JPH1090270A - Method of detecting 2-methylisoborneol - Google Patents

Abstract

PROBLEM TO BE SOLVED: To easily detect 2-MIB with high sensitivity in a short time by utilizing a camphanol having a structure similar to 2-methylisoborneol(2-MIB) which is a mold odor causing material. SOLUTION: A 2-MIB detecting sensor 1 having a gold electrode mounted on a quartz oscillator to immobilize a camphanol. OVa composite antibody, and the non-covered part of the gold electrode blocked by bovine serum albumin (BSA) is connected to an oscillating circuit 2, a frequency counter 3, and a personal computer 4, and when 2-MIB is adhered to the camphanol. OVa composite antigen, the weight can be measured as the change of frequency. The sensor 1 is set to a measuring cell having a capacity of 150mL, and air passed through activated charcoal 6 in order to a fixed measuring environment is sent in 120mL /minute by an air pump. A vessel of about 500μ l is prepared for dipping the quartz oscillator having the gold electrode, the camphanol. OVa composite solution having a concentration of 0.5mg/mL is prepared, and the camphanol. OVa composite is immobilized on the gold electrode.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for detecting 2-MIB, a musty odor substance contained in water, by an immunological reaction.

[0002]

2. Description of the Related Art Complaints and damages about off-flavors of tap water tend to increase year by year. This off-flavor is generated by a mass of algae caused by pollution of water sources and eutrophication of lakes and marshes.
-Methylisoborneol (2-methylisobobo)
(neolol: 2-MIB) and mold odor substances such as geosmin. For this reason, in recent years, the demand for safe and delicious water supply for tap water has been increasing among the people, and in response to this demand, to aim for a stable supply of higher quality tap water, "Comfortable water quality standard items" including mold odor substances are set as items that complement the water quality standards. To achieve this standard, it is important to develop sensors for odors and advanced water treatment systems.

At present, in water quality control of purified water, humans monitor by smelling the nose at regular intervals. Also,
As a means for analyzing mold odor, there are official methods such as page trap gas chromatography mass spectrometry and solid phase extraction GC / GC.
The MS method has been adopted. However, these analytical tools
There are some problems from the viewpoint of constant monitoring of water quality, and there is a need for the development of a simple and quick measurement method and apparatus without individual differences.

On the other hand, in the food industry and in the field of quality control of chemicals, "odor sensors" having a threshold close to the human nose have been developed as measuring devices for odors, and are marketed by several companies. Typical measurement principles of products include: 1) a metal oxide semiconductor sensor that detects a change in resistance, 2) a conducting polymer that detects a change in conductivity, and 3) a lipid that detects a change in weight. There is a sensor using a coated quartz oscillator.

At the research and development stage, methods for quantifying mold odor substances using luminescent genes have been reported (Yoshinobu Ishibashi et al., Proceedings of the 50th Annual Meeting of the Japan Society of Civil Engineers, p.
1302-1303, 1995), quantifiable 2-MI
The lowest concentration of B is 0.1 mg / L.

[0006]

According to the official method described above, high-precision measurement can be performed, but 1) the apparatus is expensive, and 2) one measurement time including the concentration of the sample is about 5 hours. 3) Since the operation is complicated and skill is required for accurate analysis, there are problems in that routine analysis is difficult.

[0007] Regarding commercially available odor sensors,
1) Lack of sensitivity or lack of correlation with human sensation. 2) Smell is determined by comparing the pattern of several sensor responses to a certain odor. Analysis of measurement data by processing is required. 3) Quantitative measurement is difficult. Therefore, in order to apply a commercially available odor sensor to monitoring of odorous substances in purified water, there is a problem that it is necessary to solve problems such as sensitivity, selectivity, and quantitativeness.

An object of the present invention is to solve the above-mentioned problems and to provide a measuring method capable of easily detecting 2-MIB in a short time and with high sensitivity.

[0009]

In order to achieve this object, the present invention utilizes camphor having a structure similar to that of 2-MIB, which is a substance causing mold odor, and provides specificity (high selectivity). And high sensitivity are achieved by the antigen-antibody reaction.

