JP2000292426A - Immunoassay using microtiter plate - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide immunoassay capable of more effectively performing magnetic separation of magnetic particles and permitting high sensitivity measurement by simple operation. SOLUTION: Immunoassay for measuring an antigen or antibody in a specimen qualitatively or quantitatively has a stage for reacting an antibody or antigen supported on magnetic particles with an antiboy or antigen in a specimen specifically bonded to the supported antigen or antibody in a liquid medium within the well 5 of a microtiter plate 2, a stage for arranging a magnet 4 on the lateral side of the well 5 to apply a magnetic field to the well 5 to magnetically separate and gather the magnetic particles in the liquid medium to hold them on the inner wall side surface of the well 5 and a stage measuring the optical characteristics of the supernatant soln. generated in the well 5 by holding the magnetic particles on the inner wall side surface of the well 5.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to an immunological measurement method using a microtiter plate and a magnetic separation jig used for the method.

[0002]

2. Description of the Related Art As an immunological measurement method using magnetic particles or particles containing a magnetic substance, for example, a magnetic field is applied to collect magnetic particles at a position where photometry of a container is not interrupted, and the magnetic particles are suspended in a liquid medium. By detecting insoluble carrier particles that do not contain the body by absorbance or scattered light,
There is known an immunological measurement method for qualitatively or quantitatively measuring an antigen or an antibody in a liquid medium (JP-A-1-193647). In recent years, as a method for analyzing a large number of samples, a method of optically measuring the samples in each well with a dedicated reader using a container with 96 wells (holes) called a microtiter plate has become widespread. ing.

It is conceivable to carry out the method described in Japanese Patent Application Laid-Open No. 1-193647 using a microtiter plate. However, this method is a homogeneous immunoassay and is simple in that B / F separation and washing are not required. However, there is a problem that the sensitivity is low. In addition, since no magnetic field is applied during optical measurement, aggregated magnetic particles may be released into the liquid medium, which may adversely affect the measurement.

[0004] As an immunological measurement method using a microtiter plate and magnetic particles, each well containing magnetic particles or particles containing a magnetic substance is surrounded by a magnet using a special magnet-based base device, and the magnetic particles are well-welled. A method is known in which the solution is separated on a side wall, and then the supernatant is collected and transferred to another container, followed by measurement (JP-A-1-201156).

[0005]

However, in the above method, it is necessary to collect the supernatant liquid after magnetic separation and transfer it to another container for measurement, which requires a complicated operation. Was. Therefore, an object of the present invention is to provide an immunological measurement method capable of performing magnetic separation of magnetic particles more effectively, performing B / F separation by a simple operation, and measuring at high sensitivity. Is to do.

[0006]

According to the present invention, the above object is achieved by the following method. (A) reacting an antibody or antigen carried on magnetic particles with an antibody or antigen in a sample that specifically binds to the antigen or antibody in a liquid medium in a well of a microtiter plate; One or more steps of disposing a magnet on the side of each well, applying a magnetic field to each well, and magnetically collecting and holding the magnetic particles in the liquid medium on the inner wall side of each well in the well; C) measuring the optical properties of the supernatant liquid generated in each well as a result of the magnetic particles being retained on the inner wall surface of each well as described above, An immunoassay for qualitatively or quantitatively measuring an antibody.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described more specifically. An antibody or antigen is carried on the magnetic particles, an antigen-antibody reaction is caused between the antibody and the antigen or antibody in the sample to be measured, B / F separation is performed in a subsequent step, and thereafter, directly or by another operation. Various methods are known for analyzing and qualitatively or quantifying unbound antigen or antibody present in a liquid medium through steps using any method. The method of the present invention can be applied and improved on any of such methods. The antigen or antibody measured by the method of the present invention is not particularly limited, and any antigen or antibody can be measured.

As the magnetic particles carrying the antigen or antibody, any known magnetic particles can be used. For example, commercially available magnetic particles or polymer-containing magnetic material-containing particles are available from Dynabeads manufactured by Dynal and manufactured by PE Biosystems.
BioMag, Magtex from Cortex, Magnetizable Polystyrene Particles from Polyscience, Magnetic Polystyrene Particle from Spherotech, etc. can be used. In addition, magnetic bacteria AMB-1 (FERM)
BP-5458), magnetic bacteria RS-1 (FERM P-
13283), magnetic bacteria MGT-1 (FERM P-1)
6617) and the like, and magnetic particles separated from bacteria that produce magnetic particles in the cells. The size of the magnetic particles is about 5
It is preferably about 0 nm to 100 μm.

