GB/T 38792-2020 English PDF

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GB/T 38792-2020: Technical specification for cytological evaluation of protein allergenicity
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Basic data

Standard ID: GB/T 38792-2020 (GB/T38792-2020)
Description (Translated English): Technical specification for cytological evaluation of protein allergenicity
Sector / Industry: National Standard (Recommended)
Classification of Chinese Standard: A21
Classification of International Standard: 07.080
Word Count Estimation: 7,719
Date of Issue: 2020-04-28
Date of Implementation: 2020-11-01
Issuing agency(ies): State Administration for Market Regulation, China National Standardization Administration

GB/T 38792-2020: Technical specification for cytological evaluation of protein allergenicity

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Technical specification for cytological evaluation of protein allergenicity ICS 07.080 A21 National Standards of People's Republic of China Technical specification for cytological evaluation of protein sensitization 2020-04-28 released 2020-11-01 implementation State Administration for Market Regulation Issued by the National Standardization Management Committee

Foreword

This standard was drafted in accordance with the rules given in GB/T 1.1-2009. This standard was proposed and managed by the China National Institute of Standardization. Drafting organizations of this standard. China National Institute of Standardization, Jiangnan University, Beijing Sambo Technology Co., Ltd., Shijiazhuang Junlebao Dairy Co., Ltd. the company. The main drafters of this standard. Ma Aijin, Wu Xiaoling, Xu Chuanlai, Kuang Hua, Yuan Aimeng, Liu Liqiang, Ma Wei, Xu Liguang, Wang Zhongxing, Hao Shuai, Chai Yanbing and Zhang Yaoguang. Technical specification for cytological evaluation of protein sensitization

1 Scope

This standard specifies the cytological evaluation requirements and verification methods of protein sensitization. This standard applies to the cytological evaluation of food protein allergenicity mediated by immunoglobulin E (IgE).

2 Normative references

The following documents are indispensable for the application of this document. For dated reference documents, only the dated version applies to this article Pieces. For undated references, the latest version (including all amendments) applies to this document. GB/T 6682 Analytical laboratory water specifications and test methods

3 Terms and definitions

The following terms and definitions apply to this document. 3.1 Proteinalergenicity The characteristic of allergic reactions induced by protein. Note. The protein sensitization in this standard specifically refers to the interaction of IgE bound to the surface of basophils (RBL-2H3) with proteins that cause β-aminohexine The degree of glycosidase release.

4 requirements

4.1 In accordance with relevant laws, regulations and standards, samples are received, stored, handed over, prepared, recycled, returned/destroyed, and relevant Appropriate management systems and procedures. 4.2 The laboratory should meet the first-level biosafety requirements. The cell experiment work area generally consists of a preparation room, a buffer room, a sterile room, and a cell preservation room. composition. Among them, the area of the buffer room should be no less than 3m2, and there should be a locker and an ultraviolet lamp. The intensity of the ultraviolet lamp is no less than 1.5W/m3. The distance between the UV lamp source and the ground should not exceed 2.5m, and the irradiation time for each time is 20min~30min. The minimum standard of aseptic level of the clean room should reach To ten thousand. 4.3 The type, quantity, performance, range and accuracy of the instruments and equipment used in the evaluation should be able to meet the needs of the evaluation. Utensils needed for evaluation Should go through aseptic treatment, the test operation process is aseptic operation. 4.4 Experimental water should meet the requirements of GB/T 6682, and all reagents must be sterilized before use. 4.5 After the evaluation experiment is over, the experimental materials should be treated in a harmless manner. 4.6 The evaluation results of protein sensitization are expressed as the maximum release rate of β-hexosaminidase. 4.7 The evaluation result should state the name, purity, concentration of the evaluated protein, and the maximum release rate of β-hexosaminidase.

