JP2551406B2 - How to detect aflatoxin - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method of detecting aflatoxin.

The exposure of humans and other animals to toxic substances via food, water and air and their effects is a crucial issue for our survival. Low molecular weight (1000 Daltons or less) mutagens such as aflatoxins, fluoranthene, nitropyrene, nitrofluoranthene, nitrochryserine and aminobiphenyl and / or Alternatively, it has become more important to detect carcinogens. In particular, non-invasive screening methods to assess whether humans are exposed to substances such as aflatoxins are
Both the toxin itself and its metabolites, especially covalent adducts formed with DNA and proteins in body fluids such as serum and urine, must be detectable.

Aflatoxins are representative of the toxic and carcinogenic compounds included in this group. Aflatoxin is a secondary metabolite of fungi called mycotoxin, and it includes Asparagillus flavus and Aspergillus parasiticucus.
s) is a substituted fumarine structurally containing a condensed dihydrofurofuran moiety. Aflatoxins are naturally contained in peanuts, peanut meal, cottonseed meal, corn, dried chili pepper and the like. However, the mold growth itself cannot predict the presence and concentration of this toxin. This is because the amount of aflatoxin produced depends on the growth conditions of the mold and the genetic characteristics of the species. Various aflatoxins,
That is, B ₁ , B ₂ , G ₁ , G ₂ , M ₁ , and M ₂ have been isolated and characterized. Aflatoxin type B ₁ (hereinafter AFB ₁ ) is the most biologically active of these compounds, toxic to many animal species, and acts as a mutagen and carcinogen. This mycotoxin is often found in human food in many parts of the world,
There are statistics that the frequency of human liver cancer is high in these regions, especially in Asia and Africa (Busby et al., Food-Born Infections and Intoxications).
(Lehman and Brian), Second Edition, Academic
Press, 1979, pp. 519-610), Wogan G.N., Methods Cncer Res. 7: pp.309-344 (1973)).

AFB ₁ is also highly reactive, forming covalent adducts with guanine in DNA after undergoing oxidative metabolism
2.3-becomes an epoxide. Exemplary adducts 2,3-dihydro-2 - a (N ⁷ guanyl) -3-hydroxy-aflatoxin B ₁ (hereinafter referred to as AFB ₁ -N ⁷ -Gua) (Rinra, Cancer Res. 37 : pp.4430-4438 (1977); Martin et al., Nature, (London), 267 ; pp.863-865 (197).
7)). AFB ₁ -N ⁷ -Gua adduct and its putative derivative 2,3-dihydro-2- (N ⁵ -formyl-2 ′, 5 ′,
6'-triamino-4'-oxo -N ⁵ - pyrimidyl) -
3-hydroxyaflatoxin B ₁ (hereinafter AF-N ⁷ -Gua
Is fixed in tissues and systems such as rat liver, human bronchial and colon tissue cultures, human lung cell cultures, either transiently or chronically after administration (Haugen et al. , Proc.Natl.Acad.Sci.USA 78: pp.4124-4.
127 (1987)).

Methods of quantifying metabolites, including AFB ₁ and its DNA adducts, using immunological methods and monoclonal antibodies have been studied (Herzog et al., Carcinogensis 3: pp.825-
828 (1982); Haugen et al., Proc. Natl. Acad. Sci. USA 7
8: pp.4124-4127 (1981)). Similar studies have been performed with other low molecular weight toxins found in our environment using immunological methods and reagents (Johnson et al., J. Analyt. Toxicol. 4: pp. 86-90). Hugh et al., J. Food
Prot. 47: pp.562-569 (1984)). Nevertheless, as far as is known so far, no general non-invasive screening method has been established to assess whether humans and animals are exposed to carcinogens present in such environments. .

The present invention comprises a step of loading a sample which may contain aflatoxin on an affinity column containing a solid phase adsorbent and a monoclonal antibody specific for the aflatoxin, which is immobilized thereon, and a solvent. Through the column to collect the first effluent, thereby retaining the aflatoxyl on the column by the monoclonal antibody, and passing the eluent through the column to collect the second effluent, thereby Eluting the aflatoxin from the monoclonal antibody and collecting it in the second effluent, and fluorescing by exposing the second effluent to UV light without further purification to detect the presence of the aflatoxin. A method for detecting aflatoxin in a test sample, which comprises a step of performing a measurement.

