EP2618937A1

EP2618937A1 - Magnetic cell detection

Info

Publication number: EP2618937A1
Application number: EP11779154.1A
Authority: EP
Inventors: Oliver Hayden; Michael Johannes Helou; Sandro Francesco Tedde
Original assignee: Siemens AG
Current assignee: Siemens AG
Priority date: 2010-11-03
Filing date: 2011-10-28
Publication date: 2013-07-31
Also published as: US20130224762A1; WO2012059414A1; DE102010043276A1; CN103201040A

Abstract

The invention relates to magnetic cell detection and the specific labeling of cells. The cell type to be detected is labeled, magnetic labels being bound to epitopes of a first cell-specific epitope type via antibodies of a first antibody type. Additionally, second/further magnetic labels are bound to epitopes of a second cell-specific epitope type on the cells via antibodies of a second antibody type, or the magnetic labels are bound to the antibodies of the first antibody type via antibodies of a fourth antibody type and said antibodies of the first antibody type are bound to the epitopes of the first cell-specific epitope type on the cells.

Description

description

Magnetic cell detection

The present invention relates to the magnetic Zelldetek ^¬ tion, for which the cells are marked to be detected by means of magnetic marker.

In the field of cell detection, flow measurements are known. However, these can be based on magnetic detection but more often based on optical measurement methods, such as scattered light or fluorescence spectroscopy. On the other hand, assay methods are known in which a reaction takes place in order to detect a specific substance. For flow cytometry as well as for chemical detection methods, various labeling methods are known. Fluorescent markers or antigens to be detected are bound by antibodies to the cells to be detected. In contrast to immunomagnetic detection, as described, for example, by Mujika et al., Phys. Stat. Sol. (a) 205, No. 6, 1478-1483 (2008) "Microsystem for the immunomagnetic detection of Escherichia coli 0157: H7" is known, in which a selection of the substance to de ^¬ tektierenden by immobilization on a functionalized surface takes place, provide magnetic

Flow measurements higher demands on the measuring signal. In addition to the magnetically labeled cells, unbound markers and agglomerates of unbound markers also flow in the flow and trigger a signal via the sensor. Also, the uniqueness of the labeling of a cell type is not ^always given. Especially cell types such as tumor cells have a high variance of the epitope concentration on the cell surfaces. This results in a whole spectrum of MR signals for one cell type. A magnetic Durchflusszyto ^¬ geometry has therefore only been performed with cells that have a high Epitopendichte on the cell surface. In all other cases, it was necessary to resort to optical flow measurements relating to the measurement apparatus Structure, however, are disadvantageous to magnetic Zelldetekti on.

For a sufficiently high signal-to-noise ratio in a detection with magnetoresistive sensors out of a laminar flow, the cells to be detected must have a high magnetic moment through the mark. The magnetic moment of a single cell is also decisive ^¬ DEND for the in-situ concentration and cell guide by an external magnetic field.

It is an object of the present invention to provide a method for the magnetic detection and labeling of cells be ^¬ affects a sufficiently high magnetic moment of the cells. Furthermore, a suitable measuring device should be specified.

The object is achieved by a method according to claim 1 and by a device according to claim 12.

The method according to the invention comprises magnetic cell detection and a labeling step for the specific labeling of cells. The cells to be detected are associated with a cell type which has cell-specific epitopes on the surface of the cells. The magnetic markers are attached via antibodies to the epitopes on the surface of the cells. For specific labeling, the magnetic markers are bound via antibodies of a first antibody type, which bind to epitopes of a first cell-specific epitope type. In addition, more magnetic

Label attached via antibodies of a second antibody type to epitopes of a second cell-specific epitope type on the cells. Or the magnetic markers are bound to antibodies of the first antibody type via antibodies of a fourth antibody type and these in turn to the epitopes of the first cell-specific epitope type on the cells. These methods allow specific labeling of cells. By marking the magnetic moment is increased against ^¬ over other cells. In the case of additional magnetic markers, which are connected via a second type of antibody, the magnetic marker density around the cell he ^¬ höht what happens only for cells having a first and second Epitoptyp in combination. Such a marker accordingly excludes false-positive signals by other ^¬ cells, for example, have only the first or only the second epitope type. Their magnetic moment would therefore be much lower, since only the markers with the first or only the markers with the second antibody type can bind to these cells. Or, the selectivity of the label is increased by further matching via the combination of a fourth antibody type to the first antibody type. Ins ^¬ special is the first type of antibody, a hochspezifi ^¬ shear type of antibody that binds to the epitopes of the cell. This highly specific type of antibody ensures, for example, a very small number of incorrect connections. The magnetic label can then be made, for example, via a less specific fourth antibody type. The recognition of the first antibody type is less error-prone than the recognition of the cell-specific epitopes on the surface.

