JP2004061144A - Antigen-antibody reaction detecting method and apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an antigen-antibody reaction detecting method and an apparatus which substantially improve measurement precision. <P>SOLUTION: In the antigen-antibody reaction detecting method for measuring magnetic particulates in an AC magnetic field at an antigen-antibody reaction, the direction of a magnetic moment is aligned and fixed in one direction by previously drying and fixing (a) a magnetic particulate liquid in a magnetic field prior to measurement. By performing measurement after drying, the moment of the the magnetic particulates is aligned, and the magnetic particulates are brought close to one another to form a substantially highly dense magnetic moment, and a large magnetic signal is acquired. <P>COPYRIGHT: (C)2004,JPO

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method and an apparatus for detecting an antigen-antibody reaction for detecting an antigen-antibody reaction, and more particularly to a method and an apparatus for detecting an antigen-antibody reaction which can improve efficiency in producing monoclonal antibodies such as trace enzymes and in immunoassays. .
[0002]
[Prior art]
In biotechnology research, it may be necessary to produce specific antigens (enzymes, etc.) in large quantities, but the work requires a great deal of labor, time, and money. However, by using an antibody (monoclonal antibody) linked to the specific antigen, it becomes possible to drastically reduce the labor, time and cost required for purification.
[0003]
By the way, it is known that, for example, when an antigen is injected into an animal such as a mouse, an antibody is produced in the body, but the antibody-producing cells that have produced the antibody die. Therefore, upon purification of the antigen, generally, immortal antibody-producing cells (hybridomas) prepared by a cell fusion technique are used.
[0004]
However, since there are many types of hybridomas, it is necessary to select a hybridoma that produces a monoclonal antibody that recognizes an antigen of interest.
[0005]
In this process, a plurality of small vessels (wells) to which antigens have been attached in advance are prepared, and the monoclonal antibodies produced by the hybridomas are dispensed therein, and the amount of the monoclonal antibodies reacted in each well. Find out. As a method for this, it is common to examine using a label (label) such as fluorescence or radiation.
[0006]
However, in the case of a fluorescent label, a so-called bleaching phenomenon occurs in which the fluorescence fades, so that evaluation must be performed in a short time, and there is a problem in its accuracy. In addition, the use of radiation has to be performed in a specific control area, and there is a risk of worker exposure, which is not a preferable method for safety.
[0007]
Therefore, in the antigen-antibody reaction detection device, the present inventors apply an AC magnetic field to the antibody labeled with the magnetic fine particles during the antigen-antibody reaction, and convert the signal into a SQUID (superconducting quantum interference device). A device for detecting using a magnetic sensor (Japanese Patent Application No. 2001-133458) has been invented so far. A method of applying a DC magnetic field (JP-A-11-68180) has already been proposed by those skilled in the art.
[0008]
[Problems to be solved by the invention]
However, although the above-described conventional method solves the above-mentioned problem, at present, it has not yet achieved sufficient sensitivity.
[0009]
The cause lies in the method of fixing the magnetic fine particles. That is, when measuring the magnetic fine particles under a DC magnetic field, the measurement is usually performed in a liquid state or a dry state. In this case, the magnetic moment in the fine particles is oriented in a random direction, and the distance between the fine particles is increased. Therefore, a large magnetic signal could not be obtained.
[0010]
An object of the present invention is to provide an antigen-antibody reaction detection method and apparatus capable of eliminating the above-mentioned problems and greatly improving measurement accuracy.
[0011]
[Means for Solving the Problems]
The present invention, in order to achieve the above object,
[1] In the antigen-antibody reaction detection method in which the magnetic fine particles are measured under a DC magnetic field during the antigen-antibody reaction, the direction of the magnetic moment is aligned in one direction by drying and fixing the magnetic fine particle liquid in a magnetic field before measurement. By fixing and fixing and measuring after drying, the moments of the magnetic fine particles are uniform, and the magnetic fine particles come close to each other to form a magnetic moment having substantially high density, thereby obtaining a large magnetic signal.
