JPH01214761A - Immunological analysis method using opto-acoustic spectroscopy - Google Patents

Abstract

PURPOSE:To shorten a measuring time and to enhance measuring sensitivity, by projecting intermittent light to reaction liquid wherein a labeled antigen or antibody that is bonded to a carrier and a labeled antigen or antibody that is not bonded are separated, and measuring a generated acoustic wave. CONSTITUTION:A sample incorporating a material to be measured, a labeled antigen or antibody labeled with a specified material and a carrier wherein antigen or antibody is solidified undergo antigen/antibody reaction in a reaction liquid. The reaction liquid is inputted in an opto-acoustic cell 4. Light emitted from a light source 1 is made to be intermittent light through a light chopper 2 and inputted into the cell 4. The intermittent light is absorbed with the sample in the cell 4. As a result, an acoustic signal is generated. The signal is measured by using an acoustic sensor 5. In this way, the labeled antigen or antibody bonded to a carrier and the labeled antigen or antibody that is not bonded are separately measured. The amount of the material to be measured is obtained by using a calibration curve that is prepared beforehand. The carrier to be used has a minute carrier particle having the average diameter of 0.01-100mum. It is desirable that the inactive carrier particles of 0.1-10mum are used in a solid state.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to an immunoassay method, and particularly to an immunoassay method suitable for analyzing components in a sample by photoacoustic spectroscopy.

[Conventional technology]

A receptor (antibody or anti-■) that specifically reacts with the substance to be measured in a sample, such as an antigen, antibody, or habten.
A method for determining the concentration of a substance to be measured is conventionally known by measuring the IS pattern, turbidity, or intensity of light scattering caused by the immune complex produced. However, these methods are easily affected by contaminant components in the sample.Secondly, there are problems in terms of measurement sensitivity, and when the concentration of the component to be measured is low (10-8M or less):
II! 1 cannot be determined. ``The conventional method used to measure such low concentrations of substances is the RIA method, which uses radioisotopes as labels. However, since the treatment of radioisotopes is limited, methods have been developed that use fluorescent substances or enzymes as labels instead of isotopes. These methods have advantages and disadvantages. Among measurement methods with excellent constant tolerability, methods using a homogeneous reaction system with good operability have insufficient measurement sensitivity, while measurement methods using a heterogeneous reaction system have relatively high sensitivity but are difficult to obtain results. Because it takes a long time to complete the test, it is inconvenient in ordinary clinical examinations. In a heterogeneous reaction system, the so-called B/F separation method separates the bound product of labeled antigen and antibody (abbreviated as BOUND or B) from the excess labeled antigen remaining in a free form (FREE or F). Among them, the reaction proceeds using large-sized in-phase carriers that settle in the liquid phase or the reaction tube wall as the in-phase,
A widely used method is to then wash to remove free labeled antigen and measure B activity. These in-phase methods have become popular in recent years because they are easy to handle, but these in-phase methods require several hours to obtain measurement results due to extremely low reaction efficiency. Also, in terms of measurement sensitivity, the limit was to achieve a measurement sensitivity of about 10-12M due to conflicts with the detection limits of conventional detection means such as absorbance or fluorescence intensity. In other words, it was not possible to confirm the concentration or even the presence or absence of sample components present at a lower concentration than this measurement detection sensitivity.

Furthermore, the B/F separation process directly affects the background value of the measurement results, and is therefore a major factor in reducing the reproducibility of the d1q constant value. Furthermore, in general, from the perspective of the equipment configuration, the mechanism and precision are the most required, so the fact is that the quality of the equipment's performance has a great impact on the reliability of the H1ll constant, so there is no need for B/F separation. Therefore, a highly sensitive and highly accurate analytical method is desired.

[Problem to be solved by the invention]

In the solid-phase method according to the conventional technology described above, the operation is troublesome due to the necessity of B/F separation, and it takes a long time to obtain measurement results. Furthermore, the measurement sensitivity is low due to the detection limit of absorbance or fluorescence intensity. There was a problem.

An object of the present invention is to provide an immunoassay method that can shorten operation time and increase measurement sensitivity.

[Means to solve the problem]

] - In order to achieve the above object, the immunoassay method of the present invention involves combining a sample containing a substance to be measured, a labeled antigen or antibody obtained by labeling an antigen or antibody with a predetermined substance, and a solid phase containing the antigen or antibody. by photoacoustic spectroscopy, which measures the acoustic waves generated by irradiating a reaction solution with intermittent light to react the antigen-antibody reaction with the carrier.

