JPH03223673A - Immunological detection reagent, immunological reagent, and method and apparatus for immunological detection - Google Patents

Abstract

PURPOSE:To obtain a highly sensitive reagent easy to operate by chemically bonding a fluorescent substance changing in its wavelength or intensity of fluorescence upon the approach to an antibody and the antigen specifically bonded to the antibody. CONSTITUTION:An immunological detection reagent is obtained by chemically bonding a fluorescent substance changing in its wavelength or intensity of fluorescence upon the approach to an antibody to the antigen specifically bonded to the aforementioned antibody. The antigen bonded region of the antibody develops hydrophobicity by the effect of the environment surrounding the antigen bonded region. Therefore, the free antigen present in a hydrophilic medium and the antigen bonded to the antibody are different in hydrophilic and hydrophobic environments and the fluorescence intensity of the fluorescent substance bonded to the immunological detection reagent is different. A substance to be detected can be detected by utilizing this fluorescence intensity difference. The fluorescent substance contains a dansyl group and the antigen is methaphetamine, anphetaminc or ephedrin and an antigen similar substance has a chemical structure represented by formula II (wherein R is H or a methyl group, n is 0 - 10, and m is 0 when n is 0 while m is 1 at the time of nnot equal to 0).

Description

[Detailed description of the invention] Industrial application field of the present invention (Table: Immune detection method used in highly sensitive detection technology based on immune reaction; Conventional technology related to detection method and detection device; Table: Purpose) Detected substance (antigen)
This is a measurement method using an antibody that specifically binds to L. By using an antibody, L is prevented from being interfered with by impurities when the target substance is present in an overwhelmingly smaller amount than other components. Therefore, immunodetection methods can be used to detect specific trace components in the air or blood, which are difficult to detect using conventional instrumental analyzes such as gas chromatography, liquid chromatography, and mass spectrometry. Among the effective immunological detection methods are the homogeneous method, which is relatively easy to operate and can be measured in a short time, but has low sensitivity, and the heterogeneous method, which is complicated and requires about 5 hours for measurement. The inventors have already proposed, for example, Japanese Patent Application No. 63-75447 or Japanese Patent Application No. 63-75, as an immunoassay method that has both the ease of operation of the uniform method and the high sensitivity of the non-uniform method. -1
In 84951, an excitation light in the vicinity of 28Or+m was used to generate light at around 320 nm, and the antibody's own fluorescent light emitted light at around 320 nm.Proposed to speed up the immunological detection method by utilizing the property that changes when bound to an antigen. As a representative example of the equalization method (EMIT (En
zyme Multiplied Immunoass
+& It is devised to reduce or increase the activity of the antigen-labeled enzyme by binding with the antibody. Moreover, it is easy to operate because it does not require separation from the antibody bound to anti-J-K (hereinafter referred to as B/F separation), and the measurement time is relatively short, about 5 to 20 minutes. On the other hand, the disadvantages are that the sensitivity is low at around 10''M, and the time required for measurement is not instantaneous.Therefore, in order to obtain high sensitivity, the non-uniform method is generally adopted, but in the non-uniform method, the sample l to detect the presence of antigen in
Conventionally, methods for labeling antibodies with radioactive isotopes or enzymes, and methods for B/F separation have been studied, and many methods have been proposed as so-called immunological detection methods (e.g. Day
7M Vai Collection Handbook of -L Tsukuberi Mental Immuno-1G, 'JI'A 26
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ns, 0xford)).