[0012]
[2] In an antigen-antibody reaction detection device that measures magnetic microparticles under a DC magnetic field during an antigen-antibody reaction, the magnetic moment liquid is aligned in one direction by drying and fixing the magnetic microparticle liquid in a magnetic field before measurement. And a sample stage for setting a tube having a dried fine particle sample fixed thereto, and a DC magnetic field applying means for applying a DC magnetic field in which Helmholtz coils are arranged on the left and right sides of the sample stage with respect to the antibody labeled with the magnetic fine particles. And a SQUID magnetic sensor that vertically arranges a magnetic field detection surface on a radial axis where a magnetic field component in the radial direction from the left and right Helmholtz-shaped coils of the DC magnetic field applying means is zero. .
[0013]
[3] The apparatus for detecting an antigen-antibody reaction according to the above [2], wherein the tube is a non-magnetic tube.
[0014]
[4] The apparatus for detecting an antigen-antibody reaction according to [3], further comprising: a driving device for stretching the tube; and a control device for controlling the driving device to control a pulling speed of the tube. It is characterized by the following.
[0015]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.
[0016]
First, a first embodiment of the present invention will be described.
[0017]
FIG. 1 is a schematic diagram of the detection of an antigen-antibody reaction using the magnetic fine particles according to the present invention. FIG. 1 (a) is a schematic diagram of the antigen-antibody reaction detection method, and FIG. 1 (b) is a diagram of a DNA detection method using the same. It is a schematic diagram.
[0018]
In FIG. 1A, 1 is a sample substrate, 2 is an antigen (MYOSIN: antigen, rabbit, muscle), 3 is a primary antibody (Mouse Anti-MYOSIN), and 4 is a magnetically labeled antibody.
[0019]
In FIG. 1B, 11 is a sample substrate, 12 is a gold electrode, 13 is streptavidin, 14 is biotin, 15 is a known probe DNA fragment, and 16 is an unknown target DNA fragment. , 17 are magnetic fine particles.
[0020]
As shown in FIG. 1A, an antigen 2 is attached to a sample substrate 1 in advance, and a primary antibody 3 recognizing the antigen 2 is reacted therewith.
[0021]
Next, the magnetically labeled antibody 4 labeled with magnetic fine particles is reacted with the primary antibody 3. Thereby, the primary antibody 3 that has recognized the antigen 2 is labeled with the magnetically labeled antibody 4. The magnetic signal of the magnetically labeled antibody 4 is detected by a SQUID magnetic sensor (not shown).
[0022]
Further, as shown in FIG. 1B, a known probe DNA fragment 15 is arranged on a gold electrode 12 on a sample substrate 11 via a streptavidin 13 and a biotin 14, and magnetic fine particles are formed. The unknown target DNA fragment 16 provided with 17 is reacted. Then, a magnetic signal of the magnetic fine particles 17 is detected by a SQUID magnetic sensor (not shown).
[0023]
The behavior of the magnetic fine particles by the external magnetic field used here will be described.
[0024]
FIG. 2 is a schematic diagram showing the behavior of the magnetic fine particles by such an external magnetic field, FIG. 2 (a) is a schematic diagram showing the behavior of a fine particle sample in liquid, and FIG. 2 (b) is the behavior of a zero magnetic field dry fine particle sample. FIG. 2C is a schematic diagram showing the behavior of a fine particle sample dried in a magnetic field (10.0 [Gauss: G]) according to the present invention.
[0025]
As apparent from these figures, for example, a magnetic moment 23 (FIG. 2A) in a magnetic fine particle 22 in a fine particle sample 21 in a liquid made of pure water in a Teflon (registered trademark) tube 20, and Teflon Each of the magnetic moments 26 (FIG. 2B) in the magnetic fine particles 25 in the (registered trademark) tube 20, for example, in the zero magnetic field dry fine particle sample 24, is oriented in a random direction. In the above-described embodiment, a Teflon (registered trademark) tube is shown as an example. However, the present invention is not limited to this, and a non-magnetic tube having poor wettability is desirable.
