CN113125728A - Preparation of enzyme linked immunosorbent assay kit for detecting variegated aflatoxin - Google Patents
Preparation of enzyme linked immunosorbent assay kit for detecting variegated aflatoxin Download PDFInfo
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- CN113125728A CN113125728A CN201911402008.XA CN201911402008A CN113125728A CN 113125728 A CN113125728 A CN 113125728A CN 201911402008 A CN201911402008 A CN 201911402008A CN 113125728 A CN113125728 A CN 113125728A
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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Abstract
The invention relates to an enzyme linked immunosorbent assay kit for detecting the aflatoxin and a detection method thereof, which have the advantages of sensitive, accurate and rapid detection, simple and convenient operation and strong specificity and are suitable for detecting a large number of samples. The kit comprises: the kit comprises an ELISA plate for coating a variegated aflatoxin antigen, a variegated aflatoxin standard substance, a variegated aflatoxin antibody working solution, a variegated aflatoxin enzyme-labeled secondary antibody working solution, a substrate solution A, a substrate solution B, a stop solution, a concentrated diluent and a concentrated washing solution. The principle of the enzyme linked immunosorbent assay kit for detecting the variegated aflatoxin is that a solid phase indirect competitive enzyme linked immunoreaction is carried out, an extracted sample, enzyme-labeled secondary antibody working solution and antibody working solution are added into corresponding micropores of an enzyme label plate, after incubation for a period of time, the substrate solution A and the substrate solution B are added into the plate, a color developing agent presents blue under the action of enzyme, and a stop solution is added, so that the color is changed from blue to yellow. The color depth of the color development is inversely proportional to the content of the aflatoxin in the standard or the sample. The method can be directly used for detecting the residual quantity of the aflatoxin in the dairy product.
Description
Technical Field
The invention relates to the technical field of veterinary drug residue detection, in particular to an enzyme linked immunosorbent assay kit for detecting aflatoxin in dairy products.
Background
Aflatoxins (ST) are one of the carcinogenic mycotoxins. Although the toxicity of the product is weaker than that of aflatoxin, the main toxigenic bacteria such as aspergillus versicolor and aspergillus nidulans are widely distributed in food and feed, and the proportion of toxigenic bacteria is high, so that the harm to people, livestock and poultry is not negligible. The variegated aspergillin (Sterigmatocystin) is mainly the final metabolite of Aspergillus versicolor (Aspergillus versicolor) and Aspergillus nidulans (Aspergillus nidulans), and is also the intermediate product in the later period of the process of synthesizing the flavomycin by Aspergillus flavus and Aspergillus parasiticus (Aspergillus paragonicus special). It is reported that corn infected with Aspergillus versicolor can produce more than 12g/kg of Aspergillus versicolor toxin in 21 days at 27 ℃. The rat of the aflatoxin has an oral LD 50 of mg/kg body weight, 166 as male, 120 as female and more than 800 as mouse. Monkey was injected intraperitoneally with LDso 32. As can be seen from the LDso values, primates (monkeys) are more sensitive to aflatoxins than rodents. The lethal pathological changes caused by the variegated aspergillus toxin are mainly the necrosis of liver and kidney parenchymal organs. The variegated aspergillus toxin has carcinogenicity, and can also cause bacillus subtilis and mouse cells to generate mutation reaction. ST is widely found in nature, and its toxicity and carcinogenicity are highly regarded by countries in the world because of its structure similar to aflatoxin. Heterochromotoxin the ochratoxin and the variegated aspergillus toxin
The enzyme-linked immunosorbent assay is an accurate, reliable, rapid and specific detection method, is suitable for rapid screening of a large number of samples, and has been widely applied to the food safety detection industry in recent years. The invention aims to establish an enzyme linked immunosorbent assay kit for detecting the aflatoxin and a detection method thereof.
Disclosure of Invention
In order to overcome the defects of chromatography, the invention provides an enzyme linked immunosorbent assay kit for detecting the aflatoxin and a detection method thereof. The method is sensitive, accurate and rapid, is simple and convenient to operate, has strong specificity, and is suitable for rapid detection of a large number of samples.
The enzyme-linked immunoassay kit for detecting the variegated aflatoxin comprises an enzyme label plate, a variegated aflatoxin standard substance, a working solution of a variegated aflatoxin antibody, a working solution of a variegated aflatoxin enzyme-labeled secondary antibody, a substrate solution A, a substrate solution B, a stop solution, a concentrated diluent and a concentrated washing solution.