The method of the present invention is applicable to any type of aflatoxin, and is particularly useful for testing aflatoxin B ₁ and aflatoxin M ₁ . The term "detection" as used herein includes both detection and quantification of the presence. The hybridomas obtained in the following examples have been deposited with the American Type Culture Collection,
Its deposit number is ATCC HB8719.

I. Production of Aflatoxin Immunogen A preferred immunogen is bovine gamma globulin (hereinafter BGG).
That is) to which AFB ₁ is bound.
First, BGG is pH 7.0 phosphate buffer (hereinafter referred to as PBS)
It was dissolved at a concentration of 10 mg / ml. AFB ₁ by a modified method using m-chloroperoxybenzoic acid (Martin et al., Nature (London) 267: pp.863-865).
Was bound to BGG. Typically 1 mg AFB ₁ (3.2 μmol)
Dissolved in 2.0 ml methylene chloride, this was added to a 5 molar excess of m-chloroperoxybenzoic acid (mCPBA) previously dissolved in 2.0 ml methylene chloride. In the reaction mixture, P
BGG dissolved in BS was added to produce aflatoxin in a 5 molar excess with respect to the protein content. The reaction mixture was vigorously mixed overnight at room temperature and then stopped by centrifugation at 2000 g for 20 minutes. The aqueous upper layer containing the modified BGG protein (supernatant) was extensively dialyzed against PBS pH 7.4. 362n
The absorbance at m was measured, and the concentration of modified globulin protein was measured by utilizing the fact that the molar extinction coefficient is 18000. The reaction product AFB ₁ -BGG obtained by the above method
Had 40 to 50 AFB ₁ residues bound to one molecule of BGG on average.

AFB ₁ can also be attached to other carriers using the methods described above. For example, AFB ₁ is bovine serum albumin (hereinafter, BSA
That is a useful alternative. However, the binding of AFB ₁ to bovine serum albumin is usually 20 to 30 molecules of aflatoxin to 1 molecule of BSA. Other useful carriers can also be combined with AFB ₁ using methods known in the art. Examples of such a conjugate are listed below. AFB ₁ −
BGG (mCPBA), single-stranded AFB ₁ -DNA-meBSA, AFB ₁ -N ⁷ -Gu
a-BGG-PABA (para-benzoic acid), AFB ₁ - carboxymethyl oxime-KLH (Kehoru limpet hemocyanin _{(Kehole Limpet Hemocyanin), AFB 1} - Poly -Gua-B
GG II. Immunization of Mice Female BALB / By (Jackson Laboratory) of about 16 weeks of age were immunized with AFB ₁ -BGG dissolved in PBS and emulsified with an equal volume of Freund's complete adjuvant. Immunization was 37.5 μg using 2 groups of 5 mice each
Alternatively, 12 μg of AFB ₁ -BGG (including adjuvant) in PBS (final volume 0.2 ml) was intraperitoneally administered. Five and nine weeks after the first injection, each mouse was given the same amount of AFB ₁ -BGG emulsified in Freund's incomplete adjuvant. Approximately 10 days after the second injection, the tail was bled and a serum sample was taken to measure anti-aflatoxin antibody activity by ELISA immunoassay. Mice in which specific antibody was found in serum were injected into the tail vein 3 days before sacrifice with the same AFB ₁ -BGG in PBS (0. ml).