However, in any of the above laws, LB/
Although a heterogeneous system in which antibodies or antigens are immobilized on a solid phase is used for 'F' minutes, B,・1・゛For the explanation of separation, typical examples of conventional immunoassay methods step by step through the experimental procedure of the ELISA method.) ゛C'Explanation Go to 4.
The antigen solution is prepared by dissolving the detection substance or a conjugate containing a derivative with a functional group introduced therein in a liquid solution. Pour 100 μL/well of the antigen solution into a microplate (96-well plate made of vinyl chloride or polystyrene). (B) Inject 250 μL/well of blocking BSA buffer solution.
．． Leave it at room temperature for 5-2 hours. Then, wash it 3-5 times with powder or pure water. (C) Inject the antibody reaction detection substance solution and add more antibody solution while shaking. After storing for ~5 hours, remove the antibody solution with an aspirator and wash 3 to 5 times with buffer or pure water. Injected with 0.0% at room temperature. 5-2
Leave for 3 to 5 hours and then add buffer or pure water for 3 to 5 hours.
Wash twice (E) Substrate reaction and stop color development For example, a solution (substrate solution) in which O-phenyl diamine (for selenium detection) is dissolved in a buffer and 30% hydrogen peroxide solution is added immediately before use. Inject and perform color reaction at room temperature. After 5 to 20 minutes, stop the reaction with sulfuric acid (F) Measure the absorbance at 492 nm using a microplate absorption spectrometer.Finally, the more substances to be detected, the weaker the absorbance. conduct.

The above procedure is a typical conventional immunoassay method, but it has the disadvantage of requiring significant time and effort.As mentioned above, the conventional heterogeneous immunodetection method
/F separation requires many steps, and since the reaction proceeds in a heterogeneous system where solid and liquid phases coexist, and an enzymatic reaction is used for final detection, it inherently takes a long time. Furthermore, the method of using antibody fluorescence that the present inventors have already proposed has the problem that the excitation light and emission wavelengths are close to each other, and the Raman light of water becomes noise. Therefore, the present invention 1 was developed to overcome the above-mentioned conventional problems, and is easy to operate and takes a long time to detect. An immunological detection reagent that is short, highly sensitive, and can sufficiently detect the target substance even in test samples contaminated with proteins, etc., without being affected by noise caused by water in the medium. The purpose of the present invention is to provide a detection method and device.

Means for Solving the Problems In order to solve the above problems, the present invention uses a fluorescent substance whose fluorescence wavelength or fluorescence intensity changes when it comes close to an antibody, and an antigen that specifically binds to the antibody. Chemically bonded immunodetection reagent We propose an immunoreagent that is a mixture of this immunodetection reagent and an antibody, as well as a method and device for detecting a target substance using the immunodetection reagent. The site becomes hydrophobic due to the influence of the environment surrounding the antigen binding site. Therefore, free antigen (antigen that is not bound to an antibody) exists in the hydrophilic medium.
The antigen bound to the antibody has a different hydrophilic and hydrophobic environment,
The fluorescent substances bound to the immunodetection reagent of the present invention have different fluorescence intensities.Using this difference in fluorescence intensity, the target detection substance can be detected and the fluorescent substance bound to the immunodetection reagent of the present invention can be selected. In some cases, it is possible to select long wavelengths for the excitation light and fluorescence, but for this reason, it is possible to select a wavelength longer than the wavelength range in which proteins mixed in the target detection substance emit fluorescence, and even if the target detection substance mixed with proteins Although the target substance can be detected, noise caused by Raman scattering caused by water, which is the target substance to be detected, can also be blocked.

Examples An immunodetection reagent according to the present invention (an antigen or a partially substituted antigen or an antigen bound to a functional group for binding to a fluorescent substance) and an antigen-like substance chemically bound to a fluorescent substance are used. say.

In addition, the immunological reagent as used in the present invention refers to a mixture of such an immunodetection reagent and an antibody, and includes fluorescent substances applicable to the present invention (such as dansyl chloride, fluorescamine, 0-phthalaldehyde, etc.). Covalent substances such as fluorescein isothiocyanate, or for example acridine orange.

Examples include non-conjugated substances such as 9-aminoacridine and ateprin, but no particular limitation is required. Fluorescence (51 for dansyl type)
It is preferable to use a fluorescent substance that can obtain 0 nm) because noise components such as fluorescence of proteins mixed in the target detection substance and Raman scattering of water contained in the target detection substance can be removed. We will also describe the type of antigen as the target substance to be detected (as well as various pharmaceuticals).
Examples include agricultural chemicals, explosives, and pollutants, etc. Although there are no particular limitations, the present invention is particularly useful for detecting drugs with high sensitivity and rapid detection. Moreover, among the immunodetection reagents of the present invention, for the above-mentioned reasons, when an antigen-like substance having the chemical structural formula shown below, in which methamphetamine or amphetamine and a dansyl group are bonded, is used, the following The advantage is that methamphetamine or amphetamine, which is a stimulant, can be detected easily by illegal possession or urine testing, and it can meet strong social demands. In addition to being easy to use, since the fluorescence of the dansyl group can be used, both the excitation light and the fluorescence have long wavelengths, making it possible to suppress adverse effects from proteins that may be included as impurities. Furthermore, although the adverse effects of Raman light from water can be removed, LR is hydrogen or a methyl group, and when R is hydrogen, it is a so-called amphetamine analogue, and when R is a methyl group, it is a so-called methamphetamine analogue ( CHt)-(NH)- is a methylene amino group (Yo) It is a functional group that binds a dansyl group and amphetamine or methamphetamine, n is an integer of 0 to 10, m is an integer of 0 or l, and n is Only when 0, m is also 0
In other cases of n, m is l.

