HK1008563A - Process and compounds for the magnetorelaxometric detection of analytes and use thereof - Google Patents

Description

Method and compounds for the magnetorelaxometric detection of analytes and their use

The invention relates to the subject matter of the features indicated in the claims, namely a method for the qualitative and/or quantitative magnetic relaxation detection of analytes in liquid and solid phases, compounds for the magnetic relaxation detection, and their use in analytical and immunomagnetic recording methods.

It is known that immunoscintigraphy makes it possible to detect pathological tissues in vivo by means of radiolabeled structure-specific substances (hereinafter also referred to as markers). For this purpose, antibodies or antibody fragments labelled with υ -rays are generally used. In addition, other structure-specific substances, such as peptides or oligonucleotides or polynucleic acids, can also be used or be investigated. However, the fraction of radioactivity that binds specifically in all of these methods is generally small, and thus in these studies the level of markers that do not bind specifically and therefore accumulate in the blood or in organs such as the liver, kidney, external urinary tract or bladder is high. In many cases, such high levels of background radiation prevent adequate detection of pathological tissue. Thus, Panchakashang [ immunocytobiology, 70- (1992)295] and Ziegler [ England drug communication, 324(1991)430] resorted to a method for improving immunoscintigraphy, which is also described in EP 0251494. Most methods aim to accelerate the elimination of non-specifically bound radioactivity.

Furthermore, the use of antibodies or antibody fragments conjugated to paramagnetic or superparamagnetic substances has been suggested in a variety of contexts to locate pathological tissues in vivo. So far, nuclear spin tomography or magnetometry based on changes in magnetic susceptibility (WO93/05818 and WO91/15243) have been considered as detection methods for such labeled antibodies. In these detection methods, difficulties still remain due to the unbound fraction of the label and the variable fraction of the signal due to natural changes in tissue susceptibility and relaxivity. In addition, such methods are often not sensitive enough to detect only small amounts of specifically bound labels.

However, methods are not known which are only capable of detecting the fraction of bound label and therefore are not affected to the extent that it is not bound to label.

It is also known that quantitative immunoassays, as well as other binding assays (e.g., receptor binding assays), can determine a large number of biologically relevant substances in a sample of varying combinations. However, only one parameter per sample can usually be determined in each measurement with this method. An overview of the various methods that exist is found in: t, chard [ radioimmunoassay and related techniques introduction: laboratory techniques in biochemistry and molecular biology, Vol.IV, Elsevier science Press, Amsterdam (1990) ]. All binding assays are based on the high detection sensitivity of compounds labelled with isotopes or some other method with high specificity for ligand-receptor reactions.

However, the known assay methods have the following disadvantages:

methods for the simultaneous detection of multiple analytes in the same sample are based on the binding of multiple radioactively, fluorescently or enzymatically labelled probes to the analytes. In this case, the unbound or bound activity of the analyte-detecting probe is usually measured after subsequent isolation and washing. In this case, the amount of different probe labels that can be used is very limited. Thus, for example in the case of different radioisotopes as probe labels, a so-called overlap phenomenon occurs, which can lead to a rapid decrease in the accuracy of the quantification of the individual signals. Combining multiple enzymes as probe labels leads to similar problems, for which reason reaction conditions must be investigated that allow simultaneous detection of the enzyme reactions in one system, which further hampers the feasibility of this approach.

The sensitivity of this method is limited, for example, by non-specific interactions between the substrate and the probe, or also by the limited labelling capacity (low specific activity) of the probe.

Successful performance of this method often requires processing of the resulting sample material (e.g., production of serum or plasma from whole blood, extraction of the sample with organic solvents, concentration of the analyte using chromatographic methods, etc.).

In order to successfully carry out this method, in most cases, separation and washing steps used in separating bound and unbound receptor or ligand are necessary.

In order to perform radioimmunoassays, expensive and unwieldy radionuclides must be used.

In practice, the storage of the labels used first often leads to difficulties, since they are not stable (radioimmunoassays) and therefore have to be prepared fresh on the fly, otherwise they react in a sensitive manner with environmental influencers.

It is therefore an object of the present invention to develop new methods and materials which overcome the disadvantages of the prior art, and in particular which enable the detection of retention sites without the use of radioactive materials, and the detection of the level of bound label without prior separation of unbound label.

This object is achieved by the invention.

It has been found that a qualitative and/or quantitative detection of analytes in the liquid and/or solid phase is possible if ferromagnetic or ferrimagnetic micelles are used as magnetic labels to be identified in an immunoassay or other binding assay, and their magnetization relaxation is determined as a measured variable.

The following describes for the first time a method that overcomes the drawbacks of known methods of performing immunoassays or other binding assays.

The method of the invention is based on the use of a ferromagnetic or ferrimagnetic colloidal substance (hereinafter also referred to as magnetic label) which binds to a substance to be identified (hereinafter also referred to as analyte) or to a structure-specific substance. This binding of the magnetic label according to the invention to the analyte or structure-specific substance, which will be described in more detail in this patent, is also referred to as magnetic label in the following. By the use of the terms colloidal substance or colloidal particles, it is described that the size range of the particles or substance is within the colloidal size range, i.e. the range from 1nm up to about 1000nm, and their use as a dispersed phase (in most cases aqueous) in a suitable dispersion medium. To ensure improved storage and transport capacity, the colloidal substance or particles may be provided in a dried or frozen form; however, when measurements are made, they are provided in a liquid phase in a dispersed state.