By means of the marking according to the invention, a calibration-free magnetic flow cytometry becomes possible. In an advantageous embodiment of the invention, the magnetic moment of the cells is increased in the method by addition of magnetic markers are linked via antibodies of a third antibody type to epitopes of a third cell-specific epitopept on the cells. This has the advantage partly to make a further selection against cells that also comprise epitopes of the first and second Epitoptyps in combi nation ^¬ and differ only by epitopes of a third Epitoptyps before to be detected cells. In addition, the magnetic moment of the cells is increased by the hö ^¬ here marker density on the cell surface. This may set ^¬ to a higher threshold for a positive signal.

In a further advantageous embodiment of the invention, in the method, the second and / or the third antibody type is chosen such that it does not bind to epitopes on a second cell type. Or the second and / or third type of antibody is chosen such that it only to epitopes Anbin ^¬ det which do not occur in the same concentration as in the de- tektierenden to cell type. Accordingly, a selection can take place via the choice of antibodies, which ensures a nearly exclusive detection of cells of the cell type to be detected.

In particular, an immunomagnetic marker with meh ^¬ reren antibodies against different epitopes on a cell can be done to prevent cross-selectivity to other cells with lower epitope density.

In an advantageous embodiment of the invention, in the method, the magnetic moment of the cells is increased by attaching additional magnetic markers via antibodies of a fourth antibody type to the magnetic markers of the first labeling step in a second labeling step. The marker density can therefore also be increased by a second marking of the magnetic beads already attached to the cell. The antibodies of the fourth type of antibody have to specifically bind the magnetic bead of ^¬ ers th mark. In particular, the additional magnetic markers are different from the magnetic markers of the first marking step. Thus, the Markerkon ^¬ concentration is significantly increased around the cell, and thus the magnetic moment of the cell. Thus, the magnetoresistive signal caused by the single cell is increased and ensures a high signal-to-noise ratio. b

In the method of magnetic cell detection, the specific magnetically-labeled cells are expediently detected by a magnetoresistance change. In particular, the high magnetic moment of the cells ensures that a lower threshold value for a magnetoresistance change can be set so high that the signal-to-noise ratio is at least two. In particular, the signal-to-noise ratio is at least three.

In a further advantageous embodiment of the invention, an upper threshold value for a magnetoresistance change is set so low in the method that a single cell detection takes place. This has the advantage that higher magnetoresistive change values are not assigned to individual cells but to agglomerates and are excluded as false-positive ^signals .

In a further advantageous embodiment of the invention, the magnetic detection by means of flow cytometry 20 takes place. In particular, the specifically labeled cells are detected during the flow via a sensor. In ^¬ example, the cells are guided in a laminar flow. Flow cytometry, for example, has the advantage of a high sample throughput.

2 B

In a further advantageous embodiment of the invention, magnetic markers whose diameter is less than 200 nm are used in the method. The small size of the magnetic markers, also called magnetic beads, has the advantage that it does not come to agglomerates, which arise due to the attachment of a variety of antibodies to a single large magnetic marker. The small magnetic markers with diameters of less than 200 nm can be arranged individually around the cells.

3b

In a further advantageous embodiment of the invention in the process superparamagnetic magnetic Mar ^¬ ker be used. These are in terms of comprehensibility over magnetoresistive components in the advantage over, for example, ferromagnetic beads.

In a further advantageous embodiment of the invention, in the method, the cells are guided in a gradient magnetic field and thereby enriched at the sensor. By means of a gradient magnetic field, the magnetically marked cells can be targeted via the sensor. The device according to the invention for magnetic cell detection has a sensor and an evaluation unit. The evaluation unit and the sensor are designed so that a spectrum of magnetoresistance changes can be recorded. In this case, a lower threshold value for a magnetoresistance change is stored in the evaluation unit, which is so high that the signal-to-noise ratio is at least three. Additionally or alternatively, an upper threshold for a magnetoresistance change is deposited, which is so low that a single-cell detection is feasible. The combination of both threshold values is particularly advantageous for a high specificity of the measurement.