[0010]

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[0011]

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Specifically, a transducer in which a complex antigen of camphor and protein having a structure similar to that of the above-mentioned mold odor causing substance, 2-MIB, is immobilized,
An anti-2-MIB antibody that specifically (selectively) binds to B is immersed in a solution to be measured containing a constant concentration, and unknown concentration of 2-MIB present in the solution and this camphor-protein complex antigen And the amount of anti-2-MIB antibody bound to the camphor-protein complex antigen immobilized on the transducer was determined by the output change of the transducer, and the bound antibody in the presence of 2-MIB was determined. The method is characterized in that the 2-MIB concentration in the solution to be measured is quantified as a difference from the amount.

Further, the present invention relates to a method for producing anti-2-MIB antibody, which comprises preparing camphor and bovine serum albumin (Bov).
Complex of ine Serum Album (hereinafter referred to as BSA) (hereinafter referred to as camphor-BSA complex)
Is used as an antigen. Therefore, 1) the use of a water-soluble camphor in place of 2-MIB, which is hardly soluble in water and difficult to handle, makes it easier to prepare antibodies. 2) Since camphor is a small molecule and does not show antigenicity by itself, BSA
Has the advantage that it becomes possible to have antigenicity by binding.

Further, the complex used for the competitive reaction of 2-MIB is composed of camphor and ovalbumin (Ovalbumi).
n (hereinafter referred to as Ova) complex (hereinafter referred to as camphor-O)
va complex) to obtain Camphor B
An anti-2-MIB antibody prepared using the SA complex as an antigen,
Upon reaction with the camphor-Ova complex, only camphor can be recognized and bound.

Further, according to the present invention, a quartz oscillator or a surface acoustic wave element (SAW element) is used as a transducer,
From the frequency before and after the reaction, the amount of binding of the anti-2-MIB antibody is detected as a change in weight, and 2-MIB can be quantified by the method described above.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION

[Embodiment 1] Hereinafter, a case where a transducer is a crystal unit (AT cut, 10 MHz) according to the present invention will be described.
Preparation of a camphor-Ova complex immobilized on a transducer, preparation of an anti-2-MIB antibody to be put into a solution to be measured, specificity test of the anti-2-MIB antibody and the result, detection apparatus for 2-MIB using a quartz oscillator And the preparation of a 2-MIB detection sensor, a method of detecting 2-MIB using a competitive reaction, and the preparation of a calibration curve using 2-MIB of a known concentration.

[0017] Preparation of camphor-Ova complex immobilized on a transducer: 1 g of camphor and orthocarboxymethoxylamine hemi (O-Carboxy)
methoxylamine hemi) hydrochloride 2.8
To 4 g, 14 mL of ethanol and 16.5 mL of 2N sodium hydroxide were added, refluxed for 6 hours, and left at 25 ° C. overnight. 200 mL of pure water was added thereto, and 2 mol /
adjusted to pH 9.5 with m ³ sodium hydroxide,
This was extracted three times with 30 mL of ethyl acetate, and the aqueous layer was adjusted to pH 3 with 1N hydrochloric acid. This is allowed to stand at 0 ° C. overnight, and the resulting precipitate is centrifuged (3000 rpm for 5 minutes).
And washed with 500 mL of pure water, and then dried under reduced pressure in the presence of calcium sulfate to obtain camphor-carboxy.
methyloxyme, hereinafter referred to as CMO).

Next, 18.9 mg of CMO and 12 μL of triethylamine (Triethylamine) were added for 1 m.
L tetrahydrofuran
an) and cooled at −5 ° C., followed by 12 μL of isobutyl chloroformate (Isobutyl chloroform).
The mixture was shaken at -5 ° C for 30 minutes. This reaction solution was dropped into 5 mL of a cooled 20 mg / mL Ova solution, and shaken at 4 ° C. overnight. This was dialyzed against pure water to obtain a camphor-Ova complex. Save is
Cryopreservation was performed at -20 ° C.

[0019] Preparation of anti-2-MIB antibody to be put in the solution to be measured: Since 2-MIB as an antigen is hardly soluble in water and difficult to handle, camphor having a structure similar to 2-MIB is used for preparing an antibody. did. However, since camphor has a low molecular weight and does not have antigenicity, it is necessary to bind a macromolecule such as a protein to form an antigen. As this protein, BSA having relatively low antigenicity is more suitable than Ova having strong antigenicity. Therefore, in the same manner as the camphor-Ova complex described above, camphor-
BSA complex antigen was made.