The preparation of the enzyme linked immunosorbent assay kit for detecting the variegated aflatoxin comprises the following steps: the preparation method comprises the following steps of preparing an enzyme label plate, preparing a variegated aspergillus toxin standard substance, preparing a working solution of a variegated aspergillus toxin antibody, preparing a working solution of a variegated aspergillus toxin enzyme-labeled secondary antibody, preparing a substrate solution A, preparing a substrate solution B, preparing a stop solution, preparing a concentrated diluent and preparing a concentrated washing solution.
It is further characterized in that: the ELISA plate is prepared by coating a variegated aspergillus toxin antigen, and the specific steps are that a variegated aspergillus toxin hapten is coupled with a carrier protein Bovine Serum Albumin (BSA) to obtain a coating antigen, a Carbonate (CBS) buffer solution with 0.05 mol/L and pH of 9.6 is used as a coating solution, and the variegated aspergillus toxin coating antigen is diluted into 1:40000 proportion, 100 mu L/hole, incubating for 2 h at 37 ℃ in a dark place, taking out the ELISA plate, throwing off liquid in the plate, adding diluted concentrated washing liquid 300 mu L/hole, washing the plate for 2 times and 30 s/time, then adding 0.5% Bovine Serum Albumin (BSA) sealing liquid, 150 mu L/hole, placing for 1.5 h at 37 ℃, throwing off the sealing liquid, directly drying, placing the dried ELISA plate in a constant temperature chamber (25 ℃) for airing, and storing the ELISA plate under the condition of 4 ℃ in a vacuum sealing manner after the examination is qualified.
The concentrations of the aflatoxin standard substances are respectively 0 mg/kg, 0.1mg/kg, 0.3 mg/kg, 0.9 mg/kg, 2.7 mg/kg and 8.1 mg/kg.
The working solution of the hybrid aflatoxin antibody is a monoclonal antibody obtained by immunizing a mouse with a hybrid aflatoxin artificial antigen, and is diluted into a solution with the volume ratio of 1: 60000 ratio.
The mixed aspergillus toxin enzyme-labeled secondary antibody working solution is diluted by enzyme-labeled secondary antibody and diluent to be 1: 2000, wherein the substrate solution A is a citric acid-disodium hydrogen phosphate buffer solution containing 0.5 mmol/L of carbamide peroxide, the substrate solution B is an ethanol solution of tetramethyldiphenyldiamine, the stop solution is 2 mol/L of sulfuric acid, the concentrated diluent is a 10-fold concentrated diluent, is 0.1 mol/L of PBS and has a pH value ranging from 7.0 to 7.5, and the concentrated washing solution is a 10-fold concentrated washing solution which is 0.5% of Tween-20 and is 0.1 mol/L of PBST and has a pH value ranging from 7.0 to 7.5.
An enzyme linked immunosorbent assay kit for detecting the variegated aflatoxin and a detection method thereof, based on the indirect competitive enzyme linked immunosorbent assay principle of antigen antibody, the method comprises the following steps:
(1) pretreating a sample to be tested, namely treating the sample to be tested into a liquid sample, or extracting the sample to be tested by using an organic solvent and redissolving the sample to be tested in a sample diluent;
(2) taking out the required reagent from the refrigeration environment, placing the reagent at room temperature (20-25 ℃) for balancing for more than 30 min, and shaking each liquid reagent uniformly before use;
(3) taking an ELISA plate coated with a variegated aflatoxin antigen, adding 50 mu L of standard substance/sample into corresponding micropores, adding a working solution of a variegated aflatoxin enzyme-labeled secondary antibody at 50 mu L/hole, then adding a working solution of a variegated aflatoxin antibody at 50 mu L/hole, lightly oscillating and uniformly mixing, covering the plate with a cover plate film, and reacting for 30 min in a dark environment at room temperature of 25 ℃;
(4) carefully uncovering the cover plate film, drying liquid in holes, fully washing for 4-5 times by using 260 mu L/hole of washing working liquid, wherein each time interval is 10 s, and patting to be dry by using absorbent paper (bubbles which are not cleaned after patting to be dry can be punctured by using an unused gun head);
(5) adding 50 mu L/hole of the substrate solution A and 50 mu L/hole of the substrate solution B, lightly oscillating and uniformly mixing, covering a plate with a cover plate, and reacting for 15-20 min in a dark environment at 25 ℃;
(6) adding 50 μ L of stop solution into each well, slightly oscillating, mixing, detecting with an enzyme-labeling instrument at 450 nm or double wavelength of 450/630 nm, and measuring absorbance value of each well (please read data within 5 min);
(7) and drawing a standard curve by taking the logarithm of the concentration (ppb) of the standard substance as an abscissa and the percent absorbance value of the standard substance as an ordinate, and calculating the content of the aflatoxin in the sample by contrasting the standard curve.
