Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1992-05-04
2002-10-08
Saunders, David (Department: 1644)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007940, C435S975000, C436S518000
Reexamination Certificate
active
06461825
ABSTRACT:
The present invention relates to a kit and to an immunometric assay method using this kit for the determination of surface antigens characteristic of populations or subpopulations of cells. The immunometric assay method and the corresponding kit are also intended for the determination of the cells themselves via the determination of their surface antigens. These determinations are applicable to diagnosis.
Knowledge of the antigens or markers on the cell surface has made enormous advances with the development of lymphocyte hybridization and the discovery of monoclonal antibodies by KOEHLER and MILSTEIN (Nature, 1975, 256, 495-497). In particular, monoclonal antibodies have made it possible to reveal and analyze membrane antigens or surface markers of cells of the widest possible variety of origins. These markers (or antigens) can be of different kinds: proteins, glycoproteins or glycolipids. The characterizations sought therefore apply mainly to tissue or organ markers, to markers of states of differentiation or activation of normal cells and to the identification or typing of normal or cancerous cells. A particularly important field of application is the study of the cell lines of he mopoiesis (erythrocyte, megakaryocyte, granulocyte, monocyte, lymphocyte).
Thus, for example, monoclonal antibodies have made it possible to specify the respective surface characteristics of T and B lymphocytes. The corresponding markers, by themselves or in combination, identify stages of differentiation and functional specialization of the lymphocytes. By international convention, the surface markers of human leukocytes have been classified in differentiation groups or differentiation classes (CD) defined by the IUIS-WHO subcommittee, 1984, and described in Bulletin of the World Health Organization, 1984, 62(5), 813-815.
The identification of these markers which has been made possible by monoclonal antibodies has provided access to their structure and their biological functions. For example, the molecules of the CD4 and CD8 markers participate in leukocyte adhesion functions and are present on the surface of T lymphocytes with an auxiliary and inductive function (CD4 marker) or, respectively, with a cytotoxic and suppressive function (CD8 marker).
With this knowledge established, it has been possible to use these markers, by virtue of the antibodies which recognize them, for diagnosing and following up a variety of pathologies including, in particular, malignant hemopathies (leukemias, lymphomas, etc.) and states of dysfunction of the immune system (autoimmune diseases, congenital or acquired immune deficiencies such as AIDS, etc.) (BRETON-GORIUS and VAINCHENKER, Le Biologiste, 1987, XXI, no. 167, 63-70; SHAW, Immunology Today, 1987, 8(1), 1-3).
Monoclonal antibodies are now irreplaceable tools of clinical biology applied to cell analyses.
Cell counting methods exist which use the marking of their surface antigens, but these methods are often lengthy, laborious and difficult to carry out and their results are sometimes random.
The known methods used for measuring the normal or modified expression of antigens on the surface of the cell can be separated into two groups. In the first group, the antigens are measured with the aid of complex and specialized laboratory equipment based on flow cytometry (see, in particular, PONCELET et al., J. Immunol. Methods, 1985, 85, 65-74) or quantitative microscopy techniques (POULTER et al., J. Immunol. Methods, 1987, 98, 227-234). These methods for the evaluation of cell antigens are based on the measurement of signals provided by anticell antibodies coupled directly or indirectly to a reagent labeled with fluorescent substances (or fluorochromes) such as fluorescein isothiocyanate or rhodamine isothiocyanate, or with enzymes such as peroxidase or alkaline phosphatase. The use of these fluorescent or enzyme reagents in association with appropriate washing steps then leads to the appearance of fluorescences or colorations which are strictly limited to the cell membranes and do not diffuse into the surrounding medium. Common use of these methods in the laboratory is still restricted by the need for specialized and expensive equipment (a fluorescence microscope which may or may not be associated with an image analyzer, a cryostat and a flow cytometer). Moreover, the analysis and interpretation of th immunolabeling of cells by these processes demand the competence of a specialist in cytology.
A second group of methods for the measurement of antigens is based on the quantitative evaluation of the markers of the overall cell population. These methods make it possible to measure antigens either by direct labeling or by indirect labeling, the latter most frequently being carried out in two, three or four steps. In all cases, the reagent employed in the last labeling step carries a probe which is either of isotopic character, for example iodine 125, for a determination of the radioimmunometric type (BROW et al.,J. Immunol. Methods, 1979, 31, 201; STOCKER and HEUSSER, J. Immunol. Methods, 1979, 26, 87-95), or an enzyme for a determination of the enzyme immunometric type, most frequently peroxidase, alkaline phosphatase or beta-galactosidase (VAN LEUVEN et al., J. Immunol. Methods, 1978, 23, 109-116; MORRIS, Transplantation, 1983, 36(6), 719; BAUMGARTEN, J. Immunol. Methods, 1986, 94, 91-98).
The methods in this last group are rather inconvenient, laborious and risky to apply because of the need to ash and centrifuge the cell material many times; it is sometimes necessary to take a sample of the colored medium resulting from the enzyme reaction in order to carry out the final spectrophoto-metric measurement; finally, chemical fixation of the cells, which is used in most cases, causes the irreversible destruction of certain antigens which are particularly sensitive to the customary chemical binding agents such as glutaraldehyde or methanol (DROVER et al., J. Immunol. Methods, 1986, 90, 275-281).
It is known in the literature that an antigen carrying several antigenic determinants, i.e. several epitopes, can be determined by fixing this antigen via one of its epitopes using an antibody immobilized on a solid support, and by binding, to another epitope of the antigen, another antibody carrying an enzyme or radioisotopic marker enabling the determination to be carried out.
This kind of technique, which is often referred to as the sandwich technique, is described especially in French Patent 2 487 983, French Patent 2 500 166 and European Patent Application 119 736. None of these documents describes the application of this technique to whole cells, even though the word “cell” is sometimes included in the list of antigens to which the process applies.
In the above patents, the various antigens forming the subjects of the Examples described are in all cases solely protein molecules soluble in water and physiological liquids, such as tumoral markers, enzymes or hormones in the bloodstream. On the other hand, it is clear that a cell is not a molecule and differs therefrom at least by being considerably larger and by the fact that it is not soluble in physiological media. Thus,the sandwich technique has so far never been applied to whole cells.
Furthermore, the immunocapture of cells on a solid support is described in International Patent Application 86/02091, in which the object is to remove undesirable cells from samples of bone marrow intended for transplantations. In the said patent application, capture of the cells is effected on floating microbeads and requires that the antibody used be bound to the solid support by a complex macromolecular structure, called a network-relay, which is capable of ensuring a preferential orientation of the antibody relative to that of the corresponding cell antigen. The said patent application gives no indication of an application of the technique to the quantitative determination of an antigen.
The immunocapture of cells is also described in International Patent Application 84/03151 for an analytical application. In the said patent application,
Sanofi (Societe Anonyme)
Saunders David
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