When n exceeds 10, the strength of the fluorescent substance (i.e., dansyl group) of the immunodetection reagent bound to the antigen-binding site of the antibody may be removed from the environment that makes the antigen-binding site hydrophobic. Both the immunodetectable substance bound to the antibody and the immunodetectable substance not bound to the antibody are in a hydrophilic atmosphere, and the same level of fluorescence intensity is detected, making it difficult to distinguish between the two. Desirably 6 or less
In addition, if the immunodetection reagent has any of the following structural formulas in which amphetamine or methamphetamine and a dansyl group are bonded via a butylamino group, the binding property between the antibody and the immunological detection reagent and the binding property between the antibody and the target antigen At the same time, synthetic raw materials are easily available, and synthetic reactivity is high. In this case, the synthesis procedure is the simplest and the environment surrounding the antigen-binding site of the antibody has a low degree of hydrophobicity, and the fluorescence intensity increases when the antibody and immunodetection reagent bind.
When this bond is broken and the immunological detection reagent becomes free, the fluorescence intensity decreases, which is preferable. If an aqueous solution is used, it is preferable to further adjust the pH of the aqueous solution.
For example, a phosphate-based buffer may be used for this pH adjustment (t) Furthermore, for convenience in handling and transportation, it is preferable for immunological reagents to be in solid form; in this case, they are prepared in a solution state and then lyophilized. This can be achieved by freeze-drying (above), which is preferable because it can be solidified without impairing the activity of the immunodetection reagent and antibody (i.e., the immunological reagent). The immunological detection method of the present invention will be explained by taking up the case of the following.

An immunological detection reagent in which the antigen methamphetamine (hereinafter abbreviated as MA) and a dansyl group are bonded is synthesized.

In this way, the synthetic immunodetection reagent is mixed with an antibody that specifically binds to MA to form an immunological reagent.

At this time, the antibody and the immunodetection reagent bind to each other, increasing the fluorescence intensity of the fluorescent substance in the immunodetection reagent, and the detection sample is introduced here.

If MA is contained in the detection sample, the binding between the antibody and the immunological detection reagent will be broken and the fluorescence of the immunological detection reagent will decrease. The presence or absence of MA in the measurement sample can be determined by measuring the fluorescence of the mixture mixed with the target reagent.Also, at this time, the relationship between the amount of MA in the measurement sample and the decrease in fluorescence intensity should be investigated in advance. Then, the amount of MA (that is, the target substance to be detected) contained in the measurement sample can be quantitatively measured.

By taking the above measures, the reaction proceeds in a homogeneous system (liquid phase) and the detection time can be significantly shortened because no enzymatic reaction is used. Although the difference in the wavelength of the emitted light can prevent the influence of Raman light, and since both the excitation light and the emitted light have longer wavelengths than the fluorescence of proteins, the influence of impurities can be prevented. Example 1 Dansyl chloride (hereinafter abbreviated as DNSCl (MW-26)
9,75)) 232.2mg (0,861mmol
) was dissolved in 6 ml of acetone to obtain isoN-(4-aminobutyl)methamphetamine (hereinafter AH
Abbreviated as MA (MW-220,36)) 154.7mg
(0,702a+mol) was added with the above DNSC1 solution, and anhydrous potassium carbonate (MW
-138,21) 242.8mg (1,757mm
ol) and stirred at room temperature (approximately 25°C) for 7.5 hours.
4. The progress of the reaction was measured by thin layer chromatography under the following conditions (
TLC+ Silica Gel 60 (Merck, Art,
5549.50X75x O, 2mm) Solvent: Acetone Rf: DNSCI: 0.72-0.6
6DNSABMA: 0.45-0.17ABMA:
~0 DNSCI and DNSABMA emit bright green fluorescence when irradiated with 366n111 ultraviolet light; ABMA is 254
f, which is performed by slight ultraviolet absorption and ninhydrin reaction during nm ultraviolet irradiation.