The microtiter plate used may be a commonly used 96-well type plate, but is not particularly limited thereto. The arrangement of the wells is usually linear in both the vertical and horizontal directions and is arranged at the intersection of the lattice, but this point is not limited. A slight modification may be required depending on the form of the magnetic separation jig described later.

According to the method of the present invention, a magnetic particle-antibody (or antigen) -antigen (antibody) conjugate is produced in step (A) by an antigen-antibody reaction. In the step (B), the magnetic particles collected on the inner side wall of the well by application of a magnetic field include those in the state of such a reaction product and those in the state of magnetic particles carrying an unreacted antigen or antibody. The magnetic particles are attracted and held on the inner wall of the well by the magnetic force generated by the magnets arranged on the sides of each well. Thus B /
F separation is performed, and a supernatant is obtained in the well. The supernatant may be subjected to the measurement in the step (C), or the magnetic particles held on the inner wall surface of the well may be redispersed after another processing step, and may be re-distributed through the other step (B). The magnetic separation of step ()) is performed, and the supernatant thus obtained may be subjected to step (C). What kind of operation step is performed depends on what kind of immunological method the method of the present invention is applied to. In any case, the method includes the above steps (A), (B) and (C). Various variations are possible. In the step (C), the microtiter plate is subjected to an optical measurement as usual while the magnetic particles are held on the inner side wall of the well by the magnetic force. That is, the supernatant liquid generated in each well of the microtiter plate is subjected to photometry in the vertical direction to measure optical characteristics such as transmittance, absorbance, fluorescence, chemiluminescence, and bioluminescence. At this time, since the magnetic particles are collected and held on the inner wall, they do not obstruct the optical path. In practicing the present invention, it is desirable to use the following magnetic separation jig in consideration of the fact that the microtiter plate has many wells.

That is, in the method of the present invention, the following magnetic separation jig is mounted on the upper surface of the microtiter plate in order to place a magnet on each side of the microtiter plate and apply a magnetic field to each well. Alternatively, it is mounted on the bottom surface and magnets are placed adjacent to the sides of each well.

This magnetic separation jig comprises a flat base contacting the top or bottom surface of the microtiter plate, and a plurality of magnets inserted into the microtiter plate on one side of the base contacting the microtiter plate. Wherein each of the magnets is a magnetic separation jig provided to be inserted adjacent to a side of an adjacent well of a microtiter plate.

Hereinafter, a specific description will be given with reference to the drawings. Figure
1 is a magnetic separation jig 1 used in the method of the present invention,
It is a perspective view which shows an example of the type attached to the bottom side of a microtiter plate. FIG. 2 is a plan view of the separation jig. FIG. 3 is a partial cross-sectional view of the state in which the separation jig 1 is mounted on the bottom surface of the microtiter plate 2 in contact with the bottom surface of the microtiter plate 2 along the line AA in FIG. Usually, since the microtiter plate is a plastic molded product, when viewed from the bottom side, a gap is formed between the protrusions corresponding to the wells so that a magnet can be inserted.

The jig 1 includes a base 3 and a bar-shaped magnet 4 provided vertically on one surface of the base 3.
The base 3 supports the magnet 4 and also serves as a yoke. By making the base 3 of a metal material that can be magnetized, magnetic coupling to the tray surface (metal surface) on which the microtiter plate of the microplate reader is installed can be prevented, improving operability and adversely affecting equipment. Can be prevented. Practically, it is machined, for example, from a metal such as stainless steel.

The magnet 4 is made of a material that generates a strong magnetic field, and is preferably a permanent magnet. The magnet preferably has the largest possible volume, and preferably has a height close to the liquid surface of the liquid medium when the jig is mounted on the microtiter plate. Further, the shape of the magnet is preferably a rod shape or a cylindrical shape. The stronger the magnetic field and the greater the magnetic field gradient, the better the separation takes place and the faster the separation.

