Abstract:
A magnetic immunoassay system with a mechanism for compensating the direct current residual magnetic field in the vicinity of the specimen measurement position, in a direction perpendicular to the magnetic marker direction of magnetization for the measurement target. This invention reduces the effects of the magnetic field emitted from the unbound magnetic marker due to the residual magnetic field in the specimen solution and detects with high sensitivity the signal of the bound target magnetic marker. The magnetic field at the measurement position is regulated so as to intersect the direction of magnetization of the magnetic marker for the measurement target, in order to make the magnetization direction of the magnetic marker that is unbound due to residual magnetism or remanence in the sample solution, intersect the magnetization direction of the magnetic marker for the measurement target. The signal of the bound target magnetic marker can be therefore measured with high sensitivity since it is isolated from the unbound magnetic marker signal.

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
     This application is a Divisional application of U.S. application Ser. No. 11/699,375 filed Jan. 30, 2007 now abandoned. Priority is claimed based on U.S. application Ser. No. 11/699,375 filed Jan. 30, 2007, which claims the priority of Japanese Patent Application No. 2006-063776 filed on Mar. 9, 2006, all of which is incorporated by reference. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to a system for analyzing different types of protein (immunoglobulin, tumor markers, and hormones, etc.), disease-causing germs, cancer cells, DNA and environmental toxins by utilizing magnetic nanoparticles, and relates in particular to a magnetic immunoassay system utilizing a superconducting quantum interference device (SQUID). 
     BACKGROUND OF THE INVENTION 
     Demands are increasing in recent years for high sensitivity in the examination of environmental toxins, DNA, cancer cells and disease-causing germs of different types by their immuno-reaction. This demand has spurred intensive development of immunoassay systems. The general method for making an immunoassay is an optical method that labels an antibody (for detection) selectively bound to an antigen for detection, by optically marking it with an optical fluorescent enzyme, detecting the reaction of the bound antibody-antigen as an optical signal from the optical marker to find the type and quantity of the antigen. However, this optical method does not have adequate detection sensitivity and requires a process for washing off the unbound optical marker (BF separation). 
     A magnetic method for detecting antigen-antibody reactions has been proposed in recent years that utilizes a magnetic nanoparticles and a SQUID magnetic sensor to achieve detection sensitivity surpassing the optical method. This magnetic method detects antibodies magnetically labeled with magnetic nanoparticles (hereafter called magnetic markers), by utilizing a SQUID magnetic sensor possessing extremely high sensitivity. 
     Methods for detecting magnetic markers are based on:
         (1) measuring the susceptibility,   (2) measuring the magnetic relaxation   (3) measuring the residual magnetism (remanence).       

     The above methods (1) through (3) are described next. 
     (1) Method for Measuring the Susceptibility 
     In this method, a direct current magnetic field is applied to magnetize the magnetic marker from a direction intersecting the SQUID magnetic sensor flux detection direction, and measures the change in the magnetic field occurring due to magnetic marker movement within flux detection range of the magnetic sensor (for example, patent document 1). Another variation of this method applies an alternating current magnetic field to the magnetic marker, and utilizes the SQUID magnetic sensor to detect that signal as an antigen-antibody reaction (for example, patent document 2). 
     (2) Method for Measuring Magnetic Relaxation 
     In this method, the magnetic relaxation is measured up to one second immediately after a 1 mT pulse magnetic field was applied to a magnetic marker. The measurement is made in the solution where the unbound magnetic marker is present, and the bound magnetic marker is detected (for example, in non-patent document 1 and non-patent document 2). Methods that quantitatively detects the sample in the liquid phase or solid phase by measuring the magnetic relaxation, and a method that performs magnetic relaxation measurement by utilizing chemical compounds and also utilizes them for analysis and magnetography have also been reported (for example, patent document 3). 
     (3) Method for Measuring Remanence 
     Relaxation of remanence in the magnetic nanoparticles does not occur when the size of magnetic nanoparticles becomes large. This method applies a magnetic field of approximately 0.1 T to the magnetic marker at a location separated from the SQUID magnetic sensor to generate remanence in the magnetic marker. A plate holding the sample is moved to the SQUID magnetic sensor for measuring the residual magnetism (for example, patent document 4, non-patent document 4). 
     In typical immunoassay tests, the measurement is made after B/F (bound free) separation (washing process) to eliminate the unbound magnetic markers. However, the unbound nanomagnetic particles are capable of moving randomly in the solution due to Brownian motion so that measurement can be made without a B/F separation (for example, non-patent document 3). Eliminating the B/F separation has the advantage that the testing time is short and the system can be simplified. 
     A specific example relating a method for magnetically detecting the antigen-antibody interaction is described next (for example, non-patent document 4). 