In addition, this method is based on a special measurement technique which makes it possible to determine the relaxation of the magnetization after the magnetic marker or markers have been magnetized, this measurement method of the invention (which will be described in more detail in this patent) being also referred to below as the magnetic relaxation detection method.

The main principle of the invention is that after the external magnetic field is switched off, the magnetization of the freely movable ferromagnetic or ferrimagnetic colloidal particles relaxes within the measurement time by two different mechanisms:

i) rotation of the whole colloidal particle in the surrounding liquid, whereby the time constant depends on the hydrodynamic diameter of the particle comprising the shell, the viscosity of the carrier liquid and the temperature, which mainly reflects parameters in the vicinity of the particle; this mechanism is also referred to hereinafter as Brownian relaxation or extrinsic superparamagnetic, and

ii) the rotation of the internal magnetized carriers within the colloidal particles, whereby the time constant depends in a very sensitive way on the material and shape of the particles used (anisotropy constant of the particle material used), on the volume and on the temperature. These are the fundamental intrinsic parameters of the particles; this mechanism is also referred to hereinafter as Neelian relaxation or intrinsic superparamagnetic.

The object of the invention is achieved by the fact that in immunoassays or other binding assays ferromagnetic or ferrimagnetic micelles are used as magnetic labels to be identified, which micelles are used in an unbound state under the measurement conditions and whose Brownian relaxation proceeds faster than nelian relaxation. The use of such ferromagnetic or ferrimagnetic micelles enables specific determination of the fraction of bound magnetic labels in addition to the unbound magnetic labels simultaneously present in the measurement sample due to a change in the predominant relaxation mechanism or a proportional increase in particle volume as a result of binding.

By using sensitive measurement methods, ultra-sensitive binding-specific immunoassays or other binding assays that can be performed in both liquid and solid phases can be established in the procedure of the present invention using ferromagnetic or ferrimagnetic micelles. As a particularly sensitive measurement method, after the sample is magnetized in a magnetic field and the magnetic field is switched off, the relaxation of the magnetization can be detected by means of a highly sensitive magnetic field detector, such as a superconducting quantum interference device (SQUID), an inductive coil, a saturated magnetometer, a giant magnetoresistive sensor, or a magnetoresistive transducer, or the complex susceptibility of the sample can be determined as a function of the frequency of the magnetic field.

In addition, in the method for quantitatively detecting analytes in liquid and solid phases by magnetic relaxation of the present invention, a structure-specific substance that is first bound to the analytes is labeled with ferromagnetic or ferrimagnetic colloidal particles.

These magnetically labeled structure-specific substances are applied to the liquid or immobilized sample to be measured, and the sample to be measured is magnetized by means of an externally provided magnetic field. After the external magnetic field is switched off, the relaxation of the magnetization of the magnetic labels can be measured by means of the magnetic field receptors.

The measurements were evaluated here according to methods known to the person skilled in the art, as well as in the direct assay methods described below.

The method according to the invention for the quantitative determination of analytes in liquid and solid phases by means of magnetic relaxation can also be carried out in such a way that the analytes are first of all detected by means of a magnetic relaxation

i) Is marked by ferromagnetic or ferrimagnetic colloidal particles and then

ii) applying these magnetically labelled analytes to the liquid or immobilized sample to be determined, adding a substance which binds specifically to the analytes, magnetizing the sample to be determined by means of an externally supplied magnetic field, and measuring the relaxation of the magnetization of the magnetic labels by means of the magnetic field receptors after the external magnetic field has been switched off.

The measurement results are evaluated here, also in the competitive assays described below, according to methods known to the person skilled in the art, i.e. similar to those used in immunoassays or in radioactive assays.

In both cases, the determination of the complex magnetic susceptibility of the magnetic label or of the magnetic label which changes as a result of binding can also be used as a function of frequency for analysis.

Bound and unbound labels can be distinguished by using their different relaxation mechanisms or the influence of the relaxation time of the magnetic label due to binding, which previously could only be done in special situations.

According to the invention, solid-phase bound analytes can be identified, in particular, by means of structure-specific substances which bind to the analyte, the structure-specific substances first of all

i) Labeled with ferromagnetic or ferrimagnetic micelles which relax over a measurement time, wherein the ferromagnetic or ferrimagnetic micelles are selected such that the relaxation time of the Browrian relaxation under the measurement conditions is shorter than the relaxation time of the Neeleian relaxation, and then

ii) these magnetically labelled substances are applied to the immobilised sample to be measured, the sample to be measured is magnetised by means of an externally supplied magnetic field of suitable strength, and the relaxation of the magnetisation of the magnetic label is measured by means of a magnetic field sensor when the external magnetic field is switched off, whereby the different relaxation behaviour of the solid phase bound and unbound magnetic label is used as an analysis. As a measurement variable, the complex magnetic susceptibility of the sample can also be determined as a function of frequency.

In this case, instead of the structure-specific substance, the analyte to be identified may also be bound to a magnetic label.

