JPS63153468A - Reagent and method for measuring mycotoxin - Google Patents

Abstract

PURPOSE:To measure the quantity of mycotoxin with high sensitivity by immobilizing the mycotoxin to a support such as immunoplate, adding a sample and mycotoxin measuring reagent simultaneously thereto so that a competitive monoclonal antibody reaction is completed. CONSTITUTION:The mycotoxin is immobilized to the support such as immunoplate and the sample and the mycotoxin measuring reagent are simultaneously added thereto so that the competitive monoclonal antibody reaction to the mycotoxin immobilized to the support and the myucotoxin in the sample is completed. The enzyme to label the monoclonal antibody is selected from the group consisting of peroxidase, alkali phosphatase, beta-galactosidase, catalase, glucose oxidase, lactic acid oxidase, alcohol oxidase and monoamine oxidase.

Description

DETAILED DESCRIPTION OF THE INVENTION [Technical field to which the invention pertains] The present invention relates to a reagent and method for measuring mycotoxins. More specifically, the present invention relates to a reagent for measuring mycoxin containing an enzyme-labeled monoclonal antibody and a measuring method using the reagent.

[Conventional technology]

Even if mycotoxins produced by fungi such as Penicillium, Aspergillus, or Fusarium are not acutely toxic, they may be carcinogenic or cause kidney or liver tumors.

In particular, aflatoxin B1 has strong carcinogenicity among mycotoxins. In fact, the effects of aflatoxin B1 on the human body and animals have been well studied, and it is known that aflatoxin B1 targets the liver and causes cancer.

Therefore, it is very important to measure the amount of mycotoxins in food and check in advance to prevent it from entering the mouths of humans or animals.

Conventional mycotoxin measurement methods include thin layer chromatography, high performance liquid chromatography, gas chromatography, mass spectrometry, or animal bioassays. However, these methods have practical problems in terms of measurement sensitivity and measurement/analysis time, and there is still room for further improvement.

Recently, enzyme-linked immunosorbent assay using enzyme-labeled aflatoxin B1 and its polyclonal antibody [Applied
and environmental micropiracy (^
pp1. Envi, Microbiology), first Y
, 1472 (1981)] was developed. Although this method is quite practical in terms of measurement sensitivity and measurement analysis time, the measurement sensitivity is still not sufficient, and the mycotoxin-containing reagent used for measurement must be disposed of in multiple ILs. There is still room for further improvement in terms of safety and economy.

Also, Mycotoxin Research Society Bulletin No. 21, June 30, 1985, Ikuko Ueno (υeno, rkuko: P
roecee din Hof the Japan
se 85 Society of Myco
t.

xicology No. 21, Iunel 985
) discloses a further improved enzyme immunoassay for aflatoxin B1. The measurement range of aflatoxin B by this method is 10-11000p/10μI
・assay (10g/ml~100ng/ml
).

Also, Enzyme-Linked Immunoso
rbent As5ay of＾flatoxin B
1[11HANU P, RAM and L, PATR
ICK II^RT.

J, ^5SOC, OFF, ^NAL, CHEH, (VO
L, 69, No. 5, 1986)] is also known. The measurement range of aflatoxin B1 by this method is also 1 ng/ml to 1100 n/mN. Furthermore, ^
,^,G,CandlishJ.

Monoclonal antibody to a by H. Stimson and J. E. Sm1th
flatoxin B+: detection of
themycotoxin by enzyme i
mmunoassay [Letters in＾pp
Lired Microbiology 1985,
1, 57/-61] is also known, but the measurement range of aflatoxin B1 by this method is approximately 1 to 5 ng/-.
m1. It is.

The enzyme immunoassay method described above is easy to handle;
It is very practical in this respect because it allows measurement of many samples at once.

However, the content of aflatoxin B is about 11g/
An enzyme color = 4-epidemic assay method that can quantify cases smaller than m1 has not been reported so far.

