Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-05-25
2001-08-28
Mosher, Mary E. (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007100, C435S007200, C435S007210, C435S007800, C435S030000, C436S501000
Reexamination Certificate
active
06280961
ABSTRACT:
INTRODUCTION
The present invention relates to methods of using tyramide coated cells or particles for physical separation, preferably using magnetic separation, cellular panning, column separation and/or fluorescence activated cell sorting (FACS).
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.
Physical separation of cells or particles may be achieved using a variety of methods, including magnetic separation, cellular panning, column separation, and FACS. The use of magnetic separation is described in Stanciu et al.,
Journal of Immunological Methods,
189:107-115, 1996; Luxembourg et al.,
Nature Biotechnology,
16:281-285, 1998; Sun et al.,
Journal of Immunological Methods,
205:73-79, 1997; Partington et al.,
Journal of Immunological Methods,
223:195-205, 1999; Nielson et al.,
Journal of Immunological Methods,
200:107-112, 1997; and Assenmacher et al.,
The Journal of Immunology,
161:2825-2832, 1998. The use of cellular panning is described Wysocki and Sato,
P.N.A.S.,
75(6):2844-2848, 1978. Use of column separation is described in Davis et al.,
Journal of Immunological Methods,
175:247-257, 1994. FACS described in Melamed, Lindmo, and Mendelsohn
Flow Cytometry and Sorting
Wiley-Liss, New York, 1990.
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 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 or particles 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 one or more cells or particles, preferably live cells. The invention provides a method for tyramide coating cells or particles for physical separation, wherein cells or particles are preferably exposed to a catalyzed reporter deposition system which results in specific tyramide coating of cells or particles which contain or express an analyte of interest. The invention, however, features physical separation and detection of tyramide coated cells or particles regardless of how the cell or particle is coated with tyramide and encompasses the use of any such cells or particles 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 cells or particles by use of physical separation methods. Furthermore, the present invention allows for the isolations of cells or particles that express analytes which are present in low copy number in a cell or particle sample.
The term “low copy number” means that the analyte of interest is present on or in the cell or particle 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 labeling of the cell or particle 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 a cell or particle in order for the cell or particle to be isolated by physical separation. The low copy number is preferably less than 20,000 molecules/cellular or particle surface, more preferably less than 10,000 molecules/cellular or particle surface and most preferably less than 5000 molecules/cellular or particle surface.
In a first aspect, the present invention features a method of physical separation which involves coating cells or particles with tyramide and isolating the cells or particles with a physical separation device.
By “tyramide coating” or “coating cells or particles with tyramide” is meant to relate to any process which results in cell or particle surfaces being coated with tyramide, such as the enzyme dependent deposition of tyramide on the surface of cells or particles 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 (and some particle surfaces) have an abundance of proteins, the tyramide radicals bind to the surface of the cell or particle 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, in the presence of tyramide produces tyramide radicals that coat the surface of the cell. The cells or particles 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. However, the cells or particles 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 physical separation. The cells are preferably viable during and after physical separation 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 physical separation.
By “cells” is meant the smallest unit of living structure capable of either aided or un-aided existence, composed of a membrane-enclosed interior which may contain a nucleus or nucleoid, free compact DNA, and/or other organelles such as mitochondria, the golgi apparatus, centrioles, endoplasmic reticulum, vacuoles, microsomes, lysosomes, ribosomes and the like. The cells can be bacterial cells as well as eukaryotic cells such as plant cells, yeast or fungal cells or mammalian cells. In a preferred embodiment, the cells are mammalian cells. Examples of various cells available for ph
Foley Shanon
Foley & Lardner
Mosher Mary E.
Verve, Ltd.
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