A positive MR signal from an individual cell must be able to stand out against background effects, such as unbound markers, aggregated markers or other magnetic interference fields. An individual cell must therefore achieve a magnetoresistance change above a threshold value in order to hide such background effects. In addition, an upper threshold may be set to exclude that, for example labeled cell aggregates or larger aggregates are counted by like ^¬ netic markers.

In an advantageous embodiment of the invention, the device comprises a flow-guiding system, with which the device is suitable for magnetic flow cytometry. Furthermore, the device comprises means for generating a gradient magnetic field in the flow-guiding system. Magnetically labeled cells are thereby immobilized in the gradient mag- can be enriched on the sensor. Accordingly, with ^¬ tel are designed for generating the gradient magnetic field. The means for generating the gradient magnetic field may in particular be ferromagnetic strips.

Embodiments of the present invention will be described in an exemplary manner with reference to ^FIGS . 1 to 8 of the attached drawing. FIG. 1 shows a simple magnetic marking of a

Cell A.

FIG. 2 shows a double magnetic marking of one

Cell A.

FIG. 3 shows a diagram with the distribution of the cells A with regard to the magnetoresistance change caused by them.

FIG. 4 shows a magnetically marked cell B / C.

FIG. 5 shows a diagram with the distributions of the cells A,

B and C with regard to the magnetoresistance changes they cause.

FIG. 6 shows a double magnetically marked cell A with different magnetic markers.

FIG. 7 shows a specifically labeled cell A.

FIG. 8 shows a specifically labeled cell A with a double labeling by different magnetic

Markers.

FIGS. 1, 2, 4 and 6 to 8 each show schematic representations of cells A, B, C which have epitopes 11, 12, 13 on the surface. The cell surface is represented by ei ^¬ nen large circle. The epitopes 11, 12, 13, which are shown as small circles, bind antibodies 21, 22, 23, 24, which are shown Y-shaped in the schematic drawings. In each case, one of the ends binds to an epitope 11, 12, 13 on the cell surface and another end to a magnetic marker Ml, M2. The magnetic markers M1, M2 are shown as larger circles than the epitopes 11, 12, 13. However, the magnetic markers M1, M2 much smaller diameter than the cells A, B, C. Although the figures are not to scale, a correct size ratio of the magnetic markers M1, M2 to the cells A, B, C is shown. In the case of larger magnetic markers M1, M2, aggregation and cross-linking would occur. That is to say, a plurality of antibodies 21, 22, 23, 24 would be arranged around and bind to a magnetic marker M1, M2 and thus would no longer be labeled the individual cell A, B, C, but an agglomerate of antibodies 21, 22, 23 , 24, cells A, B, C and magnetic markers M1, M2 around a very large magnetic marker.

FIGS. 3 and 5 each show a diagram in which the number of cells N that cause this MR signal is plotted over the magnetoresistance change MR. FIG. 3 shows the distribution of cells A, in FIG. 5 the distributions of cells A, B and C are shown. The cells A B call a much higher MR signal produced when the cells and these higher than the cells C. But there are also each overlapping areas in which can not be un ^¬ terschieden which cell type A, B, C, the MR signal causes. Therefore, thresholds T are ge sets ^¬ for the MR signal based on empirical values or known distributions. Then a distinction is made on the basis of the threshold value T in positive and negative events. In the diagram in FIG. 3, an upper T ₂ and a lower ΊΊ threshold value are set. Above the lower threshold value ΊΊ an MR signal is evaluated as a positive signal. Below the upper threshold T ₂ one starts from a single cell detection. Agglomerates would cause a much higher MR signal.