In the liquid phase, the analytes of the invention can be detected, in particular, by structure-specific substances which bind to the analytes, which first of all are

i) Labeled with ferromagnetic or ferrimagnetic micelles, wherein the ferromagnetic or ferrimagnetic micelles are selected such that their relaxation time under measurement conditions in Brownian relaxation is shorter than that in Neelian relaxation, and then

ii) these magnetically labelled substances are applied to the sample to be determined, which is magnetized by means of an externally provided magnetic field of suitable strength, and the relaxation of the magnetization of the magnetic labels can be measured by means of magnetic field receptors after the external magnetic field has been switched off, whereby the different relaxation behavior of the magnetic labels bound to the analyte relative to the unbound magnetic labels is used for the analysis.

As a measurement variable, the complex magnetic susceptibility of the sample can also be determined as a function of frequency.

In this case, instead of the structure-specific substance, the analyte to be determined can also be bound to a magnetic label.

A structure-specific substance is defined as any substance that specifically binds to a certain structure. Structure-specific substances can be defined in particular as antibodies, antibody fragments, biotin, or substances binding to biotin such as avidin and streptavidin, agonists binding specifically to receptors such as cytokines, lymphokines, endothelin or their antagonists, specific peptides and proteins, receptors, enzymes, enzyme substrates, nucleotides, ribonucleic acids, deoxyribonucleic acids, sugars, lipoproteins, etc. Preferred as structure-specific substances are those with binding constants in the range of 10⁵-10¹⁵(mol/l)^-1Particularly preferred are those having a binding constant in the range of 10⁷-10¹⁵(mol/l)^-1The substance of (1).

By means of methods familiar in immunochemistry, peptide chemistry and protein chemistry, it is possible to label structure-specific substances or analytes to be identified with ferromagnetic or ferrimagnetic particles. Especially advantageous is the covalent bond between the structure-specific substance or analyte to be identified and the substance forming the stable shell of the ferromagnetic or ferrimagnetic particles. Examples of particularly suitable methods are activation and coupling by means of carbodiimides [ Jacobi and Wildcer et al, enzymological methods, (1974)34], the formation of Schiff bases upon exposure of periodic acid to saccharide-containing compounds (Vitscher and Bayer et al, enzymological methods, 184: 177), their subsequent optionally reduction for further stabilization, coupling by means of glutaraldehyde (Haosman and Richardard Natt. Acad. Sci.USA 71(1974) 3537), crosslinking of bromoacetylated particles with thiolated species (Agel et al, cytology 9(1976) 81), and reductive alkylation (J.Histechem. Cytochem.24 (1976)).

Ferromagnetic or ferrimagnetic micelles can also be made in the form of a stable shell made of a structure-specific substance or analyte to be identified by directly putting the produced particles into a solution of the structure-specific substance, optionally in the presence of other adjuvants, such as proteins, carbohydrates and natural, synthetic or partially synthetic surface-active substances, etc., or also in the presence of the structure-specific substance.

Suitable colloidal particles and suspensions containing these particles are described, for example, in WO 92/12735, WO/92/22586, EP 0186616 and US 4101435.

The method according to the invention can be used, for example, for fertility, histocompatibility, allergy, infectious diseases, hygiene, genetics, virology, bacteriology, toxicology, pathology, environmental analysis and medical diagnosis.

Another object of the present invention is a compound for use in magnetic relaxation assays, which consists of a colloidal suspension of freely movable ferromagnetic or ferrimagnetic particles and a structure-specific substance or analyte to be identified, wherein the structure-specific substance is specifically defined as an antibody, an antibody fragment, biotin or a substance binding to biotin, such as avidin and streptavidin, an agonist binding specifically to a receptor, such as cytokine, lymphokine, endothelin or antagonists thereof, other specific peptides and proteins, receptors, enzymes, enzyme substrates, nucleotides, ribonucleic acids, deoxyribonucleic acids, sugars, lipoproteins, and the like.

The compounds used for the magnetomechanical relaxation detection may also consist of a combination of several ferromagnetic or ferrimagnetic particles with distinguishable relaxation times, since by using different magnetic labels with respectively very narrow relaxation times and/or magnetic moment distributions for different structure specific substances or analytes within the sample, respectively distinguishable measurements can be detected. As a result, it is possible to quantitatively detect several analytes directly and simultaneously.

As a matrix for the suspension, all liquids in which the colloidal particles can move freely are suitable. Particularly suitable are water, aqueous solutions of surface-active adjuvants, such as surfactants or oligo-or polysaccharides and proteins, and mixtures of water and alcohols, such as glycerol and polyethylene glycol. The suspension medium may additionally contain adjuvants which modify the osmotic pressure, such as crude salts. In addition, buffering substances which determine the pH, such as phosphates, may also be present.

Compounds made from ferromagnetic or ferrimagnetic micelles and structure-specific substances or analytes to be identified may also be provided in dry form, optionally in combination with other adjuvants, such as freeze-drying agents, which may, for example, promote drying or increase the stability of the dried product.

The analyte may be found with or without isolation and washing steps. In performing measurements with the step of separating bound and unbound magnetic labels, all ferromagnetic or ferrimagnetic colloidal substances can be used as magnetic labels for magnetic relaxation detection according to the present invention. In these cases, special requirements for Brownian and Neelian relaxation times are not necessary.

Due to the binding identification based on physical mechanisms, non-specific measurement signals (matrix phenomena) can be largely eliminated. The specificity of this method therefore depends only on the "true" specificity of the structure-specific substances (cross-reactivity of the antibodies, non-specific binding of the ligands).

Due to the high sensitivity of the method of the invention, it is easy to maintain under conditions below the detection limit typically encountered with binding assays.