The labeled antigen or antibody bound to the carrier and the unbound labeled antigen or antibody are separated and measured to determine the amount of the substance to be measured.

[Effect]

According to the above immunoassay method of the present invention, an antigen-antibody reaction is performed between a sample containing a substance to be measured, a labeled antigen or antibody obtained by labeling an antigen or antibody with a predetermined substance, and a carrier on which the antigen or antibody is immobilized. Then, the amount of the substance to be measured is quantified by irradiating intermittent light to the reaction solution separated into those that do not bind to the labeled antigen or antibody bound to the carrier and measuring the generated acoustic waves.

〔Example〕

An embodiment according to the present invention will be described with reference to the drawings.

First, details of the photoacoustic spectroscopy used in the present invention will be explained. In photoacoustic spectroscopy, it is essential to first absorb light. When a substance absorbs light, it means that the substance takes in the light energy and the atoms or molecules that make up the substance rise to an energy-rich state. Photoacoustic spectroscopy is a spectroscopy method that measures the process by which light energy absorbed by atoms or molecules releases heat and returns to its original state. The emitted heat generates acoustic waves, and this photoacoustic signal is detected.

The main components of a photoacoustic spectrometer are, as shown in FIG. 1, a light source 1. Light chopper 2. Lens 3, photoacoustic cell 4. Acoustic sensor 5. The amplifier 6° is the data processing section 7. The light emitted from the light source 1 passes through a spectroscope, is turned into intermittent light by an optical chopper 2, and enters a photoacoustic cell 4. In the photoacoustic cell 4, a photoacoustic signal is generated as a result of the sample absorbing the intermittent light. This is detected using an acoustic sensor 5 such as a microphone. The photoacoustic cell 4 for the sample is preferably a cell that uses the sample itself as a pressure medium and converts the heat generated in the sample into a pressure change, which is detected using a piezoelectric element. There is also a type of cell in which a sample is placed in an airtight container filled with gas, and the heat generated in the sample is converted into a pressure change in the gas, which is detected using a highly sensitive microphone. However, samples often used in clinical tests are aqueous solutions, and high-sensitivity microphones dislike water vapor, making them difficult to handle.

As a sample, a final reaction solution containing microscopic carrier particles in an amount proportional to the concentration of the component to be measured in the sample is placed in the photoacoustic cell 4 and the photoacoustic signal intensity is measured.

That is, the final reaction solution is introduced into the photoacoustic cell 4.

Irradiate incident light. Pressure changes (photoacoustic signals) generated within the sample are converted into electrical signals (voltage generation) by piezoelectric ceramics. By creating a calibration curve between the antigen concentration and the photoacoustic signal using an antigen whose concentration is known in advance, the antigen-antibody reaction can be quantitatively measured using photoacoustic spectroscopy. Antigens or antibodies can be quantified.

According to a preferred embodiment of the present invention, in conventional practice, R
Trace components that could be measured for the first time by IA can be quantitatively measured with much higher sensitivity and quickly.

The micro carrier particles used in the present invention have an average particle diameter of 0.01
Inert carrier particles of .mu.m to 100 .mu.m are suitable for the purpose, particularly preferably inert carrier particles with an average particle size of 0.1 .mu.m to 10 .mu.m are used as the solid phase. When such a carrier is reacted with a measurement sample, the reaction efficiency becomes very high, so that the measurement proceeds quickly.

By the method according to the present invention, the substance to be measured in the sample is
Needless to say, in order to perform high-sensitivity and rapid quantitative determination, it is desirable to bring the reactant supported on an inert carrier such as lux particles into contact with the reacting sample at as high a concentration as possible. .

Using insoluble carriers with various particle sizes, human IgG
When performing an antigen-antibody reaction for quantitative determination, it was found that human IgG, which is an antigen, could be quantitatively detected within 1 hour by using a carrier with an average particle size of 100 μm or less. Furthermore, it has been found that quantitative determination is possible within a reaction time of 15 minutes, especially when insoluble carrier particles of 10 μm or less are used. In analyzing the measurement sample, the permissible reaction time from the viewpoint of processing speed is 1 hour, preferably 15 hours.
It can be said that it is preferable to use inert carrier particles having an average particle diameter of 10 μm or less. Also, when comparing the measurement sensitivity and S/N ratio, the average particle size is 0.01
It has been found that a particle size of .mu.m or more, preferably 0.1 .mu.m or more is required. From the above results, it was found that inert carrier particles having an average particle diameter of 0.1 μm to 10 μm are suitable.