ELISA immunoquantification was also used to determine the presence of specific antibodies to AFB _{1 in} mouse serum (and then to immobilize specific hybridomas). These quantification methods are modifications of methods known in the art (Haugen et al., Proc. Natl. Acad. Sci. USA 78: p.
p.4124-4127 (1981), Groopeman et al., Cancer Res. 4
2: pp.3120-3124 (1982)). Briefly summarizing this method, AFB ₁ -BSA was dissolved in PBS at a concentration of 2.0 μg / ml and 50 μl of this liquid mixture was placed in each well of a polyvinyl microtiter plate and incubated at room temperature for 2-4 hours. 2 μg / in some of the wells
50 μl of PBS solution of BSA having a concentration of ml was added to serve as a control. Aspirate the liquid in each well and rinse the wells with rinsing water.
Washed times. Then add 0.2% BAS or 0.
A PBS solution containing 2% gelatin (Type IV, Sigma) was added and the plate was incubated at room temperature for an additional hour. This procedure was performed to limit non-specific binding of the antibody. The plates were then washed in rinsing water and 50 μl of mouse serum sample or hybridoma medium was placed in each well. To titrate mouse sera, dilutions in the range 1:50 to 3: 50,000 were made by serial 3-fold dilutions in PBS containing 10% fetal bovine serum. If hybridoma medium was used, 50 μl was used with the dilutions. In both cases, place the microtiter plate at 37 ° C.
Incubate for 90 minutes at 100 ° C., then wash thoroughly with plain water. The specific antibody bound to the surface of each well is
In each well, 50:20 dilution of a 1: 200 rat anti-mouse kappa antibody conjugated to alkaline phosphatase.
It was measured by adding μl and incubating at room temperature for 4 hours or at 4 ° C. overnight. The wells of each plate were then washed again with plain water. 100 μl of 1.0 mg / ml p-nitrophenyl phosphate solution made in 0.1 M diethanolamine buffer of pH 9.8 was added to each well and reacted for 1 to 2 hours. The p-nitrophenol reaction product was quantified by measuring the absorbance of the wells at 405 nm using a microtiter plate reader (Dynatech Laboratory).

The isotype of the monoclonal antibody was determined by non-competitive ELISA using a commercial kit (Boehringer-Mannheim) for immobilization of mouse immunoglobulin subtypes (ie different antibody H-chain groups were immobilized). ).

III. BALB / By CJ mice hybridoma production and isolation females immunized beforehand in complete adjuvant Freund with AFB ₁ -BGG of monoclonal antibody-producing cells have a significant anti-AFB ₁ serum timer Presence or absence was examined using the non-competitive ELISA described above. Anti-titer mice were killed and hybridomas were made according to the method described by Marshak-Rosstein et al., Journal of Immunology 122, pp.2491-2497 (1979). Dulbecco's modified Eagle medium with 20% (v / v) fetal bovine serum, 580 μg / ml glutamine, 10 units / ml penicillin, 100 μg / ml streptomycin, and a non-essential amino acid (Gibco). SP2 / 0 myeloma cell lines maintained in (hereinafter DME medium) were used for fusion cells. Mice were killed and suspensions of splenocytes were made using pH 7.2 Hank's balanced salt solution (hereinafter HPBS) buffered with 0.01 M phosphate.

Splenocytes from these mice were isolated from Gefter et al., Somati.
c Cell Genet. 3: pp. 321 (1977) and fused with SP2 / 0 myeloma cells using a modified method.
All centrifugations were performed at 700 g for 5 minutes at room temperature unless otherwise noted. Preferably, 5x10 ⁶ SP2 / 0 myeloma cells and 5x10 ⁷ immune splenocytes are placed in a round-bottomed plastic tube at 1
Combined, centrifuged, resuspended in 10 ml serum-free DME medium and centrifuged again. The supernatant was carefully discarded and the faucet water was sharply applied to the pellet to disperse the remaining cell pellet in the centrifuge tube. The cells were then thawed in serum-free DME 30
% (V / v) polyethylene glycol 1000 (baker
Chemical Co.) 0.5 ml solution for 6 minutes. During this 6 minutes the cells were gently centrifuged (150 g for 3 minutes). Then 4.0 ml of serum-free DME was added to the cell pellet and the cells were resuspended in tap water. The contents of the tubes were transferred to 100x17 mm Petri dishes and cultured for 1 day in DME medium containing 20% fetal bovine serum. The cells were then resuspended and resuspended in a growth medium containing hypoxanthine, aminopterin and thymidine (hereinafter referred to as HAT medium). Add 0.1 ml (about 10 ⁵ SP) of cells to each well of a flat-bottomed microtiter plate.
(Including 2/0 cells). After incubating for 1 week, 0.05 ml of a growth medium containing hypoxanthine and thymidine (hereinafter referred to as HT medium) was added to each well. Two weeks after fusion, for each culture,
The presence or absence of specific anti-AFB ₁ antibody was examined using the ELISA immunoassay described above.