Next, for the preparation of an anti-2-MIB antibody, the present antigen was added to PBS (Phosphate Buffered S).
alline), and 300 μL of the diluted solution was mixed with Freud's complete adjuvant (F) manufactured by “Nacalai Tesque, Inc.”
3 of complete Complete Ajuvant
An emulsion for immunization was prepared using 00 μL. This was immunized by intraperitoneal injection into a 5-week-old male “Seiwa experimental animal” BALB / C mouse. Lymphocytes were removed from the spleen of the immunized mouse, centrifuged (200-400 ×
g, 5 minutes) suspended in 10 mL of E-RDF, about 1 × 1
0 was obtained ^eight of lymphocytes. After preparing this suspension and 2 × 10 ⁷ myeloma suspensions, cell fusion was immediately performed. The cell suspension is cultured in a 96-well microplate well in a HAT medium or the like, and the well in which the hybridoma has grown is subjected to enzyme-labeled immunosorbent assay (Enzyme-
Linked Immunosorbent Asa
y, hereinafter referred to as ELISA method), and then the serum-free medium containing the antibody is centrifuged and filtered.
The fraction containing the antibody was collected and 10 mM Tris (Tri
s) After dialyzing against -hydrochloric acid buffer (pH 7.4), the solution was freeze-dried to obtain an anti-2-MIB antibody, and stored frozen at -20 ° C.

[0021] Specificity test and results of anti-2-MIB antibody: Using the serum-free culture supernatant of the obtained anti-2-MIB antibody, 2-MIB and other similar compounds were used as free antigens to measure the reactivity by competitive ELISA. As a result, a monoclonal antibody specific to 2-MIB was selected. Since the anti-2-MIB antibody was prepared using the camphor-BSA complex as an antigen, it shows specificity for both camphor and BSA. Therefore, in this test, the camphor-Ova complex was used as the immobilized antigen. By using this complex, it becomes possible to screen camphor or 2-MIB specific antibodies. First, a camphor-Ova complex as an immobilized antigen was coated on an EIA plate used in a 96-well enzyme immunoassay (Enzyme Immunoassay) and blocked. At the same time, the chemical formula is similar to the 2-MIB shown below.
Kinds of compounds, namely, camphor, camphorquinone, norcamphor, borneol, isoborneol
neol), Norbornane,
Norborneol is 10% ethanol-Tween 20
-10-fold dilution series with PBS (1000 to 0.01 μg / m
L). Equal amounts of these and a serum-free medium containing each antibody were mixed and incubated for 30 minutes. When a specificity test was performed using this reaction solution as an antibody sample,
It has little reactivity to norborneol, norcamphor, norbornane, and therefore, the anti-2
It was confirmed that the -MIB antibody has very high specificity for 2-MIB.

[0022]

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[0023]

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[0024]

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[0025]

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[0026]

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[0027]

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[0028]

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Production of 2-MIB Detector Using Quartz Crystal Resonator and 2-MIB Detection Sensor: 1) 2-MIB Detector Utilizing Quartz Resonator: FIG. FIG. 3 is a block diagram of an MIB detection device. A 2-MIB detection sensor 1 in which a gold electrode is attached to a quartz oscillator, a camphor-Ova complex antigen is immobilized, and an uncoated portion of the gold electrode is blocked with BSA.
Is connected to the oscillation circuit 2, the frequency counter 3, and the personal computer 4, and when 2-MIB is attached to the camphor-Ova complex antigen, the weight can be measured as a change in frequency. 2-
The MIB detection sensor 1 is set in a measurement cell 5 having a capacity of 150 mL. In order to maintain a constant measurement environment, air passing through activated carbon 6 is sent at a rate of 120 mL / min by an air pump.

The 2-MIB detection sensor 1 is manufactured by the following methods 2) and 3). 2) Immobilization of the camphor-Ova complex on the electrode of the quartz oscillator: FIG. 2 is a flow chart showing a 2-MIB detection sensor and a method of manufacturing the same. In each figure, each substance is schematically shown. First, the upper part of a 2 mL microtube was cut to prepare a container having a capacity of about 500 μL for immersing the crystal unit 11 having the gold electrode 12 attached thereto. 0.5mg / mL concentration camphor-Ov
a) A composite solution was prepared, and the quartz oscillator 11 with the gold electrode 12 was immersed at a temperature of 25 ° C. for 17 hours. As shown in FIG. Immobilized. Further, the substrate was washed with pure water for 35 minutes to remove unimmobilized substances, dried for 1 hour, and the frequency was measured by a 2-MIB detector shown in FIG. -Ova complex weight was calculated.