[Problem that the invention seeks to solve]

The object of the present invention is to treat manganese such as aflatoxin B The purpose of the present invention is to provide a reagent for measuring icotokin removal.

Another object of the present invention is to provide a reagent for measuring mycotoxins that has sufficient sensitivity to mycotoxins and reacts rapidly.

Still another object of the present invention is to provide a reagent for measuring mycotoxins that has sufficient stability and economical efficiency.

Still another object of the present invention is to provide an enzyme-labeled monoclonal antibody against a mycotoxin as a reagent for measuring mycotoxins having various properties as described in L.

Still another object of the present invention is to provide a method for rapidly measuring 1-) mycotoxins with high sensitivity using the reagent for measuring mycotoxins of the present invention.

Further objects and advantages of the present invention will become apparent from the description below.

[Means for solving problems]

According to the present invention, the above objects and advantages of the present invention are achieved in the first place.
This is achieved using a mycotoxin measuring reagent containing an enzyme-labeled monoclonal antibody against mycotoxin.

The present inventors first produced a monoclonal antibody that is highly specific for mycotoxins, used this unlabeled monoclonal antibody to react with mycotoxins under competitive conditions, and then used peroxidase against mouse immunoglobulins. An attempt was made to improve the method for measuring mycotoxins using an enzyme immunoassay method in which a labeled antibody reagent is reacted (see Comparative Example 1 below). As a result, the amount of mycotoxin could be detected down to the ng order, and the analysis required a long time (for example, about 6 hours or more). Due to the large number of food samples to be measured and the fact that many of them cannot be stored for long periods of time, it is necessary to improve the ability to measure mycotoxins in measurement samples more easily, quickly, and with higher sensitivity. was there.

The present inventors have conducted extensive research and have been able to create a sample for mycotoxin measurement by chemically bonding an antibody with high specificity to mycotoxins (hereinafter also referred to as anti-mycotoxin antibody) to an enzyme. Moreover, the present invention has been achieved by determining that the above-mentioned object of the present invention can be achieved by this method.

The mycotoxins targeted in the present invention are toxins produced by, for example, Penicillium, Aspergillus, or Fusarium, such as Okra I.
xin A, T-21-xin or aflatoxin B
It is 3rd prize.

Enzymes for labeling monoclonal antibodies used in the present invention include, for example, peroxidase, alkaline phosphatase, β-galactosidase, catalase,
Glucose oxidase, lactate OA-sidase, alcohol oxidase or monoamine oxidase is preferably employed.

In the present invention, mycotoxins can be measured rapidly and with high sensitivity under competitive reaction conditions using a mycotoxin measurement sample containing a monoclonal antibody labeled with an enzyme.

The mycotoxin measurement method of the present invention involves immobilizing a hemocotoxin, such as aflatoxin B, on a support such as a 96-well Immunopre 1, adding the above sample for mycotoxin measurement of the present invention and the sample together, and then adding a substrate solution. (using a substrate corresponding to the enzyme used for antibody labeling at this time), and then measuring the absorbance of the reaction coloring solution. At the step of adding mycotoxin measurement sample and sample in this operating procedure,
By causing a competitive reaction between the mycotoxin measuring reagent and the mycotoxin in the sample with the support-immobilized mycoxin (mycoxin) and the mycotoxin in the sample, the amount of mycotoxin in the sample can be measured quickly and with high sensitivity.

For immobilization of mycotoxins, in addition to immunoplates, for example, polystyrene pieces, polystyrene pieces, ABS
Resin beads etc. can be used.

I will show my son the creation of an antigen for mycotoxin measurement and the production and purification of antibodies as a reference example.

[Reference example]

Preparation of Antigen for Mycotoxin Measurement A conjugate of mycotoxin and bovine serum albumin (BSA) was prepared and used as an antigen for mycotoxin measurement to be immobilized on a 96-well plate I for immunoassay as follows.