A hybridoma secreting an IgM antibody having a high affinity for AFB ₁ was grown as an ascites tumor cell in BALB / C mice previously injected with 0.5 ml of pristen (Adrich). Hybridomas grown in mice produced large amounts of specific IgM antibodies, which were collected as ascites fluid before the mice died. Ascites fluid from these animals was stored and used as such or for further purification by saturated ammonium sulfate precipitation and dialysis against PBS for immunoassay.
Gross pathological examination revealed that all mice died as a result of widespread tumor invasion, ie, proliferation of injected hybridoma cells.

The monoclonal antibody obtained by this method was shown to be an anti-affinity IgM antibody having specificity for the AFB ₁ molecule. The affinity constant of this antibody is the Scatchard (Scat
chard plot analysis and Muller's method (J.Immunol.Met
h: 34 , pp.345-352 (1980)), 8x
10 ⁸ and 1 × 10 ⁹ liters / mole. These values are derived from the data obtained by the competitive RIA method below.

This analysis was performed using a 300 μl sample,
l was purchased from Moravec Biochemicals [ ³ H]
-Consisting of AFB ₁ tracer (specific activity 3.4 Ci / mmol). Stock solution of tracer is 1% of normal mouse serum containing 0.1% BSA.
It was diluted with PBS solution to a concentration of about 20200 cpm / 100 μl.
Monoclonal antibody was diluted to a concentration at which 30 to 50% of AFB ₁ tracer was precipitated. An antibody containing 10% fetal bovine serum PBS solution was added to the reaction mixture by 100 μl. Non-radiolabeled AFB ₁ or AF-N ⁷ -G which is the main AFB ₁ -DNA adduct
1 sample consisting of ua and its metabolites including AF-FAPyr
00 μl was added to each tube. The reaction mixture was incubated at room temperature for 2 hours and then the mixture was diluted with PBS to a volume of 1.0 ml. An equal volume of ice-cold saturated ammonium sulfate was then added and the samples mixed and placed in ice for 15 minutes. The sample was then centrifuged at 2000 g for 15 minutes and the percent inhibition of the reaction was determined using the Mueller method.

The first specificity of IgM antibody for AFB ₁ and its metabolites
Shown in Figures and 2. In addition, these results are
It was obtained using purified high affinity IgM antibody that was first fractionated by precipitation with saturated ammonium sulfate and dialyzed against PBS. The antibody was then purified by high pressure liquid chromatography. At this time, the large molecular weight of the IgM antibody (900,000 Dalton) was used to separate the antibodies by steric exclusivity. What was used in the method described here is that this purification with its specificity and high affinity
Characteristics and properties of IgM antibody. In the competitive RIA method,
For aflatoxin B ₁ , aphtoxin B ₂ , and aflatoxin M ₁ , the inhibition rate was 50% at 3.0 pmol, but for aflatoxin G ₁ , aflatoxin G ₂ , and aflatoxin Q ₁ , 60.0, 84.0, and 27, respectively.
It was 5.0 pmol. The data shown in Figure 2 is AFB
AF-, which is the _one and two major aflatoxin DNA adducts
It shows that the specificity and the inhibition rate of the IgM antibody against N ⁷ -Gua and AF-FAPyr are 50%. The 50% inhibition values were 3.0, 24.0, and 89.0 pmol, respectively. Thus, the data is the IgM antibodies indicates that about 4 times higher than the sensitivity of the sensitivity for detecting the AF-FAPyr adduct detects AF-N ⁷ -Gua adduct.

