JP3631373B2 - Immunoassay and immunoassay apparatus - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an immunoassay method and an immunoassay apparatus for detecting the concentration of organic molecules, proteins, organelles, microorganisms, etc. using an immune reaction.
[0002]
[Prior art]
Conventionally, among immunoassays, a technique using immunoagglutination has been used as a relatively simple technique. In this immunoaggregation method, it is known that the technique of the immunoaggregation method can be applied more generally by modifying the antibody to microspheres such as latex particles and causing the reaction to occur. Since this immunoagglutination method is basically a visual determination, there is a problem in terms of quantitativeness. In addition, it may take several hours until the aggregation pattern is formed, which causes a problem in time.
[0003]
An attempt to detect this agglutination reaction with a quartz crystal resonator is disclosed by Muratsugu et al. (Anal. Chem. 1992, 64, 2483). As an immunoassay method using a quartz resonator, prior to this, an antibody is immobilized on the surface of the quartz resonator, and an immunoassay is performed based on a resonance frequency change accompanying a change in weight caused by binding of an antigen to the antibody. Is disclosed. Furthermore, the present inventors have also disclosed a method of measuring a coagulation reaction based on an enzyme reaction with a resonance frequency or a resonance resistance of a crystal resonator and using it for biochemical detection. In this case, a method of increasing the resonance frequency change due to the solidification reaction by sedimentation of fine particles is also disclosed. It has also been disclosed by the present inventors that the viscoelastic change of the substance on the surface of the crystal resonator can be detected by comparing the ratio of the change in the resonance frequency and the resonance resistance.
[0004]
[Problems to be solved by the invention]
In the method of Muratsugu et al., Only the resonance frequency change of the crystal resonator during the immune aggregation reaction in the solution in contact with the crystal resonator is measured, so whether the cause of the frequency change is due to aggregation or another factor There was a problem that there was no clear standard for judging whether it was. Accordingly, it is an object of the present invention to provide an analysis method and an analysis apparatus that can clearly detect the amount of change based on the agglutination reaction.
[0005]
[Means for Solving the Problems]
In a method of analyzing the concentration of a substance to be measured by the reaction of a target substance that is a substance to be measured with a fixed microparticle of a protein having specific adsorptive properties such as an antibody or a lectin , a fine particle alone is placed on a crystal resonator. Since the viscoelastic properties are different between the case where the particles are deposited and the aggregate in which the fine particles and the target substance are combined on the quartz resonator, the ratio between the change in the resonance frequency and the change in the resonance resistance is different. Utilizing this method, we devised an immunoassay method that detects the concentration of a substance to be measured from the amount of change when the aggregate settles by judging the difference between the precipitated substances.
[0006]
As a device that realizes this method, it has a liquid holding chamber with a quartz resonator at least on the bottom, temperature control means, a measurement circuit for the resonance frequency and resonance resistance of the quartz resonator, and has specific adsorptive properties such as antibodies and lectins. Based on the reaction between the immobilized microparticles of a protein and the target substance as the substance to be measured, the matrix particles in which the microparticles and the antigenic substance are combined and the sedimentation of the microparticles are identified, and the rate and amount of change in the resonance frequency and resonance resistance are determined An immunoassay device having a data processing means for detecting and converting to the concentration of a substance to be measured has been devised.
[0007]
DETAILED DESCRIPTION OF THE INVENTION
Embodiments of the present invention will be described below with reference to the drawings.
FIG. 1 illustrates the principle of the immunoassay method of the present invention along the measurement procedure. FIG. 1A shows a liquid holding chamber 2 having a quartz resonator 1 as a bottom surface, in which a protein having a specific adsorptivity such as an antibody and a lectin shown in FIG. 1B is immobilized. A suspension of microparticles 4 such as latex beads is added (Step 1). The fine particles start to be deposited on the bottom surface and form a deposited layer indicated by A in FIG. 1D (Step 2). Subsequently, a solution containing a substance having specific adsorptivity as the object to be measured shown in FIG. 1E is added to the chamber (Step 3). As a result, the specific adsorption reaction is started as shown in FIG. 1 (f), and an immunoaggregate of the fine particles and the substance to be measured 5 is formed. The immunoaggregates start to settle, and as shown in FIG. 1 (g), deposition of the immunoaggregate deposition layer B begins (Step 4).
