Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals – Carrier is inorganic
Reexamination Certificate
1996-11-04
2001-07-24
Le, Long V. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
Carrier is inorganic
C422S105000, C435S007200, C435S007210, C435S007230, C435S007240, C435S033000, C435S395000, C436S518000, C436S525000, C436S526000, C436S809000
Reexamination Certificate
active
06265229
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 and apparatus for performing the method in different cell populations.
In biology, biochemistry and adjacent fields there 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 sophisticated methods. It is also of importance to be able to isolate the cells in viable forms.
Affinity binding is a sophisticated 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.
When such methods are used for isolation of specific cells, which are the subject for farther various examinations it is necessary that the cells should recover their function upon returning to the original conditions. This is not always the case, although it is proposed a method for providing physiological conditions such that the isolated specific cells can develop in sufficient numbers to allow further characterisation.
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 (WO91/01368) and relates to a method of connecting target cells to an insoluble support by using the abilities of hapten, antihapten 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.
Furthermore, WO91/09938 describes the use of 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. WO92/04961 comprises a method and a complicated equipment to separate cells or different molecules from a non-magnetic test medium by using colloidal magnetic paricles. In this method small (sub micron) particles are used because it is necessary to avoid precipitation of the particles in the solution and this method necessitates complicated apparatus, in which magnetic intensifying means is immersed in the test medium. This may have adverse effects on the cells.
In “Application of Magnetic Beads in Bioassays”, B. Haukanes and C. Kvam. Bio/Technology, 11:60-63. 1993,. several methods are described for use of magnetic particles to remove tumor cells from bone marrow, isolation of lymphoid cells from peripheral blood and isolation of DNA, RNA and DNA-binding proteins. All described methods have specificities which are unsuitable for the present purpose of detecting only target-cells. The above methods will in addition to target cells also bind non-target cells due to cross-reactivity and unspecific adhesion of the antibody-particle complex.
There is also described a multiwell filtration apparatus for the assay of microliter quantities (EP-A-0 098 534), a filter strip and composite assemblies for filtering microliter quantities of fluid (EP-A-0 339 769) and an assay cartridge which has a substantially rectangular base plate, a substantially rectangular top plate and four side walls (EP-A-0 131 934). None of the above apparatus are applicable for the present purpose in that they describe pore sizes which are too small for the present purpose of retaining only particle-cell rosettes. Furthermore the filters are not designed to be exposed to several examinations of the retained cells without removing them form the filter medium.
There is a need for a simple linkage to connect a target cell to an insoluble support, which does not involve compounds of a rather unspecified hapten-group, and which is directed to detection of specific target cells, with a mini-mum of unspecific cell association and which render unnecessary a later cleavage between the insoluble support and the specific target cell.
In a co-pending application by one of the applicants (WO94/07 139, filed Sep. 10, 1993) a method is described for detecting diagnostic purposes specific target cells without the problem with unspecific binding to normal cells. They represent sensitive detection methods for a variety of cell types, such that a high number of cells can be readily screened in the microscope and the procedure is rapid and simple. Furthermore, the methods 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. There is, however, a need for improvements such as isolation of the particle-bound target cells in the target cell suspension, from unbound beads, unspecifically bound non-target cells and unbound non-target cells, which is simple to perform, not time requirering and with render the target cell/particle complexes suitable to perform further analysis such as for example microscopic examinations and growing in a culture medium.
BRIEF SUMMARY OF THE INVENTION
These objects are obtained by the present invention outlined by the method, apparatus and kit characterised 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 pathogenic 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
Fodstad Øystein
Høifødt Hanne Kleppe
Rye Philip
Gabel Gailene R.
Le Long V.
Merchant & Gould P.C.
Oystein Fodstad
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