Figure 1 shows the simplest form of the magneti ^¬ rule mark. A cell A has a multiplicity of epitopes 11, 12, 13 on the cell surface. The number of epitopes may comprise about 500 epitopes on a cell. When labeling by means of an antibody 21, 22, 23, 24, which binds to a specific type of epitope 11, 12, 13, about 80% of the epitopes are covered. Ie there are free characteristics Rist epitopes 11, 12, 13, which could still receive a magnetic marker M1, M2 via a specific antibody 21, 22, 23, 24. Such a mark is not sufficient for a useful signal. That is, the markers M1, M2 connected to cells A, B, C by only one antibody type 21-24 do not increase the magnetic moment of cell A, B, C to such an extent that a sufficiently high MR signal is generated. That is, the ratio of signal to noise, for example, by unge ^¬ bound magnetic markers Ml, M2 is not sufficient for a clear positive signal. That is, the sensitivity of the Mar ^¬ kierung is too low. In addition, the selectivity of the label is too low. A cell B differs from egg ^¬ ner cell A by the number and type of epitopes 11, 12, 13 on the surface. But it can also happen that an antibody 21-24 binds incorrectly to epitopes 11-13. It also happens that cells A, B differ, but have a high degree of agreement, especially in the epitope 11 to be marked. For example, cell A and cell B have only one common epitope type 11, but at approximately the same concentration on the cell surface. Thus, cell B is similarly labeled with antibody 21 by mark M1 and is indistinguishable from cell A in the measurement. FIG. 2, in turn, shows a cell A, which, however, is now marked by several different antibody types 21, 22, 23. There are magnetic markers Ml with different antibodies 21, 22, 23 available. These bind to epitopes 11, 12, 13 on the cell surface. This first increases the magnetic moment of cell A by doubling or tripling the number of markers M1 around cell A, ie, increasing the sensitivity. Thus, as shown in FIG. 5, the MR signal of the cells A can increase significantly relative to the MR signals of the cells B or C and thus stand out from the signals of the cells B and C by a threshold limit T. In addition, therefore, an increased selectivity is achieved. Although cells B and C may have a similar number of the first epitope have 11 type. However, the second and third labeled epitope types 12, 13 do not have cells B and C in much lower concentration than cell A. Figure 6 again shows a cell A with epitopes 11, 12, 13 on the cell surface containing the cell A differ from cell B, C un ^¬. The magnetic marking is carried out in a first step via the attachment of magnetic markers Ml via antibodies 21 to the epitopes 11. A second mark is in this case not a second epitope type, but additional markers M2 with antibodies 24, which in turn to the magnetic Connect Marker Ml. Thus, the magnetic moment of the cell A is increased. Selectivity occurs via the antibody-epitope pair 21-11. The attachment of additional magnetic M2 markers is much better than using larger magnetic markers. Increasing the marker diameter or volume aggravates agglomeration effects. On a large magnetic marker over 200 nm in diameter, several antibodies bind, which then cross-link cells A, B, C and magnetic markers M1, M2.

Ideally, superparamagnetic particles having a diameter <200 nm are used for the magnetic marking. Figure 7 shows another way to increase the selectivity of the label. First, the epitopes of a first type of epitope 11 on the surface of the cell A are labeled with suitable antibodies 21. Antibodies 24 in turn bind to these antibodies 21. These antibodies 24 are linked to magnetic markers M2. Although this does not increase the magnetic moment in contrast to a marking as in FIG. 1, the connection via the combination of two antibodies 21, 24 is much more specific, which increases the selectivity of the MR measurement. To increase the sensitivity, ie to achieve a better signal-to-noise ratio, a kind of sandwich marking can again be effected, as already shown in FIG. This combination is shown in FIG. This is for a high selectivity the first magnetic label on marker M2 with antibodies 24 to the specific antibody 21 performed. The magnetic moment is then increased by a second label by label Ml via antibodies 24 which bind to the magnetic markers M2. Preferably, the labeling is carried out in two successive labeling steps.

An exemplary flow of magnetic flow cytometry will now be described. This takes place in particular in a microfluidics. Three steps are essential for efficient measurement:

1. The in-situ accumulation of the magnetically labeled cells A on the sensor,

2. the cell guidance, in particular the guidance of the magnetically marked cells A in a flow over the sensor and 3. the detection of the magnetically marked cells A by means of a magnetoresistive component. In particular, the cell transport, ie the cell flow through the microfluidics, takes place in a laminar flow. The magnetically marked cells A experience a force in an external magnetic gradient field. This gradient is aligned so that the cells A on the sensor, which is mounted, for example on or in the duct wall are attached demonstrates ^¬. For in situ enrichment and cell guidance, it is necessary that the magnetically labeled cells A have a sufficiently high magnetic moment. Only then can they be influenced and steered in the external magnetic gradient field. The external magnetic field is, for example Gra ^¬ is 100 mT or an amount of this magnitude. For the detection of the cells A having a magnetoresistive component a high stray field of the magnetic table ^¬ A labeled cells is necessary. Only if the magnetically labeled cells A sufficiently high leakage field aufwei ^¬ sen, they cause a sufficiently large change in resistance of MR in the component. By means of the method according to the invention, cells A, B, C can be labeled independently of the epitope concentration per cell surface in such a way that magnetic flow cytometry can be carried out. The epitope concentration per cell surface is typically 1000 or more. In the method, in particular superparamagnetic Parti ^¬ kel be used for labeling and so densely arranged on the cell surface, the magnetic moment, regardless of cell type and its Epitopendichte, for magnetic throughput is suitable cytometry. Due to the high marker density and therefore high magnetoresistive signal MR a suffi ^¬ accordingly high threshold T can be set for a positive signal to background effects exclude that may otherwise be recognized as false-positive signals. Insbeson ^¬ particular, a combined immunomagnetic labeling he ^¬ follow. As a result, cell enrichment is also possible in media such as whole blood and cell guidance in a gradient field. In particular, the gradient field can be generated by ferromagnetic strips, which can be arranged around the microfluidics.

Claims

claims

A method for magnetic cell detection comprising a labeling step for the specific labeling of cells (A) of a cell type to be detected, in which magnetic

Marker (M1, M2) via antibodies (21) of a first antibody type to epitopes (11) of a first cell-specific epitope type are connected, characterized in that additionally

second or further magnetic markers (M1, M2) are bound via antibodies (22) of a second antibody type to epitopes (12) of a second cell-specific epitope type on the cells (A) or

- The magnetic markers (M1, M2) via antibodies (24) of a fourth antibody type to the antibodies (21) of the first antibody type and these in turn to the epitopes (11) of the first cell-specific epitope type on the cells (A) are attached.

2. Method according to claim 1, in which the magnetic moment of the cells (A) is increased by additionally applying magnetic markers (M1, M2) via antibodies (23) of a third antibody type to epitopes (13) of a third cell-specific epitope type on the cells ( A).

A method according to any one of the preceding claims, wherein the second and / or the third antibody type (22,23) is chosen so that it does not bind to epitopes on a second cell type (B / C) or only to epitopes which are not in the same concentration as on the one to be detected

Cell type (A) occur.

4. The method according to any one of the preceding claims, wherein the magnetic moment of the cells (A) is increased by in a second marking step additional magnetic ^¬ cal marker (M2, M1), in particular of the magnetic markers (M1, M2) of the first various labeling step ^¬ dene, via antibodies (24) of a fourth type of antibody be attached to the magnetic markers (M1, M2) of the first marking step.

Method according to one of the preceding claims, in which the specifically magnetically marked cells (A) are detected via a magnetoresistance change (MR).

6. The method according to any one of the preceding claims, wherein a lower threshold value (Tl) for a magnetoresistance change ^¬ (MR) is set so high that the signal-to-noise ratio is at least 3.

7. The method according to any one of the preceding claims, wherein an upper threshold value (T2) for a magnetoresistance change ^¬ (MR) is set so low that a single cell detection takes place.

8. The method according to any one of the preceding claims for magnetic flow cytometry, in which the specific labeled cells are detected during flow via a sensor, in particular in a laminar flow.

9. Method according to one of the preceding claims, in which magnetic markers (M1, M2) with diameters of less than 200 nm are used.

A method according to any one of the preceding claims, in which superparamagnetic magnetic markers (M1, M2) are used.

11. The method according to any one of the preceding claims, in which the cells are guided in a gradient magnetic field and enriched at the sensor.

Device for magnetic cell detection with a sensor and an evaluation unit, which are designed to record a spectrum of magnetoresistance changes (MR), wherein in the evaluation unit a lower threshold value (Tl) is deposited for a magnetoresistance change (MR) which is so high that the signal-to-noise ratio is at least 3 and / or an upper threshold value (T2) for a magnetoresistance change (MR) is stored, which is so low that a single cell detection is feasible.

13. Apparatus according to claim 12 for magnetic flow cytometry with a flow-guiding system and means for generating a gradient magnetic field in the flow-guiding system, wherein the means are ausgestal ^¬ tet that magnetically marked cells can be enriched by the Gra ^¬ dientenmagnetfeld the sensor ,

Apparatus according to claim 13, wherein the means for generating the gradient magnetic field are ferromagnetic strips.