Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Patent
1993-02-26
1995-03-07
Saunders, David
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
435 724, 435 79, 435 30, 436526, 436548, G01N 33569, G01N 33577
Patent
active
053957512
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention concerns an assay for cytotoxic T cells.
BACKGROUND OF THE INVENTION
Cytotoxic T cells (or cytotoxic T lymphocytes (CTL), as they are also known) are one of the types of cells that develop from lymphoid stem cells and that play an important role in the immune system. T cells are antigen specific, and carry antigen specific receptors. T cells also carry receptors for major histocompatability complex (MHC) proteins, and will only function in the presence of the relevant antigen and the appropriate MHC protein. Cytotoxic T lymphocytes interact with a target cell carrying the relevant antigen (e.g. a virus protein fragment on the surface of a cell infected with a particular virus) and also carrying the appropriate MHC Class 1 protein (or human leucocyte antigen (HLA) Class 1 molecule), and the CTL release cytotoxic enzymes which act to kill the target cell.
SUMMARY OF THE INVENTION
According to the present invention there is provided a method of detecting in a sample CTL specific for a particular virus, comprising contacting the sample with a support carrying immobilized antibodies to a surface antigen on T cells; separating the support and attached materials; contacting the separated support with target cells matched to the HLA type of the source of the sample and with HLA matched peptide epitope of the virus that interacts with the CTL of interest; and monitoring lysis of the target cells.
Any CTL in the sample will bind to the immobilised antibodies and hence the support, and will be separated from other material in the sample on separation of the support. Only CTL specific for the virus of interest will act to cause lysis of HLA matched target cells in the presence of the relevant peptide epitope, so that lysis of the target cells is indicative of the presence of the CTL of interest in the sample. By comparing results with those obtained from known standards a measure of the quantity of CTL in the sample can be obtained by monitoring target cell lysis.
The method has been shown to work well in detecting human immunodeficiency virus (HIV) specific CTL, using peptide fragments of HIV such as those disclosed in European Patent Specifications Nos. 0346022 and 0412766, and International Application No. PCT/GB91/00013. The invention is also applicable to detection of CTL specific for other viruses including HIV 1, HIV 2, related HIV viruses etc.
There is evidence that HIV specific CTL activity in patients infected with HIV varies with development of acquired immunodeficiency syndrome (AIDS) or related conditions: many healthy HIV seropositive patients have a vigorous anti-HIV CTL response, but there is evidence that HIV specific CTL activity declines as disease progresses. Measurement of HIV specific CTL in samples from patients infected with HIV may thus provide useful information in following disease progression.
The antibodies are conveniently immobilized in conventional manner on a suitable support as will be well known to those skilled in the art. The preferred support comprises a plurality of magnetic beads, such as Dynabeads (Trade Mark) produced by Dynal, Norway. The beads (and attached materials) are readily separated from a sample by magnetic attraction, providing an easy, simple separation technique.
The antibodies are preferably monoclonal antibodies, which may be produced using known techniques. Antibodies to any suitable T cell surface antigen may be used. The presently preferred antibody is monoclonal antibody to the CD8 marker on lymphocytes, but it will be clear that antibodies to other markers may also be used.
The sample may comprise, for example, suitable body fluid from a patient, such as blood, cerebrospinal fluid, lung fluid etc. Good results have been obtained in detection of HIV specific CTL in whole blood, without prior treatment or separation (e.g. of peripheral blood mononuclear cells). The technique can thus involve only a small number of processing steps, which is an advantage in infection containment and in reducing the risk of infection
REFERENCES:
patent: 4554088 (1985-11-01), Whitehead et al.
F. Sanchez-Madrid et al, Proc. Nat'l. Acad. Sci., USA, 79, 7489-7493, 1982.
Gotch Frances M.
McMichael Andrew J.
Nixon Douglas F.
Saunders David
United Biomedical Inc.
LandOfFree
Assay for cytotoxic T cells does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Assay for cytotoxic T cells, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Assay for cytotoxic T cells will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-1405462