       FIG. 1A  through  FIG. 1E  are drawings of the procedure in a typical method for measuring the residual magnetism or remanence in a magnetic immunoassay method of the related art without a B/F separation and utilizing the antigen-antibody interaction. 
     In a pre-process as shown in  FIG. 1A , an antibody  2 , which is bound to an antigen serving as the detection target, is fixed in the bottom  1   a  of the sample container  1 . Hereafter this is referred to as a capturing antibody. Next, a block reagent  3  is applied to prevent nonspecific adsorption onto the side walls, and this blocking reagent  3  covers the inner walls of the container. When a sample  5  containing a fixed amount of a substance for measurement is injected into the sample container  1  prepared in this way, an antigen  6  in the sample binds to the capturing antibody  2  via an antigen-antibody interaction as shown in  FIG. 1B . When a solution containing a magnetic marker for binding with the antigen  6  is next injected into the sample container, a portion of the magnetic marker  7   b  binds by an antigen-antibody interaction to an antigen  6   a  linked to the capturing antibody  2  as shown in  FIG. 1C . A portion of the marker  7   a  also binds to an antigen  6   b  in the solution that is not bound to the capturing antibody  2 . An unbound marker  7   c  not bound to an antigen, and a magnetic marker  7   b  bound to the antigen  6   b  in the solution, move randomly by Brownian motion. 
     When an external magnetic field is applied to the sample in this state, in the direction of the large arrow shown in  FIG. 1D , all of the magnetic markers are magnetized in the direction that the magnetic field is applied. The small arrows in the drawing show the direction of magnetization of each marker. When using magnetic markers containing magnetic nanoparticles that possess remanence (residual magnetism), the remanence still remains even when there is no longer a magnetic field. 
     However, when no magnetic field was applied as shown in  FIG. 1E , the unbound magnetic marker  7   c , and the magnetic marker  7   b  bound to the antigen  6   b  in the solution move randomly in the solution due to Brownian motion so that the direction of magnetization (direction of magnetic momentum) also becomes random, and the magnetic signals from these magnetic markers cancel each other out, so that normal magnetic signals cannot be generated. In other words, there is no need to eliminate the unbound magnetic markers in a washing process. Brownian motion however does not occur in the magnetic marker  7   a  bound to the antigen  6   a  bound to the capturing antibody  2  so that remanence is retained in the direction of magnetization even when no magnetic field was applied, and a magnetic signal is generated that is proportional to the amount of antigen  6   a  bound to the capturing antibody  2 . The amount of antigen  6   a  bound to the capturing antibody  2  is dependent on the amount of antigen contained in the sample so that the amount of an antigen to be investigated can be measured by detecting this magnetic signal with a high-sensitivity SQUID magnetic sensor. 
     In view of the fact that immunoassays using optical markers require a washing process (B/F separation) process, immunoassays using magnetic markers possess a large advantage since one characteristic feature is that no washing process is required. 
     [Patent document 1] JP-A No. 33455/2001 
     [Patent document 2] JP-A No. 133458/2001 
     [Patent document 3] JP-A No. 513551/1998 
     [Patent document 4] JP-A No. 257425/2005 
     [Patent document 5] JP-A No. 508031/1999 
     [Non-patent document 1] Y. R. Chemla, et al.: Proc. National Acad. Sciences of U.S.A. 97, 14268 (2000) 
     [Non-patent document 2] A. Haller, et al.: IEEE Trans. Appl. Supercond. 11, 1371 (2001) 
     [Non-patent document 3] R. Kotitz, et al.: IEEE Trans. Appl. Supercond. 7, 3678 (1997) 
     [Non-patent document 4] K. Enpuku, et al.: IEEE Trans. Appl. Supercond. 13, 371 (2003) 
     SUMMARY OF THE INVENTION 
     Performing immunoassay analysis possessing the advantage that remanence or magnetic relaxation is measured without a washing (B/F separation) process, requires that the direction of magnetic monemtum of each unbound magnetic marker within the solution be random by Brownian motion. The magnetic signals from the unbound magnetic marker  7   c  in this case cancel each other out so that no magnetic signals are detected from the solution ( FIG. 1E ). However, results from experiments conducted by the present inventors revealed that unbound magnetic markers emit magnetic signals from within the solution due to the unbound magnetic markers being aligned in the direction of a residual magnetic field within the magnetic shield section. The residual (magnetic) field within the periphery of the sensor and the sample could be reduced by magnetic shielding but completely reducing this magnetic field to zero was impossible. 