I'RL shows that all ABMA has reacted. After removing potassium carbonate by filtration, acetone is distilled off. 2
The remaining 97.4 og of yellow liquid was dissolved in approximately 2 ml of acetone and subjected to preparative TLC (Merck, Ar
5717.200x200x2mm) and developed with acetone.The DNSABMA portion was scraped off and extracted with methanol to obtain 140 mg of the final purified product (9) as a pale yellow liquid (D
NS-ABMA (MW=453.65.0.309mm
ol), yield 44.0%).

The above synthesis process is shown below.

(Space below) (DNSCI > (ABMA) DNS-ABMA phosphate buffered saline (PBS) solution (10-'M) 2.5 + nl monoclonal antibody (IgG) solution (2.5x) prepared under the following conditions 10
Samples in which the emission spectrum was measured under the following conditions after adding 20 or 40 μm of 20 or 40 μm of DNS-ABMAI DNS-AI3MA in PBS and stirring carefully with a nine Pasteur pipette: [DNS-ABMAI DNS-AI3MA was diluted with PBS, M and 1, 0.45 μm filtered circle [1 gG] PBS of a monoclonal antibody with a binding constant of 10-' against MA prepared by the inventors
Pour the solution through a 0.45μ filter to 280
The absorption intensity in μm is 0.563 and μ I% concentration of Ig
As As5s-15 of G, [IgG]-2,5x t
Measurement conditions: [Excitation wavelength] 528 μm [Pantopasco excitation side + 2Or++++ Emission side: lOnm [Scanning speed] 120 μm/win [Emission wavelength] 400-620 nm [Intensity range 0-20] Shown in Figure 1. With the addition of antibodies, the DNS
-The fluorescence intensity of ABMA increased to 550 at the same time.
The maximum emission wavelength of DNS-ABMA in ne is 523
This is a phenomenon that often occurs when the polarity of the environment surrounding the fluorescent substance decreases.
The MA portion of NS-ABMA binds to the antibody, and in this case, the DN
It is thought that this phenomenon is caused by the fact that the S part is in close proximity to the protein (antibody), so the polarity is lower than in a free aqueous solution. An experiment was conducted to detect MA by inhibition of MA against the binding of antibodies to and antibodies.The results of this experiment are described below.
) 546 μl, antibody solution 24 μl, MA solution 3 of each concentration
0 μl was placed in a quartz microcell, stirred for 30 seconds, and the fluorescence intensity was measured. - ABMA was diluted with PBS to 10-'M and Lo, and transferred to a 45 μm filter.
) was filtered through a 0.45 μm filter at 9280 n f
The absorption intensity of fl was 0.563 for 1s concentration of IgG.
Apes-157 [IgG]-2,5x 1
[Excitation wavelength] 328na+ [Band pass] Excitation side: 20r+m Emission side: 20μm [Measurement wavelength] 530μm Figure 2 shows the fluorescence of DNS-ABMA plotted against the MA concentration. In Figure 2, the MA concentration that gives a 50X change is approximately 1
This value is EL
The sensitivity was the same as when MA was detected by ISA. In this method, the time used for the actual reaction was
Therefore, according to the present invention, it is possible to increase the speed of the conventional immunodetection method without sacrificing the sensitivity of the conventional immunodetection method. The difference in wavelength of light emission is 200 μm.
Raman light (approximately 36 On (transition) at this time had no effect at all. In addition, excitation light (328 nm), emission (530 nm), co-protein fluorescence (excitation 281) nII+, emission 320 nm were included because the wavelength was longer than that. Example 2 By carrying out the same synthesis using MA instead of ABMA in Example I, DNS-MA with the following molecular structure was synthesized. The results of the same evaluation as in Example I using DNS-MA are described below.

