Abstract:
The present invention relates to an immunoassay and an apparatus for the same. A method of the present invention comprises following processes; (1) an analyte is labeled with a magnetic label to detect antigen-antibody reaction, (2) the magnetic material label is magnetized by a magnetic field, (3) the magnetized magnetic material label detected by a SQUID which detect a magnetic field having right angle to the magnetic field. At the same time, the present invention contains an apparatus to execute the method provided by the present invention. The apparatus comprises a magnetic field generation means that generates a magnetic field to magnetize the labels. The apparatus comprises a SQUID that measures magnetic field.

Description:
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS  
       [0001]    This application is a division of Application Ser. No. 09/621,341, filed Jul. 21, 2000, now pending, and based on Japanese Patent Application No. 11-206248, filed Jul. 21, 1999, by Keiji ENPUKU. This application claims only subject matter disclosed in the parent application and therefore presents no new matter. 
     
    
     
       BACKGROUND OF THE INVENTION  
         [0002]    1. Field of the Invention  
           [0003]    The present invention relates to an immunoassay and an apparatus for the same. More specifically, the present invention relates to a method and an apparatus for an immunoassay with a magnetic label and a SQUID.  
           [0004]    2. Detailed Description of Invention  
           [0005]    An immunoassay is a method to detect an antigen or an antibody (mentioned with word “analyte” in this specification). For identification or measurement, a label is attached to antibody of antigen-antibody reaction. Various labels and detection method are executed frequently and proposed.  
           [0006]    Particularly, various optical methods are known well. In these methods, labels with light, fluorescence or color are used. However, optical methods have short sensitivity for requirement.  
           [0007]    As an another method, method with radioactive label is known. However, this method is pointed out a problem about safety and limited its execution.  
           [0008]    Furthermore, there is methods with magnetic labels as a reemergence measurement or a magnetic relaxation method. However, in this method, grain size of the label influences to measured value seriously. Therefore, accuracy of measurement of this method should not be stable.  
           [0009]    On the other hand, a SQUID was put to practical use recently. The SQUID comprises a circular current road and one or two Josephson junction(s) on the road. The SQUID has a very high sensitivity compared with a Hall device or a flux gate and is used as a magnetism sensor.  
           [0010]    Then, a new assessment method with magnetic label occurs to us. In this method, it is expected that labels are detected by a SQUID with high accuracy. However, there is no practical method with a SQUID. Magnetic label has to be magnetized for detection by a SQUID. However, a strong magnetic field of dozens of gauss dimension is necessary for label to be magnetized.  
           [0011]    On the other hand, a SQUID has very high sensitivity. Therefore, a serious problem occurs that a SQUID receives affect of magnetic field of magnetization means and measured value changes.  
           [0012]    Furthermore, an analyte is treated with prepared slide actually. But, a strong magnetic field magnetizes a prepared slide. Therefore, it is difficult to detect only a label.  
         SUMMARY OF THE INVENTION  
         [0013]    Then the present invention provides a method for an immunoassay with magnetized label and SQUID, which comprising following processes;  
           [0014]    (1) an analyte is labeled with a magnetic material label to detect antigen-antibody reaction,  
           [0015]    (2) the magnetic material label is magnetized by a magnetic field,  
           [0016]    (3) the magnetized magnetic material label detected by a SQUID which detect a magnetic field having right angle to the magnetic field.  
           [0017]    In method of the present invention, labels are magnetized and detected by a SQUID. According to a preferable embodiment of the present invention, the magnetic field for magnetization is a static magnetic field.  
           [0018]    According to another preferable embodiment of the present invention, an analyte is inspected while moving parallel to the flux forming the magnetic field inside the detection region of the SQUID. Then, the SQUID detects a variation of magnetic field occurred by the moving labels magnetized in particular direction.  
           [0019]    At the same time, the present invention contains an apparatus to execute the method provided by the present invention. The apparatus comprises a magnetic field generation means that generates a magnetic field to magnetize the labels. The apparatus comprises a SQUID that measures magnetic field.  
           [0020]    It is preferable that the apparatus comprises a transportation means which moves the analyte with magnetized label parallel to the magnetic field generated by the magnetic field generation means.  
           [0021]    Furthermore, the apparatus comprises magnetic field compensation means preferably. The compensation means generates a magnetic field parallel to the detection direction of the SQUID. The magnetic field for compensation cancels the magnetic field that has right angle to the magnetic field for magnetization. Because, the magnetic field for magnetization contains component that has right angle to the desired magnetic field and the SQUID has very high sensitivity to detect the component.  
           [0022]    According to the preferable embodiment of the present invention, the SQUID is formed of an oxide superconducting thin film having a high critical temperature. By the way, the sensitivity of a SQUID is in proportion to 3 power of distance between a SQUID and an analyte. The oxide superconducting materials can be used with a small cooling-systems. The use of the oxide superconducting materials is advantageous in this point.  