     The state of magnetic markers in the case where a magnetic field is completely blocked and sufficient Brownian motion is occurring, and the case where a residual magnetic field is present are shown in the drawings in  FIG. 2A  and  FIG. 2B . In the case as shown in  FIG. 2A  where the magnetic field is completely blocked, each nanoparticle is moving randomly due to Brownian motion so that the magnetism from each of the magnetic markers mutually cancel each other out so that no macro magnetic signal is generated. The arrow  10  shown in the vicinity of the magnetic marker  11 , indicates the direction of magnetic momentum retained by the respective markers  11 . However, in the residual magnetic field as shown in  FIG. 2B , a bias is applied in that direction so the movement is not completely random. The strength of this magnetic alignment varies with the size of the residual magnetic field but tends to array in the direction of the residual magnetic field. Macro magnetic signals are therefore generated in the direction of that residual magnetic field. The magnetic signals from these unbound magnetic markers become background signals (noise) causing a drop in the signal-to-noise (SN) ratio and an increase in the minimum detection limit when attempting to measure a fixed magnetic marker. 
     Even though the residual magnetic field can be lowered to the required level by utilizing a magnetic shield with a highly sealed multi-layer structure, the magnetic shield should ideally be as simple as possible in view of the need to lower equipment costs and provide operating features such as replacement of samples, etc. 
     Whereupon, an object of the present invention is to provide a method capable of easily eliminating the effects of residual magnetic fields that cannot be reduced by magnetic shields, and also to provide a high-sensitivity magnetic immunoassay system capable of sufficiently reducing the effect of signals from unbound markers. 
     The inventors perceived that the above described problems could be resolved by making the magnetization direction of the magnetic marker bound to a capturing antibody, and the magnetization direction of an unbound magnetic marker mutually intersect each other. 
     The magnetic signal waveform detected in the SQUID when a magnetic sample is made to pass under a SQUID magnetometer is described while referring to  FIG. 3 . The coil surface of the pickup coil is arranged along a flat x-y plane, and the magnetic sample passes in the x direction directly beneath the pickup coil parallel to the x-y plane. The x-y plane view and the x-z plane view in  FIG. 3  show the positional relation between the pickup coil and magnetic sample and the direction of movement of the magnetic material are shown. The same figure also shows the signal waveform, which is x position dependence due to the magnetic orientation of the magnetic sample. The (a), (b), (c) in this figure, show the case where the magnetic sample is respectively magnetized in the x direction, y direction, and z direction. 
     As the magnetic sample approaches the SQUID while magnetized in the x direction, the flux generated from the magnetic sample starts to bind to the pickup coil section of the squid from bottom to top so that the SQUID detects the flux, and the SQUID output signal changes. When the sample reaches the exact center of the pickup coil, the flux from the sample binds to the pickup coil from bottom to top but the SQUID output signal becomes zero since the same amount of return flux to the sample binds to the pickup coil from top to bottom at the same time. Moreover, as the sample moves farther away from the SQUID, the flux from the magnetic material binds from top to bottom to the SQUID pickup coil so that the output signal changes to the minus direction. The signal once again becomes zero as the sample moves even further away. Setting the x position above the sensor as the origin point, makes the waveform symmetrical around the origin point. The polarity of the magnetic signal varies according to the magnetic polarity and the polarity of the SQUID pickup coil so that the upward and downward directions of the waveform that is obtained might sometimes be reversed. 
     Next, when the magnetic sample is approaching the SQUID while the magnetic sample is magnetized in the y direction, the flux generated from the magnetic sample that entered the SQUID from one side, come out from the other side of the SQUID. In this case there is no regular magnetic signal bound to the SQUID, and the output signal is always zero. 
     Next, when the magnetic sample is approaching the SQUID while the magnetic sample is magnetized in the z direction, the flux generated from the magnetic sample starts to bind to the SQUID from top to bottom so that the SQUID detects the flux, and the SQUID output signal changes. When the sample moves to directly below the pickup coil, the flux binds to the pickup coil from bottom to top so that the SQUID output signal appears extremely large in the reverse direction. Moreover, when the sample moves away from the SQUID, the flux again binds to the SQUID from top to bottom so that an output signal is generated in the original direction. 
     As can readily be understood from viewing the magnetic signals for cases (a), (b), (c), the magnetic signal is symmetrical around the origin point utilizing the pickup coil center-point serving as the boundary in the (a) case. Therefore, after the signal was obtained, it (the signal) is divided up in the x direction and if the difference between each signal is found, then a signal can be obtained that is twice the amplitude of a signal on one side of the origin point. In the (c) case however, the signal varies symmetrically on an origin point along the vertical axis so that processing it in the same way yields a signal of zero. If the processing uses the sum of the signals, then the signal will have twice the amplitude in the (c) case, versus a signal of zero in the (a) case. 
     The desired signal can therefore be obtained by processing by adding or subtracting according to the magnetization direction of the signal to be measured. 
     Therefore if the captured magnetic marker to be measured is magnetized in the x or the z direction, and the magnetization direction of the unbound magnetic marker in the solution causing the noise is controlled to intersect the magnetization direction of the bound magnetic marker, then the waveforms that are obtained will be two waveforms with different symmetry, or in other words, the sum of the captured marker waveform and the waveform of the marker within the solution. The waveform of the captured marker can be easily obtained by utilizing the difference in symmetry between these measured waveforms. 
