Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-06-12
2003-02-25
Jones, W. Gary (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S007500, C435S007900, C536S024310, C536S025320
Reexamination Certificate
active
06524798
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to new methods for characterizing the genotype and phenotype of a cell in a sample, and finds application in medicine, genetics, histology, developmental biology and cell biology.
BACKGROUND OF THE INVENTION
Determination of cell phenotype by immunophenotyping or immunocytochemistry (ICC) and determination of cell genotype using hybridization-based methods (e.g., in situ hybridization assays, “ISH”) are important processes in the diagnosis and study of disease. Although both ISH and ICC have been widely used for analysis, they have only infrequently been used in combination (e.g., on the same cells or different aliquots of cells from a sample). In part this is because the methods now available for simultaneously determining phenotype by antibody staining and determining genotype by ISH are cumbersome, inefficient, and of limited accuracy.
The conditions required for ISH and ICC are generally incompatible or non-optimal for the practice of the other. Carrying out ISH analysis following ICC may be inefficient due to the blocking effects of the reporting molecules used in ICC (e.g., substrate precipitate or fluorochromes). Conversely, carrying out ISH prior to ICC is unsatisfactory, e.g., because the elevated temperature and denaturation steps used for ISH are not compatible with subsequent ICC. Thus, attempts to carry out the two methods in combination have been limited. Neft et al., 1997,
Acta Cytologica
41:1769-1773, carried out ISH after ICC staining for the intermediate filament protein (cytokeratin). In this study, centromeric repetitive sequences from chromosome 7 were used for the ISH DNA probes, but no data relating to hybridization efficiency and accuracy were provided. Schouten et al., 1995,
Brit. J. Haematology
91:162-166, described the use of ISH with RNA probes in combination with anti-CD30 immunocytochemistry. They reported low efficiency in identifying Y chromosome containing cells and poor results when using multiple different ISH probes. Pazouki et al. 1996,
Acta histochem
(
Jena
) 98:29-37 described using ICC with an anti-hemoglobin antibody and a second-antibody detection system followed by ISH using repetitive-sequence probes (from chromosome X and Y centromeric sequences) to identify fetal cells. However, the authors provided no information on the efficiency of the method or the number of cells correctly typed.
None of these references provided a method that is specific and accurate when used for immunostaining of many types of cell antigens (e.g., cytoplasmic, membrane-associated, etc.) and many types of ISH probes (e.g., centromeric probes and unique sequence probes).
The present invention provides new and surprisingly efficient methods for analysis of cell phenotype and genotype, which may be carried out on the same cells in a sample.
SUMMARY OF THE INVENTION
In one aspect, the invention provides a method of simultaneously determining a cell phenotype and genotype by:
(a) contacting the cell with an antibody bound to a ligand, wherein the antibody is specific for an antigen suspected of being associated with the cell;
(b) contacting the cell with polynucleotide probe under conditions that allow the probe to hybridize to a target sequence in a cellular nucleic acid, thereby forming a complex of the polynucleotide probe and cellular nucleic acid;
(c) contacting the cell with a detectably labeled anti-ligand under conditions where the detectably labeled anti-ligand binds to the ligand;
(d) detecting the presence of the labeled anti-ligand;
(e) detecting the presence of the complex of the polynucleotide probe and cellular nucleic acid; and,
(f) correlating the presence of the anti-ligand with the presence of the antigen in the cell thereby determining the cell phenotype, and correlating the presence of said complex of the polynucleotide probe and cellular nucleic acid with the presence of the target sequence in the cell, thereby determining the cell genotype. According to the invention, step (a) of the method precedes step (b), and step (b) precedes step (c).
In preferred embodiments, the cell is exposed to a fixative between step (a) and step (b).
In various embodiments of the method, the cell is a human cell, such as a human fetal cell; the antibody is monoclonal; the ligand is biotin; the anti-ligand is avidin or streptavidin, which may be labeled with a reporter molecule that is fluorescent, a radioisotope, or an enzyme; the antigen is hemoglobin; and/or the polynucleotide probe is DNA, such as a probe that specifically hybridizes to sequences of human chromosomes X, Y. 13, 18 or 21. In some embodiments of the invention, signal detection is accomplished by microscope analysis.
In certain embodiments, the disclosed method is used to detect or diagnose a chromosomal abnormality in a fetal cell, a malignant cell, or a cell suspected of being malignant.
In a related aspect, the invention provides a method of simultaneously determining a cell phenotype and genotype comprising:
(a) contacting the cell with an antibody specific for an antigen suspected of being associated with the cell;
(b) contacting the cell with polynucleotide probe under conditions that allow the probe to hybridize to a target sequence in a cellular nucleic acid, thereby forming a complex of the polynucleotide probe and cellular nucleic acid;
(c) contacting the cell with a binding molecule that specifically binds the antibody of step (a);
(d) detecting the presence of the binding molecule;
(e) detecting the presence of the complex of the polynucleotide probe and cellular nucleic acid; and,
(f) correlating the presence of the binding molecule with the presence of the antigen in the cell thereby determining the cell phenotype, and correlating the presence of said complex of the polynucleotide probe and cellular nucleic acid with the presence of the target sequence in the cell, thereby determining the cell genotype. Step (a) of the invention precedes step (b), and step (b) precedes step (c). In preferred embodiments, the cell is exposed to a fixative between step (a) and step (b).
In various embodiments of the invention, the binding molecule is an anti-immunoglobulin antibody, protein A or protein G, which may be detectably labeled, for example with a reporter molecule that is fluorescent, a radioisotope, or an enzyme.
In various embodiments of the method, the cell is a human cell, such as a human fetal cell; the antibody is monoclonal; the antigen is hemoglobin; and/or the polynucleotide probe is DNA, such as a probe that specifically hybridizes to sequences of human chromosomes X, Y, or 21.
The invention further provides kits useful for practicing the combined immunocytochemistry and in situ hybridization method of the invention.
DETAILED DESCRIPTION
I. Introduction
In one aspect, the invention provides a highly efficient method for determining the phenotype and genotype of a cell by carrying out both antibody staining to detect antigens in the cell or population (e.g., immunocytochemistry) and in situ hybridization to determine the genetic composition or karyotype of the cell.
Although methods for carrying out ISH and ICC are well established, the art lacks efficient and effective methods for carrying out ISH and ICC on the same cells. For optimal utility in diagnostic and research applications, a combined ICC/ISH method, should work with antigens from different cell compartments (e.g., cytoplasmic, extracellular, etc.), should be amenable to use with both repetitive-sequence and non-repetitive-sequence ISH probes, and should be highly specific and accurate.
In one aspect of the invention disclosed here, cells are analyzed by binding a cell-antigen specific antibody to a target cell, exposing the cell to a fixing agent, carrying out ISH on the cell, and subsequently detecting the antibody bound to the cell antigen. The bound antibody can be detected using a reporter molecule that binds (e.g., through an anti-ligand) to a ligand associated with the cell-antigen specific antibody. Alternatively, the bound antibody (i.e., the primary antibody
Goldbard Simon
Hong Tsai-Hsia
Lin Emily
Zoccoli Michael A.
Applied Imaging Corporation
Jones W. Gary
Townsend and Townsend / and Crew LLP
LandOfFree
High efficiency methods for combined immunocytochemistry and... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with High efficiency methods for combined immunocytochemistry and..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and High efficiency methods for combined immunocytochemistry and... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3169494