Ligand-aminodextran-marker conjugates and uses thereof

Details Ligand-aminodextran-marker conjugates and uses thereof Ligand-aminodextran-marker conjugates and uses thereof

1999-10-27

2002-05-14

Chin, Christopher L. (Department: 1641)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S007220, C435S007230, C435S007240, C435S007250, C435S007200, C436S172000, C436S512000, C436S513000, C436S529000, C436S800000, C436S805000, C530S391100, C530S391300, C530S391500

Reexamination Certificate

active

06387622

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to methods for analyses of cell populations using fluorescent labels or markers.
BACKGROUND OF THE INVENTION
Multiplex labeling of cells for analysis of mixed cell populations by flow cytometry has progressed in various ways. Only a finite number of fluorescence emission colors of known organic fluorophores can be squeezed into the visible-near-UV-near-IR spectral regions in which flow cytometry measurements are made. The limitations have been dictated by the bandwidths of emission bands, the spectral overlap between these emission bands, and the excitation wavelength requirements. Up to eight colors requiring three laser lines have been introduced in various stages: two- to four-color [H. M. Shapiro in
Practical Flow Cytometry
, 3rd ed., Wiley-Liss, Inc., New York, N.Y., 1995, Chap. 7, p. 291]; five color [A. J. Beavis and K. J. Pennline,
Cytometry
, 15: 371-376 (1994); M. C. O'Brien et al,
Cytometry
, 21:76-83 (1995); M. Roederer et al,
Cytometry
, 21: 187-196 (1995)]; six color [M. Roederer et al,
Cytometry
, 24: 191-197 (1996)]; seven color for imaging [T. Ried et al,
Proc. Natl. Acad. Sci. USA
, 89: 1388-1392 (1992); A. Gothot et al,
Cytometry
, 24: 214-225 (1996)]; and eight color [M. Roederer et al,
Tissue Antigens
, 48: 485 (1996), abstract TC-6-02]. The six and seven color cases appear to represent the present upper limit for flow cytometry applications in which known organic dyes are used as fluorescent labels, since the eight-color example cannot as yet be considered to be of clinical significance due to severe overlap between emission bands of the fluorochromes.
As the upper limit in the number of usable colors was reached, other methods, based on fluorescence intensity differences, either intrinsic to analyzed cell populations or contrived by various means, have been described. Mutually exclusive pairs of targeted white blood cell populations with widely different, intrinsic numbers of receptors per cell can be labeled by a single color marker and analyzed by flow cytometry [U.S. Pat. No. 5,538,855]. U.S. Pat. No. 4,499,052 describes a method of distinguishing multiple subpopulations of cells by labeling specific antibodies with fluorescent polymers containing different, pre-selected ratios of fluorescein and rhodamine [See also, A. M. Saunders and C.-H. Chang,
Ann, N.Y. Acad. Sci
., 468:128 (1986)]. H. M. Shapiro in
Practical Flow Cytometry
, 1st ed., Alan R. Liss, Inc., pp. 127-128 (1985) describes a method using three different antibodies labeled with fluorochrome A, B, and a combination of A and B. U.S. Pat. No. 5,206,143 describes saturated and sub-saturated amounts of marker mixed with the sample of cells. Quantitative differences in fluorescence intensity of one or two fluorochromes used for labeling cells were obtained. Each subset to be analyzed was labeled with a different amount of fluorochrome, exhibiting fluorescence intensities within a distinguishable range. Mixtures of fluorescein- and phycoerythrin-labeled and unlabeled antibodies were used to produce fluorescence intensity differences of several orders of magnitude among various cell subsets [P. K. Horan et al,
Proc. Natl. Acad. Sci. USA
, 3: 8361-8365 (1986)]. Use of this method to identify helper and suppressor/cytotoxic T cells, NK and B cells, and monocytes in whole blood was shown [C.-M. Liu et al,
Am. J. Clin. Path
, 92: 721-728 (1989)]. Further, eight leukocyte subsets in whole blood were analyzed with six monoclonal antibodies linked with one of three fluorochromes [P. Carayon et al,
J. Immunol. Methods
, 138: 257-264 (1991)].
