Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1997-11-24
2001-04-17
Saunders, David (Department: 1644)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007940, C435S007950, C436S506000
Reexamination Certificate
active
06218132
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to detection of soluble factor secretion by activated T-cells. In particular, this invention relates to modifications of the standard ELISPOT assay.
Therapeutic strategies ranging from vaccine design to T cell specific immunosuppression require identification of immunodominant T cell epitopes and enumeration of T cell frequency. Several assays are currently employed to provide this information. Modified proliferation assays have been used to identify T cell epitopes based on stimulation indices of ≧2.0 (Plebanski, M., and Burtles, S. S.,
J. Immunol. Meth.
170:15 (1994)), but this assay is extremely sensitive to variations in serum and often proves difficult for large scale clinical screenings. The limiting dilution assay (LDA) employs relatively large PBMC quantities and two rounds of in vitro stimulation to detect the T cell response to whole antigens or peptides (Sharrock, C. E. M. et al.,
Immunol. Today
11:281-286 (1990)). This assay has provided estimates of antigen specific CD4+ T cell frequencies ranging from approximately 1/10
3
-1/10
5
for alloreactive T cells (Sharrock supra) to 10
6
-1/10
7
for autoreactive T cells (Weiner, H. L. et al.,
Science
259:1321 (1993)). The LDA has been used to monitor efficacy in clinical trials, but the quantities of PBMC's (peripheral blood mononuclear cells) required limit the application of this assay in cases requiring frequent blood draws or the screening of large numbers of candidate peptides. Several flow cytometric methods can detect T cell activation by upregulation of characteristic markers such as CD69. Activation-induced T cell lymphokine production can be measured by flow cytometry using a monensin block of secretion, saponin permeabilization, and indirect immunofluorescent staining (Jung, T. et al.,
J. Immunol. Meth.
159:197 (1993)), or by trapping of secreted lymphokines on the surface of the secreting cell (Manz, R. et al.,
Proc. Natl. Acad. Sci. USA
92:1921 (1995)). These flow cytometry techniques are sufficiently sensitive when a relatively high frequency of T cells respond, as occurs in alloreactivity or superantigen stimulation, but they cannot detect most rare antigen-specific T cells. ELISA assays of lymphokine secretion are similarly limited to cases in which the responses of primed T cells, T cell clones, or high frequency T cells are measured. In situ hybridization of lymphokine mRNA is sufficiently sensitive to detect antigen-specific T cells with frequencies in the range of 1/10
4
-1/10
5
(Link, J. et al.,
Neurol.
44:728 (1994); Link, J. et al.,
Ann. Neurol.
35:197 (1994)), but this technique is not readily scalable to large sample numbers.
A modification of the ELISA assay (enzyme-linked immunosorbent assay), termed the immunospot or ELISPOT assay, has been developed to detect lymphokine secretion by individual T cells following antigen stimulation (Czerinsky, C., et al.,
J. Immunol. Methods
110:29-36 (1988); Olsson, T. et al.,
J. Clin. Invest.
86:981-985 (1990)). However, the sensitivity of the standard ELISPOT assay is low. For example, for many multiple sclerosis (MS) patients, the standard ELISPOT assay of T cell responses to autoantigens can only be detected in cells sampled from the CSF, which entails difficult sampling and low cell yield. Identifying peptide epitopes within autoantigens such as MBP (myelin basic protein) by this assay is even more difficult given the relatively low precursor frequency. Furthermore, counting ELISPOT sample wells under light microscopy is slow and somewhat subjective. It would be desirable to have improved methods of measuring lymphokine secretion by activated T-cells, particularly those which occur at low frequency. This invention fulfills this and related needs.
SUMMARY OF THE INVENTION
One aspect of the invention provides a method for detecting an antigen reactive T-cell in a biological sample suspected of containing said T-cells. The method comprises:
(a) stimulating the T-cells in the biological sample with the antigen for a first time period sufficient to permit T-cell expansion;
(b) restimulating the T-cells with an effective amount of a combination of the antigen and antigen presenting cells to induce secretion of a soluble factor;
(c) detecting the presence of the soluble factor by capturing the soluble factor on a solid support; and
(d) relating the presence of the soluble factor on the solid support to the presence of the antigen reactive T-cell.