2) Blocking of camphor-Ova complex with BSA: A 0.5 mg / mL BSA solution was added to a camphor-Ova complex at a temperature of 25 ° C. and a humidity of 55% for 17 hours.
The quartz resonator 14 with a gold electrode on which the Ova composite was fixed was immersed, and the uncoated portion 15 of the electrode of the quartz resonator was subjected to blocking treatment with BSA 16 as shown in FIG.
Further, after washing with pure water for 55 minutes and drying for 1 hour, 2- in FIG.
The frequency was measured by an MIB detection device, and the weight of the immobilized BSA was determined from the frequency change from the initial value.

Through the above steps, the camphor-Ova complex 13 was immobilized on the gold electrode 12 of the quartz oscillator 11 to produce the 2-MIB detection sensor 1. Next, a method for detecting 2-MIB using the present sensor will be described. . FIG. 3 shows a conceptual diagram of a method for detecting 2-MIB using a competitive reaction.

Firstly, the anti-2-MIB in the 2-MIB 17 and known weight W _A known concentration C _M (= known concentration C _A × volume V)
The mixed solution of antibody 18 was reacted. The 2-MIB detection sensor 1 described above was immersed in the reaction solution, and allowed to stand at a temperature of 30 ° C. to perform a competitive reaction. Next, after washing with pure water and drying for 1 hour, the frequency was measured by the 2-MIB detector of FIG. From this measurement and the initial measurement, the amount of change in frequency can be determined, and the camber-Ov on the 2-MIB detection sensor 1 can be determined.
aWeight of anti-2-MIB antibody 19 bound to complex 13
_B is required. 2-MI of known concentration C _M The results
The weight W _C of the anti-2-MIB antibody reacted with B17 is W _C =
It is obtained as W _A -W _B.

[0034] Preparation of calibration curve with known concentration of 2-MIB: FIG. 4 shows the weight W of anti-2-MIB antibody bound to known concentration _CM of 2-MIB by a competitive reaction, prepared from the frequency measurement result of the quartz oscillator. The relationship with _C was shown.
Anti 2-MIB antibody concentration C _A in the solution is 0.04 mg / m
L is constant. As is apparent from FIG.
By varying the concentration C _M and 0.005 to 5 / L, it can be seen bound to 2-MIB anti 2-MIB antibody amount W _C will change proportionally. Accordingly, it was found that it is possible to quantify the unknown 2-MIB concentration from the amount of bound antibody W _C of anti 2-MIB in the antibody of this calibration curve based.

Embodiment 2 Next, an embodiment of the 2-MIB detection method according to the present invention will be described with reference to FIGS. 5 and 6 in the case where the transducer is a surface acoustic wave element (SAW element). FIG. 5 shows a 2-MI using a SAW element.
FIG. 3 is a circuit diagram showing a measurement system of B. As shown in this figure, the SAW element includes a piezoelectric substrate 21, a metal thin film 22, a surface acoustic wave propagation surface 23, an amplifier 24, and a mixer 25. Using a method similar to that of the first embodiment, a camphor-Ova is formed on the propagation surface 23 of the SAW element (for example, 160 MHz).
The complex was immobilized and a competitive reaction was performed under the same conditions as in Example 1.

The resultant 2-MIB concentration and the known 2-MIB concentration were determined by measuring the frequency of the resulting SAW device.
FIG. 6 shows the relationship with the amount of MIB antibody. This figure shows the same results as in FIG. 4 using a quartz oscillator, and it was found that it is possible to quantify the unknown 2-MIB concentration from the amount of bound antibody based on this calibration curve. Furthermore, it can be easily analogized that odor substances other than 2-MIB, for example, geosmin, can be quantified using the same method.

The numerical conditions such as the amount of the material used, the processing time, and the processing temperature used in the above two embodiments are merely examples, and the present invention is not limited to these conditions.

