GB/T 5009.118-2003 Enzyme-linked immunosorbent assay (ELISA) for T-2 toxin in wheat

This standard specifies the enzyme-linked immunosorbent assay (ELISA) method for T-2 toxin in wheat. This standard is applicable to the determination of T-2 toxin in wheat and its products. The detection limit of this method is 1μg/kg. GB/T 5009.118-2003 Enzyme-linked immunosorbent assay (ELISA) for T-2 toxin in wheat GB/T5009.118-2003 Standard download decompression password: www.bzxz.net

ICS67.040
National Standard of the People's Republic of China
GB/T5009.118--2003
Replaces GB/T14933-1994
Determination of T-2 toxin in wheat withenzyme-linked immunosorbent assay (ELISA)
Determination of T-2 toxin in wheat withenzyme-linked immunosorbent assay (ELISA)2003-08-11Promulgated
Implementation on 2004-01-01
Ministry of Health of the People's Republic of China
Administrative Committee of Standardization of the People's Republic of China
GB/T5009.118—2003
This standard replaces GB/T14933—1994 "Enzyme-linked immunosorbent assay of T-2 toxin in wheat (ELISA)". Compared with GB/T14933-1994, this standard has the following major revisions: The Chinese name of the standard has been revised and changed to "Enzyme-linked immunosorbent assay (ELISA) for T-2 toxin in wheat": - The structure of the original standard has been revised according to GB/T20001.4--2001 "Standard Preparation Rules Part 4: Chemical Analysis Methods".
This standard is proposed and managed by the Ministry of Health of the People's Republic of China. The drafting unit of this standard: Food Hygiene Supervision and Inspection Institute of the Ministry of Health. The main drafters of this standard: Yang Chuanhe, Luo Xueyun, Ji Rong. The original standard was first issued in 1994, and this is the first revision. 88
1 Scope
Enzyme-linked immunosorbent assay (ELISA) for T-2 toxin in wheat
This standard specifies the enzyme-linked immunosorbent assay (ELISA) method for T-2 toxin in wheat. This standard is applicable to the determination of T-2 toxin in wheat and its products. The detection limit of this method is 1μg/kg.
Method 1
Indirect method
2 Principle
GB/T5009.118-2003
Adsorb the known antigen on the surface of the solid phase carrier, wash away the unadsorbed antigen, add a certain amount of antibody and a mixture of the extract of the sample to be tested (containing the antigen), and after competitive incubation, an antigen-antibody complex is formed on the surface of the solid phase carrier. Wash away the excess antibody components, then add the enzyme-labeled antiglobulin second antibody conjugate, combine with the antigen-antibody complex adsorbed on the solid surface, and then add the enzyme substrate. Under the catalytic action of the enzyme, the substrate undergoes a degradation reaction to produce a colored product. The amount of enzyme substrate degradation is measured by the enzyme label detector, thereby inferring the amount of antigen in the sample to be tested.
3 Reagents
3.1 Methanol.
3.2 Petroleum ether.
Triazine.
Absolute ethanol.
Ethyl acetate.
Dimethylformamide.
Tetramethylbenzidine (TMB).
Tween-20.
30% hydrogen peroxide (30% H2O.).
3.10 Antibody: Specific monoclonal antibody against T-2 toxin produced by hybridoma cell line 1D7. Antigen: Conjugate of T-2 toxin and carrier protein-bovine serum albumin (BSA). 3.11
Conjugate of rabbit anti-mouse immunoglobulin and horseradish peroxidase (enzyme-labeled secondary antibody). 3.12
3.13 ELISA buffer system.
3.13.1 The coating buffer is a carbonate buffer with a pH of 9.6. Weigh 1.59g sodium carbonate (NazCO,) and 2.93g sodium bicarbonate (NaHCO,) and dilute to 1000mL with water. 3.13.2 The washing solution is a pH 7.4 phosphate buffer (PBS-T) containing 0.05% Tween-20. The preparation method is as follows: weigh 0.2g potassium dihydrogen phosphate (KH2PO4), 2.9g disodium hydrogen phosphate (Naz2HPO4,·12H2O), 1.0g sodium fluoride (NaCl), 0.2g potassium chloride (KCl), 0.5mL Tween-20, and add water to 1000mL. 3.13.3 The substrate buffer is a phosphate-citrate buffer with a pH of 5.0. The preparation method is as follows: 0.1 mol/L citric acid (CHO, ·H, O), that is, weigh 19.2 g of citric acid, add water to 1000 mL, which is liquid A; 0.2 mol/L disodium hydrogen phosphate (NaHPO.), that is, weigh 71.7 g of disodium hydrogen phosphate, add water to 1000 mL, which is liquid B; 89
GB/T5009.118—2003
Take 24.3 mL of liquid A and 25.7 mL of liquid B, add water to 100 mL. 3.13.4 Substrate solution: Take 50 μL TMB (10 mg TMB dissolved in 1 mL dimethylformamide) solution + 10 mL substrate buffer + 10 μL 30% hydrogen peroxide, mix well.