IV. Affinity Matrix Containing Immobilized Anti-AFB ₁ IgM Monoclonal Antibody This purified IgM monoclonal antibody was used to prepare an affinity matrix material by the following method described by Pharmacia Fine Chemicals. It was Sufficient amount (2.0 mg) of monoclonal antibody was added to 0.1M NaHCO ₃ 0.5M NaC.
It was dissolved in a binding buffer (pH 8.3) containing 1 and 2. This antibody solution was added to Sepharose-4B (Sigma) activated by cyanogen bromide which had been pre-incubated in 8.0 ml of 0.001 M HCl. After allowing Sepharose and the antibody solution to react with each other for 1 hour, the solid-phase adsorption material was treated with 1.0 M
The unbound sites of the gel to which the antibody was bound were blocked by incubating with ethanolamine (pH 8.5) for 1 hour. The affinity matrix was formed by the IgM antibody immobilized on the solid phase adsorbent material, which was then used in a volume of 1-2 ml.

The preferred solid phase adsorbent material is activated Sepharose 4B.
Although a gel, it is recognized that many other materials can be used as the solid phase material. Mention may be made, for example, of other agarose gel compositions, dextrans, carbon and silicon particulate formulations including glass plates. Similarly, methods of immobilizing anti-affinity IgM antibodies on their respective chemical compositions are well known and described in the art.

V. Method for detecting and isolating aflatoxin in liquid sample Aflatoxin and aflatoxin-DN in test sample
The method for detecting the A adduct is an affinity chromatography method including the following steps. (1) A uniform affinity matrix in which an anti-affinity IgM antibody specific for aflatoxin or an aflatoxin-DNA adduct of interest is immobilized on a solid-phase adsorption material is produced. (2) The test sample is contacted with the affinity matrix so that the aflatoxin in the test sample is bound and retained by the IgM antibody of the affinity matrix. (3) Add an eluent for releasing aflatoxin from the IgM antibody to the affinity matrix. This eluent is an aqueous solution of an aprotic solvent selected from the group consisting of dimethyl sulfoxide, dimethylformamide and dimethylacetamide, at a concentration of 50% (v / v) or higher. (4) Identify if aflatoxin is present in the recovered effluent from the affinity matrix.

An important feature of the anti-aflatoxin IgM monoclonal antibody is not only its specificity for the AFB ₁ antigen, but also its high affinity constant (at least about 1 × 10 ⁸ liter / mol, preferably 1 × 10 ⁹ liter / mol). This is an important feature.
Therefore, after the target aflatoxin is bound to the antibody covalently bound in the affinity column, a special eluent must be used to elute it. This is demonstrated in the next example. That is, in the following examples, the antibody bound to the adsorbent shows not only the ability to bind the target aflatoxin, but also after the aflatoxin has been adsorbed to the affinity column, it is conventionally dissociated. It is shown that this cannot be done with the lysing agent.

Example 1 The first experiment was anti-AF immobilized on Sepharose 4B.
This was done to determine the ability of the B ₁ IgM antibody to bind AFB ₁ . In these experiments, 10 ml of radiolabeled ³ H-AFB ₁ was used.
Used in PBS at a concentration of 1 ng. This concentration is the concentration of aflatoxin expected to be detected in body fluids of humans exposed to contaminated food. First, 100μ
l ³ H-AFB ₁ tracer, 0.1% BSA, 10 mM NaN ₃
Then, 10.0 ml of PBS containing 0.1 ml of normal mouse serum and 3 μl of ³ H-AFB ₁ was added to a gel bed of about 0.1 ml. The effluent column flow rate was slightly higher than 0.25 ml / min. About 200 μl PB
S was added to the tracer and transferred to the column by lowering the column until the top of the affinity matrix was exposed to air (without drying the column). further
Wash out the tracer from the column by adding 2.0 ml of PBS,
The effluent was collected as a single fraction in Calasbin (No.
1). In this way, a tracer of about 24486 CPM was added to the column, and the content of the radiolabeled substance was measured by scintillation after each fraction was collected. Then different eluents were added to the affinity matrix and the ability to release the AFB ₁ tracer was measured. These results are shown in Table 1 below.