     The magnetic orientation of the magnetic marker in the solution can be controlled so as to intersect the magnetic direction of the bound magnetic marker, by setting the residual magnetic field component along the magnetic moment of the bound magnetic marker to zero. Another method is to apply a magnetic field in a direction intersecting the magnetic direction of the marker, to control the magnetic direction of the unbound magnetic marker within the solution so as to intersect the magnetic direction of the fixed magnetic marker. As can be seen from the example shown in  FIG. 3 , if the magnetic direction of the unbound magnetic marker within the solution is controlled in the y direction, then there is no need to isolate the (magnetic) signals since the SQUID cannot detect magnetic signals from a magnetic marker magnetized in the y direction. Yet another method is to align the magnetic direction of the unbound marker within the solution to intersect the magnetic direction of the fixed magnetic marker even when using a magnetic shield structure where the direction of the residual magnetic field within the shield intersects the direction of magnetization of the fixed magnetic marker. A combination of these methods may also be used. 
     In  FIG. 4 , a SQUID gradiometer containing a first order planar gradiometric pickup coil is utilized instead of a SQUID magnetometer.  FIG. 4  shows the signal waveforms detected in the SQUID when a magnetic sample has passed over it. The x-z planar surface is omitted in  FIG. 4 . The coil surface of the gradiometric pickup coil is also arrayed along the x-y planar surface in  FIG. 4 , and the magnetic sample passes along the x direction, directly beneath the pickup coil parallel with the x-y planar surface. The direction perpendicular to the pickup coil is z, the sample direction of movement is x, and here y is a direction perpendicular to x and z. The (a), (b), and (c) in  FIG. 4  show the case where are each the same as (a), (b), and (c) in  FIG. 3 , and the case is shown where the magnetic sample is magnetized in the x, y, and z directions. The differential direction of the gradiometric pickup coil is the x direction, and the solid line and the dotted line of the pickup coil in the drawing show that the pickup coil polarity is reversed. Unlike  FIG. 3 , the signal waveforms detected in the SQUID when the magnetic sample is magnetized in the x direction, y direction and the z direction are bilaterally symmetrical, zero and are central symmetrical. Just as with the magnetometer shown in  FIG. 3 , the signal waveforms detected in the SQUID from the sample magnetized in the z direction, and the sample magnetized in the x direction possess different symmetry so that the signals can be easily isolated. The magnetic signals from the sample magnetized in the y direction, as expected, cannot be detected so there is no need to isolate the signals. 
     Therefore, just as with the magnetometer, the magnetic orientation of the unbound magnetic marker in the solution can be controlled so as to intersect the magnetic direction of the bound magnetic marker, by setting the residual magnetic field component along the direction of the magnetic moment of the bound magnetic marker to zero. Another method is to apply a magnetic field in a direction perpendicular to the magnetic direction of the magnetic marker, to control the magnetic direction of the unbound magnetic marker within the solution so as to intersect the magnetic direction of the fixed magnetic marker. Yet another method is to align the magnetic direction of the unbound magnetic marker within the solution to intersect the magnetic direction of the fixed magnetic marker even when using a magnetic shield structure where the direction of the residual magnetic field within the shield intersects the direction of the fixed magnetic marker. A combination of these methods may also be used. 
     If a signal with a different symmetry can be obtained from the different magnetization direction of the sample, then the desired signal waveform can be obtained with the method of this invention even if utilizing a higher order gradiometer or a pickup coil with a different structure or shape. 
     Reducing the direct current field to zero in all directions with a magnetic shield is impossible but the magnetic field in one direction can easily be controlled, as used in this invention. A compensation coil can be installed for example in the vicinity of the SQUID magnetic sensor and the residual magnetic field components in the vicinity of the measurement position canceled in one direction out by adjusting the direct current flowing to the compensation coil. Moreover, controlling the magnetization direction of unbound magnetic markers in the solution to a direction perpendicular to the magnetization direction of bound magnetic markers is comparatively simple. A permanent magnet or a coil for example can be installed to control the magnetization direction of unbound magnetic markers in the solution by applying a magnetic field. Controlling the direction of the residual magnetic field within the shield is also comparatively simple. In the case of a cylindrical magnetic shield for example, the shield rate along the center axis of the tube is lower than a direction perpendicular to the center axis so that a magnetic field can be left remaining along the center axis in the center of the cylindrical magnetic shield. 
     Moreover, by combining with the above method, a direct current magnetic field left remaining in the vicinity of the sample measurement position of this invention can be controlled in a direction perpendicular to the magnetization direction of the magnetic nanoparticles in the object for measurement. 