[0008]
A more detailed view of this deposited layer is shown in FIG. The A layer in which only the fine particles are deposited has a relatively dense particle accumulation and is relatively high in viscosity. The B layer on which the immune aggregates are deposited is deposited in a relatively small state in terms of viscosity because the immune aggregates are bonded in a structurally rough state. Furthermore, depending on the type of the substance to be measured, an immune aggregation layer as shown as a C layer may be formed in a dense state on the surface of the deposit of the A layer in the initial stage of the immune reaction. In the C layer, the bond is dense and the viscous property is high.
[0009]
Based on the measurement results when the nitrifying bacteria antibody is immobilized on latex particles and the measurement is performed on a nitrate bacteria sample, the changes in the resonance frequency and resonance resistance of the crystal resonator in the above-described process are handled as follows. Let me explain. FIG. 3 (a) shows the change over time of the resonance frequency, and FIG. 3 (b) shows the change over time of the resonance resistance. First, due to the injection of Step 1 (S1) into the chamber, the resonance frequency is greatly reduced and the resonance resistance is increased due to the viscosity of the liquid. Next, the deposition of the fine particles of Step 2 (S2) causes a change in the weight of the surface of the crystal resonator, the resonance frequency decreases slowly, and the resonance resistance gradually increases due to the viscous nature of the deposited layer. Furthermore, when a solution of the substance to be measured is added (Step 3: S3), in the subsequent deposition of immune aggregates in Step 4 (S4), the resonance frequency rapidly decreases due to the weight change due to the deposition, and then the sedimentation converges. Along with this, it will decrease gradually. This is because sedimentation of immune aggregates occurs in a relatively short time. The resonance resistance also increases rapidly after increasing rapidly due to the increase in viscous properties due to the accumulation of immune aggregates. Eventually, there will be little increase in either.
[0010]
If the above change is described in the graph plotting the resonance frequency and the resonance resistance shown in FIG. 3C, it can be seen that the slope of the Step 2 portion is different from the slope of the Step 4 portion. This difference in slope shows the difference in the degree of change in the resonance resistance that reflects the change in the viscosity of the surface with respect to the change in the resonance frequency that reflects the change in the weight. it can. That is, the Step 2 portion is due to the deposition of only fine particles, and the Step 4 portion is due to the deposition of immune aggregates. It can be seen that there are two more inclined portions Step 4a and Step 4b in the Step 4 portion of FIG. This Step 4a corresponds to the C layer of the dense immune aggregate shown in FIG. 2, and the portion of Step 4b corresponds to the B layer in FIG. It can be seen that the slope of the Step 4a portion is larger than the slope of Step 2, and the slope of the Step 4b portion is smaller than Step 2. As an index of the amount of change, the amount of change in the resonance frequency or resonance resistance of this Step 4 portion can be obtained, and the concentration can be calculated from a calibration curve obtained in advance.
[0011]
Next, FIG. 3D shows a resonance frequency-resonance resistance plot when a blank solution of the substance to be measured is used. In FIG. 3D, the slope of the Step 2 portion representing the deposition of fine particles is observed in the same manner as in FIG. 3C, but it can be seen that the Step 4 portion is greatly different. The slope of the portion of Step 4 is a value close to the slope of Step 2, and here, it can be seen that immune aggregates are not formed and deposition of fine particles continues. Here, the slight difference in slope between Step 2 and Step 4 is due to a change in the composition of the solution.
[0012]
Next, FIG. 4 shows a schematic diagram of an immune analyzer for performing this immunoassay. In FIG. 4, the immunoassay device has a liquid holding chamber 21 having a quartz resonator placed on the bottom surface, a temperature control means 22, a measurement circuit 23 for the resonance frequency and resonance resistance of the quartz resonator, an antibody, a lectin and the like. The matrix particles in which the microparticles and the target substance are bound and the sedimentation of the microparticles are identified by the reaction between the immobilized microparticles of a protein and the antigenic substance to be measured, and the rate and change of the change in the resonance frequency and resonance resistance. It comprises data processing means 24 for detecting the amount and converting it to the concentration of the substance to be measured, a display device 25, and a key switch 26. By installing a plurality of chambers 21 and corresponding measurement circuits 23, it is possible to simultaneously measure a plurality of samples.