As can be seen, about 10% of the original tracer was not adsorbed on the affinity matrix, and the effluent fraction was
It was recovered as No.1. This is due to non-specific tritium exchange in the tracer. Subsequent reactions with the listed individual eluents resulted in tracer AFB in the flow-through fractions 2-7 due to matrix IgM antibody.
It was found that the release of ₁ did not occur substantially. A large amount (90%) of AFB ₁ tracer was released from the affinity matrix only after using 50% dimethyl sulfoxide (hereinafter referred to as DMSO). Also, when DMSO was added to the affinity column, a clear color change in the matrix became visible and the column bed itself became more translucent. When PBS was added to the affinity matrix after collecting the DMSO effluent fraction,
The gel bed returned to its original opaque state.

The affinity matrix returned to its original state without damaging or modifying (denaturing, precipitating, etc.) the IgM antibody bound to the Sepharose gel. This was shown by continuing the experiment with DMSO releaser and subsequent PBS washes.

Second 100 μl tracer ³ H dissolved in 200 μl PBS
-AFB ₁ was added to the same column previously used to isolate the _first ³ H-AFB ₁ tracer sample. After adding the second tracer, rinse the column with 2.0 ml PBS and fractionate the effluent.
It was collected as No. 8. Then 2.0 ml of DMSO in PBS was added and the effluent was collected as Fraction No. 9. 2 columns next
It was washed twice with 2.0 ml PBS each and the effluent was collected as fractions 10 and 11. The results are shown in Table 2 below.

The second tracer had a total radioactivity of about 24486 cpm, but also about 10% of it was not retained in the affinity matrix. Fraction 9 also contains a large amount of AFB ₁ tracer (approximately
60%) is eluted with DMSO and the rest is collected in fraction 10 by the first PBS wash with some residual DMSO.

Furthermore, at least 50% to bring about the elution effect
A variation of this experiment was performed to show that it was necessary to use different concentrations of DMSO. 100 used in this experiment
μl of ³ H-AFB ₁ total about 27000 to 28000 cpm of ³ H-A
Included FB ₁ . 100 μl 3 tracer was loaded onto the affinity column and washed with 200 μl PBS to remove unretained unretained tracer from the column. The matrix was then washed with 1.0 ml increasing concentrations of DMSO. The results are shown in Table 3 below.

It can be seen from Table 3 that ³ H-AFB ₁ is not substantially eluted from the affinity matrix before using the 50% solution of DMSO. In addition, even if the concentration of DMSO is greater than 50%,
It can be seen that the release of AFB ₁ from the affinity matrix does not detectably increase. For this reason, DMSO, whose concentration is at least 50%, is sufficient for use as an eluent in this method. Other suitable eluents for use in this method are dimethylformamide and dimethylacetamide. To be effective eluents, these eluents must also be used at a concentration of at least 50%. With these eluents, ie, more commonly aprotic solvents, the affinity matrix can be reused repeatedly. Elution with such an aprotic solvent does not damage the matrix material. This is important for industrial detection and measurement of mycotoxins. When carrying out this method industrially, it is necessary to reuse the affinity matrix for economic reasons, but elution with an aprotic solvent such as DMSO meets this requirement.

On the other hand, elution can also be carried out using lipophilic solvents such as methanol or ethanol, but the affinity matrix or column is virtually unusable. In this aspect of the invention, preferably 50 to 100%, particularly preferably 100% methanol or ethanol is added to the DMS.
It can be used in the same manner as the above-mentioned method except that these are used instead of O.

VI. In Vitro Isolation of Aflatoxins from Human Urine, Serum, and Breast Milk As demonstrated above, the ability of the IgM antibody affinity matrix to bind AFB ₁ as demonstrated by radiotitration or absorbance measurement, AFB ₁ 1.0 from 10 ml PBS per 1 ml column bed
Or 1.3 μg. These parameters can be used to isolate and detect aflatoxin in vitro from human urine, serum and breast milk samples as described below.

Human serum (10 ml each) or human breast milk (10 m each)
It can be applied directly to the antibody matrix without pretreatment. In both cases, aflatoxin in the liquid sample bound quantitatively to the antibody matrix, these were:
It is quantitatively recovered by using 50% DMSO as eluent. This means that ³ H
-Experimentally found by adding ₁ ng of AFB1 directly to serum or breast milk samples. Therefore, when testing serum or breast milk, no preparatory steps are required to quantitatively recover aflatoxin.