3.14T-2 toxin standard solution
Use methanol to prepare 1mg/mL T-2 toxin stock solution and store in a refrigerator at -20℃. On the day of the test, accurately aspirate the stock solution and dilute it with 20% methanol in PBS (preparation method is the same as PBS-T, without Tween-20) to the required concentration for preparing the standard curve. 4 Instruments
All glassware are soaked in sulfuric acid solution and rinsed with tap water and steamed water. 4.1 ELISA detector.
4.2 ELISA plate (40 wells or 96 wells).
4.3 Electric oscillator.
4.4 Electric constant temperature water bath.
4.5 10mL small concentration bottle with 0.2mL tail tube. 5 Analysis steps
5.1 Extraction
Weigh 20 g of the sample that has been crushed and passed through a 20-mesh sieve, place it in a 200-mL stoppered conical flask, add 8 mL of water and 100 mL of chloroform-anhydrous ethanol (4+1), stopper tightly, shake for 1 h, filter through filter paper, take 25 mL of the filtrate and put it in an evaporator, place it in a 90°C water bath for ventilation and evaporate to dryness. Use 50mL of petroleum ether to dissolve the residual in the evaporated blood in portions, wash into a 250mL separatory funnel, then wash with 20mL of methanol-water (4+1) in portions, transfer to the same separatory funnel, shake for 1.5min, let stand for about 15min, collect the lower layer of methanol-water extract and purify it through a chromatography column (equipment of the chromatography column: plug about 0.1g of absorbent cotton at the connection between the lower end of the chromatography column and the small tube, plug it as tightly as possible, first fill it with 0.5g of neutral alumina, knock it flat, then add 0.4g of activated carbon, and knock it tightly). Pour the eluate after passing through the column into the evaporated blood and concentrate it to dryness in a water bath. Add 3mL of ethyl acetate while it is hot, heat to boiling, select dryness, repeat once, and finally add 3mL of ethyl acetate. Cool to room temperature and transfer it to a concentration bottle. Wash the evaporating dish with an appropriate amount of ethyl acetate and put it into the concentration bottle. Place the concentration bottle in a 95℃ water bath, evaporate and cool, and then make up the volume with PBS containing 20% ​​methanol for ELISA detection. 5.2ELISA test
5.2.1 Coat the ELISA plate with T-2-BSA (4μg/mL), 100pL per well, overnight at 4℃; 5.2.2 Wash the ELISA plate with PBS-T for 3 times, 3 minutes each time, add a mixture of different concentrations of T-2 standard solution (to prepare a standard curve) or sample extract (to detect the toxin content in the sample) and antibody solution (1+1, 100μL per well, the mixture should be prepared the day before use and kept at 4℃ overnight), and place at 37℃ for 1h; 5.2.3 Wash the ELISA plate for 3 times, 3 minutes each time, add enzyme-labeled secondary antibody, 100μL per well, and place at 37℃ for 1.5h; 5.2.4 After washing as above, add substrate solution, 100μl per well, and place at 37℃ for 30min; 5.2.5 Terminate the reaction with 1mol/L sulfuric acid solution, 50pL per well, and measure the absorbance at 450nm. 6 Result calculation
Calculate according to formula (1):
Wherein:
X---T-2 concentration, in nanograms per gram (ng/g); the amount of T-2 toxin measured on the ELISA plate (ng), according to the standard curve: Vi--the volume of the sample extract, in milliliters (mL): 90
. (1)
V2-the volume of the added sample solution, in milliliters (mL), D-the total dilution multiple of the sample solution;
m--the mass of the sample, in grams (g). 7 Precision
GB/T5009.118—2003
The absolute difference between two independent determination results obtained under repeatability conditions shall not exceed 20% of the arithmetic mean. Second method Direct method
8 Principle
The known antigen is adsorbed on the surface of the solid phase carrier, the unadsorbed antigen is washed away, and a certain amount of enzyme-labeled antibody and a mixture of the sample (containing the antigen) extract are added. After competitive incubation, an antigen-antibody-enzyme complex is formed on the surface of the solid phase carrier. The excess is washed away and the enzyme substrate is added. Under the catalytic action of the enzyme, the substrate undergoes a degradation reaction to produce a colored substance. The amount of enzyme substrate degradation is measured by an enzyme-labeled detector, thereby inferring the amount of antigen in the test sample.