Reference example 1-1. A conjugate of ochratoxin A (,0TA) produced by Aspergillus bacteria and BSA (Ol” A-
BSA) was created by the method shown below. First, O
5 mg of TA was dissolved in 0.12 mN of ethanol and 3 ml of 0.1M phosphate buffer (pH 7.0). Next, 0,1
1-ethyl-3 (3-d
imet, l+yla+ninopropyl)car
bodiimide hydrochloride
(E D P C) was added to this mixture and heated at 20°C for 24 hours.
Stir in the dark for an hour. After dialysis with distilled water, freeze-drying 1~
30g of 0TA-BSA was obtained.

Reflection example 1-2. A conjugate of T-2 toxin (T-2) produced by Fusarium bacteria and BSA (T-1-BSA) was prepared by the method shown below.

First, by reacting the hydroxyl group of T-2 with succinic anhydride,
2 hemisuccinate (T-2-H8) was synthesized. Using this T-2-H8, (T-2-H8-BSA) was produced in the same manner as in the case of OTA-BSA production.

Reference example 1-3. A combination of aflatoxin B (AFB) produced by Aspergillus bacteria and BSA (AFB)
+BSA) was created by the method shown below.

20 mg of AFB (manufactured by Sigma) was dissolved in methanol by heating to about 60°C. To this was added carboxymethoxyamine hydrochloride (manufactured by Tokyo Kasei) 200+ag. 60 while adjusting the pH to 7 with 5 N-Na0tl.
Oxime at ~70°C for 2-3 hours. This oxime formation was carried out according to Chu's method (Reference 1).

Next, AFBl-oxime was purified and from this 10ml
? was collected and dissolved by adding ITI (l) of dimethylformamide.
．． EDPA i8.0m! dissolved in 1-Oml of 1M phosphate buffer (pH 7,2). ? Add to this -
11 A F B +-oxime solution was added dropwise.

Further, 5.2 mg of EDPC was added to this mixed solution. 0
．． The mixture was stirred at room temperature for 24 hrs in the dark while adjusting pi-15.5 with 1M hydrochloric acid. After dialysis with distilled water, it was freeze-dried to obtain 45 mS of AFB+-oxime-BSA.

This preparation was carried out according to Cheney's method (Reference 1) and Tsuchi and No's method (Reference 2).

(Reference 1) Chu, F, S,, H4, S, l1sia4nd P,
Sun,] 977.

Preparation and character
Ization of aflatoxin 11. −
0-Carboxy+oetbyloxime, J, ^
5soc, off, 8nal.

Chem, 60 Near 9l-794 (Reference 2) Chu, F. S., and 1. Ueno, 1977,
Production ofanl, it+ody
aHair+st a"1toxin B+,
8 ppl, Environ.

1l- Hicrobiol, 33; 1125~l 12
8゜Reference Example 2 Production and Purification of Antibodies JP-A-61-171,500 and JP-A-61-
A monoclonal antibody with high specificity for OTA, T-2, or AFB owned by the present inventor was produced and purified by the method described in Japanese Patent No. 229,899.

For the production of monoclonal antibodies, 107 cell lines suspended in phosphate M street fluid were incubated with BALB/c mice (
The test was carried out by intraperitoneally administering pristane (0.5 ml of pristane was administered intraperitoneally to male mice, 8 weeks old, and 2 weeks previously).

A remarkable increase in mouse body weight was observed from around the first week, and from 1 to
Ascites was removed as appropriate on the third week. The monoclonal antibody titers thus obtained were 106-108 for the monoclonal antibody against OTA, 104-106 for the monoclonal antibody against T-2, and 106-108 for the monoclonal antibody against AFB+.

Purification of monoclonal antibodies from the obtained ascites was performed as follows. First, ascites was dialyzed with L. Liny's hydrochloric acid buffer (PH 7,4), and D
It was applied to an EAE-cellulose column. 5 fractions passing through
Salting out with 0% saturated ammonium sulfate, and adding the resulting precipitate to phosphoric acid M solution (
pH 7.4) and dialyzed. The purity of the monoclonal antibodies against OTA, T-2, and A F B + thus obtained was determined by slab gel electrophoresis using SDS polyacrylamide, and it was found that all the antibodies were highly pure.