[0026]
On the other hand, the direction of the magnetic moment 29 in the magnetic fine particles 28 in the fine particle sample 27 dried in the magnetic field (10.0 [G]) in the Teflon (registered trademark) tube 20 according to the present invention is one direction. It is aligned in the direction [FIG. 2 (c)]. Here, the magnetic field strength is exemplified as 10.0 [G], but it is sufficient that the magnetic moment can be substantially oriented, and the upper limit is set in consideration of the price and size of the equipment. Just fine. For example, about 0.5 [G] to about 500 [G] is desirable.
[0027]
That is, by preliminarily drying and fixing the magnetic fine particle liquid in a magnetic field, the direction of the magnetic moment 29 can be aligned in one direction and fixed. By measuring after drying, the magnetic fine particles 28 approach each other. Since the magnetic moment 29 having a substantially high density is formed, it has been found that a magnetic signal which is several times larger than that of the conventional method can be obtained.
[0028]
As a result, a smaller amount of the antigen-antibody reaction can be detected, and highly sensitive detection of environmental trace substances such as environmental hormones and dioxins becomes possible.
[0029]
Here, MYOSIN, Anti-MYOSIN,... Are used, but these are merely examples, and the present invention is not limited to these, and may be appropriately selected depending on the application form. It is important to label.
[0030]
FIG. 3 is an overall configuration diagram of an antigen-antibody reaction detection device showing an embodiment of the present invention.
[0031]
In this figure, 100 is a Teflon (registered trademark) tube, 101 is a magnetic fine particle sample, 102 and 103 are Helmholtz coils disposed on the left and right sides of the magnetic fine particle sample, and 104 is a direct current connected to the Helmholtz coils 102 and 103. A power source, 105 is a sample stage on which the magnetic fine particle sample 101 is set, and 106 is provided on the sample stage 105, and a magnetic field is applied on a radial axis where the radial magnetic field component from the left and right Helmholtz coils 102, 103 becomes zero. SQUID magnetic sensor having a detection surface arranged vertically, 107 is a SQUID drive circuit, 108 is a personal computer, 109 is an XY pen recorder, 110 is a liquid nitrogen tank for cooling the SQUID magnetic sensor 106, and 111 is Teflon ( (Registered trademark) A wire for stretching the tube 100 Over, 112 a motor as a drive device for tensioning the wire 111, 113 is a control device of the motor 112, it is possible to adjust the tension of the wire 111.
[0032]
FIG. 4 is a forward DC magnetic field dependence characteristic diagram of various magnetic fine particle samples. The horizontal axis indicates the magnetic field [G], and the vertical axis indicates the magnetic flux [Φ ₀ ], as shown in the right column. , A indicates a sample dried in a magnetic field, b indicates a liquid sample, and c indicates a sample dried in a zero magnetic field.
[0033]
As is clear from this figure, the signal of the sample a dried in the magnetic field is several times as large as the other samples. This is because a large magnetic moment was obtained because the axis of easy magnetization was aligned at the time of drying and the distance between fine particles was narrowed.
[0034]
As described above, conventionally, there was a problem in the method of fixing the magnetic fine particles. That is, when measuring the magnetic fine particles under a DC magnetic field, the measurement is usually performed in a liquid state or a dry state, but in this case, the magnetic moment in the fine particles is in a random direction, and the fine particles are separated from each other Therefore, a large magnetic signal could not be obtained.
[0035]
In contrast, in the present invention, the magnetic fine particle liquid is dried and fixed in a magnetic field in advance before measurement, whereby the direction of the magnetic moment can be fixed in one direction, and the measurement can be performed after drying. As a result, the magnetic fine particles approach each other, and a magnetic moment having a substantially high density is formed, so that a magnetic signal that is several times or more as large as that of the conventional method can be obtained.