The inert carrier particles include an organic polymer latex which is substantially insoluble in the liquid medium used for measuring biological fluids and has the above-mentioned average particle size.

cells, or for example silica, silica alumina.

Inorganic oxides such as alumina and other mineral powders.

Metal etc. are used.

In photoacoustic spectroscopy, it is possible to distinguish and extract signal intensities of substances with different particle diameters. For this purpose, it is sufficient to select and irradiate a wavelength corresponding to one particle diameter. In the analysis method according to the present invention, carrier particles of a fixed particle size to which a labeled antibody is bound may be used as the target particle size, and there is no need to extract the signal of the remaining labeling substance.

Next, a specific embodiment according to the present invention will be described.

First, an anti-α-fetoprotein antibody sensitized latex reagent (anti-human AFP latex reagent) is prepared as follows.

Anti-human AFP antibody in glycine buffer (0.1M.

p I (6, 5) 10 m Q, the average particle size is Q, 2
2 μm polystyrene latex (solids concentration, 10
After adding 1 mQ of weight%) and stirring at room temperature for 3 hours, the mixture was stirred at 12.00 rpm for 40 minutes under cooling at 2 to 4°C.
It was centrifuged at The obtained precipitate was suspended in 10 mM phosphate buffer (pH 6.5) containing 0.5% bovine serum albumin, 0.1M NaCQ, and 2mM MgCQz, and the sensitized latex particles were added to an antimicrobial solution with a concentration of 1% by weight. AFP latex reagent was prepared.

Next, 100 μQ of sample solution, 50 μQ of anti-AFP latex reagent, 501 tΩ of labeled antibody solution, and 100 μQ of reaction buffer were mixed and heated at 37°C.

The anti-antigen was reacted for 20 minutes. As a result, an immune reaction complex consisting of the solid-phase antibody, the antigen, and the labeled antibody, and the remaining labeled antibody depending on the amount of the antigen are formed.

Next, the reaction solution was introduced into a flow-type photoacoustic cell, and the photoacoustic signal was measured. An alboion laser was used as the light source.

The schematic configuration of the measuring device is as shown in FIG.

Furthermore, a calibration curve prepared for human FP measurement is shown in FIG. As is clear from FIG. 2, it is possible to quantitatively measure the low concentration region below IQmg/mQ, which could not be measured by conventional methods.

As described above, when measured by photoacoustic spectroscopy, the immunoreaction complex and the remaining labeled antibody have significantly different particle sizes, making it easy to distinguish them on the measurement. moreover,
Since B/F separation of the reaction solution is not necessary, operability is improved.

〔Effect of the invention〕

According to the present invention, since the method is as described above, the following effects can be achieved.

Antigen-antibody reactions can be quantitatively measured using photoacoustic spectroscopy without the need for B/F separation, which allows the target antigen or antibody to be quantified, increases measurement sensitivity, and allows detection in low-concentration areas. It has the effect of being able to measure Furthermore, the immobilized carrier is not limited to latex, but any material such as artificial cells with a uniform molecular weight of 1 can be used, and by setting the particle size of the carrier in the range of 0.01 to 100 μm, antigen This method has excellent effects such as the short reaction time of the antibody reaction, shortening operation time, and the ability to quantitatively measure trace components quickly and with high sensitivity.

[Brief explanation of the drawing]

FIG. 1 is a schematic diagram of a photoacoustic spectrometer used in an embodiment of the immunoassay method according to the present invention, and FIG. 2 is a diagram showing a calibration curve for AFP measurement. ■...Light source, 2...Light chopper, 3...Lens,
4... Photoacoustic cell, 5... Acoustic sensor, 6...
Amplifier, 7... data processing section.

Claims (1)

[Claims] 1. A reaction solution in which a sample containing a substance to be measured is subjected to an antigen-antibody reaction with a labeled antigen or antibody labeled with a predetermined substance and a carrier on which the antigen or antibody is immobilized. The labeled antigen or antibody bound to the carrier and the unbound labeled antigen or antibody are separated and measured by photoacoustic spectroscopy, in which the acoustic waves generated by irradiating intermittent light are measured. An immunoanalysis method using photoacoustic spectroscopy, which is characterized by determining the amount. 2. The immunoassay method using photoacoustic spectroscopy according to claim 1, wherein the particle size of the carrier is within the range of 0.01 to 100 μm.