     This invention is capable of easily isolating signals from bound magnetic markers even when magnetic signals are emitted from magnetic markers in a liquid state due to a residual magnetic field. The present invention can therefore eliminate the effects from magnetic signals emitted by magnetic markers in a liquid state and provide a high sensitivity immunoassay. Moreover this invention does not require a magnetic shield with good multilayer sealing that was required for high sensitivity immunoassays so this invention can reduce equipment costs and improve operability. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1A  through  FIG. 1E  are drawings showing a typical procedure for the immunoassay method of the related art; 
         FIG. 2A  and  FIG. 2B  are drawings showing the state of the magnetic markers when the magnetic field is completely blocked, and the magnetic marker state when there is a residual magnetic field; 
         FIG. 3A ,  FIG. 3B  and  FIG. 3C  are drawings showing the relation between the magnetization direction of the sample when using a magnetometer, to the detected magnetic signal waveform; 
         FIG. 4A ,  FIG. 4B  and  FIG. 4C  are drawings showing the relation between the magnetization direction of the sample when using a first-order planar gradiometer, to the detected magnetic signal waveform; 
         FIG. 5  is a drawing showing a typical structure of the immunoassay system including a compensation coil for the first embodiment of this invention; 
         FIG. 6  is a drawing showing a sample container used in the first embodiment; 
         FIG. 7  is a drawing showing the high-temperature superconducting SQUID used in the embodiment of this invention; 
         FIG. 8  is a flow chart showing the test protocol used in the embodiment; 
         FIG. 9A  is a magnetic signal waveform obtained from a sample containing 100 pg of IgE measured with no electrical current flowing in the compensation coil; 
         FIG. 9B  is a magnetic signal waveform obtained from a reference sample not containing IgE and measured with no electrical current flowing in the compensation coil; 
         FIG. 10  is magnetic signal waveforms from the reference sample showing the compensation coil current dependence; 
         FIG. 11A  is a magnetic signal waveform obtained from a sample containing 100 pg of IgE measured with an electrical current of 200 μA flowing in the compensation coil; 
         FIG. 11B  is a magnetic signal waveform obtained from a reference sample not containing IgE measured with an electrical current of 200 μA flowing in the compensation coil; 
         FIG. 12A  is a waveform obtained from a sample where bilaterally symmetrical components were eliminated from the magnetic signal waveform measured with an electrical current of 200 μA flowing in the compensation coil; 
         FIG. 12B  is a waveform of the reference sample not containing IGE and measured with an electrical current of 200 μA flowing in the compensation coil; 
         FIG. 13  is a drawing showing the structure of the immunoassay system of the second embodiment; 
         FIG. 14A  is a magnetic signal waveform obtained in the second embodiment from a sample containing 100 pg of IgE; 
         FIG. 14B  is a magnetic signal waveform obtained in the second embodiment from a reference sample not containing IgE. 
         FIG. 15  is a drawing showing the structure of the immunoassay system of the third embodiment; and 
         FIG. 16  is magnetic signal waveforms obtained in the third embodiment from a sample (solid line) containing 100 pg of IgE and a reference sample (dotted line) not containing IgE. 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     First Embodiment 
     The following description utilizes antibodies labeled with magnetic nanoparticles (magnetic markers) containing residual magnetic signals, and the description of the embodiment of this invention utilizes a magnetic immunoassay system for detecting magnetic signals from a sample reacting with a test reagent in a first-order planar SQUID gradiometer as an example. The following disclosure is nothing more than an embodiment of this invention, and does not limit the technical scope of this invention. 
       FIG. 5  is a cross sectional view showing the structure of the first-order gradiometer SQUID of the first embodiment of this invention. In order to reduce the input of magnetic environmental noise into the SQUID, the cooler device (made up of an outer tank  21 , a vacuum layer for thermal insulation  22  and inner tank  23 ) for cooling the SQUID is enclosed by an RF shield  19  and magnetic shields  30 ,  31 . The RF shield  19  is made from metal material with low electrical resistance such as aluminum. The magnetic shields  30 ,  31  are made from high permeability material such as permalloy. A portion of the magnetic shield  30  is formed with a slot  38  to allow insertion of the sample container  71 . 
     A sample container  71  in the nonmagnetic disk shaped sample stage  32  is clamped to a rotating shaft  35  by a clamping screw  33 . A flat (plan) view of the sample container  71  is shown in  FIG. 6 . This container is made from highly non-magnetic material such as plastic. The container  71  is formed in a circular shape and contains cone-shaped recess sections  70  at 12 points on the outer circumference. A hole  72  is drilled in the center for clamping to the equipment. The diameter of the recess sections in the bottom is 5 millimeters. 