9 Reagents
9.1 Methanol.
9.2 Petroleum ether.
9.3 Chloroform.
9.4 Anhydrous ethanol.
9.5 Ethyl acetate.
9.6Dimethylformamide.
9.7Tetramethylbenzidine (TMB).
9.8Tween-20.
9.930% hydrogen peroxide (30%H,02). 9.10Anti-T-2 toxin monoclonal antibody and horseradish peroxidase conjugate. Antigen: T-2 toxin and carrier protein-bovine serum albumin conjugate (T-2-BSA). 9.11
9.12ELISA buffer system.
The coating buffer is a carbonate buffer with a pH of 9.6. Weigh 1.59g sodium carbonate (NazCO.), 2.93g sodium bicarbonate 9.12.1
(NaHCO) and dilute to 1000mL with water.
9.12.2 The washing solution is a phosphate buffer solution of pH 7.4 (PBS-T) containing 0.05% Tween-20. The preparation method is as follows: weigh 0.2g potassium dihydrogen phosphate (KH,PO.), 2.9g disodium hydrogen phosphate (Na,HPO.·12H,O), 8.0g sodium chloride (NaCI), 0.2g potassium chloride (KCI), 0.5mL Tween-20, and add water to 1000mL. 9.12.3 The substrate buffer is a phosphate-citrate buffer with a pH of 5.0. The preparation method is as follows: 0.1 mol/L citric acid (CHsO,.HO), that is, weigh 19.2g of citric acid, add water to 1000mL, which is liquid A; 0.2 mol/L disodium hydrogen phosphate (NazHPO,), that is, weigh 71.7g disodium hydrogen phosphate, add water to 1000mL, which is liquid B; take 24.3mL of liquid A and 25.7mL of liquid B, add water to 100mL. 9.12.4 Substrate solution: Take 50μLTMB (10mgTMB dissolved in 1mL dimethylformamide) solution + 10mL substrate buffer + 10uL 30% hydrogen peroxide, mix well.
9.13T-2 toxin standard solution
Use methanol to prepare 1mg/mL T-2 toxin stock solution and store in a refrigerator at -20℃. On the day of the test, accurately pipette the stock solution and dilute it with PBS containing 20% ​​methanol (prepared in the same way as PBS-T, without Tween-20) to the required concentration for preparing the standard curve. 91
GB/T5009.118—2003
10 Instruments
All glassware should be soaked in sulfuric acid solution and rinsed with tap water and distilled water. 10.1 ELISA detector.
10.2 ELISA plate (40-well or 96-well).
10.3 Electric shaker.
10.4 Electric constant temperature water bath.
10.5 10mL small concentration bottle with 0.2mL tail tube. 11 Analysis steps
11.1 Extraction
Weigh 20 g of the sample that has been crushed and passed through a 20-mesh sieve, place it in a 200-mL stoppered conical flask, add 8 mL of water and 100 mL of trinitroform-anhydrous ethanol (4+1), stopper tightly, shake for 1 h, filter through filter paper, take 25 mL of the filtrate and put it in evaporating blood, place it in a 90°C water bath for ventilation and shake to dry. Dissolve the residue in the evaporating dish with 50 mL of petroleum ether, wash it into a 250 mL separatory funnel, and then wash it with 20 mL of methanol-water (4+1) in batches, transfer it to the same separatory funnel, shake it for 1.5 minutes, let it stand for about 15 minutes, collect the lower layer of methanol-water extract and purify it through a chromatography column (equipment of the chromatography column: plug about 0.1 g of absorbent cotton at the connection between the lower end of the chromatography column and the small tube, plug it as tightly as possible, first fill it with 0.5 g of neutral alumina, flatten the surface, then add 0.4 g of activated carbon, and tap it tightly). Pour the eluate after passing through the column into the evaporating blood, and concentrate it to dryness in a water bath. Add 3 mL of ethyl acetate while it is hot, heat it to boiling, evaporate it, repeat it once, and finally add 3 mL of ethyl acetate, cool it to room temperature, and transfer it to a concentration bottle. Wash the evaporating dish with an appropriate amount of ethyl acetate and put it into the concentration bottle. Place the concentrate bottle in a 95℃ water bath, evaporate and cool, and then use PBS containing 20% ​​methanol to make up the volume for ELISA detection. 11.2ELSA detection
.11.2.1 Coat the ELISA plate with T-2-BSA (4μg/mL), 100uL per well. Incubate at 4℃ overnight. 11.2.2 Wash the ELISA plate with PBS-T 3 times, 3 minutes each time, add a mixture of different concentrations of T-2 standard solution (to make a standard curve) or sample extract (to detect the content of cystin in the sample) and antibody-enzyme conjugate solution (1+100) (1+1, 100uL per well, the mixture should be prepared the day before use and stored at 4℃ overnight for use), and place at 37℃ for 1.5h. 11.2.3 Wash the ELISA plate 3 times, 3 minutes each time, and add the substrate solution. 100μL per well, 37℃ for 30min. 11.2.4 Use 1mol/L sulfuric acid solution to terminate the reaction, add 50μL to each well, and measure the absorbance at 450nm. 12 Result calculation bZxz.net