OTA, T-2 or AFB obtained as described above,
A mycotoxin measurement method (ELISA method) using a mycotoxin measurement reagent prepared by enzymatically labeling a purified monoclonal antibody against the mycotoxin has made it possible to measure mycotoxins with high sensitivity and speed.

The present invention will be explained in detail below.

(i) Preparation of reagent for measuring mycotoxin Enzyme labeling of antibodies can be carried out using known methods, such as the one-step method using glutarartethyl [immunochemistry (T+n
munocl+e+n1stry), 6.4.3(1
969)] or the two-step method [Immunochemistry (Im
munochemistry), shadow, 1175 (1
971)] periodic acid oxidation method [Methods in Engymology
), 37.133 (1,975) and the maleimide method [
Journal of the Biochemistry
nal of the rliochemistry)
, 7-Ratio, 235 (1975)].

The latter two methods are preferred. After binding oxygen and antibodies, it can be used directly for mycotoxin measurement, but to further increase sensitivity, 5ephadex, 5eph
It is preferable to use an enzyme-labeled antibody purified by gel filtration using acryl or the like in 17 as the mycotoxin measurement reagent. The enzyme-labeled antibody fraction was prepared using phosphate buffer (pH 7,
4) Or dialyze with Tris-HCl buffer (pH 7, 4), freeze-dry or filter with a sterilization filter, and use as a mycotoxin measurement reagent.

(ii) Measurement of Myco1 to Toxin Antigen (
The antigen to be immobilized on a 96-well flat-bottomed plate for immunoassay) contains a polymeric protein different from the mycotoxin-USA BSA used as an immunogen to obtain anti-mycotoxin antibodies [for example, Jinkasagai hemocyanin (KL).
H) , ovalbumin (OVA)] combined (
Mycotoxin-KLH, Mycotoxin-0, respectively
VA) etc. The mycotoxin is left to stand and immobilized at the rate to be measured. Wash this antigen-treated well and
Blocking is performed to prevent non-specific binding of the antibody against mycotoxin to the part of the well where no antigen is bound. Next, this well is washed, and equal volumes of the sample (or a known amount of mycotoxin is used for making foot traces) and the mycotoxin measurement reagent described in (i) are added and left to stand for a certain period of time. do. The wells were thoroughly washed, a substrate solution (containing a substance that develops a color when the enzyme reaction occurs) corresponding to the enzyme used to label the antibody in the mycotoxin measurement reagent was added, and the enzyme reaction was allowed to occur for a certain period of time. The absorbance of the reaction solution is measured at the wavelength at which the color shows the maximum absorbance. In this way, a calibration curve is created from the results using known amounts of mycotoxins, and the OT in the sample is determined.
You can know the amount of mycotoxins such as A, T-2, APR, etc.

〔Example〕

Examples of the present invention will be specifically described below.

Note that these Examples are for illustrating the present invention, and do not limit the scope of the present invention.

Example 1 Preparation of mycotoxin measurement reagent The three purified monoclonal antibodies described in Reference Example 2 (OTA, T-2
and AFB) using a known enzyme labeling method as shown below to prepare a mycotoxin measurement reagent.

First, dissolve 7.32 mg of horseradish peroxidase in 1 m of distilled water, and add 20 mg of 0.1 M thorium periodate.
0μ! In addition, it was allowed to stand at room temperature for 30 minutes. This enzyme solution was dialyzed overnight at 4°C using 1mM acetate buffer (pH 4, 5), and then diluted with 0.2M carbonic acid. J Uh M Street liquid (pH
9,5) The pH was adjusted to 9.5 by adding 100 μN. On the other hand, 8.78 mg of monoclonal antibodies (OTA, T
-2, for AFBl) was dialyzed against 0.01 M sodium carbonate buffer (pH 9,5) overnight at 4°C.

