Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals – Carrier is inorganic
Reexamination Certificate
1997-06-24
2001-02-06
Chin, Christopher L. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
Carrier is inorganic
C435S002000, C435S007100, C435S007200, C435S007230, C435S007240, C435S007250, C435S007500, C435S007800, C435S007940, C435S040000, C435S052000, C435S174000, C435S181000, C435S961000, C436S513000, C436S518000, C436S523000, C436S532000, C436S534000, C436S538000, C436S540000, C436S824000, C436S828000
Reexamination Certificate
active
06184043
ABSTRACT:
The present invention relates to an immunomagnetic method for detection of specific target cells in cell populations and solutions of cell populations. The invention also relates to a kit for performing the method in different cell populations.
In biology, biochemistry and adjacent fields it is considerable need for methods in which chemical entities are linked together. Such methods have an increasing importance in research regarding both normal and abnormal cell populations. Especially when solid supports are used cells can be immobilized, detected and isolated and purified. Furthermore, the cell content may be examined using a range of sofisticated methods. It is also of importance to be able to isolate the cells in viable forms.
Affinity binding is a sofisticated way of linking chemical/biochemical entities together. In such a method a pair of binding partners, which for example are attached to the substances to be linked, bind to each other when brought in contact. One of the binding partners in such a linkage may be represented by a molecule on the cell surface. Several such binding partner systems are known, such as antigen-antibody, enzyme-receptor, ligand-receptor interactions on cells and biotin-avidin binding, of which antigen-antibody binding is most frequently used. A hapten/anti-hapten binding pair has also recently been suggested (WO 91/01368).
When such methods are used for isolation of specific cells, which are the subject for further various examinations, it is necessary to reverse the linkage without producing destructive effects on the binding partners, which ideally should recover their function upon returning to the original conditions. This is not always the case, although it is proposed a method for adequately cleaving antigen/anti-antigen and hapten/anti-hapten linkages (PCT/EP91/00671, PCT/EP90/01171).
Methods are known in which one of the binding partners is attached to an insoluble support, such as paramagnetic particles, and by which isolation of target cells in a mixed cell population is performed as negative isolation or positive isolation. In a negative isolation procedure the unwanted cells can be removed from the cell preparation by incubating the cells with antibody-coated particles, specific for the unwanted cells. Following the incubation the cell/antibody/particle-complex can be removed using the particles, leaving the wanted target cells behind. This result is often not satisfactory, since the wanted cells are left in the cell population, more or less purified, and also since the intention of the isolation procedure is to examine and/or perform further studies on the specific target cells. Attempts have been made to use particles for positive isolation, in which the wanted target cells are removed from the mixed cell population. These procedures have, however, been directed to certain target cells and are not suited for all target cell systems. A positive isolation procedure involving the hapten/anti-hapten linkage system has recently been proposed (PCT/EP90/01171) and also a method for isolating haemopoietic progenitor cells from bone marrow (PCT/EP90/02327). The latter is directed to use a combination of positive and negative selection for the purpose of isolating and possibly growing specific cells, i.e. haematopoietic progenitor cells, in the bone marrow, and is dependent upon removal of the particles.
PCT/EP90/01171 relates to a method of connecting target cells to an insoluble support by using the abilities of hapten, anti-hapten antibodies and anti-cell antibodies to bind to each other, thus constructing a linkage between the insoluble support, i.e. particle, and the target cell, consisting at least of hapten and anti-hapten antibody or combinations of hapten and anti-hapten antibodies and anti-anti-hapten antibodies or secondary anti-cell antibodies. The later cleavage of the complex is performed by again exposing it to hapten or hapten analogue. Thus the constructed link always consists of hapten in addition to 1 or more elements. The method is directed to unspecified target cells and is directed to isolation of target cells and release of the insoluble support.
There is a need for a simple linkage to connect a target cell to an insoluble support, which do not involve compounds of a rather unspecified haptene-group, and which is directed to detection of specific target cells, with a minimum of unspecific cell association and which render unnecessary a later cleavage between the insoluble support and the specific target cell.
Thus the object of the present invention is to detect for diagnostic purposes specific target cells when used in a blood and bone marrow, without the problem with unspecific binding to normal cells. It represents a sensitive detection method for a variety of cells types, such that a high number of cells can be readily screened in the microscope and the procedure is rapid and simple. Furthermore, the present method can be used for isolation of cells for biochemical, biological and immunological examination, and for studying of specific genes at the nucleotide or protein level, in addition to culturing the cells, without the need for cleaving the cell-particles complex. A further object of the invention is to provide a kit for performing the method as characterized in the claims.
The intensions of the inventions are obtained by the method and kit characterized in the enclosed claims.
The method for immunomagnetic detection of target cells in a mixed cell population and physiological solutions containing cell populations is suitable for detection, but may also be used in positive isolation of specific types of both normal cells and patogenic cells. The method creates a linkage between a specific target cell and an insoluble support, such as paramagnetic particles, which consists of one or two elements. The particle is either coated with an anti-cell antibody of murine or human origin, directed to the specific antigen determinants in the membranes of the wanted target-cells, or the particles are coated with a polyclonal anti-mouse or anti-human antibody capable of binding to the Fc-portions of the specific anti-cell antibody directed to the antigen determinants in the target-cell membranes. Instead of using the polyclonal anti-mouse/anti-human antibody for coating the particles, a monoclonal rat anti-mouse/anti-human antibody may be used. This last antibody, due partly to its monoclonal origin, may provide a more specific binding to the anti-cell antibody and reduce the risk for possible cross-reactions with other cells in solutions, such as blood. Furthermore, incubation of the cell suspension with a mild detergent and/or second set of antibodies or antibody fragments, prelabeled or not with fluorescent agents, metallocolloids, radioisotopes, biotin-complexes or certain enzymes allowing visualization, will dramatically increase the specificity of the method.
In the following a more detailed disclosure of the method is presented, using cancer cells as the target-cells for detection and possible isolation. The method is, however, not limited to cancer cells and the disclosure shall not limit the method to this particular field of use, since the method is suitable within a range of cytological research areas.
In the management of cancer patients, the staging of the disease with regards to whether it is localized or if metastatic spread has occurred to other tissues, is of utmost importance for the choice of therapeutic alternative for the individual patient. Malignant cells spread by direct invasion into the surrounding tissue, through the lymphatics or by the distribution of tumor cells in the blood to distant organs, including the bone marrow and the central nervous system and the cerebrospinal fluid.
Detection of metastatic tumor cells has, until recently, relied on morphological methods using light and electron microscopy on biopsied tumor specimens, on smears of bone marrow and peripheral blood, and on slides prepared after cytosentrifugation of various body fluids. Since the advent of monoclonal antibodies r
Fodstad Øystein
Kvalheim Gunnar
Chin Christopher L.
Merchant & Gould P.C.
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