Calculate according to formula (2):
Wherein:
T-2 concentration, in nanograms per gram (ng/g) X-
-the amount of T-2 toxin measured on the ELISA plate (ng), obtained according to the standard curve: Vi——the volume of the sample extract, in milliliters (mL); V,——the volume of the added sample solution, in milliliters (mL); D—the total dilution factor of the sample solution;
-the mass of the sample, in grams (g)).
13 Precision
The absolute difference between two independent measurement results obtained under repeatability conditions shall not exceed 15% of the arithmetic mean. 92
(2)13T-2 toxin standard solution
Prepare 1mg/mL T-2 toxin stock solution with methanol and store in a refrigerator at -20℃. On the day of the test, accurately pipette the stock solution and dilute it with 20% methanol in PBS (prepared in the same way as PBS-T, without Tween-20) to the required concentration for preparing the standard curve. 91
GB/T5009.118—2003
10Instruments
All glassware should be soaked in sulfuric acid solution and rinsed with tap water and distilled water. 10.1 ELISA detector.
10.2 ELISA plate (40-well or 96-well).
10.3 Electric shaker.
10.4 Electric constant temperature water bath.
10.5 10mL small concentration bottle with 0.2mL tail tube. 11 Analysis steps
11.1 Extraction
Weigh 20 g of the sample that has been crushed and passed through a 20-mesh sieve, place it in a 200-mL stoppered conical flask, add 8 mL of water and 100 mL of trinitroform-anhydrous ethanol (4+1), stopper tightly, shake for 1 h, filter through filter paper, take 25 mL of the filtrate and put it in evaporating blood, place it in a 90°C water bath for ventilation and shake to dry. Dissolve the residue in the evaporating dish with 50 mL of petroleum ether, wash it into a 250 mL separatory funnel, and then wash it with 20 mL of methanol-water (4+1) in batches, transfer it to the same separatory funnel, shake it for 1.5 minutes, let it stand for about 15 minutes, collect the lower layer of methanol-water extract and purify it through a chromatography column (equipment of the chromatography column: plug about 0.1 g of absorbent cotton at the connection between the lower end of the chromatography column and the small tube, plug it as tightly as possible, first fill it with 0.5 g of neutral alumina, flatten the surface, then add 0.4 g of activated carbon, and tap it tightly). Pour the eluate after passing through the column into the evaporating blood, and concentrate it to dryness in a water bath. Add 3 mL of ethyl acetate while it is hot, heat it to boiling, evaporate it, repeat it once, and finally add 3 mL of ethyl acetate, cool it to room temperature, and transfer it to a concentration bottle. Wash the evaporating dish with an appropriate amount of ethyl acetate and put it into the concentration bottle. Place the concentrate bottle in a 95℃ water bath, evaporate and cool, and then use PBS containing 20% ​​methanol to make up the volume for ELISA detection. 11.2ELSA detection
.11.2.1 Coat the ELISA plate with T-2-BSA (4μg/mL), 100uL per well. Incubate at 4℃ overnight. 11.2.2 Wash the ELISA plate with PBS-T 3 times, 3 minutes each time, add a mixture of different concentrations of T-2 standard solution (to make a standard curve) or sample extract (to detect the content of cystin in the sample) and antibody-enzyme conjugate solution (1+100) (1+1, 100uL per well, the mixture should be prepared the day before use and stored at 4℃ overnight for use), and place at 37℃ for 1.5h. 11.2.3 Wash the ELISA plate 3 times, 3 minutes each time, and add the substrate solution. 100μL per well, 37℃ for 30min. 11.2.4 Use 1mol/L sulfuric acid solution to terminate the reaction, add 50μL to each well, and measure the absorbance at 450nm. 12 Result calculation
Calculate according to formula (2):
Wherein:
T-2 concentration, in nanograms per gram (ng/g) X-
-the amount of T-2 toxin measured on the ELISA plate (ng), obtained according to the standard curve: Vi——the volume of the sample extract, in milliliters (mL); V,——the volume of the added sample solution, in milliliters (mL); D—the total dilution factor of the sample solution;
-the mass of the sample, in grams (g)).