     A sample  14  containing a magnetic marker is inserted in the recess section  70  in the sample container. The sample stage  32  rotated via the rotating shaft  35  connected to a rotation mechanism  34 . The rotation mechanism  34  is supported to allow movement in three dimensions on the motion stages  36 ,  37 . The movement of the motion stages  36 ,  37  via the rotation mechanism  35  makes a section of the sample container  71  pass through an insertion slot  31 , move to the internal section of the magnetic shield  30 , and adjust the position so that the bottom of the recess section  70  approaches a sapphire window  29 . As described using  FIG. 1 , an antibody  2  bound to an antigen and serving as the object for detection, is clamped in the bottom of the recess section  70  of the sample container, and is covered on the peripheral walls by a blocking reagent  3 . 
     The pickup coil of the SQUID  28  is installed in the lower part of the sapphire window  29  in order to shorten the distance between the sample  14  and the SQUID  28  pickup coil, and to boost the spatial resolution and detection sensitivity for magnetic signals generated by the sample. The rotation of the sample container  71  causes the multiple samples  14  to pass above the pickup coil of the SQUID  28 , and the magnetic signal is measured at that time. The sapphire window  29  is clamped to the nonmagnetic cylindrical piece  17  and is capable of being positioned upward or downward. A compensation coil  16  is wound around this cylindrical piece  17  for compensating the residual magnetic field. Making an electrical current flow in this coil applies a compensating magnetic field in a direction perpendicular to the SQUID pickup coil. The center axis of the compensation coil  16  passes along the center of the SQUID pickup coil. A solenoid coil with a simple structure was utilized here, however a Helmholtz coil or other coil shapes providing a more uniform magnetic field distribution may also be utilized. In other words, the effect of this invention can be obtained if the magnetic field for the sample detection position above the SQUID can be compensated. 
     The SQUID  28  is installed in vacuum layer for thermal insulation  22  of the cooling container and is indirectly cooled by the liquid nitrogen  24  via a sapphire rod  13  and a copper rod  26  possessing high thermal conductivity. The outer tank  21  and an inner tank  23  in the cooling device are made non-magnetic material such as SUS and FRP (fiber reinforced plate). Interposing the sapphire rod  13  between the SQUID  28  and the copper rod  26  has the effect of reducing the effect of magnetic noise generated from the copper rod  26 . 
     A high-temperature superconducting SQUID gradiometer was utilized as the SQUID  28  in the immunoassay system of the first embodiment.  FIG. 7  is a flat (plan) view showing the structure of the high-temperature superconducting SQUID gradiometer. The pickup coil  62  and the SQUID ring  64  were fabricated by patterning high-temperature superconductive material such as YBa 2 Cu 3 O x  formed on a bicrystal substrate  60  of single crystal such as SrTiO 3  or MgO with a shifted crystal orientation and bonded to a bicrystal plane  61 . The SQUID ring  64  cuts across the bicrystal plane  61  formed on the bicrystal substrate  60 . A grain boundary Josephson junction  65  was formed in the superconducting thin film formed on the bicrystal plane  61 , to consequently form two grain boundary Josephson junctions  65  on the SQUID ring  64 . On the SQUID used here, two SQUID rings  64  and  64 ′ coupled to the same pickup coil are formed on one substrate. Among these two SQUID rings, the ring having the better characteristics is used here. 
     The pickup coil  62  is made up of a gradiometric pickup coil in a figure “8” shape of two loops of 5 millimeters on one side. When magnetic flux enters the pickup coil  62  the differential between the shielding currents flowing in each loop of the two loops flows into the SQUID rings  64 ,  64 ′ via the center section  66  of the pickup coil. This electrical current is detected as a flux. The feedback coils  67  and  67 ′ are formed as circuit patterns on the substrate  60  so as to enclose one loop on the pickup coil  62 . One among the two feedback coils  67 ,  67 ′ were used. The gold wiring pads  63 ,  63 ′ and  68 ,  68 ′ were patterned on the superconducting thin film when a wiring connection was required. The wiring pad  63  was electrically connected to the SQUID ring  64 , and the wiring pad  68  was electrically connected to the feedback coil  67 . 
     The sample container passes above the SQUID along the arrow direction  69  in  FIG. 7 . The sample piece is magnetized in a direction perpendicular to the bottom of the container. This magnetizing direction is perpendicular to the pickup coil surface. The relation between the SQUID and the magnetic signal emitted from the bound magnetic marker, as well as the magnetic signal waveform that is obtained therefore correspond to those shown in  FIG. 4C . 
     The total IgE (immunoglobulin E) was detected in the first embodiment. The test protocol is shown in  FIG. 8 . First of all, in step  81 , a capturing antibody (anti IgE antibody) was fixed to the bottom of the recess section  70  of the non-magnetic reaction chamber  71 . The capturing antibody was directly attached to the reaction chamber in the example of the first embodiment. However, the same measurement can be performed by binding the capturing antibody to a solid such as polymer beads or cellulose string having a size of several microns or larger and incapable of large movement during measurement, and placing this in the recess section  70  of the non-magnetic reaction chamber  71 . 