           [0023]    It is an important characteristic of the present invention that the magnetic field for magnetization has right angle to the magnetic field detected by the SQUID. That is to say, in a prior art, the magnetic field for magnetization and the magnetic field detected are parallel each. Therefore, the SQUID detects a magnetic field for magnetization, too.  
           [0024]    On the contrary, in apparatus of the present invention, the magnetic fields are arranged right angle each. The SQUID detects a flux having right angle to its circular current road and never detects a flux parallel to the circular current road. Therefore, in apparatus provided by the present invention, the SQUID does not detect the magnetic field for magnetization. In a method with a SQUID by prior art, a magnetic field for magnetization is alternative and a noise is offset by using a lock in amplifier.  
           [0025]    According to a preferable embodiment of the present invention, a static magnetic field can be used. Because, the static magnetic field can be easily compensated by simple means with a solenoid.  
           [0026]    However, because the SQUID has very high sensitivity, even using the magnetic field for compensation will not compensate the magnetic field for magnetization perfectly. Then, according to a preferable embodiment of the present invention, the SQUID detects a variation of magnetic field. This variation of magnetic field is occurred by a motion of the magnetized label in the detection field. This variation itself is not influenced magnetic field of perimeter.  
           [0027]    The above and other objects, features and advantages of the present invention will be apparent from following description of preferred embodiments of the invention with reference to the accompanying drawings. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0028]    [0028]FIG. 1 is a perspective view showing a principle of the method provided by the present invention.  
         [0029]    [0029]FIG. 2 is a sectional view showing a basic construction of the apparatus provided by the present invention.  
         [0030]    [0030]FIGS. 3A and 3B show labels and antibodies.  
         [0031]    [0031]FIG. 4 is a graph showing an output signal of the SQUID.  
         [0032]    [0032]FIG. 5 is a graph showing a relationship between concentration of an antibody and the output of the SQUID.  
         [0033]    [0033]FIG. 6 shows the antigen-antibody reaction labeled with a magnetic label.  
         [0034]    [0034]FIG. 7 is a graph showing measured resultant in comparison with a resultant by a prior art. 
     
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS  
       [0035]    In the method of the present invention, as shown in the FIG. 1, an analyte  2  supported on a support  1  with label is magnetized at first by a magnetic field shown with arrow A parallel to surface of the support  1 , and is detected by a SQUID  3  at last.  
         [0036]    The SQUID  3  comprises a ringed current road that is arranged parallel to the surface of the support  1 . Therefore, a magnetic flux detected by the SQUID  3  has a right angle to the surface of the support  1 . Namely, a region under the SQUID  3  becomes a detection region of the SQUID  3 . On the contrary, the magnetic field for magnetization is parallel to the surface of the support  1 . Therefore, the SQUID  3  has no sensitivity to the magnetic field A for magnetization substantially.  
         [0037]    Furthermore, the support  1  moves parallel to the magnetic field A with fixed velocity X. When the analyte  2  passes into the detection region of the SQUID  3 , the magnetic field of the detection region changes and the SQUID  3  detects the change of the magnetic field. By the way, at the same time, the support  1  is magnetized too. Therefore, it is preferable that the length L and the width W of support  1  are large sufficiently so that the detection region is met by support  1  while no analyte  2  is in the detection region.  
         [0038]    The method mentioned above can be executed with an apparatus shown by FIG. 2. This apparatus comprises magnetic shields  101   a,    101   b,  SQUID  103 , coils for magnetization  106   a,    106   b, a  compensating coil  107  and a transportation means  105 .  
         [0039]    The magnetic shields  101   a,    101   b  surround the whole apparatus and the measurement is done within the magnetic shields  101   a,    101   b.  SQUID  103  is taken into a container  102  filled with liquid nitrogen  102   a  and arranged horizontally. The magnetization coils  106   a,    106   b  are placed parallel mutually and have right angle to the SQUID  103 .  
         [0040]    The compensating coil  107  is placed in the lower part of the SQUID  103  and arranged parallel to the SQUID  103 . Vertical component of the magnetic field generated by the magnetization coils  106   a,    106   b  is canceled with the magnetic field formed by the compensating coil  107 . Then the magnetic field inside the detection region includes only horizontal flux substantially.  
         [0041]    The transportation means  105  comprises an arm that moves to X-Y direction in horizontal and conveys a sample  104 . Transportation means  105  can carry sample  104 . The sample  104  is inserted into the magnetic shields  101   a,    101   b  from side by the transportation means  105  and passes inside the coil  106   a,    106   b.  Then the sample  104  is magnetized by the coil  106   a,    106   b.  In next, the sample  104  arrives the detection region.  
         [0042]    We assembled the apparatus mentioned above with elements below.  
         [0043]    The SQUID  103  was made of patronized oxide superconducting thin film on a SrTiO 3  substrate. The magnetic shields  101   a,    101   b  were made of Permalloy.  