13 Precision
The absolute difference between two independent measurement results obtained under repeatability conditions shall not exceed 15% of the arithmetic mean. 92
(2)13T-2 toxin standard solution
Prepare 1mg/mL T-2 toxin stock solution with methanol and store in a refrigerator at -20℃. On the day of the test, accurately pipette the stock solution and dilute it with 20% methanol in PBS (prepared in the same way as PBS-T, without Tween-20) to the required concentration for preparing the standard curve. 91
GB/T5009.118—2003
10Instruments
All glassware should be soaked in sulfuric acid solution and rinsed with tap water and distilled water. 10.1 ELISA detector.
10.2 ELISA plate (40-well or 96-well).
10.3 Electric shaker.
10.4 Electric constant temperature water bath.
10.5 10mL small concentration bottle with 0.2mL tail tube. 11 Analysis steps
11.1 Extraction
Weigh 20 g of the sample that has been crushed and passed through a 20-mesh sieve, place it in a 200-mL stoppered conical flask, add 8 mL of water and 100 mL of trinitroform-anhydrous ethanol (4+1), stopper tightly, shake for 1 h, filter through filter paper, take 25 mL of the filtrate and put it in evaporating blood, place it in a 90°C water bath for ventilation and shake to dry. Dissolve the residue in the evaporating dish with 50 mL of petroleum ether, wash it into a 250 mL separatory funnel, and then wash it with 20 mL of methanol-water (4+1) in batches, transfer it to the same separatory funnel, shake it for 1.5 minutes, let it stand for about 15 minutes, collect the lower layer of methanol-water extract and purify it through a chromatography column (equipment of the chromatography column: plug about 0.1 g of absorbent cotton at the connection between the lower end of the chromatography column and the small tube, plug it as tightly as possible, first fill it with 0.5 g of neutral alumina, flatten the surface, then add 0.4 g of activated carbon, and tap it tightly). Pour the eluate after passing through the column into the evaporating blood, and concentrate it to dryness in a water bath. Add 3 mL of ethyl acetate while it is hot, heat it to boiling, evaporate it, repeat it once, and finally add 3 mL of ethyl acetate, cool it to room temperature, and transfer it to a concentration bottle. Wash the evaporating dish with an appropriate amount of ethyl acetate and put it into the concentration bottle. Place the concentrate bottle in a 95℃ water bath, evaporate and cool, and then use PBS containing 20% ​​methanol to make up the volume for ELISA detection. 11.2ELSA detection
.11.2.1 Coat the ELISA plate with T-2-BSA (4μg/mL), 100uL per well. Incubate at 4℃ overnight. 11.2.2 Wash the ELISA plate with PBS-T 3 times, 3 minutes each time, add a mixture of different concentrations of T-2 standard solution (to make a standard curve) or sample extract (to detect the content of cystin in the sample) and antibody-enzyme conjugate solution (1+100) (1+1, 100uL per well, the mixture should be prepared the day before use and stored at 4℃ overnight for use), and place at 37℃ for 1.5h. 11.2.3 Wash the ELISA plate 3 times, 3 minutes each time, and add the substrate solution. 100μL per well, 37℃ for 30min. 11.2.4 Use 1mol/L sulfuric acid solution to terminate the reaction, add 50μL to each well, and measure the absorbance at 450nm. 12 Result calculation
Calculate according to formula (2):
Wherein:
T-2 concentration, in nanograms per gram (ng/g) X-
-the amount of T-2 toxin measured on the ELISA plate (ng), obtained according to the standard curve: Vi——the volume of the sample extract, in milliliters (mL); V,——the volume of the added sample solution, in milliliters (mL); D—the total dilution factor of the sample solution;
-the mass of the sample, in grams (g)).
13 Precision
The absolute difference between two independent measurement results obtained under repeatability conditions shall not exceed 15% of the arithmetic mean. 92
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