The thus obtained peroxidase solution and monoclonal antibody solution were mixed and allowed to stand at room temperature for about two and a half hours. Add 0.4% by weight of tetrahydroboric acid to this reaction solution.
200 μp was added, and the mixture was left standing at 4° C. for 2 hours. The peroxidase-labeled monoclonal antibody thus obtained was
The mixture was thoroughly dialyzed at 4° C. using a phosphoric acid wt solution (pH 7.4), and used as it was or after lyophilization as a mycotoxin measurement reagent for mycotoxin measurement.

Example 2 Quantification of OTA produced by Aspergillus bacteria
The mixture was dispensed into μβ portions and left to stand overnight at 4° C. for immobilization. After washing twice with a washing solution (pH 7.4 phosphate buffer containing 0.1% Tween 20), the plate was washed with the same washing solution containing 10% bovine serum at room temperature to prevent non-specific adsorption of antibodies to the plate. It was left standing for 30 minutes.

Next, after washing twice with the same washing solution, mycotoxin measurement reagent (103, 104, 3 x 104.6X) diluted with the same washing solution was used.
50 μl of 10', 105, 106, 107 times diluted solution)
Standard OTA solution L7 prepared with the same washing solution [(Opg ~ 5μ
g) 50 μp of 150 μm] were added to each well at the same time and left to stand at room temperature for 30 minutes (in the case of creating a calibration curve). After washing 4 times with washing solution, add substrate solution (0-phenylenediamine 20m
g, 10 μp of 35% H2O2 at pH 5.0. IM citrate M solution (dissolved in 50 ml of citrate solution)) was dispensed into 100 μρ portions, and the mixture was left standing at room temperature for 30 minutes while shielding from light with aluminum foil.

Finally, the enzyme reaction was stopped by dispensing 50 μβ of 2N sulfuric acid, and the absorbance of the reaction stop solution was measured at 500 nm using a microplate photometer.

The results are shown in Figure 1. In this case, 3×104
A few pg of mycotoxin measurement reagent diluted twice,
can be measured with high sensitivity. Then 50 μl of 0.1% BS
Measured values were determined for a sample that calculated 50 pg of OTA in solution A. The measurement operation was performed according to the method shown in Example 2. However, 0 'I' A in this sample
In the measurement, 50 μl of mycotoxin measurement reagent diluted 3×10′ was used. The measured value thus obtained was 52 pg, and the calculated value and measured value almost matched.

The measurement time was very short, approximately 1 hour.

Example 3 Quantification of T-2 produced by Fusarium bacteria
100 μe each was dispensed and left to stand at 4° C. overnight for immobilization. Washing solution (pH containing 0.1% Tll1een 20)
After washing twice with phosphate buffer (7,4), the plate was left standing at room temperature for 30 minutes with the same washing solution containing 10% bovine serum to prevent non-specific adsorption of antibodies to the plate. Next, after washing twice with the same washing solution, mycotoxin measurement reagents (102, 103, 2X10', 104.2X10') diluted with the same washing solution were used.
', 105 times diluted solution) at 50 μf and standard T-2 solution prepared with the same washing solution [(Opg ~ 5 μ1150 μm] 50 μp
was added to each well at the same time and left to stand at room temperature for 30 minutes (in the case of creating a calibration curve). After washing four times with the washing solution, the substrate solution (0
-Phenylenediamine 20mg, 35% 820210
50ml of 0.1M citrate buffer at pH 5.0
(dissolved in ) was dispensed in 100μβ portions, and the mixture was left standing at room temperature for 30 minutes while shielding from light with an aluminum boiler. Finally add 2Ng sulfuric acid to 50
The enzyme reaction was stopped by dispensing μρ portions, and the absorbance of the reaction stop solution at 500 nm was measured using a microplate photometer. The results are shown in Figure 2. In this case, a mycotoxin measurement reagent diluted 2×], 0′ can measure amounts up to several p8 with high sensitivity. Next, a calculated 50 p
The measured value of the sample containing fl T-2 was determined.