[0016] The magnet preferably applies a magnetic field to the adjacent well, most preferably one magnet applies a magnetic field to the surrounding, usually four wells. Each magnet is magnetized in any direction, and is preferably magnetized in a direction parallel to the base surface. More preferably, the direction is parallel to the base surface as shown in FIG. 6 (that is, the horizontal direction at the time of use). Most preferably, as illustrated in FIG. 7A, a pair of adjacent magnets are magnetized so that the same magnetic poles are opposed to each other (that is, in the direction of repulsion). With this arrangement, the magnetic field gradient applied to the wells increases, and the application conditions for all the wells become equal.

Referring back to FIG. 3, the base 3 is preferably sized to be accommodated in the area of the bottom surface of the microtiter plate 2, for example, a standard 96-well microtiter plate (85 mm long × 127 mm wide). In this case, it is preferable that the height is 75 mm or less and the width is 115 mm or less. The base 3 has a hole 6 at a position corresponding to the well 5 of the microtiter plate 2 so that photometry passes for optical measurement. In the case of a standard microtiter plate, the size of this hole is preferably at least 4 mm in diameter, particularly preferably 7 to 8 mm.

The magnets 4 must be provided such that there is at least one magnet adjacent to each well. FIG.
In this example, one magnet is provided in four wells, and each magnet is arranged so as to be surrounded by four wells (four-well application type). For this type of arrangement,
One magnet will be adjacent to one well. FIG. 9 shows an example (full set type) in which magnets are arranged at all positions (usually at the center) surrounded by any four adjacent wells. In the full set type, four magnets are adjacent to one well.

As shown in FIG. 3, the length of the magnet 4 when it is mounted on the microtiter plate 2 is desirably substantially the same as the water level 7a of the liquid 7 placed in the well 5.

The jig used in the present invention may be of a type mounted on the upper surface of a microtiter plate. FIG. 4 is a partial cross-sectional view showing this type of configuration mounted on a microtiter plate. In this case, a hole 8 for inserting a magnet is formed in a predetermined position on the upper surface of the microtiter plate. The length of the magnet is preferably substantially the same height as the bottom of the well 5, as shown in FIG. If the length is too short, the magnetic field does not effectively act on the sample solution placed in the well.

FIG. 5 is a partial sectional view of a jig of a type mounted on the upper surface side of a microtiter plate, showing a modification of a magnet. In this example, a cylindrical supporting portion 4a is provided on a base, and a magnet 4b is fixed to a tip of the supporting portion 4a. The support portion 4a may be made of a material such as a synthetic resin or ceramic. When the jig is attached to the microtiter plate, it is preferable that the connecting portion between the support portion 4a and the magnet 4b is substantially the same height as the liquid surface 7a of the sample liquid 7 in the well 5, as shown in FIG.

The method of the present invention can be applied to the following method for performing B / F separation using an antigen or antibody carried on magnetic particles. As the antigen-antibody reaction method, the measurement can be performed in a reaction format by a sandwich method, a one-step sandwich method, or a competitive method. When a biotin-labeled antibody or antigen is used, a biotin-avidin system can be used, and avidin labeled with an enzyme, a fluorescent substance, or a chemiluminescent substance can also be used for the reaction. The signal is measured after adding a substrate to the enzyme remaining in the well of the microplate and reacting it for a certain period of time. By changing the substrate to be added, it is possible to select a measurement method such as colorimetry, fluorescence, chemiluminescence, bioluminescence and the like and detection sensitivity.

Measurement by colorimetry is the most common method for measuring the absorbance or transmittance of a chromogenic substrate of an enzyme-labeled antibody, and various chromogenic substrates are commercially available. Enzymes such as alkaline phosphatase, peroxidase, glucose oxidase, and β-galactosidase, which are enzymes used in ordinary enzyme immunoassays, can be used in which enzymes or antigens are labeled.

In the measurement by fluorescence, the fluorescence of a fluorescent substance generated in a solution by reacting with a substrate by an enzyme labeled with an antibody or an antigen is measured. Enzymes and substrates such as 4-methylumbelliferyl phosphate for alkaline phosphatase, p-hydroxyphenylpropionic acid + hydrogen peroxide for peroxidase, and 4-methylumbelliferyl-β-D-galactoside for β-galactosidase. Used as a combination. An antibody or an antigen labeled with a complex of rare earth elements such as Eu, Tb, Sm, and Dt can also be used. These rare earth complexes have a long fluorescence lifetime and can be measured after the background has disappeared, so that measurement can be performed with high sensitivity. In the fluorescence measurement, a homogeneous immunoassay can also be performed by measuring a fluorescent label remaining in the solution without binding to the magnetic particles due to the antigen-antibody reaction. In the case of an antibody or an antigen that is labeled with a substance that emits fluorescence such as FITC, it is possible to leave unbound fluorescently labeled substance in the solution by magnetically separating magnetic particles on the side of the well. Measurement is also possible without B / F separation.