As magnetic labeling substance, all ferromagnetic or ferrimagnetic materials that can be colloidally dispersed in a medium suitable for magnetic relaxation detection can be used. When using a magnetic relaxation detector, which does not require a separation step of bound and unbound magnetic labels, the Neelian relaxation time of the magnetic labels under the measurement conditions must be longer than the Brownian relaxation time of the magnetic labels. Particularly suitable is a Brownian relaxation time in aqueous medium in the range of 10^-8-10^-1Second, Neelian relaxation time greater than 10^-8In seconds. For all ferromagnetic or ferrimagnetic colloidal particles to be measured without a separation step, the viscosity of the dispersion medium used must be matched to the relaxation times and measurement times of the ferromagnetic and ferrimagnetic particles, since the suspension medium fundamentally determines the time constant of the Brownian relaxation.

Particularly preferred are ferromagnetic or ferrimagnetic colloidal particles made of iron, iron oxides, barium ferrate, strontium ferrate, cobalt, nickel ferrate, cobalt ferrate and chromium dioxide, which have a Neelian relaxation time longer than the Brownian relaxation time.

It is generally advantageous to use magnetic labels having a narrow particle size and/or magnetic moment distribution. Magnetic labels can be separated to have a narrow particle size distribution by, for example, chromatography or special filtration methods (e.g., glass capillary systems or tangential filtration), by using molecular sieves, or by centrifugation. Magnetic markers with a magnetic moment that is as uniform as possible can be produced by methods such as sorting in a gradient magnetic field.

The ferromagnetic and ferrimagnetic materials may be stabilized by a shell made of oligo-or polysaccharides, proteins, peptides, nucleotides, surfactants, other mono-, oligo-or polymers and/or lipids.

The ferromagnetic and ferrimagnetic substances advantageously have a particle size of between 1nm and 400nm, particularly preferably between 1nm and 100 nm.

According to this method, the magnetic relaxation detection is carried out in a measurement arrangement which first makes it possible to magnetize the sample to be investigated by means of a suitable magnetic field and subsequently measures the magnetic relaxation of the magnetic labels. The measurement arrangement for the magnetic relaxation detection of analytes used in the examples is shown in fig. 1. In contrast to all other known processes (JP-235774 and WO 91/1. omega. 43). In the magnetic relaxation measurement of the method according to the invention, instead of the static magnetization in the presence of a magnetic field, its change with time in the absence of a magnetic field is determined. Only in this way can data be obtained on the bound state of the label, and, in addition, the influence of diamagnetic or paramagnetic components or impurities on the measurement signal can thus also be avoided, and the sensitivity of the measurement is decisively increased.

The frequency-dependent magnetization of the marker (taking the complex susceptibility as a function of frequency) can be further measured in the presence of a magnetic field by means of a highly sensitive sensor such as a SQUID, due to a suitably switchable magnetic field. In this case, the special frequency dependence of the magnetic susceptibility of the magnetic marker is used, in contrast to the frequency dependence of the paramagnetic or diamagnetic component, which can be determined separately. This method is also different from the method proposed in WO91/15243 for determining the magnetic susceptibility of superparamagnetic substances. In WO91/15243, neither the frequency dependence of the magnetic susceptibility of the magnetic labels nor a method for using this property is described.

Another aspect of the invention relates to methods for determining the retention sites and levels of specifically bound labels independently of the labels circulating in the blood. In these methods, the use of radioactive substances is avoided, which in the past could not be avoided when carrying out the scintillation recording method of the prior art.

The method of the present invention is based on the fact that the difference in relaxation time between bound and unbound magnetic labels in a liquid and the change in the predominant relaxation mechanism due to the binding of the magnetic labels to the solid phase can also be used for magnetic relaxation detection of substances or structures in vivo. This method is also referred to hereinafter as immunomagnetic recording.

The in vivo measurement of the spatial distribution of relaxing magnetic markers for the human body over the measurement time range can be carried out by two different measurement methods:

1. the magnetic field is generated as homogeneous as possible in an advantageous volume, the magnetic field is switched off and the spatial distribution of the magnetic field being relaxed is measured by means of a multi-channel sensor. The sensor should enclose the measuring object as completely as possible. In order to generate sufficient measurement information, the measurement object can also be repeatedly measured with successive gratings.

2. The spatially limited local magnetic field is continuously generated, the magnetic field is switched off, and the spatial distribution of the relaxing magnetic field is measured by means of a single-channel sensor. Multi-channel sensors may also be used.

In both methods, it is preferable to measure both the magnetization of the object and the resulting magnetic field strength in all three spatial directions in order to obtain as much data as possible.

The above measurements can be described by a model, which is also used in the analysis of the bioelectric current magnetic field. The magnetic dipole, multipole or multi-dipole model is used herein as a basis. By suitable approximation, the deviation between the measured data and the model parameters can be minimized, and the specific parameters of the model, in particular the location of the dipole or multipole, can be determined. These parameters provide information about the spatial distribution of the magnetized particles.

A similar method is known, which has proven useful for analyzing the magnetic field of bioelectric currents.

As methods and compounds suitable for immunomagnetic recording, all the methods and substances cited by the method of magnetometric relaxation detection can be used.

Particularly suitable for immunomagnetic recording are magnetic labels, which are biodegradable and compatible. Magnetic labels consisting of iron oxide are particularly advantageous.