Optionally, a second soluble factor such as, for example, a cytokine(s) and/or growth factor(s) may be added to facilitate continued T-cell expansion. This second soluble factor may be the same or different to the soluble factor whose detection is related to the presence of the antigen reactive T-cell.
The methods disclosed herein can be used to detect rare T-cells, especially those reactive to autoantigens and occurring at low frequencies, as low as 1 T-cell per 10
5
PBMCs. A related aspect of the invention uses frozen T-cells as an internal control to validate the assay.
Also provided are methods for:
(1) identifying an antigen which stimulates T-cells in a patient biological sample,
(2) identifying a patient having T-cells reactive to an autoantigen,
(3) screening for putative drugs capable of inducing deletion or unresponsiveness of T-cells,
(4) the screening of potential blood donors for use in generating antigen specific clones,
(5) the identification of T cell epitopes
(6) monitoring of drugs/treatments which may induce a generalized state of immunosuppression, for safety or efficacy, and
(7) monitoring of the immune response to antigen(s) over time.
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patent: 5750356 (1998-05-01), Spack et al.
patent: WO 90/04182 (1990-04-01), None
McCutcheon et al, Jour. Immunol. Meth; 210(2), 149-166, 1997.*
Czerkinsky, C. et al., “Reverse ELISPOT Assay For Clonal Analysis of Cytokine Production, I. Enumeration of Gamma-Interferon-Secreting Cells”J. Immunol. Meth., 1988, vol. 110, pp. 29-36.
Link, H. et al., “Myasthenia Gravis: T and B Cell Reactivities to the &bgr;-Bungarotoxin Binding Protein Presynaptic Membrane Receptor”J. Neuorological Sci., 1992, vol. 109, pp. 173-181.
Link et al. “The T-cell repertoire in myasthenia gravis involves multiple cholinergic receptor epitopes”Scand. J. Immunol., 1992, vol. 36, pp. 405-414.
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Lu et al. “Interleukin-2 secreting cells in multiple sclerosis and controls”J. Neurol. Sci., 1993, vol. 120, pp. 99-106.
Menz et al., “Analysis and sorting of live cells according to secreted molecules, relocated to a call-surface affinity matrix”Proc. Natl. Acad. Sci. USA, 1995, vol. 92, pp. 1921.
Olsson, T. et al., “Increased numbers of T cells recognizing multiple myelin basic protein epitopes in multiple sclerosis”Eur. J. Immunol., 1992, vol. 22, pp. 1083-1087.
Olsson, T. et al., “Autoreactive T Lymphocytes in Multiple Sclerosis Determined by Antigen-Induced Secretion of Interferon-&lgr;”J. Clin. Invest, 1990, vol. 86, pp. 981-985.
Plebanski et al., “In vivo primary responses of human T cells to soluble protein antigens”J. Immunol. Meth., 1994, vol. 170, pp. 12.
Sharrock et al., “Limiting dilution analysis of human T cells: a useful clinical tool”Immunol. Today, 1990, vol. 11, pp. 281-286.
Söderström, M. et al., “Optic Neuritis and Multiple Sclerosis: The T Cell Repertoires to Myelin Proteins and MBP Peptides Change With Time”Acta Neurological Scandinavica, 1994, vol. 90, pp. 10-18.
Söderström, M. et al., “T Cells Recognizing Multiple Peptides of Myelin Basic Protein Are Found in Blood and Enriched in Cerebrospinal Fluid in Optic Neuritis and Multiple Sclerosis”Scand. J. Immunol., vol. 37, pp. 355-368, 1993.
Sun, J.-B. et al., “T cells responses to human recombinant acetylcholine receptor-&agr; subunit in myasthenia gravis and controls”Eur. J. Imm
McCutcheon Michael A.
Spack Edward G.
Wehner Nancy G.
Anergen Inc.
Saunders David
Townsend and Townsend / and Crew LLP
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