For measurement by chemiluminescence, an antibody or antigen labeled with, for example, a luminol derivative, lucigenin, acridinium derivative, dioctacene derivative, oxalic acid derivative or the like can be used as a chemiluminescent substance.

The measurement by bioluminescence can be performed by, for example, adding luciferin as a substrate and ATP, oxygen, and magnesium ions to a luciferase-labeled antibody or antigen to measure luminescence. In addition, the luminescence of an antibody or antigen labeled with aequorin can be measured by adding calcium ions.

[0027]

EXAMPLES Example 1 Magnetic Separation of Magnetic Particles Using a Magnetic Separation Jig A 0.5 μg / ml suspension of magnetic particles of 100 μm was added to each well of a 96-well microtiter plate (Imulon 600 manufactured by Greiner).
Then, a magnetic separation jig was attached to the bottom of the microtiter plate, and magnetic separation was performed for about 1 minute. The four magnetic separation jigs shown in Table 1 were used. During magnetic separation, the plate was shaken with a plate mixer (MPX-96, manufactured by Iwaki Glass Co., Ltd.) to promote the separation. Then, with the magnetic separation jig attached, the microtiter plate was transferred to a microplate reader (MPR-A4i, manufactured by Tosoh Corporation),
The absorbance of the supernatant was measured at 05 nm. Table 1 shows the results.
The absorbance of the magnetic particle suspension before the separation is 1.7 to 1.9, and the absorbance (background) of the microtiter plate in which only the solvent to which the magnetic particles are not added is dispensed.
0.03 to 0.04.

[0028]

[Table 1]

From the results shown in Table 1, it was confirmed that the model of the arrangement of the repulsion direction in the 4-well application mode has the best separability, and the variation due to the positional factor is low. In the same direction arrangement, the applied magnetic force is stronger than the repulsion direction arrangement, but the results of the magnetic field analysis show that the portion of the uniform magnetic field is relatively large, and it is considered that the separability is somewhat weak because the magnetic field gradient is low. .

Example 2 Immunological Measurement Using Magnetic Particles Derived from Magnetic Bacteria (One-Step Sandwich Method) (Preparation of Reagent) Anti-human Fab antibody 4 mg per 20 mg of magnetic particles extracted from magnetic bacteria After physical adsorption of 1
% Bovine serum albumin / phosphate buffer (pH 7.4). (Operation method) 1% bovine serum albumin / phosphate buffer (pH
In each well of the 96-well microtiter plate subjected to the blocking treatment according to 7.4), 100 μl of human Fab diluted to various concentrations as an antigen, 50 μg of magnetic particles carrying an anti-human Fab antibody, and 100 μl of alkaline phosphatase-labeled anti-human Fab antibody Was dispensed. After shaking and reacting at room temperature for 60 minutes, the magnetic separation jig shown in FIG. 1 was attached to the bottom of the microtiter plate as shown in FIG. 3, and magnetic separation was performed for about 2 minutes. Transfer the microtiter plate to a microplate washer (Nippon Bio-Rad Co., Ltd., 1575) with the magnetic separator attached, remove the supernatant, and wash 5 times with 0.05% Tween 20 / phosphate buffer (pH 7.4) did. Once the magnetic separation jig was removed from the microtiter plate, a luminescent substrate (Moss pNPP-10) was added to each well.
Add 00 μl and shake for 60 minutes to develop color. Again, a magnetic separation jig was attached, magnetic separation was performed, the sample was transferred to a microplate reader (MPR-A4i, manufactured by Tosoh Corporation), and the absorbance of the supernatant was measured at a wavelength of 405 nm. Human Fab antigen concentration 1-1000ng / ml
When the absorbance was graphed in the range, a good calibration curve shown in FIG. 10 was obtained. Measurement accuracy was high and reproducibility was also recognized.