However, urine must be subjected to pretreatment, including chromatography, before isolation and recovery of aflatoxin to remove interfering substances such as salts in the sample. This is shown schematically in FIG.
Here, a Sep-Pak C ₁₈ cartridge (Waters Associates) was used to remove interfering substances. If the urine sample was first passed through a preparative low pressure liquid chromatography cartridge to remove proteins and other interfering substances, 90-95% of the aflatoxins present in human urine samples were consistently detected by the method of this invention. It can be collected quantitatively.

Moreover, the use of high-affinity IgM antibodies is specific for AFB ₁ is, AFB _1-DNA adducts are useful for quantitatively detecting and recovering AF-N ⁷ -Gua. These aflatoxin DNA adducts are detected and quantitatively recovered from serum and other body fluids without any pretreatment. When urine from humans or animals is used as the test sample, it is preferable to pretreat the sample by high pressure liquid chromatography through a carbon chromatography cartridge before adding the sample to the affinity matrix. In this way, AFB ₁ and its metabolites from complex biological samples from circumscribed people can be quantitatively and qualitatively isolated and purified. Furthermore, this method also serves as an analytical method for detecting aflatoxin itself.

From the above, it can be seen that the present invention can easily and accurately detect various concentrations of aflatoxin in a sample. An important aspect of this invention is the affinity matrix or column to which the specific monoclonal antibody is bound. Using such an affinity matrix comprising an antibody immobilized on a solid phase adsorbent provides a rapid and efficient means for purifying a test sample. Due to the high affinity and selectivity of the monoclonal antibody, the crude extract of the sample is passed through the antibody matrix column, whereby the toxin of interest is bound to the antibody and all other components of the sample extract pass through. . The original crude sample is eluted and recovered in quantitative yield with the highly purified toxin by eluting with a suitable eluent such as DMSO or methanol. Such an efficient one-step purification method is particularly advantageous in the field of toxicological analysis.

After purifying aflatoxin with the antibody matrix material, the aflatoxin is detected or quantified by fluorescence analysis. In any case, the present invention provides a simple and rapid purification method using an affinity matrix to which a monoclonal antibody is bound and passing the sample for analysis and detection once through the affinity matrix.

Aflatoxin is also detected or quantified by fluorescence analysis. That is, when the purified sample is exposed to ultraviolet rays (365 nm), aflatoxin emits fluorescence at 425 nm. This type of analytical method is particularly preferred due to its analytical performance and the ability to do some quantitative analysis. Such a fluorescence analysis operation is performed by the AOAC method (AOAC Method, Chapter 26, pp. 4).
14-429 (1980)).

Since the antibody binding matrix allows for one-step purification of samples containing toxicants, the present invention finds application in examining various toxicants contained in industrial wastes and food products such as corn and peanuts, and body fluids. be able to.
Taking the corn sample as an example, the overall operation method is shown below.

As can be seen from the above, it is clear that a common and important step is the purification of the sample by using an affinity column to which the specific monoclonal antibody is bound. Once the sample is purified, the concentration or presence of the toxicant can be qualitatively or quantitatively measured by various methods.

Qualitative Analysis According to the invention, poisons can first be detected qualitatively. For example, aflatoxins in corn samples can be detected by the following steps.

(1) Weigh 50 g of ground corn.

(2) Place the ground corn in the mixer.

(3) Add methanol to the mixer.

(4) Mix for 1 minute.

(5) Add distilled water to the mixture and mix.

(6) Take the extract from the mixer through a filter.

(7) The extract is loaded on an aflatoxin affinity column.

(8) Pass distilled water through an aflatoxin affinity column.

(9) Methanol is passed through an affinity column (preferably contained in a syringe) and collected in a reaction container having a fluorescent lighting means.

(10) Take a container having fluorescent lighting means and put it in a device for visually measuring (not quantitative) the amount of fluorescence.

(11) Take the container and discard.

Quantitative Analysis On the other hand, the concentration of the toxic substance can be quantitatively measured by the following procedure. Again, the measurement of aflatoxin in corn is taken as an example.