For binding-specific magnetic relaxation assays in vivo, the Brownian relaxation time of the combination of ferromagnetic or ferrimagnetic substances and structure-specific substances introduced into body fluids at body temperature must be shorter than the Neelian relaxation time.

In immunomagnetic recording, structure-specific substances are defined in particular as all substances which bind specifically to the human tissue to be identified. Particularly suitable are antibodies, antibody fragments, agonists binding specifically to the receptor or antagonists thereof, specific peptides and proteins, receptors, enzymes, enzyme substrates, nucleotides, ribonucleic acids, deoxyribonucleic acids, carbohydrates or lipoproteins. Among the agonists binding to the receptor, cytokines, lymphokines or endothelins are particularly suitable.

Very suitably, the binding constant is in the range of 10⁵-10¹⁵(mol/l)^-1All structure-specific substances of (1). Particularly suitable is a binding constant in the range of 10⁷-10¹⁵(mol/l)^-1All structure-specific substances of (1).

The following examples are intended to explain the objects of the invention in more detail without intending to restrict them to this purpose.

Example 1

Mu.g of a collagen III monoclonal antibody (hereinafter referred to as anti-collagen III) was dissolved in 500. mu.l of a 0.1M sodium bicarbonate solution, and 1ml of a dextran-coated magnetite suspension (Mei) containing 1mol Fe/l and having a particle size of about 40nm was buffered with 0.1M sodium bicarbonate by means of a Sephadex column (Pharmacia PD10)to Sangyo), 0.5ml of a solution containing 10mmol of sodium periodate is added to the suspension, the solution is maintained in the dark for 2 hours, then it is eluted by PD10 with a 0.1M sodium bicarbonate solution, the above-mentioned anti-collagen III solution is added to the suspension, the mixture is maintained in the dark at 4 ℃ for 3 hours, 5mg of NaBH are added₄The mixture was maintained in the dark for 8 hours at 4 ℃ with a solid and slight rotation, and magnetite-labeled anti-collagen III (hereinafter referred to as magnetic-anti-collagen III) was eluted through a PD10 column with a phosphate-buffered crude salt solution (hereinafter referred to as PBS, pH 7.4).

200 μ l of buffer containing 5 μ g collagen III was incubated in a polystyrene sampling tube (phosphate buffered crude salt solution (PBS) and the liquid phase was then discarded and washed with a solution containing 0.1% Tween^(R)20 phosphate buffered crude salt solution (hereinafter referred to as PBST) the sample tube was rinsed three times, 200. mu.l of a solution containing 5. mu.l of magneto-anti-collagen III in PBST was added to the sample, incubated at room temperature for 1 hour, and the sample was magnetized in a magnetic shielding chamber (see FIG. 1) under a superconducting quantum interference device at a strength of 2mT 4 cm. Relaxation measurements were taken within 100 seconds 400 milliseconds after switching off the magnetic field, and relaxation was identified in the sample from the disappearing magnetic field. The relaxation signals of the collagen III containing samples are shown in figure 2. Example 2

Mu.l of a solution containing 5. mu.g of collagen V in PBS buffer (pH7.4) was incubated in a polystyrene sampling tube, the liquid phase was then discarded, the sampling tube was washed three times with PBST wash buffer pH7.4, and 5. mu.l of the magnetic-anti-collagen III solution prepared according to example 1 in 200. mu.l of PBST was added to the sample. After 1 hour incubation at room temperature, the sample was magnetized in a magnetic shielding chamber (see FIG. 1) under a superconducting quantum interference device in a magnetic field of 2mT 4cm intensity. The sample is measured after the magnetic field is switched off. Relaxation measurements were taken within 100 seconds after the field was switched off for 400 milliseconds. In the samples containing collagen V, the disappearing magnetic field was not detected within the limits of the measurement reliability (fig. 3). Example 3

Mu.l of glutaraldehyde solution (3% aqueous solution) was added to a solution of 1ml of PBS containing 100. mu.g of collagen III, and the solution was stirred at 4 ℃ for 24 hours and then centrifuged. The precipitate contains precipitated crosslinked collagen III. Cross-linked collagen III (sample 1) was suspended in 1ml PBS. Mu.l glutaraldehyde solution (3% aqueous solution) was added to a solution of 100. mu.g collagen V in 1ml PBS, and the solution was stirred at 4 ℃ for 24 hours and centrifuged, and the precipitate contained precipitated crosslinked collagen V. Cross-linked collagen V (sample 2) was suspended in 1ml PBS. Mu.l of the magneto-anti-collagen III suspension of example 1 were added to samples 1 and 2, respectively, and incubated at 37 ℃ for 1 hour, after which both samples were magnetized by means of a shielded chamber of a SQUID detector in a magnetic field of strength 2 mT. Relaxation measurements were taken 400 milliseconds after the field was turned off. The vanishing magnetic field was detected in sample 1, and the vanishing magnetic field was not detected in sample 2. Example 4

From a 10ml solution of 1.9mg/ml collagen III in PBS (pH7.4), 5ml portions of a solution were prepared with the following dilutions: 10,000ng/ml, 1.00ng/m l, 100ng/ml, 10ng/ml, 1 ng/ml.

1ml of each dilution was pipetted into a polystyrene tube (2.5ml volume) three times in total, allowed to stand at 37 ℃ for 1 hour, the contents of the tube were discarded, and each tube was washed three times with 1ml of PBST.