[Example 3] Immunological measurement using commercially available magnetic particles containing antibodies (one-step sandwich method) (Preparation of reagent) Magnetic particles containing anti-human IgG antibodies (manufactured by PE Biosystems) BioMag) is dispersed in 1% bovine serum albumin / phosphate buffer (pH 7.4). (Operation method) 1% bovine serum albumin / phosphate buffer (pH
In each well of the 96-well microtiter plate subjected to the blocking treatment according to 7.4), 100 μl of human IgG diluted to various concentrations was used as an antigen, 50 μg of magnetic substance-containing particles with an anti-human IgG antibody, and 100 μl of alkaline phosphatase-labeled anti-human IgG antibody. Was dispensed. After shaking and reacting at room temperature for 60 minutes, the magnetic separation jig shown in FIG. 1 was attached to the bottom of the microtiter plate, and magnetic separation was performed for about 2 minutes. The sample was transferred to a microplate washer (Nippon Bio-Rad Co., Ltd., 1575) with the magnetic separation jig attached, and after removing the supernatant, the plate was washed five times with 0.05% Tween 20 / phosphate buffer (pH 7.4). Once the magnetic separation jig is removed from the microtiter plate, a luminescent substrate (Moss pNPP-10) is added to each well.
Was added and shaken for 60 minutes to develop color. The magnetic separation jig is attached to the microtiter plate again to perform magnetic separation, and the microplate reader (MP manufactured by Tosoh Corporation)
R-A4i), and the absorbance of the supernatant was measured at a wavelength of 405 nm. When the absorbance against the human Fab antigen concentration was graphed, the calibration curve shown in FIG. 11 was obtained. It could be used for measurement at a concentration of 10 ng / ml or more.

Example 4 Immunological measurement using commercially available magnetic substance-containing particles (antibody-free product) (one-step sandwich method) (Preparation of reagent) Magnetic substance-containing particles (DYNABEA, manufactured by DYNAL)
DSM450) and anti-human Fab antibody
Disperse in 1% bovine serum albumin / phosphate buffer (pH 7.4). (Operation method) 1% bovine serum albumin / phosphate buffer (pH
In each well of the 96-well microtiter plate subjected to the blocking treatment according to 7.4), 100 μl of the human Fab diluted to various concentrations was used as an antigen, 50 μg of magnetic substance-containing particles with an anti-human Fab antibody, and 100 μl of an alkaline phosphatase-labeled anti-human Fab antibody. Was dispensed. After shaking and reacting at room temperature for 45 minutes, a magnetic separation jig was attached to the bottom of the microtiter plate, and magnetic separation was performed for about 2 minutes. Transfer to a microplate washer (Nippon Bio-Rad Co., Ltd. 1575) with the magnetic separator attached, remove the supernatant,
The plate was washed five times with 0.05% Tween 20 / phosphate buffer (pH 7.4). Once the magnetic separation jig was removed from the microtiter plate, 100 wells of luminescent substrate (Moss pNPP-10) were added to each well.
μl was added and shaken for 45 minutes to develop color. The magnetic separation jig was mounted again to perform magnetic separation, transferred to a microplate reader (MPR-A4i, manufactured by Tosoh Corporation), and the absorbance of the supernatant was measured at a wavelength of 405 nm. When the absorbance against the human Fab antigen concentration was graphed, the calibration curve shown in FIG. 12 was obtained. It could be used for measurement at a concentration of 25 ng / ml or more.

[0033]

According to the method of the present invention, after the immunoreaction using the microtiter plate, the magnetic fine particles can be agglutinated on the inner wall side surface of the well. Since the absorbance of the supernatant can be measured, the immunological measurement can be easily performed. More specifically, the following advantages can be obtained. 1. It is possible to perform from reaction to measurement in the same reaction vessel called the well of a microtiter plate. 2. Measurement can be performed using a commonly-used spectrophotometer or microplate reader while maintaining magnetic separation. 3. Since the absorbance of the supernatant is measured, there is an advantage that the measured value is not affected by the suspension state of the magnetic particles. 4. If adjacent magnets are arranged in the horizontal and repulsive directions, and a four-well application type arrangement is used, it is easy to increase the applied magnetic force and magnetic field gradient, and quick and reliable magnetic separation is possible. . 6. The same magnetic field can be applied to all wells. 7. Since the base for fixing the magnet also functions as a yoke, there is almost no leakage of the magnetic field to the jig bottom. 8. When all materials are metal, they are robust and can be used semi-permanently without any fear of damage.

[Brief description of the drawings]

FIG. 1 is a perspective view showing an example of a magnetic separation jig used in the method of the present invention, which is mounted on a bottom side of a microtiter plate.

