinetics, drug screening, diagnostics and detection of soluble agents, and DNA sequence detection or analysis. They describe developments such as advances in automated sample handling, molecular approaches for incorporating affinity tags or fluorescent probes into proteins and the availability of microsphere reagents that enable multiplexing.
Flow cytometric analysis of cell surface molecules is a technology used in both medical diagnostic laboratories and biomedical research laboratories. In clinical practice flow cytometry is used for samples derived from patients infected with human immunodeficiency virus type 1, patients with leukemias and lymphomas, and patients with primary immunodeficiences.
Lollini et al., “Flow Cytometry on Intracellular Antigens After Tyramide Signal Amplification”, Immunological Blackboard:
Bulletin of the Gruppo Di Cooperazione in Immunologia,
Vol. 1, Number 2 (1998), which is incorporated herein by reference in its totality, including any drawings, describes tyramide signal amplification (TSA) for detection of intracellular antigens by flow cytometry and indicates that TSA is not superior to conventional techniques for detecting surface antigens on live cells. Lollini et al. states on page 5, “(t)he main problem appeared to be a high level of spontaneous activation and non-specific binding of the fluorescent substrate to live cell membranes”.
Various methods have been described for assaying biological samples with amplified reporter systems. Bobrow et al., U.S. Pat. Nos. 5,196,306, 5,583,001 and 5,731,158, which are all herein incorporated by reference in their totality including any drawings, describe methods for detecting or quantitating analytes using an analyte dependent enzyme activation system as well as catalyzed reporter deposition methods. Specifically, Bobrow et al. describe calorimetric and fluorometric solid phase enzyme immunoassays which are enhanced by amplification of the reporter molecules.
Chao et al., “Immunofluorescence Signal Amplification By The Enzyme-Catalyzed Deposition Of A Fluorescent Reporter Substrate (CARD)”,
Cytometry
23:48-53 (1996), describe a CARD system that uses horseradish peroxidase substrate Cy3.29-tyramide to deposit fluorogen molecules onto fixed tissues and cells as well as proteins bound to nitrocellulose membranes, with up to a 15 fold increase over standard indirect immunofluorescence methods.
Malisius et al., “Constant Detection of CD2, CD3, CD4, And CD5 In Fixed and Paraffin-Embedded Tissue Using The Peroxidase-Mediated Deposition Of Biotin-Tyramide”,
The Journal of Histochemistry and Cytochemistry,
Vol. 45(12):1665-1672, (1997), describe a method for enhancing detection of leukocyte antigens in formalin-fixed tissue samples.
SUMMARY OF THE INVENTION
This invention features methods for enhancing the detection and/or quantitation of an analyte of interest on a live cell in flow cytometric analysis. The invention provides a method for tyramide coating live cells for flow cytometry, wherein live cells are preferably exposed to a catalyzed reporter deposition system which results in specific tyramide coating of cells which contain or express an analyte of interest. The invention, however, features flow cytometric detection of tyramide coated live cells regardless of how the cell is coated with tyramide and encompasses the use of any such cells which can be prepared using various techniques known by those skilled in the art. Thus, the present invention allows for increased detection of an analyte of interest in a sample of live cells by flow cytometric methods. Furthermore, the present invention allows for detection of analytes which are present in low copy number in a live cell sample.
The term “low copy number” means that the analyte of interest is present on or in the cell but is not represented in an easily detectable amount. An aspect of the present invention is that rare, hard to detect analytes may be readily detected by the increase in the staining of the cell caused by the amplification of the labeling molecule. Hence, a low copy number analyte, such as the Fas ligand, would not have to be over-expressed in order to be detected by flow cytometry. The low copy number is preferably less than 20,000 molecules/cellular surface, more preferably less than 10,000 molecules/cellular surface and most preferably less than 5000 molecules/cellular surface.
In a first aspect, the present invention features a method of flow cytometry which involves coating live cells with tyramide and analyzing the cells with a flow cytometric device.
By “tyramide coating” or “coating live cells with tyramide” is meant to relate to any process which results in cell surfaces being coated with tyramide, such as the enzyme dependent deposition of tyramide on the surface of cells containing the analyte of interest. In the presence of oxygen radicals, short lived tyramide radicals are formed which form covalent linkages with aromatic molecules such as certain amino acids (tyrosine and tryptophan for example) found in most proteins. Since cell surfaces have an abundance of proteins the tyramide radicals bind to the surface of the cell to which it is in closest proximity. The generation of oxygen radicals, by the catalytic activity of the enzymatic portion of the second binding partner and the appropriate substrate, over a period of time, produces tyramide radicals that coat the surface of the cell. The live cells preferably are not fixed before contacting with the binding partner specific for the analyte of interest, and have not been treated with a conventional fixation procedure such as methanol fixation. See, Lollini et al., supra, page 2. However the cells may be fixed by procedures known in the art after contacting with the binding partner which is specific for the analyte of interest.
What is meant by “live cells” is that the cells to be assayed for an analyte of interest are viable when contacted with the binding partner for the analyte of interest. In certain embodiments the cells are viable during flow cytometric analysis. The cells are preferably viable during and after flow cytometric analysis to allow for selection and/or sorting of cells which have or do not have the analyte of interest, if desired, and used for therapeutic and/or research methods. It is known by those of skill in the art that the cells may also be manipulated to remain in a certain stage of the cell cycle during analysis. It is also understood that the cells may be fixed for analysis after contact with the binding partner specific for the analyte of interest.
By “viable” is meant that the cells are capable of being grown, cultured, or further propagated at the time at which contact with the binding partner for the analyte of interest occurs. Essentially, viable cells are alive and capable of mitotic or meiotic division and further growth after contact with the binding partner specific for the analyte of interest. In a preferred embodiment of the invention, the cells are capable of being grown, cultured, or further propagated after being analyzed by flow cytometry.
B
Foley & Lardner
Gabel Gailene R.
Le Long V.
Verve, Ltd. c/o James Bell
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