The enzyme linked immunosorbent assay kit for detecting the aflatoxin and the detection principle of the detection method thereof of the invention are as follows: the method comprises the steps of competing antibodies of the aflatoxin in a sample and antigen specificity fixed on an enzyme label plate, adding an enzyme-labeled secondary antibody, reacting with the antibodies, developing color through enzyme catalysis of a color developing agent, and judging the content of the aflatoxin in the sample according to the color depth of the color development. If the content of the aflatoxin in the sample is low, the color development is deep; otherwise, the color development is light. The kit detection method is simple and convenient to operate, sensitive, accurate and rapid in detection, and is suitable for rapid detection of large-batch samples.
Detailed Description
Preparation of the aflatoxin protein conjugate:
obtaining the carboxyl-containing aflatoxin hapten derivative by adopting a succinic anhydride method, and then taking 0.05 mmol of the derivative and a carrier protein BSA according to the weight ratio of 10: 1 in 0.05 mol/L of Carbonate Buffer (CBS) at pH 9.6, then adding 0.15 mmol of carbodiimide, stirring and standing at room temperature for reaction for 24 h, finally dialyzing in 0.2 mol/L of PBS buffer at pH 7.6 for two days, removing unreacted hapten, and storing the obtained protein conjugate solution at-20 ℃ for later use.
Preparation of the aflatoxin antibody:
selecting healthy adult pure BALA/C mice, mixing 50 mu g of immune antigen prepared by coupling protein with an equal amount of complete Freund's adjuvant, performing primary immunization by intraperitoneal injection, performing secondary and tertiary immunization by intraperitoneal injection by using the same amount of immune antigen and the equal amount of incomplete Freund's adjuvant every 3 weeks, performing tail vein blood sampling after 6 days of each immunization to determine antiserum titer to a certain titer, performing final immunization by using the same amount of adjuvant-free antigen, performing cell fusion on spleen cell suspension prepared by taking spleen after 3 days, screening out required hybridoma cell lines for cloning, selecting hybridoma cells in logarithmic growth phase for cryopreservation, preparing ascites, performing intraperitoneal injection of 0.5 ml of liquid paraffin to BALB/C mice for sensitization, and performing intraperitoneal injection of 1 multiplied by 10 after 2 weeks6Inoculating hybridoma cells, generating ascites after 7-10 days, collecting ascites with injector when ascites is as much as possible, centrifuging at 4000 rpm for 15 min, collecting supernatant, purifying monoclonal antibody with octanoic acid-ammonium sulfate method, lyophilizing to obtain lyophilized powder, and storing at-20 deg.C.
Preparing an ELISA plate coated with a variegated aspergillus toxin coating antigen:
the coating antigen is obtained by coupling a aflatoxin hapten and carrier protein Bovine Serum Albumin (BSA), 0.05 mol/L Carbonate (CBS) buffer solution with pH of 9.6 is used as a coating solution, the aflatoxin antigen is diluted into a ratio of 1:40000 and 100 mu L/hole, the mixed solution is placed at 37 ℃ for 2 hours, an ELISA plate is taken out, liquid in the plate is thrown away, diluted concentrated washing solution is used for 300 mu L/hole, the plate is washed for 2 times and 30 s/time, then 0.5% Bovine Serum Albumin (BSA) is added for sealing, the 150 mu L/hole is placed at 37 ℃ for 1.5 hours, the sealing solution is discarded, the blotted ELISA plate is placed at a constant temperature (25 ℃) for airing, and the ELISA plate is placed at 4 ℃ after being qualified by sampling inspection.
The preparation concentrations of the aflatoxin standard substance are respectively 0 mg/kg, 0.1mg/kg, 0.3 mg/kg, 0.9 mg/kg, 2.7 mg/kg and 8.1 mg/kg.
Preparation of a variegated aflatoxin antibody working solution: adopting a variegated aspergillus toxin artificial antigen to immunize a mouse to obtain a monoclonal antibody, and diluting the monoclonal antibody into 1: 60000 ratio.
The mixed aspergillus toxin enzyme-labeled secondary antibody working solution is diluted by enzyme-labeled secondary antibody and diluent to be 1: 2000, substrate solution A is citric acid-disodium hydrogen phosphate buffer solution containing 0.5 mmol/L carbamide peroxide, substrate solution B is ethanol solution of tetramethyl diphenyldiamine, stop solution is 2 mol/L sulfuric acid, concentrated diluent is 10 times of concentrated diluent, 0.1 mol/L PBS, pH value ranges from 7.0 to 7.5, concentrated washing solution is 10 times of concentrated washing solution, the concentrated washing solution is PBST containing 0.5% Tween-20 and 0.01mol/L, and the pH value ranges from 7.0 to 7.5.