Optimal excitation wavelength: 328nm Maximum emission wavelength: 550n (without antibody) 530nm
nm (When adding antibody) MA concentration at 50% change in fluorescence: 10-aJ 1 bottle Practical Color Plague Detection Reagent L It was found to be effective in detecting MA as in Example 1.9 Example 3 By performing similar synthesis using N-(4-aminobutyl)amphetamine (ABAP) in place of ABMA in Example 1, DNS-ABAP with the following molecular structure was synthesized.9 Using DNS-ABAP, The results of the same evaluation as in Example 1 are described below.

Optimal excitation wavelength: 328r+m Maximum emission wavelength: 550r+m (without antibody)5
30 nm (when adding antibody) MA concentration at 50% change in fluorescence = 101・IM Immunodetection reagent k of this example Example 4, which was found to be effective for MA detection similar to Example 1 Implementation Amphetamine (AP) instead of ABMA in Example 1
DNS-AP having the following molecular structure was synthesized by performing the same synthesis using 9 DNS-AP, and the results of the same evaluation as in Example 1 are described below.

Optimal excitation wavelength: 328r+m Maximum emission wavelength: 550r+m (without antibody)5
30 nm (when adding antibody) MA concentration at 50% change in fluorescence: 10-'・1M Immunodetection reagent L of this example As in Example 1, it was found to be effective for detecting MA. Examples 1-4
Examples of the detection principles included in the present invention are shown in Figures 1 and 2.
Antibody solution Immunodetection reagent The strength is such that it is possible to introduce each sample into the device individually.If the antibody solution and immunodetection reagent are mixed at the optimal concentration from the beginning and prepared as a mixture of immunodetection reagents, In addition, when carrying out the present invention, the pH of all solutions should be controlled to around 6.5 to 7.5 using, for example, a phosphate buffer.
It is preferable because it improves reproducibility and the shelf life of the antibody. In order to improve the shelf life, a mixture of Ji antibody and an immunological detection reagent (phosphate buffer solution) is freeze-dried and refrigerated in a powder state. , the shelf life increases dramatically.

Example 5 FIG. 3 shows the configuration of an example in which the measurement principles of Examples 1 to 3 above are applied to an automatic measuring device.

In FIG. 3, 1 to 3 are metering pumps, metering pump 1 is an immunological detection reagent solution (concentration 10-'M), metering pump 2 is an antibody solution (concentration 10-'M), metering pump 3 is
is connected to the sample solution, and 4 is a mixer/valve, which switches the flow paths of the solution of metering pumps 1 to 3 and is connected to the sample solution.
It is a fluorescence intensity detector with a built-in flow cell of 32 m.
The fluorescence intensity of 530r+m using 8nm excitation light can be continuously monitored.

Simultaneously with the start of measurement, each of the metering pumps 1 to 3 operates to feed each solution into the mixer 4 by 50 μm.

After sufficient mixing, the mixed liquid is sent to the fluorescence detector 5, but no fluorescence is emitted because the inside of the fluorescence detector 5 is filled with air or pure water for cleaning. When the mixed solution is fed from the mixer 4, fluorescence due to the immunodetection reagent is emitted, and this fluorescence intensity is 1. If the so-called target detection substance such as MA is present in the sample, the same principle as in the experiment of Example 1 will be used. Therefore, the presence or absence of the target substance to be detected in the sample can be determined by the decrease in the height of the fluorescence peak. FIG. 4 shows the results of automatically detecting MA using this apparatus as an example.

In Figure 4, curve a is pure water that does not contain MA as a sample, curve l is a sample containing MA at a concentration of 10-'M, curve C is a sample containing MA at a concentration of 10-'M, and curve d is a sample containing MA at a concentration of 1.
A sample containing 0-6M MA was used.The vertical axis shows fluorescence intensity, and the horizontal axis shows time (minutes). Example 6: MA could be detected within 1 minute with high sensitivity In Example 5, three solutions of the anti-antibody immunodetection substance and the sample were operated independently using three pumps, but the antibody solution and the immunodetection substance solution By using a mixture of the two pumps, it is possible to obtain the same effect with two pumps. The configuration of this embodiment is shown in FIG.