     After adding the antibody, the blocking process was performed in step  82  using BSA (Bovine serum albumin). After washing, the test sample including IgE was placed in step  83 , and a reaction between the capturing anti IgE antibody and IgE was made to occur. A PBS (phosphate buffered saline) solution of 50 μl containing 100 pg of IgE was used in the test sample. A PBS solution of 50 μI not containing IgE was utilized as the reference sample. After 30 minutes a magnetic marker was placed from above in step  84  and a reaction between the IgE and magnetic marker made to occur. The first embodiment utilized a magnetic marker with a structure where the anti IgE antibody was attached to the surface of polymer-coated Fe 3 O 4  (magnetite) nanoparticles with a diameter of 25 nm. Thirty minutes later in step  85  a permanent magnet (neodymium magnet, diameter 30 mm, surface flux density 300 mT) was placed on the bottom of the reaction chamber for one minute, and magnetization in the z direction was performed as described in  FIG. 4C . After magnetization, the sample container was mounted in the measurement system and the magnetic signals generated from the sample were measured in step  86 . The conditions for each of these steps were recorded to the side of each step. 
       FIG. 9A  shows the magnetic signal waveform obtained from the sample containing 100 pg of IgE; and  FIG. 9B  shows the magnetic signal waveform obtained from the reference sample not containing IgE. Magnetic signals were obtained from the reference sample that supposedly should not generate regular magnetic signals because of Brownian motion, showing that a magnetic field that could not be blocked by the magnetic shield remained in the vicinity of the sample measurement position. The amount of change in both waveforms was approximately the same with a difference of only 0.2 mφ 0 . In this data, detecting 100 pg of IgE was impossible because the variations in the reference sample was 1 mφ 0  or larger. 
       FIG. 10  shows the magnetic signal waveform of the reference sample when measured while an electrical current flowed in the compensation coil  16 . The waveform gradually neared bilateral symmetry as the current increased in the compensation coil  16 . A bilaterally symmetrical waveform was obtained when an electrical current of 200 μA flowed in the coil. When the current in the compensation coil  16  was increased still further, the offset from the bilaterally symmetrical waveform of the reference sample was inverted centering on the axis. 
     The magnetic signal waveform obtained when an electrical current of 200 μA flowed in the compensation coil  16  is corresponding to the waveform shown in  FIG. 4A . Therefore, this result indicates that magnetic field in the z direction is canceled by the compensation coil  16 . The compensation coil at this time applies a magnetic field of approximately 50 nT to the vicinity of the sample. 
       FIG. 11A  and  FIG. 11B  show magnetic signal waveforms obtained from a sample in  FIG. 11A  containing 100 pg of IgE measured in a state where 200 μA of electrical current is flowing in the compensation coil  16 ; and a sample in  FIG. 11B  for a reference sample not containing IgE. In this state a difference of 0.9 mφ 0  was obtained. The magnetic signal waveform from the magnetic marker in the solution is bilaterally symmetrical due to the effect of the compensation coil  16  and therefore that eliminating the symmetrical signal components to extract just the nonsymmetrical components was attempted. 
       FIG. 12A  and  FIG. 12B  show waveforms where the bilaterally symmetrical components at the reaction chamber center position in the  FIG. 11A  and  FIG. 11B  waveforms were eliminated. This process can be performed by subtraction as described for  FIG. 3  and  FIG. 4 . As can be seen in  FIG. 12A , in contrast to the sharp signal with an amplitude of 5 mφ 0  in the sample material containing 100 pg of IgE; the waveform in the reference sample in  FIG. 12B  with a maximum amplitude of 1.3 φ 0  is unclear, and the effect of the unbound magnetic marker in the solution has largely been eliminated. The optimal current of the compensation coil  16  can in this way be evaluated beforehand in the first embodiment, and by allowing an appropriate amount of electrical current flow, the bound magnetic markers contained in tiny amounts in unbound magnetic markers in the liquid can be measured. 
     Second Embodiment 
     An example of the second embodiment is described utilizing a magnetic immunoassay system for detecting magnetic signals from a sample that reacted with a test reagent in a first-order planar SQUID gradiometer, using antibodies labeled with magnetic nanoparticles having remanence. 
       FIG. 13  is a cross sectional view showing the structure of the magnetic immunoassay system of the second embodiment. A permanent magnet  18  and a position adjuster  20  for the permanent magnet are installed instead of the compensation coil  16  for the system used in the first embodiment shown in  FIG. 5 . The permanent magnet  18  applies a magnetic field in a direction perpendicular to the magnetization direction (z direction) of the bound magnetic marker at the measurement position. The other parts of the structure are identical to the first embodiment. 