FIG. 2 is a plan view of the separation jig.

FIG. 3 is a partial sectional view taken along line AA of FIG. 2;

FIG. 4 is a cross-sectional view showing an example of a magnetic separation jig used in the method of the present invention, which is mounted from the upper surface side of a microtiter plate.

FIG. 5 is a partial cross-sectional view illustrating one embodiment of a magnet portion of a magnetic separation jig of a type mounted on an upper surface side of a microtiter plate.

FIG. 6 is a perspective view showing a magnetizing direction of a magnet.

FIGS. 7A and 7B are diagrams for explaining the arrangement direction of the magnets, where A indicates the repulsion direction arrangement and B indicates the same direction arrangement.

FIG. 8 is a diagram illustrating a four-well application type arrangement in which magnets are arranged so that one magnet is adjacent to four wells.

FIG. 9 is a view for explaining a full-set type arrangement in which magnets are arranged at the entire center surrounded by any four adjacent wells.

FIG. 10 shows a calibration curve of Fab concentration-absorbance obtained in Example 2.

FIG. 11 shows a calibration curve of IgG concentration-absorbance obtained in Example 3.

FIG. 12 shows a calibration curve of Fab concentration-absorbance obtained in Example 4.

[Explanation of symbols]

 1. Magnetic separation jig 2. Microtiter plate 3. Base 4. Magnet 5. Well 6. Hole 7. Liquid 8. hole

Claims (12)

[Claims] (A) reacting an antibody or antigen carried on magnetic particles with an antibody or antigen in a sample that specifically binds to the antigen or antibody in a liquid medium in a well of a microtiter plate (B) placing a magnet on the side of each well, applying a magnetic field to each well, and magnetically separating and holding magnetic particles in the liquid medium in the well on the inner wall side surface of each well. The above stages,
(C) measuring the optical properties of the supernatant liquid generated in each well as a result of the magnetic particles being held on the inner wall surface of each well as described above, wherein the antigen in the sample is Alternatively, an immunological measurement method for qualitatively or quantitatively measuring an antibody. 2. The method according to claim 1, wherein a magnet is arranged on a side of each well of the microtiter plate, and a magnetic field is applied to each well. 2. The method according to claim 1, wherein the microtiter plate is mounted on the top or bottom surface of the microtiter plate, and the magnet is arranged adjacent to the side of each well. A jig for applying a magnetic field from the side to the well of the microtiter plate, the flat base being in contact with the top or bottom surface of the microtiter plate, and the microtiter on one side of the base in contact with the microtiter plate. A plurality of magnets inserted into the plate, each of said magnets being inserted adjacent to a side of an adjacent well of a microtiter plate. Claim 1, wherein the provided it as
The method described in. 3. The method according to claim 2, wherein all magnets provided on the base are magnetized in a direction parallel to the base. 4. The method according to claim 3, wherein any one of the magnets is arranged so that the same magnetic pole faces at least one adjacent magnet. 5. The method according to claim 2, wherein the base is made of a magnetizable metal. 6. The method according to claim 1, wherein one magnet is provided for every four wells, and one magnet is surrounded by four adjacent wells. A method characterized by being arranged. 7. The method according to claim 1, wherein the magnet is provided at a position on the microtiter plate surrounded by any four adjacent wells. A method comprising: 8. The method according to claim 1, wherein said magnet is a neodymium iron boron magnet. 9. A magnetic separation jig for applying a magnetic field to a plurality of wells of a microtiter plate from the side, comprising: a flat base contacting an upper surface or a bottom surface of a microtiter plate; On one side of the side in contact with the titer plate, a plurality of magnets are inserted into the microtiter plate, and each of the magnets is inserted adjacent to a side of an adjacent well of the microtiter plate. All the magnets are magnetized in a direction parallel to the base, and any magnet is arranged so that the same magnetic pole faces at least one adjacent magnet. A magnetic separation jig characterized by the above. 10. The magnetic separation jig according to claim 9, wherein said base is made of a magnetizable metal. 11. The magnetic separation jig according to claim 9, wherein one magnet is provided for four wells, and one magnet is arranged so as to be surrounded by four adjacent wells. A magnetic separation jig characterized by: 12. The magnetic separation jig according to claim 9, wherein said magnets are provided at all positions on a microtiter plate surrounded by any four adjacent wells. A magnetic separation jig characterized by being used.