Based on the prepared reagent, the enzyme linked immunosorbent assay kit for detecting the aflatoxin comprises the following materials:
(1) 96-hole enzyme label plate is multiplied by 1 block;
(2) standard solution × 6 bottles: (1 mL/bottle) 0 mg/kg, 0.1mg/kg, 0.3 mg/kg, 0.9 mg/kg, 2.7 mg/kg, 8.1 mg/kg;
(3) 7 mL of antibody working solution;
(4) 7 mL of enzyme-labeled secondary antibody working solution;
(5) 7 mL of substrate solution A;
(6) 7 mL of substrate solution B;
(7) 7 mL of stop solution;
(8)10 × 40 mL of concentrated diluent;
(9)10 × 40 mL of concentrated washing solution;
when the kit is used for detecting the residual quantity of the aflatoxin in a dairy product sample, the method is implemented by the following steps: sample pretreatment, detection by using the kit and result analysis.
(1) The kit of the invention is used for detecting the residual quantity of the aflatoxin in a sample to be detected
Taking an ELISA plate coated with a variegated aflatoxin antigen, and adding 50 muL/hole of a standard product/sample into the corresponding micropore; adding an enzyme-labeled secondary antibody working solution into 50 muL/hole, then adding a variegated aspergillus toxin antibody working solution into 50 muL/hole, lightly oscillating and uniformly mixing, and placing the mixture in a light-tight environment at room temperature of 25 ℃ for reaction for 30 min after a cover plate is covered by a cover plate film; carefully uncovering the cover plate film, spin-drying liquid in the holes, fully washing the holes for 4 times by using 300 muL/hole of washing working liquid, soaking the holes for 15-30 s, and patting the holes dry by using absorbent paper; adding 50 muL/hole of substrate liquid A and 50 muL/hole of substrate liquid B, lightly oscillating and uniformly mixing, and placing the mixture in a dark environment at 25 ℃ for reaction for 15 min after covering a cover plate with a cover plate film; adding 50 muL of stop solution into each hole, slightly oscillating and uniformly mixing, setting an enzyme-labeling instrument at 450 nm or double-wavelength 450/630 nm for detection, and determining the absorbance value of each hole (please finish reading data within 5 min); and (3) comparing the absorbance values of the sample to be detected and the standard substance, and quantitatively analyzing the residual quantity of the aflatoxin in the sample to be detected.
(2) Analysis results
Calculation of the percent absorbance value, the percent absorbance value of the standard or sample being equal to the average of the absorbance values of the standard or sample (double well) divided by the absorbance value of the first standard (0 standard) and multiplied by 100%, i.e.
Percent absorbance value (%) ═ B/B0×100%
Wherein B is the average absorbance value of the standard solution or sample solution, B0-average absorbance value of 0 ppb standard solution.
And drawing a standard curve by taking the logarithm of the concentration (ppb) of the standard substance of the aflatoxin as an abscissa and the percentage absorbance value of the standard substance as an ordinate, and solving a linear equation. And substituting the percent absorbance value of the sample into the standard curve, reading out the concentration corresponding to the sample from the standard curve, and multiplying the corresponding dilution multiple by the concentration corresponding to the sample to obtain the actual concentration of the aflatoxin in the sample.
Claims (7)
1. The enzyme linked immunosorbent assay kit for detecting the variegated aflatoxin comprises an enzyme label plate, a variegated aflatoxin standard substance, a working solution of a variegated aflatoxin antibody, a working solution of a variegated aflatoxin enzyme-labeled secondary antibody, a substrate solution A, a substrate solution B, a stop solution, a concentrated diluent and a concentrated washing solution.
2. An enzyme linked immunosorbent assay kit for detecting the variegated aflatoxin and a detection method thereof comprise the following steps: the preparation method comprises the following steps of preparing an enzyme label plate, preparing a variegated aspergillus toxin standard substance, preparing a working solution of a variegated aspergillus toxin antibody, preparing a working solution of a variegated aspergillus toxin enzyme-labeled secondary antibody, preparing a substrate solution A, preparing a substrate solution B, preparing a stop solution, preparing a concentrated diluent and preparing a concentrated washing solution.
