Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-04-14
2003-10-07
Housel, James (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S005000, C435S007900, C435S030000, C436S526000, C436S548000
Reexamination Certificate
active
06630316
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to methods for measuring the function of lymphocytes and their responses to mitogens or specific antigens. The methods are suitable for measurement of the responses of T lymphocytes when they are a subpopulation of cells, and also for measuring the function of specific subsets of T lymphocytes, each subpopulation or subset of a subpopulation having characteristic determinants on their cell surface. The invention also relates to test kits used in performing such methods. The methods of the invention facilitate screening of complex biological fluids, such as whole blood, by means of incubating a sample of the fluid with a mitogen or antigen, separating the selected subset of interest, e.g., via affinity separation, and detecting the presence of an internal cellular component, advantageously ATP, that is increased as a result of the response.
BACKGROUND OF THE INVENTION
The immune system is central to control of infectious diseases and cancer. Lymphocytes, a class of white blood cells, are critical cell types that are responsible for the activities of the immune system. Lymphocytes are divided into two major categories, T lymphocytes and B lymphocytes. Overall assessment of the function of the immune systems and, in particular, lymphocytes is important in assessment of immunodeficiency caused by: genetic factors, infectious disease such as (HIV), drugs following transplantation, stress, aging, or nutritional deprivation.
Lymphocytes express receptors on the cell surface that bind with specific antigens or epitopes. Exposure to the antigen results in expansion in the population of the lymphocytes that are reactive to that antigen. Measurement of the response of the immune system to a specific antigen can be useful in diagnosis of infectious disease, hypersensitivity to certain agents, exposure to immunologically reactive drugs, or response to vaccination.
The function of B lymphocytes or their response to specific antigen can be assessed by measuring the level of specific antibody in bodily fluids such as blood, saliva or urine. The function of T lymphocytes or their response to specific antigens is more difficult to measure. Measurement of the functions of T lymphocytes or T cells is complicated by a number of factors. First, there are several different subsets of T cells with different functions. These subsets have been classified in part by the expression of characteristic cell surface markers and in part by a variety of functional assays including measurement of cytokines. Second, T cells respond to antigens only when they are presented by other cells in the context of major histocompatibility antigens on the surface of the presenting cell. Third, many of the functions of T cells depend on cell-cell contact with effector cells or the functions are fairly localized. Current methods for measuring immune function are tedious, time consuming, and poorly adapted to the clinical laboratory setting.
Methods that are currently used for measurement of Immune function include: methods based on counting the number of T cells or different subsets; methods based on measuring the proliferation of lymphocytes, methods based on measurement of cytotoxic activity or secretion of cytokines, and methods used in vivo such as skin tests and adoptive transfer. These methods are described in detail in the literature (see for example Groeneveld et al., Journal of the International Federation of Clinical Chemistry, 6: 84-94; 1994; Clough and Roth, JAVMA 206:1208-1216, 1995).
The methods most commonly used in the clinical laboratories are based on counting the number of T cells or subsets. A variety of techniques have been described including immunofluorescence microscopy, immunocytochemistry, enzyme immunoassay, and flow cytometry. Flow cytometry, in particular, is widely used in clinical laboratory settings. Flow cytometry is particularly useful in measurement of subsets of interest within a complex population of cells. For example, U.S. Pat. No. 4,727,020 to Recktenwald describes the use of two fluorescent channels to detect cells in a subpopulation specifically labeled with two different immunofluorescent agents. U.S. Pat. No. 4,284,412 to Hansen, et al. describes the use of fluorescence channels to detect forward and right angle light scatter of cells of different subpopulations in blood. Major disadvantages of flow cytometry are that it requires complex and expensive equipment, each sample must be run and analyzed individually and the results require interpretation and are frequently not repeatable. These disadvantages are particularly acute in a clinical laboratory which must process multiple patient specimens daily and where the need for consistent and reliable results is extremely important.
U.S Pat. No. 5,385,822 to Melnicoff et. al. and U.S. Pat. No. 5,374,531 to Jensen disclose alternative methods to flow cytometry for counting the number of lymphocytes or of a subset of lymphocytes within a mixed population of cells. The methods described in these patents involve coupling a detectable reporter substance to the bio-membrane or incorporating the reporter substance into the cell, then separating the subset or population of interest and detecting the reporter substance. These methods utilize affinity separation to isolate populations of interest from a complex mixture of cells. This technique offers improvements over flow cytometry but it is still based on cell counting techniques.
The major difficulty with all cell counting techniques is that they do not measure the function of specific cells or their responses to specific antigens or mitogens. Cells that respond to mitogens or antigens have unique cell surface markers found only on the responding cells. Methods for counting the number of cells exhibiting these markers have been described but these methods are relatively insensitive due to the fact that the responding cells are generally a small fraction of the total population. These methods are also tedious and subject to poor reproducibility.
Direct measurement of responses of lymphocytes have included lymphoproliferation assays, cytotoxicity assays, and measurement of cytokines. In general, these methods require separation of white cells from the original sample followed by incubation with antigen or mitogen. Measurement of the function of specific subsets of lymphocytes requires extensive manipulations prior to the assay. The requirement for antigen presenting cells then means that additional cells have to be added back to the culture. Lymphoproliferation assays are based on division of responding cells and are typically performed using radioactive isotopes. Because they evaluate the division of a small population of cells and require tissue culture, the assays take 3-10 days and are subject to significant variability based on the specific technique and the reagents used in the assay. Cytotoxic tests also require significant cell manipulation and are similarly highly variable depending on the specific conditions used. Cytokine assays can also be performed, but require many steps and separation of subsets of interest prior to stimulating the cells. U.S. Pat. No. 5,344,755 to McMicheals describes a modification of the cytotoxic assay based on initial immunomagnetic separation of T lymphocytes, but this method still requires extensive manipulation of effector cells. U.S. Pat. No. 5,344,755 provides an example of use of cytokine measurements to assess immune status in HIV positive patients but is tedious and requires multiple steps. These methods have required separation of critical cell types, long incubation times, and in some cases use of radioactive substances. For these reasons, these methods have not been suitable for clinical applications.
Affinity separation of cells using protein-coated magnetic particles or other types of solid supports such as polystyrene particles is known and is used as part of several of the methods cited above, see U.S. Pat. Nos. 5,374,531, 5,385,822, and 5,344,755. Various methods for sorting biological populations
Brown Stacy S.
Cylex, Inc.
Housel James
Whitham, Curtis & Christofferson, Inc.
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