6.7 are metering pumps, and the metering pump 6 pumps a mixed solution of an antibody and an immunological detection substance (i.e., an immunological reagent).
The metering pump 7 is a pump for pumping the sample solution. 4 and 5 are a mixer/valve and a fluorescence intensity detector as in Example 5. By performing the same operation as in Example 5, it can be used for the same purposes such as MA. Compared to Example 5, which achieved the effect of detecting the substance to be detected, the device configuration is simpler.Although this example is considered to be more effective, the above example shows an example of a drug. The invention is not limited to, but is mainly applicable to the detection of pathogenicity or disease markers in clinical tests, as well as in the field of detecting trace amounts in a wide range of industrial applications. The immunodetection reagent is an immunodetection reagent in which a fluorescent substance whose fluorescence intensity changes and an antigen that specifically binds to the antibody are chemically bonded. The immunodetection method, which uses fluorescence, shortens the immunodetection time, which conventionally required more than 5 hours, to less than 1 minute.
In addition, it has become possible to increase the sensitivity (ξ By widening the wavelength difference between the excitation wavelength and the emission wavelength in fluorescence measurement, it has become possible to exclude the influence of Raman light from water, and the wavelength of both the excitation and emission wavelengths has been increased. By doing so, it becomes possible to reduce the effects of impurities.

[Brief explanation of drawings]

Fig. 1 is a graph showing the change in fluorescence spectrum when an antibody is added to a solution of an example of the immunodetection reagent of the present invention, and Fig. 2 is a graph showing the fluorescence intensity of the immunological reagent of an example of the present invention. Figure 3 shows the flow path of an embodiment of the immunodetection device of the present invention. Figure 4 shows the relationship between the concentration of the target detection substance and the concentration of the target detection substance. Figure 5 shows the relationship between fluorescence intensity and measurement time detected using the present invention.・Quantitative pulse 4...Mixer and pulse 5...Fluorescence intensity detector

Claims (13)

[Claims] (1) It is an antigen-like substance that chemically combines a fluorescent substance whose fluorescence wavelength or fluorescence intensity changes when it comes close to an antibody and an antigen that specifically binds to the antibody. immunodetection reagent. (2) The immunodetection reagent according to claim 1, wherein the fluorescent substance contains a dansyl group represented by the following structural formula. ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ (3) The immunological detection reagent according to claim 1, wherein the antigen is methamphetamine, amphetamine, or ephedrine. (4) The immunological detection reagent according to claim 1, wherein the antigen-like substance has the chemical structural formula of the following general formula. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ However, R is hydrogen or methyl group, 0≦n≦10,
m is 0 or 1; when n=0, m=0;
When is other than 0, m is 1. (5) The immunological detection reagent according to claim 4, wherein the antigen-like substance has the following structural formula. ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ (6) The immunological detection reagent according to claim 4, wherein the antigen-like substance has the following structural formula. ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ (7) The immunological detection reagent according to claim 4, wherein the antigen-like substance has the following structural formula. ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ (8) The immunological detection reagent according to claim 4, wherein the antigen-like substance has the following structural formula. ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ (9) An immunological reagent characterized in that it is a mixed solution containing a solution of the immunological detection reagent according to any one of claims 1 to 4 and a solution of an antibody that specifically binds to an antigen. (10) The immunological reagent according to claim 9, wherein the mixed solution contains a buffer solution. (11) An immunological reagent obtained by freeze-drying the liquid mixture according to claim 9 or 10. (12) using an antibody that specifically binds to the target detection substance and the immunological detection reagent according to any one of claims 1 to 4;
Detecting the target detection substance by increasing the fluorescence of the immunodetection reagent by mixing the antibody and the immunodetection reagent, and then introducing the target detection substance and measuring a decrease in the fluorescence. An immunological detection method characterized by: (13) A test solution, a solution containing the immunodetection reagent according to any one of claims 1 to 4, an antibody that specifically binds to the target detection substance and the immunodetection reagent, and the test solution. The immunological detection method is characterized by comprising a means for feeding the immunological reagent, a mixing section for mixing the test solution and the immunological detection reagent, an excitation light source, and a fluorescence intensity detection section. Detection device.