Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-12-15
2004-12-07
Le, Long V. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S004000, C435S007200, C435S007210, C435S007800, C435S007900, C435S030000, C435S007230, C436S501000, C436S546000, C436S063000, C436S064000
Reexamination Certificate
active
06828109
ABSTRACT:
The present invention relates in part to methods of using tyramide in order to enhance the detection of analytes by flow cytometry, preferably using catalyzed reporter deposition and amplification staining. In preferred embodiments, the analytes detected according to the instant methods are cellular antigens, which may be intracellular or expressed on the surface of cells. The invention also relates in part to methods and compositions for use in amplification staining of analytes.
BACKGROUND OF THE INVENTION
The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to 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 cellular antigens is a technology used in both medical diagnostic laboratories and biomedical research laboratories. In clinical practice flow cytometry is used, e.g., for samples derived from patients infected with human immunodeficiency virus type 1, patients with leukemias and lymphomas, and patients with primary immunodeficiences.
Various methods have been described for assaying biological samples with amplified reporter systems. Bobrow et al., U.S. Pat. No. 5,196,306, U.S. Pat. No. 5,583,001 and U.S. Pat. No. 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 colorimetric 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.
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 entirety, including any drawings, describes tyramide signal amplification (TSA) for detection of intracellular antigens by flow cytometry. Lollini et al. indicates that TSA is not superior to conventional techniques for detecting surface antigens on live cells, stating for example 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.”
Karkmann et al., “Enzymatic Signal Amplification For Sensitive Detection Of Intracellular Antigens By Flow Cytometry,”
J. Immunol. Meth.
230:113-120 (1999), which is incorporated herein by reference in its entirety, including any drawings, describes TSA for detection of intracellular cytokines by flow cytometry. The Karkmann et al. reference states that direct detection of fluorochrome-labeled tyramide is problematic, “probably due to hydrophobic interactions with the cell membrane” Thus, the Karkmann et al. reference uses a complicated indirect staining method, in which antibody selective for an antigen of interest, coupled to peroxidase, catalyzes the deposition of biotin-tyramide, which is detected by fluorescently labeled streptavidin.
Despite progress towards catalyzed reporter deposition methods for use in detecting analytes, there remains a great need in the art for materials and methods that provide enhanced detection of analytes by enzymatic signal amplification, particularly for use in flow cytometry.
SUMMARY OF THE INVENTION
The instant invention features materials and methods for enhancing the detection and/or quantitation of an analyte of interest by flow cytometric and immunostaining analysis. In preferred embodiments, an analyte is a cellular antigen, which can be either intracellular or expressed on the surface of cells.
The invention provides methods for catalyzed reporter deposition staining of intracellular antigens that are simple, and that provide signal amplification levels of 10-fold or more in comparison to standard flow cytometry methods. Moreover, the invention also provides a method for tyramide coating cells for flow cytometry, wherein 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. Additionally, the invention further provides materials and methods for improving catalyzed reporter deposition staining of antigens generally, including the staining of fixed sections, fixed and permeabilized cells, particles, and live cells.
Thus, in a first aspect, the present invention discloses materials and methods that provide an enhanced signal for detection of an intracellular analyte of interest in a cell sample by flow cytometric methods. The methods disclosed comprise the steps of fixing the cells in the cell sample, permeabilizing the cells, and catalyzing the deposition of tyramide in those cells that comprise the analyte of interest. The present invention allows for detection of analytes which are present in low copy number in the cell sample by increasing the signal from an analyte of interest from 5-fold to 50-fold or more in comparison to standard flow cytometric methods.
The terms “fix” and “fixing” as used herein with regard to cells, refers to various chemical and physical methods well known to the skilled artisan that render the contents of a cell insoluble. Preferred fixation methods, which generally rely on crosslinking and/or rapid dehydration, can include the use of one or more chemicals such as formaldehyde, paraformaldehyde, glutaraldehyde, acetic acid, methanol, ethanol, and acetone.
The term “permeabilize” as used herein with regard to cells, refers to various methods that allow an increased amount of one or more agents to pass into the interior of a cell. The skilled artisan will understand that a cell is ordinarily surrounded by a semipermeable plasma membrane. Permeabilizing agents can act to open the plasma membrane, allowing molecules to enter the cell and to reach a concentration within the cell that is greater than that which would ordinarily be attained. Permeabilizing can also allow molecules to enter a cell that would ordinarily not enter the cell, for example due to a large molecular size or because the molecule is highly charged. Preferred permeabilizing agents are detergents such as CHAPS, cholic acid, deoxycholic acid, digitonin, n-dodecyl-&bgr;-D-maltoside, glycodeoxyc
Bell, Jr. James R.
Cheu Changhwa J
Foley & Lardner LLP
Le Long V.
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