The measurement operation was performed according to the method shown in Example 3. However, in the measurement of T-2 in this sample, 2
], mycotoxin measurement reagent diluted 0' times
0 μl was used. The measurement value obtained in this way is 47p
g, and the calculated value and measured value almost matched.

The measurement time was very short, approximately 1 hour.

Example 4 AFB produced by Aspergillus bacteria.

Quantification of A F B + B S A (0,5J-19/
m (1) in a 96-well flat-bottom plate 1 for immunoassay.
~ (manufactured by Nunc) 1. Dispense OOjt Il,
It was fixed by standing at 4°C overnight.

Washing solution (pH 7 containing 0.05% Tu+een20,
After washing twice with phosphate buffer solution (No. 4), the plate was left standing at room temperature for 30 minutes with the same washing solution containing 10% bovine serum to prevent non-specific adsorption of antibodies to the plate.

Next, after washing twice with the same washing solution, add 50μ of Aphta 1 to Xin B+ measurement reagent (104 times diluted solution) diluted with the same washing solution.
Add 50 μl of a standard AFB solution [(Opg ~ 5 ng) 150 μl'] prepared with l and an extract for toxin analysis in fertilizer feed or food samples (e.g., 50% methanol aqueous solution) to each well, and incubate at 37°C for 30 minutes. Alternatively, it was allowed to stand at room temperature for 2 hours (in the case of creating a calibration curve).

After washing four times with washing solution, 100 μβ of the substrate solution (20 my of O-phenylenediamine, 10 itl of 35% H2O dissolved in 50 ml of 0.1M citrate buffer, pH 5.0) was added.
Dispense in portions and incubate at 37°C for 15 minutes or at room temperature, protected from light.
It was left standing for 0 minutes. Finally, dispense 50 μl of 2N sulfuric acid to stop the enzyme reaction, and measure the absorbance of 4921 using a microplate photometer. The results are shown in FIGS. 1 and 2. As a result, it is possible to measure with high sensitivity down to 6.9 pg/assay or less, and the measurement time is extremely short.
It was about 1 hour. FIG. 5 shows a calibration curve depicting the absorbance as a percentage of the competitive reaction (room temperature, 2 hours and 37° C., 30 minutes) when the mycotoxin measuring reagent was diluted 104 times.

Comparative Example 1 Quantification of OTA produced by Aspergillus bacteria 0TA-BSA (20 μg/ml) was dispensed in 100 μβ portions into a 96-well flat-bottomed microplate, and left to stand at 4° C. overnight to immobilize. After washing twice with a washing solution, the plate was left standing at room temperature for 30 minutes with a washing solution containing 10% bovine serum to prevent non-specific adsorption of antibodies. Next, after washing twice with cleaning solution,
Highly specific anti-mycotoxin monoclonal antibody solution (102, 103, 104, 10
5,5 x 105.106.5 x 106.107.10'
Standard OTA prepared with 50 μl of 1-fold diluted solution and the same washing solution.
50μp of solution [(Opg~5μy>15oμm]) was simultaneously added to each well and left to stand at room temperature for 30 minutes (for creating a calibration curve).After washing three times with washing solution, peroxidase-labeled antibody against mouse immunoglobulin (10% bovine Diluted 103 times with serum-containing diluent) was dispensed into 100μβ portions and allowed to stand at room temperature for 2 hours. After washing 4 times with washing solution, substrate solution (07x nylenediamine 20my, 35% H2
= 23-0210μl of 0.1M citrate buffer pH 5.0 5
0ml) to 100ml. The solution was dispensed into JN portions and left standing at room temperature for 30 minutes while shielding from light with aluminum foil.

Finally, 50 μl of 2N sulfuric acid was dispensed to stop the enzyme reaction, and the absorbance of the reaction stop solution was measured at 500 nm using a microplate photometer.

