CN113125729A - Enzyme linked immunosorbent assay kit for detecting citrinin and detection method thereof - Google Patents
Enzyme linked immunosorbent assay kit for detecting citrinin and detection method thereof Download PDFInfo
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- CN113125729A CN113125729A CN201911407160.7A CN201911407160A CN113125729A CN 113125729 A CN113125729 A CN 113125729A CN 201911407160 A CN201911407160 A CN 201911407160A CN 113125729 A CN113125729 A CN 113125729A
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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Abstract
The invention relates to an enzyme linked immunosorbent assay kit for detecting citrinin 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: an enzyme-linked immunosorbent assay plate coated with an citrinin antigen, an citrinin standard substance, an citrinin antibody working solution, an citrinin 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 citrinin is that 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 enzyme-labeled plate micropores, after incubation for a period of time, substrate solution A and substrate solution B are added into a washing 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 is inversely proportional to the content of citrinin in the standard or sample. The method can be directly used for detecting the residual quantity of the citrinin in the dairy products.
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 citrinin in dairy products.
Background
The citrinin is a toxic metabolite of penicillium citrinum and the like, and mainly pollutes rice; the citrinin is yellow crystal, and the melting point is 172 ℃; is soluble in dilute alkaline solution and organic solvent, and is insoluble in water. It is a kidney poison. LD50(mg/kg (body weight)) mice from various animals were injected both subcutaneously and intraperitoneally at 35, 110 per os; rat subcutaneous and abdominal cavities were 67; the rabbit vein is 19; guinea pig subcutaneous 37; the combination of citrinin and N- (3, 5-dichlorophenyl) -succinimide can induce renal tumor in rat, and has genetic toxicity.
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 citrinin 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 citrinin 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 immunosorbent assay kit for detecting the citrinin comprises an enzyme label plate, an citrinin standard substance, an citrinin antibody working solution, an citrinin 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 preparation of the enzyme linked immunosorbent assay kit for detecting the citrinin comprises the following steps of: the preparation method comprises the steps of preparing an enzyme label plate, preparing an citrinin standard substance, preparing an citrinin antibody working solution, preparing an citrinin enzyme-labeled secondary antibody working solution, 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 enzyme label plate is prepared by coating an citrinin antigen, and the specific steps are that an citrinin 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 citrinin coating antigen is diluted into a 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 standard product of the citrinin are 0 mg/kg, 0.1mg/kg, 0.3 mg/kg, 0.9 mg/kg, 2.7 mg/kg and 8.1 mg/kg respectively.
The citrinin antibody working solution is prepared by immunizing a mouse with an citrinin artificial antigen to obtain a monoclonal antibody, and diluting the monoclonal antibody into a solution with the antibody diluent of 1: 60000 ratio.
The citrinin 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 citrinin and a detection method thereof are based on the principle of indirect competitive enzyme linked immunosorbent assay of antigen antibody, and 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 enzyme label plate coated with an citrinin antigen, adding 50 mu L of standard substance/sample/hole into a corresponding micropore, adding an citrinin enzyme-labeled secondary antibody working solution with 50 mu L/hole, then adding an citrinin antibody working solution with 50 mu L/hole, slightly oscillating and uniformly mixing, covering the plate with a cover plate film, and then placing the plate in a dark environment at the room temperature of 25 ℃ for reaction for 30 min;
(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 citrinin in the sample by contrasting the standard curve.
The enzyme linked immunosorbent assay kit for detecting the citrinin and the detection principle of the detection method thereof are as follows: the citrinin in the sample and the antigen specificity competitive antibody fixed on the enzyme label plate are added with enzyme label secondary antibody to react with the antibody, enzyme catalysis color developing agent is used for developing color, and the content of the citrinin in the sample is judged according to the color depth of the color developing. If the content of the citrinin in the sample is low, the color is dark; 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 citrinin protein conjugate:
obtaining the citrinin hapten derivative with carboxyl by adopting a succinic anhydride method, and then taking 0.05 mmol of the citrinin hapten 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 an citrinin 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 6 days after 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 3 days after and preparing the spleen cell suspension with myeloma cells, screening out a required hybridoma cell line for cloning,selecting hybridoma cells in logarithmic growth phase, freezing, and injecting 0.5 ml liquid paraffin into BALB/C mouse to sensitize, and injecting 1 × 10% into abdominal cavity 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 enzyme label plate coated with an orange penicillin coating antigen:
the coating antigen is obtained by coupling an orange penicillin hapten and carrier protein Bovine Serum Albumin (BSA), 0.05 mol/L Carbonate (CBS) buffer solution with pH 9.6 is used as a coating solution, the orange penicillin antigen is diluted into a ratio of 1:40000 and 100 mu L/hole, the coating solution is placed at 37 ℃ for 2 hours, an enzyme label plate is taken out, liquid in the plate is thrown off, 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 solution is 150 mu L/hole, the plate is placed at 37 ℃ for 1.5 hours, the sealing solution is discarded, the patted-dried enzyme label plate is placed at a constant temperature (25 ℃) for airing, and the enzyme label plate is placed at 4 ℃ after vacuum sealing is qualified through sampling inspection.