A variety of antibody-aminodextran conjugates are described in U.S. Pat. No. 5,527,713 and U.S. Pat. No. 5,658,741 described the preparation of antibody-aminodextran conjugates containing two or more antibody molecules per conjugate. Recently, polymeric carriers containing the divinyl sulfone moiety for covalent attachment of protein and other molecular species were described in European Patent No. 0 594 772 B1.
The human genome project has been a driving force behind the development of new detection and sequencing methods for nucleic acids. Several non-radioactive gene probes, oligos with attached fluorescent dye, that hybridize, or bind to sample DNA, have been described [L. M. Smith et al.,
Nature
, 321:674-679(1986) and L. M. Smith et al.,
Nucleic Acids Res
., 13:2399-2412(1985)] and are being used. Automated DNA sequencers use four fluorescent dyes with non-overlapping emission bands, one per nucleotide base. Electrophoretic mobilities of the fluorescent dye-oligo primer conjugates need to be similar for all four conjugates. Also, the molecular weights of the conjugates cannot be too high otherwise they will not move through the polyacrylamide or agarose gel used in the electrophoresis. Molecules do not travel through gel pores but follow a tortuous path through entanglements of gel fibers by a process called forced reptation [G. W. Slater et al.,
Macromolecules
, 24:6715-6720 (1991); P.-G. de Gennes,
J. Chem. Phys
., 55:572 (1971)]. Large molecules can become stuck in the gel in forms such as “hernias”, “cul-de-sac”, or “impaled spirals” in gel pores. However, electrophoretic methods using pulsed electric fields [U.S. Pat. No. 4,971,671] have been successfully applied to separate DNA fragments containing up to about 5,000,000 base pairs.
An alternative method for sequencing DNA is a carry-over from the Southern hybridization technique [E. M. Southern,
J. Mol. Biol
., 98: 503 (1975)], wherein DNA is digested with one or more restriction enzymes, and the resulting fragments are separated according to size by electrophoresis through an agarose gel. The DNA is denatured in situ and transferred from the gel to a solid support, usually a nitrocellulose filter or nylon membrane. Without the use of a radiolabel as the probe, the DNA attached to the filter is hybridized to fluorescence-labelled DNA or RNA [E. M. Southern,
Trends Genet
., 12: 110-115 (1996)], which allows detection of the positions of bands complementary to the probe.
The gene chip probe does not depend on the use of gels or electrophoresis. This solid state surface probe has been described in Z. Strezoska et al.,
Proc. Natl. Acad. Sci. USA
, 88: 10089-10093 (1991); R. Drmanac et al.,
Science
, 260: 1649-1652 (1993); A. B. Chetverin and F. R. Kramer,
Biotechnology
, 12: 1093-1099 (1994); T. Studt,
R
&
D Magazine
(February 1998), and is being designed to allow up to 400,000 oligos per chip for simultaneous DNA/RNA analysis.
The SER-gene probe [T. Vo-Dinh et al,
Anal. Chem
. 66: 3379-3383 (1994) and U.S. Pat. No. 5,306,403] uses a non-fluorescent dye conjugated to an oligonucleotide to bind nucleic acid on a nitrocellulose membrane. After hybridization the SER-Gene probe-DNA complex is transferred onto a surface-enhanced Raman scattering (SERS) active substrate to detect SERS spectra from the dye.
The need for increased sensitivity of probes used in automated DNA sequence analysis by attaching multiple dye molecules per oligonucleotide primer were recognized as early as in L. M. Smith et al,
Nature
, 321: 674-679 (1986). However, only a limited degree of fluorescence enhancement has been possible for dye-oligo conjugates that are constrained to low molecular weight for separation by gel electrophoresis. The use of two dye molecules per oligo in which the pair of dye molecules is related by donor-acceptor type energy transfer to enhance fluorescent intensities from 2- to 6-fold has recently been described in J. Ju et al.,
Proc. Natl. Acad. Sci. USA
, 92: 4347-4351 (1995); J. Ju et al.,
Anal. Biochem
., 231: 131-140 (1995); M. L. Metzker et al.,
Science
, 271: 1420-1422 (1996).
The measurement of antigen-specific T cells requires recognition by a polymorphic surface T cell receptor which is unique for each different antigen. The ligand for the TCR is an antigenic

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