1ml of a 1: 100 dilution of magnetite-labeled antibody prepared as in example 1 was added to each tube, the tubes were allowed to stand at room temperature for 1 hour, the samples were magnetized (2mT) using the measurement arrangement outlined in FIG. 1, and relaxation was measured within 100 seconds after the magnetic field was turned off. The evaluation of the difference between the measured magnetic flux densities 1200 ms and 100 ms after switching off the magnetic field, based on the collagen concentration in the sample, is reproduced in fig. 4. Example 5

Mu.g of a monoclonal antibody to collagen III (hereinafter referred to as anti-collagen III) was dissolved in 500. mu.l of a 0.1M sodium bicarbonate solution, 1ml of a magnetite suspension containing 1mol of Fe/l and having a dextran coating with a particle size of about 40nm was buffered with 0.1M sodium bicarbonate through a Sephadex column (Rharmacia PD10), 0.5ml of a 10(mmol solution of sodium periodate) solution was added to the suspension, the solution was allowed to stand in the dark for 2 hours, and then eluted with 0.1M sodium bicarbonate solution through PD10, and the resulting solution was washed with waterAdding the above anti-collagen III solution to the suspension, standing the mixture at 4 deg.C in the dark for 3 hours, and adding 5mg NaBH₄The solid was swirled slightly, the mixture was allowed to stand in the dark at 4 ℃ for 8 hours, and magnetite-labeled anti-collagen III (hereinafter referred to as magnetic-anti-collagen III) was eluted through a PD10 column with a phosphate-buffered crude salt solution (PBS, pH 7.4).

Mu.l each of the magnetic-anti collagen III suspensions was diluted with 390. mu.l of a phosphate buffered crude salt solution (pH7.4) additionally containing 0.1% PBST, and the suspensions were filled into three sampling tubes made of polyacrylic acid, each tube having a volume of 500. mu.l, 100. mu.l of an aqueous solution of human serum albumin (1mg albumin/ml) was added to the first sampling tube (sample 1), 100. mu.l of a PBST solution of collagen V (1. mu.g collagen V/ml) was added to the second sampling tube (sample 2), and 100. mu.l of a PBST solution of collagen III (1. mu.g collagen II/ml) was added to the third sampling tube (sample 3). 200 seconds after the addition of the protein solution, the samples were magnetized (2mT) according to the measurement schedule outlined in FIG. 1, and after switching off the magnetic field for 20 milliseconds, the magnetic relaxation in each tube was determined starting from 1 second. In samples 1 and 2, the disappearing magnetic field was not detected within the limits of measurement reliability, while in sample 3, the disappearing magnetic field was detected. After the coupons were emptied and rinsed three times with each 500 μ l PBST, the measurements were repeated, at which time the disappearing magnetic field was not detected in all coupons within the limits of measurement confidence. Example 6

Mu.g of avidin was dissolved in 500. mu.l of 0.1M sodium bicarbonate solution, 1ml of a dextran-coated magnetite suspension having a particle size of about 404nm and containing 1mol of Fe/l was buffered with 0.1M sodium bicarbonate by means of a Sephadet column (Pharmacia PD10), 0.5ml of a solution containing 10mmol of sodium periodate was added to the suspension, the solution was allowed to stand in the dark for 2 hours, then eluted with 0.M sodium bicarbonate solution by means of PD10, the above-mentioned avidin solution was added to the suspension, the mixture was allowed to stand in the dark for 3 hours at 4 ℃ and 5mg of NaBH was added₄The mixture was allowed to stand in the dark for 8 hours at 4 ℃ with a little swirling, and then the magnetite-labeled avidin (bottom) was eluted by passing a phosphate-buffered crude salt solution (PBS, pH7.4) through a PD10 columnReferred to herein as magnetic avidin).

1mg of bovine serum albumin was coupled with biotin-N-hydroxysuccinimide (hereinafter referred to as biotin albumin) and diluted with PBS to a concentration of 1. mu.g/ml.

The above 1ml biotin albumin dilution was incubated for 3 hours at room temperature in a polystyrene sampling tube, and then the liquid phase was discarded and washed with a solution containing 0.1% Tween^(R)20(PBS) in phosphate buffered crude salt solution the sample tube was rinsed three times, 5. mu.l of the above-described magnetic avidin was added to the sample, incubated at room temperature for 1 hour, and the sample was magnetized in a magnetically shielded chamber under a superconducting quantum interference device in a magnetic field of 2mT 4cm intensity (see FIG. 1). Relaxation measurements were taken within 100 seconds 400 milliseconds after switching off the field and the disappearing field was measured in the sample.

1ml of bovine serum albumin dilution in PBS (0.1mg/ml) in a polystyrene sampling tube was incubated for 3 hours at room temperature, and then the liquid phase was discarded and washed with a solution containing 0.1% Tween^(R)20(PBST) phosphate buffered crude salt solution the sampling tube was rinsed three times, 5. mu.l of the above described magnetic avidin was added to the sample, incubated at room temperature for 1 hour, and the sample was magnetized in a magnetic shielding chamber (see FIG. 1) under a superconducting quantum interference device at a strength of 2mT 4 cm. The relaxation prediction was carried out within 100 seconds 400 milliseconds after the magnetic field was turned off, and the vanishing magnetic field was not detected in the sample within the limit of the measurement reliability.