(1) Weigh 50 g of ground corn.

(2) Place the ground corn in the mixer.

(3) Add methanol to the mixer.

(4) Mix for 1 minute.

(5) Add distilled water to the mixture and mix.

(6) Take the extract from the mixer through a filter.

(7) The extract is loaded on an aflatoxin affinity column.

(8) Pass distilled water through an aflatoxin affinity column.

(9) Methanol is passed through the affinity column and collected in a test cuvette included in the measuring device.

(10) Read the measured value.

(11) Remove the sample and discard.

[Brief description of drawings]

Figure 1 shows IgM using ³ H-AFB ₁ and various aflatoxins.
FIG. 2 is a graph showing the results of competitive radioimmunoassay for measuring the specificity of the antibody, and FIG. 2 shows the use of IgM antibody using ³ H-AFB ₁ tracer and aflatoxin B ₁ and the main metabolic aflatoxin-DNA adduct. FIG. 3 is a schematic flow chart outlining the general method for detecting and isolating toxins, which is a graph showing the results of competitive radioimmunoassay.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI Technical indication location G01N 33/543 597 G01N 33/543 597 (72) Inventor Jiyoung Dee Grapesman Linfield Mellow Road, Massachusetts, USA 25 (72) Inventor The Gerard N Nuorgan 125 Belmont Claflin Street, Massachusetts, USA 125 (72) Inventor Anne Marshyak-Rosstein, Newton Lois Road, Massachusetts, United States 40 (56) ． Natl. Acad. Sc i. USA, 81 (1984) p. 7728-7731

Claims (11)

(57) [Claims] 1. A step of loading a sample that may contain aflatoxin onto an affinity column containing a solid phase adsorbent and a monoclonal antibody specific for the aflatoxin, which is immobilized thereon. Pass the solvent through the column,
Collecting a first effluent, thereby retaining the aflatoxin on the column with the monoclonal antibody, and passing an eluent through the column to collect a second effluent, thereby transferring the aflatoxin to the monoclonal. Eluting from the antibody and collecting in the second effluent, and performing a fluorescence measurement by exposing the second effluent to UV light without further purification to detect the presence of the aflatoxin. A method for detecting aflatoxin in a test sample containing. 2. The method according to claim 1, wherein the eluent is a solution of an aprotic solvent having a concentration of 50% or more. 3. The method according to claim 2, wherein the aprotic solvent is dimethyl sulfoxide, dimethylformamide or dimethylacetamide. 4. The eluent is 50 to 100% of a lipophilic solvent.
The method according to claim 1, which is a solution. 5. The lipophilic solvent is 100% methanol or 100%.
The method according to claim 4, which is% ethanol. 6. The antibody according to any one of claims 1 to 5, wherein the antibody is specific for the aflatoxin to be detected and has an affinity constant of at least about 1 × 10 ⁸ liter / mole. The method according to item 1. 7. The method according to claim 6, wherein the aflatoxin is aflatoxin B ₁ or aflatoxin M ₁ . 8. The method according to any one of claims 1 to 7, wherein the sample is serum, urine or food. 9. The method according to claim 1, wherein the second effluent is exposed to ultraviolet rays having a wavelength of 365 nm.
The method described in the section. 10. A method according to any one of claims 1 to 9 wherein the second effluent is subjected to a quantitative analysis to detect the amount of aflatoxin in the sample. 11. Aflatoxin B ₁ or aflatoxin M ₁
And a solid phase adsorbent immobilized thereon and specific for the aflatoxin, the affinity constant of which is at least 1 × 10 ⁹
Loading the sample on an affinity column containing IgM antibody in liters / mole; passing the solvent through the column;
Collecting the effluent of the aflatoxin, thereby retaining the aflatoxin on the column with the antibody, and passing an eluent through the column to collect a second effluent, thereby eluting the aflatoxin from the antibody. And recovering in the second effluent, and performing a fluorescence measurement by exposing the second effluent to UV light without further purification to detect the presence of the aflatoxin. The method according to claim 1, which detects aflatoxins in a sample.