         [0044]    Sample  104  was supported by a glass plate having dimension of 20 mm*80 mm as a support  1 . The glass plate is produced by Nalge Numc International company (USA). The glass plate passed 1.5 mm lower part of the SQUID.  
         [0045]    We prepared two kinds of antibody for preparation samples.  
         [0046]    One is a A type antibody named “MACS” provided from Miltenyi Biotec company (Germany). The MACS is a particle of gamma-Fe 2 O 3    14   a  coated by a polymer  14   b  and antibody  14  sticks to the polymer  14   b  as shown in FIG. 3 ( a ). Average particle diameter of the A type antibody is 50 nm and weight of A type antibody is approximately 4*10 −16  g.  
         [0047]    Another one is a B type antibody named “dynabeads” provided by Dynal company (Norway). Plural magnetic material ultrafine particle  14   a  is contained in a polymer graining  14   c  as shown in FIG. 3( b ) and an antibody  14  sticks to polymer  14   c.  Average particle diameter of B type antibody is 4.5 μm and weight of B type antibody is approximately 14.3*10 −12  g.  
       EXAMPLE 1  
       [0048]    Sample mentioned above was inspected with apparatus shown by FIG. 2. We used a decentralized liquid of A type antibody (rat/anti mouse Ig G 1 ). In stock solution, concentration was indicated 0.2 mg/ml and Average particle diameter was 50 nm, 5.2 g/cm 3 . According to the inference, weight of magnetic material particle is 3.4*10 −16  g and the particle is contained during stock solution at 5.8*10 11 /ml. Then we diluted the stock solution with PBS into {fraction (1/10)} and put it on the glass plate as an analyte. The sample on the glass plate occupied a region with 2 mm diameter and its amount was 2μ liter. Accordingly, this sample contains 1.2*10 8  magnetic particles and general mass of the magnetic particles is 40 ng.  
         [0049]    The acidity of magnetic field for magnetization was 8*10 −4  T and the drift speed of analyte was 8 mm per second. Output signal of the SQUID  103  was recorded through a band-pass filter having range from 0.1 Hz to 5 Hz. Recorded output signal is shown in FIG. 4.  
         [0050]    As shown in the FIG. 4, extremely clear variation of the magnetic field was recorded. Sensitivity of SQUID depends on the distance between a SQUID and an analyte. Therefore, the sensitivity of the apparatus can be regulated by the distance.  
       EXAMPLE 2  
       [0051]    A relation between the concentration and the detection resultant of the sample is shown in FIG. 5.  
         [0052]    Circles plotted in the FIG. 5 show determination resultant of the sample that was labeled with the A type antibody and diluted with PBS in various concentrations. Rectangles plotted in the FIG. 5 show determination resultant of the sample that was labeled with the B type antibody and diluted with PBS in various concentrations. The sample was rat/anti mouse Ig G 1  and diluted with PBS. As shown in the FIG. 5, high correlation between the detected magnetic signals and the quantity of the labeled antibody can be seen for the both cases  
       EXAMPLE 3  
       [0053]    Another Sample was prepared. As shown in FIG. 6, in this sample, antigen  11  is fixed by a first antibody  12  to the support  1 . Then, a second antibody  13  sticks selectively to the antigen  11 . Furthermore, a third antibody  14  labeled with magnetic material  14   a  sticks to the second antibody  13 . The SQUID detects the magnetic material  14   a.    
         [0054]    The sample put on the region having a diameter of 8 mm on the support. At first, we fixed a “mouse/anti humans interferon β monoclonal antibody (YMASA company, JAPAN) as the first antibody  12  in region. Next, we let a humans-interferon β as the antigen  11  react to the region at 37 degrees Centigrade for 3 hours. Then we prepared a rabbit-anti human interferon β/polyclonal antibody (Bio-Rad company, U.S.A.) as second antibody  13  and goat/anti rabbit Ig G as the third antibody  14 . The Goat/anti rabbit Ig G has been labeled with a magnetic material ultrafine particle and was reacted to the region at 37 degrees Centigrade for 1 hours.  
         [0055]    Determination effect by measuring the sample above is shown in FIG. 7. The determination resultant is plotted with circles. At the same time, rectangles are plotted in the FIG. 7. These rectangles means resultant surveyed by an optical method according to a prior art, ELISA system type  11  by Biotrak company. In this method, at first, an antibody is reacted to an antigen and next, a stroma is added them. Then the coloring antibody can be detected. For reference, the same goat/anti rabbit Ig G was used as an antibody.  
         [0056]    As shown in the FIG. 7, this optical method shows good correlation with specified field where the concentration is more than 1 unit/ml. However, the correlation becomes worsen with lower field than the specified field. On the contrary, a good correlation is maintained by the method of the present invention. Then we understood the method of the present invention is clearly superior to the prior art. As explained, the method of the present invention can realize high sensitivity and high accuracy. Furthermore, a magnetic material can be smaller than 600 pg, therefore, the sensitivity of the present invention should be improved easily.