Claims (42)

1. Method for the qualitative and/or quantitative detection of analytes in a liquid and/or solid phase, characterized in that ferromagnetic or ferrimagnetic micelles are used as magnetic labels to be identified in an immunoassay or other binding assay, the relaxation of their magnetization being determined as a measurement variable.

2. Method for the quantitative detection of analytes in liquid and solid phases by means of magnetic relaxation measurements, wherein ferromagnetic or ferrimagnetic micelles are used as magnetic labels to be identified in immunoassays or other binding assays, their Brownian relaxation proceeding faster than nelian relaxation under the measurement conditions.

3. Method for the quantitative detection of analytes in liquid and solid phases by measuring the complex susceptibility as a function of frequency, wherein ferromagnetic or ferrimagnetic micelles are used as magnetic labels to be identified in immunoassays or other binding assays, whose Brownian relaxation proceeds faster than nelian relaxation under the measuring conditions.

4. Method for the quantitative detection of analytes in liquid and solid phases, in which a structure-specific substance which binds to the analyte is first of all

i) Is marked by ferromagnetic or ferrimagnetic colloidal particles and then

ii) applying these labelled structure-specific substances to the liquid or immobilized sample to be measured, magnetizing the sample to be measured by means of an externally supplied magnetic field, and measuring the relaxation of the magnetization of the magnetic labels by means of the magnetic field receptors after the external magnetic field has been switched off.

5. Method for the quantitative detection of analytes in liquid and solid phases, in which the analytes are first of all

i) Is marked by ferromagnetic or ferrimagnetic colloidal particles and then

ii) these magnetically labelled analytes are applied to the liquid or immobilized sample to be measured, a substance is added thereto which specifically binds to the analyte, the sample to be measured is magnetized by means of an externally supplied magnetic field, and after the external magnetic field has been switched off, the relaxation of the magnetization of the magnetic labels is measured by means of magnetic field receptors.

6. Method for the quantitative detection of analytes bound to a solid phase, in which a structure-specific substance bound to the analyte is first of all detected

i) Labelled with ferromagnetic or ferrimagnetic micelles which relax over the measuring time, whereby said ferromagnetic or ferrimagnetic micelles are selected in such a way that their Brownian relaxation times are shorter than the Neelian relaxation times under the measuring conditions, and then

ii) applying these magnetically labelled substances to the immobilised sample to be measured, the sample to be measured being magnetised by means of an externally supplied magnetic field, the relaxation of the magnetisation of the magnetic labels being measurable by means of a magnetic field sensor when the external magnetic field is switched off, whereby the different relaxation behaviour of the magnetic labels bound to the analyte relative to unbound magnetic labels is used for the analysis.

7. A method according to claim 6, wherein the analyte to be identified is labelled with ferromagnetic or ferrimagnetic micelles, which undergo relaxation over a measurement time range, the relaxation time of the Brownian relaxation being shorter than the relaxation time of the Neelian relaxation under the measurement conditions, and wherein a substance specifically binding to the analyte is added to the sample to be measured or is present immobilized on the sample.

8. Method for the quantitative detection of analytes present in a liquid phase, in which a structure-specific substance which binds to the analyte is first of all

i) Labelled with ferromagnetic or ferrimagnetic colloidal particles having a Brownian relaxation time shorter than the Neelian relaxation time under the measurement conditions, and then

ii) applying these magnetically labelled substances to the sample to be measured, magnetizing the sample to be measured by means of an externally supplied magnetic field, and measuring the relaxation of the magnetization of the magnetic labels by means of magnetic field receptors after the external magnetic field has been switched off, whereby the different relaxation behavior of the magnetic labels bound to the analyte relative to the unbound magnetic labels is used for the analysis.

9. A method according to claim 8, wherein the analyte to be identified is labelled with ferromagnetic or ferrimagnetic micelles, which have a shorter relaxation time for Brownian relaxation than for nelian relaxation under the measurement conditions, and wherein a substance specifically binding to the analyte is added to the sample to be measured.

10. A method according to claims 1 and 4 to 9, wherein for detection a measurement of the complex magnetic susceptibility as a function of frequency is used.

11. Method according to claims 1 to 10, wherein the structure-specific substances are antibodies, antibody fragments, biotin or substances specifically binding to biotin, such as avidin and streptavidin, agonists specifically binding to receptors or their antagonists, specific peptides and proteins, receptors, enzymes, enzyme substrates, nucleotides, ribonucleic acids, deoxyribonucleic acids, sugars or lipoproteins.

12. The method according to claim 11, wherein the agonist that binds to the receptor is a cytokine, a lymphokine, or an endothelin.

13. A method according to claims 11 and 12, wherein the binding constant of the structure-specific substance is in the range of 10⁵-10¹⁵(mol/l)^-1。

14. A method according to claims 11 and 12, wherein the binding constant of the structure-specific substance is in the range of 10⁷-10¹⁵(mol/l)^-1

15. The method according to claims 1 to 14, wherein superconducting quantum interference devices (SQUID), inductive coils, saturated magnetometers, giant magnetoresistive sensors or magnetoresistive transducers are used as magnetic field receptors.

16. A method according to claims 1 to 15, wherein two or more different analytes in a sample are determined.

17. A method according to claim 16, wherein two or more ferromagnetic or ferrimagnetic substances with distinguishable Brownian or Neelian relaxation times are used.