[0038]

According to the present invention, a transducer having a camphor-protein complex immobilized thereon is immersed in a test solution containing a constant concentration of an anti-2-MIB antibody, and an unknown concentration of 2% contained in the test solution is contained. -MIB competitively competes with the camphor-protein complex on the transducer.
The amount of the antibody bound to the complex on the transducer is determined by the change in the output of the transducer. By preparing a calibration curve based on the known concentration of 2-MIB and the amount of bound antibody, the amount of unknown 2-MIB in the solution to be measured can be quantified. Since this reaction utilizes an antigen-antibody reaction, 1) low concentration 2-MIB can be detected with high sensitivity without the need for concentration, as compared with the conventional quantification method. 2) 2-MIB can be selectively detected due to the specificity of the antibody, and the operation of analyzing a plurality of data by statistical processing is not required. In addition, there is an advantage that 3) the amount can be determined by an easy operation.

[Brief description of the drawings]

FIG. 1 shows a 2-MIB using frequency measurement of a quartz oscillator.
Block diagram of detection device

FIG. 2 is a flowchart showing a 2-MIB detection sensor and a method for manufacturing the sensor.

FIG. 3 is a conceptual diagram of a method for detecting 2-MIB using a competitive reaction.

FIG. 4 shows a known 2-MIB obtained by measuring the frequency of a quartz oscillator.
Relationship between concentration and amount of anti-2-MIB antibody bound to 2-MIB

FIG. 5 is a circuit diagram showing a 2-MIB measurement system using a SAW element.

FIG. 6 shows a known 2-MIB obtained by measuring the frequency of a SAW element.
Relationship between concentration and amount of anti-2-MIB antibody bound to 2-MIB

[Explanation of symbols]

1: Sensor for detecting dimethyl isoborneol (2-MIB) 2: Oscillation circuit 3: Frequency counter 4: Personal computer 5: Measurement cell 6: Activated carbon 7: Air pump 11: Quartz crystal oscillator 12: Gold electrode 13: Kanfa-Obo Albumin (Ova) complex 14: Complex immobilized quartz oscillator 15: Uncoated portion of gold electrode 16: Bovine serum albumin (BSA) 17: 2-MIB 18: Anti-2-MIB antibody 19: Campha-Ova complex Antibody bound to body 21: Piezoelectric substrate 22: Metal thin film 23: Propagation surface 24: Amplifier 25: Mixer

 ────────────────────────────────────────────────── ─── Continuing on the front page (72) Inventor Takahisa Miyamoto 3-5-901 Hyodomichihama, Sawara-ku, Fukuoka City, Fukuoka Prefecture (72) Inventor Shikako Kawada 2-2-1 Nagasaka, Yokosuka City, Kanagawa Prefecture (72) Inventor Yoshiharu Tanaka 2-2-1 Nagasaka, Yokosuka City, Kanagawa Prefecture Incorporated Fuji Electric Research Institute (72) Inventor Tokio Oto 2-2-1 Nagasaka, Yokosuka City, Kanagawa Prefecture No. Inside Fuji Electric Research Institute

Claims (5)

[Claims] (1) 2-methylisoborneol (hereinafter referred to as 2-M
IB) and an antigen-antibody reaction between an anti-2-MIB antibody prepared using a complex of camphor and a protein having a similar structure to that of the 2-MIB to be measured and a camphor-protein complex antigen 2-MIB detection method. 2. A camphor having a structure similar to 2-MIB.
The transducer on which the protein complex antigen is immobilized is immersed in the solution to be measured prepared so that the anti-2-MIB antibody has a constant concentration, and the unknown concentration of 2-MIB contained in the solution to be measured is added to the transducer. The antigen-immobilized camphor-protein complex antigen is allowed to competitively react with the anti-2-MIB antibody, and as a result, the transducer by the anti-2-MIB antibody bound to the camphor-protein complex antigen is used. A method for detecting 2-MIB, which quantifies the concentration of 2-MIB in a solution to be measured based on a change in output. 3. A method for detecting 2-MIB, wherein the anti-2-MIB antibody according to claim 1 or 2 is prepared using a complex of camphor and bovine serum albumin. 4. The method for detecting 2-MIB, wherein the protein of the camphor-protein complex antigen according to claim 1 or 2 is ovalbumin. 5. The method according to claim 2, wherein the transducer is a quartz oscillator or a surface acoustic wave device (SAW device).