Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals – Carrier is inorganic
Reexamination Certificate
1999-09-03
2002-11-26
Ponnaluri, Padmashri (Department: 1627)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
Carrier is inorganic
C436S501000, C436S149000, C436S173000, C436S806000, C435S007100
Reexamination Certificate
active
06485985
ABSTRACT:
This invention relates to processes for magnetorelaxometric qualitative and/or quantitative detection of analytes in the liquid and solid phases, compounds for magnetorelaxometric detection, and their use in analysis and immunomagnetography.
It is already known that immunoscintigraphy makes it possible to detect pathological structures in vivo with the aid of radiolabeled structure-specific substances, which are also referred to below as markers. To this end, antibodies that are labeled with &ggr;-rays or antibody fragments are usually used. In addition, other structure-specific substances, such as, e.g., peptides or oligonucleic or polynucleic acids are also used or are being researched. The portion of specifically bound radioactivity is, however, generally small in all these processes. Consequently, in the case of these studies, the levels of markers that are not specifically bound and thus circulate in the blood or accumulate in organs such as the liver, kidney, efferent urinary passages, or bladder are very high. In many cases, this high background radiation impedes adequate detection of pathological structures. In EP 0251494, in Panchapakesan et al., 1992 Immunol. Cell Biol. 70:295 and in Ziegler et al., 1991, New England Journal of Medicine 324:430, reference is therefore made to ways of improving immunoscintigraphy. The goal of most of the processes is to accelerate the elimination of radioactivity that is not specifically bound.
In addition, the use of antibodies that are conjugated with paramagnetic or superparamagnetic substances or antibody fragments for locating pathological structures in vivo has been proposed on various occasions. To date, nuclear spin tomography or the magnetometry that is based on changes in susceptibility (WO93/05818 and WO91/15243) have been considered as detection processes for such labeled antibodies. In the case of these detection processes, the problem of the variable portion of the signal owing to unbound portions of the marker as well as owing to natural variations in the susceptibility and relaxivity of the tissue also remains present. In addition, the methods often are not sensitive enough to be able to detect just small amounts of specifically bound markers.
A process that makes it possible to detect only the portion of bound markers and thus is not influenced by the extent of the unbound markers is not known, however.
One of the objects of this invention is therefore to develop new processes and substances that are superior to the above-mentioned prior art and that make it possible to detect the retention site without using radioactive substances and the extent of the bound markers without the influence of markers that circulate in the blood.
In addition, it is also already known that quantitative immunoassays as well as other binding assays (e.g., receptor binding assays) make it possible to determine a very large number of substances that can also be of biological relevance in samples of varying composition. Generally, however, only one parameter per sample in an assay is determined in this way. An existing survey of the various processes is: T. Chard; An Introduction to Radioimmunoassay and Related Techniques: Laboratory Techniques in Biochemistry and Molecular Biology, 4th ed., Elsevier Science Publishers, Amsterdam (1990). The basis of all binding assays is the high detection sensitivity of compounds that are labeled with isotopes or by some other means with the high specificity of ligand-receptor reactions.
The known assay processes have the following drawbacks, however:
1. The processes for simultaneous determination of various analytes within the same sample are based on the binding of various radio-, fluorescence- or enzymologically-labeled probes to the analytes. In this case, the unbound or bound activity of the probes for quantitative determination of the analyte is generally measured after subsequent separation and washing. In this case, the amount of usable different probe labels is very limited. Thus, for example, in the case of different radioisotopes as probe labels, so-called overlapping phenomena occur which lead to a rapid loss of the quantitative accuracy of individual signals. The combination of various enzymes as probe labels causes comparable problems, whereby the feasibility here is further hampered by the necessary search for reaction conditions that allow the simultaneous determination of enzyme reactions in a system.
2. The sensitivity of the process is limited by, for example, non-specific interactions between matrix and probe, or else by limited labeling capability on the part of the probe (low specific activity).
3. The successful implementation of the process often requires that the sample material obtained be worked up (e.g., production of serum or plasma from whole blood, extraction of samples with organic solvents, concentration of the analyte using chromatographic processes, etc.).
4. For successful implementation of the processes, separation and washing steps, which are used in the separation of bound and unbound receptors or ligand, are essential in most cases.
5. To carry out radioimmunoassays, the use of radiating nuclides, which are costly and complicated to handle, is necessary.
6. In practice, the storage of previously used markers often causes problems since they are either unstable (radioimmunoassays) and must therefore constantly be made up fresh or else react in a sensitive manner to environmental influences.
Another object of this invention is therefore to develop novel, economical processes and substances that overcome the drawbacks of the above-mentioned prior art.
First processes are now described that overcome the drawbacks of the known processes for implementing immunoassays or other binding assays.
The processes according to the invention are based on the use of colloidal ferromagnetic or ferrimagnetic substances, also referred to below as magnetic labeling, which are combined with substances to be identified—also referred to below as analytes—or structure-specific substances. Such combinations, according to the invention, of magnetic labelings with analytes or structure-specific substances, which are described in more detail in this patent, are also referred to below as magnetic markers. Through the use of the term colloidal substances or colloidal particles, both the range of sizes of the particles or substances in the size range of colloids, i.e., the range of 1 nm up to about 1000 nm, and their use as a dispersed phase in a suitable dispersion medium, which is aqueous in most cases, is described. To ensure improved storability and transportability, the colloidal substances or particles can also be present in dried form or frozen; while measurements are being made, however, they are present in the liquid phase in the dispersed state.
In addition, the processes are based on special measuring techniques, which make it possible to determine the relaxation of magnetization after the magnetic labeling or the magnetic markers are magnetized. Such measuring processes according to the invention, which are described in more detail in this patent, are also referred to below as magnet-relaxometry or magnetorelaxometry or magnet-relaxometric detection.
An important principle of the invention is that after an external magnetizing field is turned off, the magnetization of freely movable ferromagnetic or ferrimagnetic colloidal particles relaxes by two different mechanisms:
i) Turning of the whole colloidal particle inside the surrounding liquid, whereby the time constant depends on the hydrodynamic diameter of the particles including the shell, the viscosity of the carrier liquid, and temperature, which mainly reflects parameters of the environs of the particles; this mechanism is also referred to below as Brownian relaxation or extrinsic superparamagnetism,
and
ii) Turning of the internal magnetizing vector inside the colloidal particles, whereby the time constant depends in a very sensitive manner on material and shape (the anisotropy constants of the particle material used), volume and
Bunte Thomas
Kottiz Roman
Trahms Lucz
Weitschies Werner
Millen White Zelano & Branigan P.C.
Ponnaluri Padmashri
Schering Aktiengesellschaft
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