18. Compounds for the quantitative detection of analytes in liquid and solid phases by means of magnetic relaxation measurements or by means of measurements with complex magnetic susceptibility as a function of frequency, wherein they consist of ferromagnetic or ferrimagnetic micelles bound to a structure-specific substance or to a substance to be identified.

19. Compounds for the quantitative detection of analytes in liquid and solid phases by means of magnetic relaxation measurements or by means of measurements with complex susceptibility as a function of frequency, wherein they consist of ferromagnetic or ferrimagnetic micelles bound to a structure-specific substance or to be identified, whose Brownian relaxation under the measurement conditions proceeds faster than Neelian relaxation.

20. A method according to claims 1 to 17, wherein the ferromagnetic and ferrimagnetic substances have a particle size in the range of 1 to 400 nm.

21. A method according to claims 1 to 17, wherein the ferromagnetic and ferrimagnetic substances have a particle size in the range of 1 to 100 nm.

22. A method according to claims 1 to 17, wherein the ferromagnetic and ferrimagnetic substances are stabilized by having a shell made of oligo-or polysaccharides, proteins, peptides, nucleotides and/or lipids.

23. A compound according to claims 18 and 19, wherein the particle size of the ferromagnetic and ferrimagnetic substances is in the range of 1 to 400 nm.

24. A compound according to claims 18 and 19, wherein the ferromagnetic and ferrimagnetic substances have a particle size in the range of 1 to 100 nm.

25. The compound according to claims 18, 19, 23 and 24, wherein the ferromagnetic and ferrimagnetic substances are stabilized by having a shell made of oligo-or polysaccharides, proteins, peptides, nucleotides, surfactants, polymers and/or lipids.

26. Compound according to claims 18, 19, 23 and 24, wherein the structure-specific substances are antibodies, antibody fragments, biotin or substances binding to organisms such as avidin and streptavidin, agonists binding specifically to receptors or their antagonists, specific peptides and proteins, receptors, enzymes, enzyme substrates, nucleotides, ribonucleic acids, deoxyribonucleic acids, sugars or lipoproteins.

27. Use of the method according to claims 1-17 and 20 to 22 for fertility, histocompatibility, allergy, infectious disease, hygiene, genetics, virology, bacteriology, toxicology, environmental analysis and medical diagnosis.

28. Use of a compound according to claims 18, 19, 23 and 24 for fertility, histocompatibility, allergy, infectious disease, hygiene, genetics, virology, bacteriology, toxicology, environmental analysis and medical diagnosis.

29. Use of a combination of ferromagnetic and ferrimagnetic substances with structure-specific substances or a combination of ferromagnetic or ferrimagnetic substances with an analyte to be identified in a method according to claims 1-17 and 20 to 22.

30. Method for detecting ferromagnetic or ferrimagnetic micelles introduced into the human body by means of magnetic relaxation, wherein the Neelian relaxation of the magnetization of these particles is measured after switching off the magnetic field.

31. Immunomagnetic recording method, wherein the structure-specific substance is first

i) Is marked by ferromagnetic or ferrimagnetic colloidal particles and then

ii) these labelled structure-specific substances are introduced into a living organism, the organism under investigation is magnetized by means of an externally supplied magnetic field, and the relaxation of the magnetization of the magnetic labels is measured by means of magnetic field receptors after the external magnetic field has been switched off.

32. The method according to claims 30 and 31, wherein the structure specific substance is an antibody, an antibody fragment, biotin or a substance specifically binding to biotin, such as avidin or streptavidin, an agonist specifically binding to a receptor or an antagonist thereof, a specific peptide and protein, a receptor, an enzyme substrate, a nucleotide, a ribonucleic acid, a deoxyribonucleic acid, a carbohydrate or a lipoprotein.

33. The method of claim 32, wherein the receptor-binding agonist is a cytokine, lymphokine, or endothelin.

34. The method according to claims 32 and 33, wherein the binding constant of the structure-specific substance ranges from 10⁵-10¹⁵(mol/l)^-1。

35. The method according to claims 32 and 33, wherein the binding constant range of the structure-specific substance is10⁷-10¹⁵(mol/l)^-1。

36. A method according to claims 30 to 35, wherein superconducting quantum interference devices (SQUID), inductive coils, saturated magnetometers, giant magnetoresistive sensors or magnetoresistive transducers are used as magnetic field receptors.

37. A method as claimed in claims 30 to 36, wherein relaxation measurements are replaced by measurements of complex magnetic susceptibility as a function of frequency.

38. Use of a compound according to claims 17 and 21 to 24 in a method according to claims 30 to 37.

39. Use of a ferromagnetic or ferrimagnetic substance in combination with a structure-specific substance in a method according to claims 30 to 37.

40. Compounds for use in the method according to claims 30-37, wherein they consist of biodegradable ferromagnetic or ferrimagnetic substances in combination with structure specific substances.

41. Compounds for use in the method according to claims 30-37, wherein they consist of biodegradable ferromagnetic or ferrimagnetic substances bound to structure specific substances, the Brownian relaxation time of the binding of said ferromagnetic or ferrimagnetic substances to structure specific substances in body fluids being shorter than the Neelian relaxation time at body temperature.

42. A compound according to claim 41, wherein the ferromagnetic or ferrimagnetic substance is biodegradable iron oxide.