3. The ELISA kit for detecting aflatoxin according to claim 2 and the detection method thereof, characterized in that: the preparation method of the ELISA plate comprises the steps of coupling the aflatoxin hapten and carrier protein Bovine Serum Albumin (BSA) to obtain an aflatoxin coating antigen, using 0.05 mol/L Carbonate (CBS) buffer solution with pH of 9.6 as a coating solution, diluting the coating antigen into 1:40000 proportion, 100 mu L/hole, incubating at 37 ℃ for 2 h, taking out the ELISA plate, throwing off liquid in the plate, adding diluted concentrated washing liquid 300 mu L/hole, washing the plate for 2 times and 30 s/time, then adding 0.5% Bovine Serum Albumin (BSA) for sealing, 150 mu L/hole, placing at 37 ℃ for 1.5 h, discarding the sealing liquid for direct patting, placing the patted-dry ELISA plate in a constant temperature room (25 ℃) for airing, and storing the ELISA plate under the condition of 4 ℃ in a vacuum sealing manner after the sampling inspection is qualified.
4. The ELISA kit for detecting aflatoxin according to claim 2 and the detection method thereof, characterized in that: the concentrations of the aflatoxin standard substances are respectively 0 mg/kg, 0.1mg/kg, 0.3 mg/kg, 0.9 mg/kg, 2.7 mg/kg and 8.1 mg/kg.
5. The ELISA kit for detecting aflatoxin according to claim 2 and the detection method thereof, characterized in that: the working solution of the aflatoxin antibody is a monoclonal antibody obtained by immunizing a mouse with an artificial antigen of aflatoxin, and is diluted into a solution with the volume ratio of 1: 60000 ratio.
6. The ELISA kit for detecting aflatoxin and the detection method thereof according to claim 2 are characterized in that: the mixed aspergillus toxin enzyme-labeled secondary antibody working solution is diluted by enzyme-labeled secondary antibody and diluent to be 1: 2000 proportion, the substrate liquid A is a citric acid-disodium hydrogen phosphate buffer solution containing 0.5 mmol/L of carbamide peroxide, the substrate liquid B is an ethanol solution of tetramethyl diphenyldiamine, the stop solution is 2 mol/L of sulfuric acid, the concentrated diluent is 10 times of concentrated diluent which is 0.1 mol/L of PBS and has a pH value ranging from 7.0 to 7.5, and the concentrated washing liquid is 10 times of concentrated washing liquid which is 0.5 percent of Tween-20 and 0.01mol/L of PBST and has a pH value ranging from 7.0 to 7.5.
7. The ELISA kit for detecting aflatoxins and the detection method thereof as claimed in claim 2, which is based on the principle of indirect competitive enzyme-linked immune reaction of antigen and antibody, and is characterized in that: pretreating a sample to be detected, taking an ELISA plate coated with a variegated aflatoxin antigen, sequentially and respectively adding a standard substance/sample, a variegated aflatoxin enzyme-labeled secondary antibody working solution and a variegated aflatoxin antibody working solution into corresponding micropores at each rate of 50 mu L per hole, lightly oscillating and uniformly mixing, placing the cover plate with a cover plate membrane in a dark environment at the room temperature of 25 ℃ for reaction for 30 min, spin-drying the liquid in the holes, fully washing with a washing working solution for 4-5 times at an interval of 10 s every time, adding a substrate solution A of 50 mu L per hole and a substrate solution B of 50 mu L per hole after drying, lightly oscillating and uniformly mixing, placing the cover plate membrane in a dark environment at the temperature of 25 ℃ for reaction for 15-20 min, adding a stop solution of 50 mu L per hole, lightly oscillating and uniformly mixing, setting an ELISA instrument for detection at the position of 450 nm or at the double wavelength of 450/630 nm, and determining the absorbance value of each hole (please finish, and drawing a standard curve by taking the logarithm of the concentration (ppb) of the standard substance as an abscissa and the percent absorbance value of the standard substance as an ordinate, and calculating the content of the aflatoxin in the sample by contrasting the standard curve.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114806888A (en) * | 2022-04-06 | 2022-07-29 | 浙江工业大学 | Aspergillus versicolor ZJUTE2 and application thereof in preparation of aflatoxin |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114806888A (en) * | 2022-04-06 | 2022-07-29 | 浙江工业大学 | Aspergillus versicolor ZJUTE2 and application thereof in preparation of aflatoxin |
| CN114806888B (en) * | 2022-04-06 | 2023-09-05 | 浙江工业大学 | Aspergillus versicolor ZJUTE2 and application thereof in preparation of aflatoxin |
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Application publication date: 20210716 |