[0036]
This makes it possible to detect a smaller amount of an antigen-antibody reaction, for example, a detection of an antigen-antibody reaction of DNA, and to perform highly sensitive detection of environmental trace substances such as environmental hormones and dioxins.
[0037]
It should be noted that the present invention is not limited to the above embodiments, and various modifications are possible based on the spirit of the present invention, and they are not excluded from the scope of the present invention.
[0038]
【The invention's effect】
As described above, according to the present invention, the following effects can be obtained.
[0039]
(A) By preliminarily drying and fixing the magnetic fine particle liquid in a magnetic field, the direction of the magnetic moment can be aligned in one direction and fixed. The approach and the formation of substantially dense magnetic moments have resulted in magnetic signals that are several times greater than conventional methods.
[0040]
(B) Detection of a smaller amount of an antigen-antibody reaction, for example, detection of an antigen-antibody reaction of DNA, enables highly sensitive detection of environmental trace substances such as environmental hormones and dioxins.
[Brief description of the drawings]
FIG. 1 is a schematic diagram of detection of an antigen-antibody reaction using magnetic fine particles according to the present invention.
FIG. 2 is a schematic diagram showing behavior of magnetic fine particles by an external magnetic field.
FIG. 3 is an overall configuration diagram of an antigen-antibody reaction detection apparatus according to an embodiment of the present invention.
FIG. 4 is a graph showing a DC magnetic field dependence characteristic of various magnetic fine particle samples in a forward direction.
[Explanation of symbols]
1,11 sample substrate 2 antigen 3 primary antibody 4 magnetically labeled antibody 12 gold electrode 13 streptavidin 14 biotin 15 known probe DNA fragment 16 unknown target DNA fragment 17, 22, 25, 28 magnetic fine particles 20, 100 Teflon ( (Registered trademark) tube 21 Fine particle sample in liquid 23, 26, 29 Magnetic moment 24 Zero magnetic field dried fine particle sample 27 Fine particle sample 101 dried in magnetic field 101 Magnetic fine particle sample 102, 103 Helmholtz coil 104 DC power supply 105 Sample stand 106 SQUID magnetism Sensor 107 SQUID drive circuit 108 Personal computer 109 XY pen recorder 110 Liquid nitrogen tank 111 Wire 112 Motor 113 Motor control device

Claims (4)

In the antigen-antibody reaction, in the antigen-antibody reaction detection method of measuring the magnetic fine particles under a DC magnetic field,
Before the measurement, the magnetic fine particle liquid is dried and fixed in a magnetic field beforehand, so that the direction of the magnetic moment is fixed in one direction, and the measurement is performed after drying, so that the moments of the magnetic fine particles are aligned and the magnetic fine particles approach each other. And forming a magnetic moment having substantially high density to obtain a large magnetic signal. In the antigen-antibody reaction, in the antigen-antibody reaction detection device that measures the magnetic fine particles under a DC magnetic field,
(A) a sample table for setting a tube having a dry fine particle sample in which the direction of the magnetic moment is fixed in one direction by previously drying and fixing the magnetic fine particle liquid in a magnetic field before measurement;
(B) DC magnetic field applying means for applying a DC magnetic field in which Helmholtz coils are arranged on the left and right sides of the sample table with respect to the antibody labeled with the magnetic fine particles;
(C) a SQUID magnetic sensor that vertically arranges a magnetic field detection surface on a radial axis where a magnetic field component in the radial direction from the left and right Helmholtz-shaped coils of the DC magnetic field applying means is zero. Antigen-antibody reaction detector. 3. The apparatus according to claim 2, wherein the tube is a non-magnetic tube. The antigen-antibody reaction detection device according to claim 3, further comprising: a driving device that stretches the tube; and a control device that controls the driving device to control a pulling speed of the tube. Antigen-antibody reaction detector.

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