The results are shown in FIG.

In the case of an immunoassay method using such an unlabeled anti-mycotoxin monoclonal antibody, the monoclonal antibody against OTA is diluted 106 times, and approximately 6
Even if it took a long time, only an amount of up to several ng could be detected.

Comparative Example 2 AFB produced by Aspergillus bacteria.

Quantification of A F B + B S A (0,5 μg/m
1) in a 96-well flat-bottom microplate at 100μ
The mixture was dispensed into 300ml portions and left to stand overnight at 4°C for immobilization. After washing twice with washing solution, add 1 ml to prevent non-specific adsorption of antibodies.
The plate was left standing at room temperature for 30 minutes with a washing solution containing 0% bovine serum.

Next, after washing twice with washing solution, highly specific anti-AFB monoclonal antibody solution diluted with washing solution (103,1
04,105,5X10', 106.107 times diluted solution)
Standard AF prepared with 50μp and an extract for toxin analysis in fertilizer feed or food samples (e.g. 50% methanol aqueous solution)
B. Add 50 μp of solution [(Opg ~ 5 ng) 150 μm] to each well at the same time and leave it at room temperature for 2 hours (for creating a calibration curve).

After washing three times with washing solution, peroxidase-labeled antibody against mouse immunoglobulin (1
(Used after diluting 0.03 times) was dispensed into 100 μl portions and allowed to stand at room temperature for 2 hours. After washing four times with the washing solution, the substrate solution (0
-Phenylenediamine 20mg, 35% H2O2101
50ml of 0.1M citrate buffer at pH'5.0
Dissolved in 100 AI) and incubate at room temperature for 3 minutes in the dark.
It was left standing for 0 minutes. Finally, the enzyme reaction was stopped by dispensing 50 μp of 2N sulfuric acid, and the absorbance of the reaction stop solution at 492n+a was measured using a microplate photometer. The results are shown in FIG. Similarly to Example 4, when the absorbance was expressed as a percentage, the calibration curve was as shown in FIG.

In the case of such a two-step immunoassay method using unlabeled anti-AFB monoclonal antibodies, monoclonal antibodies against aflatoxin B1 are diluted 5x105 times, and even if the measurement takes about 6 hours, only several hundred pg/g ass
Only amounts up to ay could be detected.

Example 5 Experiment of Addition and Recovery of Aflatoxin B to Peanuts According to the one-step method of Example 4, an experiment of addition and recovery of aflatoxin B was attempted using peanuts.

The samples of peanuts were from Japan and China.

These peeled peanuts were pulverized using a homogenizer to form particles of a size that could pass through a III-11 sieve. This powder sample was added with standard aflatoxin B111.
It was impregnated with 25 μN of 1tty/ml methanol concentration solution.

25 ml of 50% aqueous methanol solution was added to this, and mixed and extracted for 3 minutes using a biomixer. Next, Toyo Roshi No. 14
Or whatma J' paper No. 4 or equivalent filter paper was used. This tear fluid and aflatoxin B1
The samples without additives were measured in accordance with Example 4 below. The results are shown in Figure 9.

As a result, the amount of aflatoxin B1 in peanuts was already 3.4 ppb (67.5 pg/assay), 4
．． It contained 8 ppb (95, Opg/assay). Koreni, 50% methanol extract (25mn)
1 ], , 1 p++b (556 pg/a
ssay) Aflatoxin B at 94 degrees was added.

This measured value is 573 pg/assay, , 625 p
Spike recoveries were 92% and 96% from g/assay.
I got almost satisfactory results.

As described above, according to the present invention, by immobilizing myco-1 to toxine on a support such as Immunopre I, and adding a sample and a mycotoxin measurement reagent at the same time, the mycotoxin immobilized on the support and the mycotoxin in the sample can be combined. It is characterized by completing the competitive monoclonal antibody reaction against. Therefore, the method of the present invention can measure the amount of mycotoxin more easily in a shorter time and with higher sensitivity than conventional measuring methods, and is an excellent measuring method with further improved safety and economical efficiency.