     A sample identical to that for the first embodiment was measured using this system. The method for making the samples was identical to that of the first embodiment. The magnetic field applied to the sample measurement position was approximately 1 nT to 100 μT. The optimal magnetic field depends on the characteristics of the magnetic marker and the strength of the residual magnetic field. Therefore just as with the first embodiment, it is important to evaluate the magnetic signal waveform produced by the permanent magnet in terms of the difference in magnetic field strength, and use a permanent magnet possessing optimal magnetic field strength. A comparatively weak rubber magnet was utilized here as the permanent magnet however an electromagnet of course may also be used. 
       FIG. 14A  shows a magnetic signal waveform obtained from a sample containing 100 pg of IgE.  FIG. 14B  shows a magnetic signal waveform obtained from a reference sample not containing IgE. The amount of change in the magnetic signal from the reference sample is one/tenth that of the waveform for the reference sample in  FIG. 9B . The reason for the smaller change is that the y-direction magnetic field from the permanent magnet  18  aligns the magnetic markers in the solution in the y-direction so that the number of unbound magnetic markers aligned in the x and z directions, which are detected by SQUID, is decreased. The sample containing 100 pg of IgE can therefore be clearly distinguished from the reference sample as shown in  FIG. 14A  and  FIG. 14B  thus confirming the effect of this invention. 
     Third Embodiment 
     An example of the third embodiment is described utilizing a magnetic immunoassay system for detecting magnetic signals from a sample that reacted with a test reagent in a first-order planar SQUID gradiometer, using antibodies labeled with magnetic nanoparticles having remanence. 
       FIG. 15  is a cross sectional view showing the structure of the system. The system utilizes a dual layer cylindrical magnetic shield made from a cylindrical magnetic shield  43  with an inner diameter of 40 centimeters and length of 1 meter, and a cylindrical magnetic shield  44  with an inner diameter of 30 centimeters and length of 90 centimeters. The shield  43  and  44  material is 2 millimeter thick permalloy. A sample container  45  is a non-magnetic reaction chamber with an 8 by 12 array of 96 holes. The sample container  45  is supported by a sample container holder  47  so that the measurement position is at the center of the magnetic shield, and a 3-axis manipulator  48  moves the desired sample to the measurement position. A SQUID  41  utilizes a gradiometer with the structure shown in  FIG. 7 . 
     A pulse tube cryocooler  49  cools the SQUID  41  via a sapphire rod  50 . The SQUID at low temperature is located within a vacuum insulation chamber  42 . The temperature of the SQUID is controlled in a range of 65 to 80 K within a variation of ±0.1 K. The longitudinal direction of the gradiometer pickup coil matches the x (horizontal) direction of the sample container holder  47 . Antibodies are affixed to the sample container  45 . A sample  46  is a liquid sample containing unbound magnetic markers and magnetic markers bound to the target substance. After being magnetized in advance, the sample  46  is sealed into the sample container  45 . 
     Each of the 96 hole samples were moved along the x direction to the measurement position over the SQUID by moving the entire sample container  45  and the change in magnetic signals at that time was measured. The measurement time for one row (12 samples) was from 0.1 second to several seconds. Multiple measurements were made by moving the samples back and forth over the SQUID, and the data averaging was then performed. When measurement of one row (12 samples) was completed, the entire sample container  45  was shifted along the y direction by a width equal to one row, and the same measurement made after movement to that next row. 
       FIG. 16  shows the magnetic signal waveform (solid line) obtained from a sample containing 100 pg of IgE; and a magnetic signal waveform (dotted line) obtained from a reference sample not containing IgE. A magnetic signal fluctuation of approximately 6 mφ 0  was detected in the sample containing 100 pg of IgE. A fluctuation width approximately 4 times greater than that in the reference sample was obtained. The reason is that the magnetic field remaining along the y direction within the cylindrical magnetic shield, aligned the magnetic markers in the solution along the y direction so that fewer magnetic markers along the x and z direction were detected as magnetic signals by the SQUID. 
     The description of the embodiments described detection of IgE as one type of antibody related to allergies. However this invention is of course not limited to IgE and may by applied to tests for substances usually measured by general immunoassay systems including for example, tests for biological substances such a hormones, cytokine, and tumor markers, as well as environmental toxins including dioxins, etc. Moreover, the examples in the embodiments were described using the sandwich method for capturing antibodies, but this invention can also be applied to the quantitative competition method and bridge method utilized in general immunoassay inspections. This invention is also effective for cases using other types of magnetic markers such as when using magnetic elements other than magnetite. The effect of this invention is also obtained when using magnetic sensors other than the high-temperature superconducting SQUID, such as when using high-sensitivity magnetic sensors including light-pumping flux meters and low-temperature superconductor (for example, Nb and MbG 2 , etc.) SQUID, and inductive coils, flux gate flux meters, proton flux meters, MR sensors, GMR sensors, and Mi sensors, etc.