Detection and determination of solid phase-associated factors

Details Detection and determination of solid phase-associated factors Detection and determination of solid phase-associated factors

1998-12-18

2003-10-14

Le, Long V. (Department: 1641)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

C435S006120, C435S007240, C435S007200, C435S007310, C435S007900, C435S962000, C435S968000, C435S013000, C435S077000, C435S007920, C435S810000, C436S063000, C436S069000, C436S172000, C436S501000, C436S519000, C436S548000, C252S700000

Reexamination Certificate

active

06632599

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention relates to procedures for the detection or for the determination of solid phase-associated factors, which are multiply associated with the same solid phase. According to the invention, the sample is brought into contact with a transmitter particle, on which at least one ligand having binding affinity for a solid phase-associated factor and a transmitter are immobilized, and a receiver particle, on which at least one ligand having binding affinity for said solid phase-associated factor and a receiver is immobilized, and then the signal is determined which results when transmitter and receiver are brought sufficiently close to one another. In particular, the invention relates to the detection of cell surface receptors which can be used for the typing of cells or for the determination of cell activation states. It is thus possible to replace the hitherto widely customary flow cytometry by a more simple procedure.
The differentiation of blood cells, in particular of leucocytes granulocytes, monocytes and lymphocytes) is a routinely used and important procedure in diagnostics. It is based, inter alia, on the fact that different cell types are characterized by different surface antigens, such as, for example, membrane proteins of the integrins family (Hynes R O. Integrins: a family of cell surface receptors. Cell 1987; 48: 549-554) (incorporated herein by reference). Most of these membrane proteins are designated by CD numbers (cluster designation numbers).
Membrane proteins can also only be exposed on the surface after stimulation of the cells or secreted by fusion of intracellular vesicles with the surface, such as, for example, proteins from the selectins group (Bevilacqua M P and Nelson R M. Endothelial-Leukocyte adhesion molecules in inflammation and metastasis. Thromb. Haemost. 1993; 70: 152-154) (incorporated herein by reference).
In the case of platelets, for example, GMP-140 (P selectin; DC62P) is an activation marker. Furthermore, in activation states characteristic complexes of receptors of cells and ligands can result, such as, for example, on activated platelet complexes of the glycoproteins GP Ib/IX or GP IIb/IIIa, which bind von Willebrand factor or fibrinogen (see, for example, Clemetson K J. Biochemistry of platelet membrane glycoproteins, Prog. Clin. Biol. Res. 1988; 283:33-75) (incorporated herein by reference). After activation, phosphatidylserine-containing lipid membranes, to which clotting factors or other phospholipid-binding protein (for example from the annexins family) can bind, are also exposed on platelets.
In previous procedures, labeled antibodies or other labeled reactive ligands, for example annexins, against these surface antigens were added to blood for the detection of phosphatidylserine-containing lipid membranes (Römisch J et al., Anticoagulant properties of placenta protein 4 (annexin V); Thromb. Res. 1990; 60: 355-366) (incorporated herein by reference). By means of flow cytometry, the cells are then sorted according to their size and in the course of this a conclusion is drawn at the same time via the detection of the labeling on the number and proportions of one or several cell types in parallel. Labels used are substances known per se to the person skilled in the art, in particular chemiluminescent compounds (for a general survey see, for example, Michelson, A. D. and Barnard, M. R., U.S. Pat. No. 5,552,290) (incorporated herein by reference).
The abovementioned flow cytometry is an established procedure, but has the disadvantage that it can be used only for the specific purpose of cell counting and/or typing. Usually, it is therefore also only established in specific laboratories. Wider use of the differentiation of cells and their activation states would be desirable, however, for clinical problems. For routine use, application in customary clinicochemical analyzers or other automated equipment is necessary for the routine laboratory.
The invention was therefore based on the object of making available an alternative to the previously customary flow cytometry methods, which allows the determination of cell surface antigens in a homogeneous, immunochemical procedure.
A number of homogeneous, immunochemical procedures for the determination of antigens and antibodies are already known, such as, for example, the FRAT
1
System (Syva), the EMIT
1
System, enzyme channeling immunoassays, fluorescence energy transfer immunoassays (FETI, e.g. TRACE
1
Technology; CIS bio International), enzyme inhibitor immunoassays (Hoffmann LaRoche, Abbott Laboratories) or fluorescence polarization immunoassays (Dandlicker). These homogeneous procedures were developed in order to offer methods which can be carried out without separation and/or washing steps. Some of these procedures have only a limited sensitivity or are not suitable for the determination of high molecular weight analytes having multiple epitopes.
The expression scintillation proximity assay (SPA) was introduced by Hiram E. Hart and Elaine B. Greenwald (Molecular Immunology 1979; 16: 265-267) (incorporated herein by reference) in order to describe a specific homogeneous radioimmunoassay. In this procedure, two different types of polymeric beads are employed, which are loaded with specific binding components. The first of these bead types is additionally loaded with a dye while the second bead type additionally carries tritium. The dye has the property of emitting light pulses as soon as it is stimulated by the
1
H &bgr;-radiation (Auger electrons). This radiation, however, only has a range of a few micrometers in aqueous solutions, so that in dilute suspensions which contain both bead types, only a few beads of the one type are found in sufficiently close to beads of the other type. As a result, all in all only a small fluorescence signal can result. By means of the addition of reactants which can react with the specific binding components of the two bead types, however, an aggregation of the beads takes place which brings many of the beads of the first type (tritium beads) into the vicinity of beads of the second type (fluorophore beads), so that an altogether higher signal results. The resulting signal is detected in a scintillation counter. A further development of this procedure by use of
125
iodine-labeled specific binding components was described by Udenfriend, S. et al. (Proc. Natl. Acad. Sci. 1985; 82: 8672-8676) (incorporated herein by reference).
A further procedure is described (EP-0 515 194 A2; Ullman et al., Proc. Natl. Acad. Sci. 1994; 91: 5426-5430 (incorporated herein by reference); Ullman et al., Clinical Chemistry 1996; 42: 1518-1526) (incorporated herein by reference) as a luminescent oxygen channeling immunoassay (LOCI). In this, two particle types are used, one of which contains a photosensitizer (sensitizer beads) and the other a chemiluminescent component (acceptor beads). The photosensitizer generates singlet oxygen and activates the chemiluminescent component if it is sufficiently close. The activated chemiluminescent component generates light which can be detected as a measuring signal.
Bystrak, S. et al. (Analytical Biochemistry 1995; 225: 127-134) (incorporated herein by reference) describe a homogeneous procedure in which a photooxidation of a fluorescent substrate, which is bonded to a unilaminar vesicle, by singlet oxygen takes place. Specific binding components are covalently bound to the surface of the vesicle.
These procedures all comprise specific binding of particles to binding components. As a rule, the binding of these binding components is carried out via the coating of the particles with appropriate specific ligands, such as, for example, antigens or antibodies for immunochemical detection. Up to now, these procedures were only used for the detection of soluble (humoral) factors. On binding to these factors (for example proteins), transmitter and receiver particles are brought into a spatial vicinity which allows a transfer of the energy emitted by a transmitter to a receiver particle. Use for the detect

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