[0013]
As an actual sample, nitrifying bacteria antibody is immobilized on latex particles, and the relationship between the change in resonance frequency of Step 4 portion and the nitrifying bacteria concentration is measured with respect to the nitrate bacteria sample in FIG. The measurement was completed in about 30 minutes, and the measurement time could be greatly reduced as compared to the conventional time of several hours. Moreover, the measurement sensitivity could be improved as compared with visual observation.
[0014]
Furthermore, although the present embodiment has been described by using a shear vibration mode crystal resonator, a shear vibration mode piezoelectric element other than the crystal resonator may be used.
[0015]
【The invention's effect】
With the immunoassay method and the immunoassay apparatus of the present invention, it has become possible to clearly identify the accumulation of immune aggregates, and to measure the immunoaggregation method with high accuracy and accuracy. Furthermore, according to the present invention, the measurement time can be greatly shortened, the measurement can be quantified, and the measurement sensitivity can be improved.
[Brief description of the drawings]
FIG. 1 is an explanatory diagram of the principle of an immunoassay method of the present invention.
FIG. 2 is an explanatory diagram of the principle of the immunoassay method of the present invention.
FIG. 3 is an explanatory diagram of the principle of the immunoassay method of the present invention.
FIG. 4 is a configuration diagram of an immune analyzer according to the present invention.
FIG. 5 shows a measurement example of the immunoassay method of the present invention.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 Crystal oscillator 2 Liquid holding chamber 4 Protein fixed microparticle 5 Measuring substance 21 Liquid holding chamber 22 Temperature control means 23 Resonance frequency and resonance resistance measurement circuit 24 Data processing means 25 Display device 26 Key switch

Claims (4)

A method of analyzing the concentration of a substance to be measured by the reaction between the substance to be measured and a microparticle on which a protein having specific adsorptivity is immobilized, and measuring temporal changes in the resonance frequency and resonance resistance of the piezoelectric vibrator. Then, the sedimentation of the aggregate in which the fine particles and the substance to be measured are combined is identified from the ratio of the change in the resonance frequency of the piezoelectric vibrator and the change in the resonance resistance, and the amount of change in the resonance frequency or resonance resistance based on the aggregation reaction An immunoassay that detects the concentration of a substance to be measured from A step (A) of adding the fine particle solution to a sample chamber in which the piezoelectric vibrator is disposed on the bottom surface and measuring a change in a resonance frequency and a resonance resistance of the piezoelectric vibrator; was added, the and a step (B) measuring the change in the resonant frequency and resonant resistance of the piezoelectric vibrator, with respect to the rate of change of the resonance resistance and the resonance frequency in the step (a), the step ( B) determining that the agglutination reaction has progressed when the rate of change between the resonance frequency and the resonance resistance is different, and detecting the concentration of the substance to be measured from the amount of change in the resonance frequency or resonance resistance based on the agglutination reaction. The immunoassay method according to claim 1, wherein At least a liquid holding chamber having a piezoelectric vibrator disposed on the bottom surface, temperature control means, a measurement circuit for measuring temporal changes in the resonance frequency and resonance resistance of the piezoelectric vibrator, and a protein having specific adsorptivity is immobilized. Agglomerates in which the microparticles and the substance to be measured are combined by the reaction between the microparticles and the substance to be measured, and sedimentation of the microparticles are identified from the ratio of change in the resonance frequency and the resonance resistance, and the resonance frequency based on the aggregation reaction Alternatively, an immunoassay device having data processing means for obtaining the concentration of a substance to be measured from the amount of change in resonance resistance. Said piezoelectric vibrator, the immune analyzer according to claim 3, characterized in that the quartz oscillator of shear vibration mode.

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