The preparation concentrations of the standard product of the citrinin 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.
Preparing an orange penicillin antibody working solution: immunizing a mouse by using an artificial antigen of citrinin to obtain a monoclonal antibody, and diluting the monoclonal antibody into a solution with an antibody diluent, wherein the solution is prepared by the following steps of 1: 60000 ratio.
The citrinin 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 citrinin 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 citrinin in the dairy product sample, the kit 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 citrinin in a sample to be detected
Taking an enzyme label plate coated with an orange penicillin antigen, and adding 50 muL/hole of a standard substance/sample into a corresponding micropore; adding an enzyme-labeled secondary antibody working solution into 50 muL/hole, then adding an orange penicillin antibody working solution into 50 muL/hole, lightly oscillating and uniformly mixing, and placing the mixture in a dark environment at 25 ℃ at room temperature for reaction for 30 min after covering a plate with 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 citrinin 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 citrinin 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 a standard curve, reading the concentration corresponding to the sample from the standard curve, and multiplying the concentration by the corresponding dilution multiple to obtain the actual concentration of the citrinin in the sample.
Claims (7)
1. An enzyme linked immunosorbent assay kit for detecting citrinin and a detection method thereof comprise an enzyme label plate, an citrinin standard substance, an citrinin antibody working solution, an citrinin 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.
2. An enzyme linked immunosorbent assay kit for detecting citrinin and a detection method thereof comprise the following steps: the preparation method comprises the steps of preparing an enzyme label plate, preparing an citrinin standard substance, preparing an citrinin antibody working solution, preparing an citrinin enzyme-labeled secondary antibody working solution, 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 enzyme linked immunosorbent assay kit for detecting citrinin and the detection method thereof according to claim 2, wherein the enzyme linked immunosorbent assay kit comprises: the preparation method of the ELISA plate comprises the steps of coupling an citrinin hapten with a carrier protein Bovine Serum Albumin (BSA) to obtain an citrinin coating antigen, using a Carbonate (CBS) buffer solution with 0.05 mol/L and pH of 9.6 as a coating solution, and 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 enzyme linked immunosorbent assay kit for detecting citrinin and the detection method thereof according to claim 2, wherein the enzyme linked immunosorbent assay kit comprises: the concentrations of the standard product of the citrinin are 0 mg/kg, 0.1mg/kg, 0.3 mg/kg, 0.9 mg/kg, 2.7 mg/kg and 8.1 mg/kg respectively.
5. The enzyme linked immunosorbent assay kit for detecting citrinin and the detection method thereof according to claim 2, wherein the enzyme linked immunosorbent assay kit comprises: the citrinin antibody working solution is prepared by immunizing a mouse with an citrinin artificial antigen to obtain a monoclonal antibody, and diluting the monoclonal antibody into a solution with the antibody diluent of 1: 60000 ratio.
6. The ELISA kit for detecting citrinin and the detection method thereof according to claim 2, wherein the ELISA kit comprises: the citrinin 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 enzyme linked immunosorbent assay kit for detecting citrinin and the detection method thereof as claimed in claim 2, based on the principle of indirect competitive enzyme linked immunosorbent assay of antigen antibody, the method is characterized in that: pretreating a sample to be detected, taking an ELISA plate coated with an citrinin antigen, sequentially and respectively adding a standard substance/sample, 50 mu L of an citrinin enzyme-labeled secondary antibody working solution and 50 mu L of an citrinin antibody working solution into corresponding micropores, lightly oscillating and uniformly mixing, placing the cover plate in a dark environment at the room temperature of 25 ℃ for reaction for 30 min, drying the liquid in the pores, fully washing for 4-5 times by using a washing working solution at an interval of 10 s every time, adding 50 mu L of a substrate solution A/pore and 50 mu L of a substrate solution B/pore after drying, lightly oscillating and uniformly mixing, placing the cover plate in a dark environment at the temperature of 25 ℃ for reaction for 15-20 min, adding 50 mu L of a stop solution/pore, lightly oscillating and uniformly mixing, setting an ELISA reader to detect at the position of 450 nm or at the double wavelength of 450/630 nm, and determining the light absorption value of each pore (please read data in 5 min), 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 citrinin in the sample by contrasting the standard curve.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115160282A (en) * | 2021-11-26 | 2022-10-11 | 国家食品安全风险评估中心 | Citrinin hapten, preparation method thereof, artificial antigen, enzyme linked immunosorbent assay kit and detection method |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115160282A (en) * | 2021-11-26 | 2022-10-11 | 国家食品安全风险评估中心 | Citrinin hapten, preparation method thereof, artificial antigen, enzyme linked immunosorbent assay kit and detection method |
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