[Brief explanation of the drawing]

In Figure 1, OTA-BSA (20 μm/ml) was immobilized on a 96-well flat-bottom plate for immunoassay, and mycotoxin measurement reagents diluted to various concentrations and standard OTA solutions of various concentrations were placed on the plate. This is a calibration curve created by adding the solution to each well and carrying out an enzyme reaction, and finally measuring the absorbance of the reaction stop solution at 50OnI11 on the plate using a microplate photometer. Figure 2 shows T-2-H8-BS instead of 0TA=BSA.
This is a calibration curve created in the same manner as in Fig. 1 using A (20 μ!?/ml > and using standard T-2 solution instead of standard OTA solution. Fig. 3 shows 0TA- AFBI-BSA instead of BSA
(0,5,czy/ml>) and a standard AFB solution instead of the standard oTA solution.A calibration curve was created in the same manner as in Figure 1 (However, the competitive reaction was performed in 50% methanol. Figure 4 shows a calibration curve similar to Figure 3, except that the substrate reaction was carried out for 30 minutes at room temperature and the absorbance was measured at 492 nm. The competition reaction was carried out at 37°C for 30 minutes and the substrate reaction was carried out at 37°C for 15 minutes. , and are diagrams showing the absorbance converted into 100 parts.Curve a is for the case of FIG. 3, and curve l) is for the case of FIG. 4. FIG. 6 is a calibration curve chart prepared in Comparative Example 1. FIG. 7 is a calibration curve chart prepared in Comparative Example 2. Figure 8 shows the 105 times and 10 times the calibration curve in Figure 7.
It is a figure showing the curve of 6-fold dilution, converting the absorbance into 100 parts. FIG. 9 shows the results of an A F B + addition and recovery experiment to peanuts. In FIG. 9, A and B indicate AFB, additive-free Japanese peanuts, and Chinese peanuts, respectively. Figure 1: Addition to ochratoxin & (weight/As5ay) 2nd
Figure 5〃9 50ng 500pg 5pg
O'-2) V"z74 degrees (if / As5ay
)Fig.
Figure 5: Aflatoxin 81 heritage (Amount / As5ay) Figure 6: Degree of removal to Okratoyashin (Weight / As5ay) 7th
Figure 8

Claims (1)

[Scope of Claims] 1. A mycotoxin measuring reagent containing an enzyme-labeled monoclonal antibody against mycotoxins. 2. Measurement of mycotoxin according to claim 1, wherein the enzyme labeling the monoclonal antibody is selected from the group consisting of peroxidase, alkaline phosphatase, β-galactosidase, catalase, glucose oxidase, lactate oxidase, alcohol oxidase, and monoamine oxidase. reagent. 3. The reagent for measuring a mycotoxin according to claim 1, wherein the mycotoxin is produced by a bacterium of the genus Penicillium, a bacterium of the genus Aspergillus, or a bacterium of the genus Fusarium. 4. Claim 1 in which the mycotoxin is ochratoxin A, T-2 toxin or aftraxin B_1
A reagent for measuring mycotoxins as described in Section 1. 5. A method for measuring mycotoxins, which comprises measuring mycotoxins under competitive reaction conditions using an enzyme-labeled monoclonal antibody against mycotoxins. 6. Measurement of mycotoxin according to claim 5, wherein the enzyme labeling the monoclonal antibody is selected from the group consisting of peroxidase, alkaline phosphatase, β-galactosidase, catalase, glucose oxidase, lactate oxidase, alcohol oxidase, and monoamine oxidase. Law. 7. The method for measuring a mycotoxin according to claim 5, wherein the mycotoxin is produced by a Penicillium genus, an Aspergillus genus, or a Fusarium genus. 8. Claim 5 in which the mycotoxin is ochratoxin A, T-2 toxin or aflatoxin B_1
Method for measuring mycotoxins as described in section.