5 verification method

5.1 Determination of β-hexosaminidase release rate 5.1.1 Sample preparation 5.1.1.1 Solid samples 5.1.1.1.1 Plant samples After crushing the sample to be tested and filtering it with a 450μm microporous membrane, accurately weigh 5.00g into a polypropylene centrifuge tube and add it to the centrifuge tube 40mL water, shake well and mix well. Place the centrifuge tube horizontally in the shaker, 100 times/min at room temperature, shake for 12h, and separate at 5000r/min. Heart for 10min, carefully pipet the supernatant into another clean polypropylene centrifuge tube, freeze-dry in vacuum, the solid protein powder can be stored at 2℃~ Store at 8℃ for no more than 24h. 5.1.1.1.2 Animal samples After the sample to be tested is stirred and homogenized, accurately weigh 5.00g, place it in the funnel, add 50mL petroleum ether for 3 consecutive rinses, and put it Dry in the oven. Place the dried sample in a polypropylene centrifuge tube, and add 10 mL of 15% trichloroacetic acid solution to the centrifuge tube. Integral), shake and mix thoroughly, and let stand for 10 min. Centrifuge the sample solution at 5000r/min for 10min, carefully aspirate the supernatant on another dry In a clean polypropylene centrifuge tube, vacuum freeze-drying, the solid protein powder can be stored at 2℃~8℃ for no more than 24h. 5.1.1.2 Liquid or semi-liquid samples Weigh 5.00g liquid sample, place it in a funnel, add 50mL petroleum ether for 3 consecutive rinses, and put it into an oven to dry. will Place the dried sample in a polypropylene centrifuge tube, add 40 mL of water, shake and mix well, and place the centrifuge tube horizontally in the shaker. 100 times/min at temperature, shaking overnight for 12h, centrifuge at 5000r/min for 10min, carefully pipette the supernatant on another clean polypropylene In the heart tube, vacuum freeze-drying, the solid protein powder can be stored at 2℃~8℃ for no more than 24h. 5.1.2 Sample solution preparation Weigh 50.0 mg of the above protein sample, dissolve it with PBS buffer (see Appendix A) and dilute to a 50 mL volumetric flask to prepare A stock solution with a mass concentration of 1 mg/mL. Use Tyrode's solution (see Appendix A) to dilute to a certain concentration of working solution during the test. Use now. 5.1.3 Cell culture Place RBL-2H3 cells (see Appendix A) in a cell culture flask, add 10 mL of cell culture medium (see Appendix A), Incubate in a 37℃, 5% carbon dioxide cell incubator for 48h~72h, and observe the cell growth under an inverted microscope. When the cells grow to When 80%~90% of the flask is incubated, cell count and cell inoculation are performed. 5.1.4 Cell seeding Aspirate the cell culture fluid from the cell culture flask, and wash the cells once with PBS buffer. Add 2.0 mL of pancreas to the cell culture flask Protease solution (see Appendix A), digest at 37°C for 2min~4min. Observe cell digestion under an inverted microscope. When the circle is close to peeling off, pour out the trypsin solution. Add 10 mL of cell culture medium and pipette with a pipette to detach the cells to make a cell suspension liquid. Aspirate the cell suspension and add.200μL/well to the cell culture plate to make the cell seeding density 1×105 cells/mL. 37℃,5% Cultivate in a carbon dioxide cell incubator for 24 hours to adhere to the wall. 5.1.5 Cell activation Aspirate the cell culture medium from the cell culture plate, and wash the cells three times with PBS buffer. Add.200μL/well to the cell culture plate Into the cell culture medium containing 50μg/mLIgE (see Appendix A), 37℃, 5% carbon dioxide cell incubator and continue to culture for 24h. 5.1.6 Stimulation of cells and proteins 5.1.6.1 Sample group Aspirate the cell culture medium from the cell culture plate, and wash the cells three times with PBS buffer. Add 50μL/well to the cell culture plate Add different concentrations of the protein to be tested working solution, 37 ℃, 5% carbon dioxide cell incubator to react for 45 minutes. After the reaction is over, put in ice The reaction is terminated in water. All the above items are provided with 3 multiple holes. 5.1.6.2 Release all groups Aspirate the cell culture medium from the cell culture plate, and wash the cells three times with PBS buffer. Add 50μL/well to the cell culture plate Cell lysate (see appendix A), gently pipette to make the lysate and RBL-2H3 cells fully contact, after 45 minutes of reaction at 4 ℃, aspirate the whole Part of the cell culture supernatant was centrifuged at 1200r/min for 5min, and the cell supernatant was collected. Set up 3 multiple holes above. 5.1.6.3 Blank control group Aspirate the cell culture medium from the cell culture plate, and wash the cells three times with PBS buffer. Add 50μL/well to the cell culture plate Tyrode's solution, 37℃, 5% carbon dioxide cell incubator react for 45min. After the reaction is over, put into ice water to stop the reaction. Above Set 3 complex holes. 5.1.7 Determination Aspirate the reaction solution after action in 5.1.6, transfer it to a new cell culture plate at 30 μL/well, and transfer it to the cell culture plate at 50 μL/well Add PNP-NAG solution (see Appendix A), and after reacting at 37°C for 1 hour, add.200 μL/well of sodium carbonate stop solution (see Appendix A) to the end Stop the reaction and measure the absorbance value of each well with a microplate reader at a wavelength of 405nm. The absorbance of the sample group is labeled A1, and the absorbance of the blank control group The degree is marked as A0, and all release groups are marked as Am. 5.2 Calculation of β-hexosaminidase release rate results The β-hexosaminidase release rate is calculated according to formula (1). R= (A1-A0) (Am-A0)× 100% (1) Where. R ---β-hexosaminidase release rate; A1 --- the absorbance of the sample group; A0 --- the absorbance of the blank control group; Am---the absorbance of all release groups. The average value of the parallel sample is the final release rate value, and the calculation result is kept to two decimal places.

Appendix A

(Informative appendix) Solution preparation A.1 Water Grade 2 water specified in GB/T 6682. A.2 0.01mol/L phosphate buffer (PBS buffer) Weigh 7.90g sodium chloride, 1.44g disodium hydrogen phosphate, 1.80g potassium dihydrogen phosphate, 0.20g potassium chloride, dissolve it with 980mL water, and use salt Adjust the pH to 7.2 with acid, then add water to make the volume to 1000mL, filter and sterilize with 0.22μm microporous membrane, store at 4℃ for later use or use similar products Chemical products. A.3 0.001mol/L p-nitrobenzene-N-acetyl-β-D-glucosamine solution (PNP-NAG solution) Weigh 34.2mg PNP-NAG, add water to dissolve and dilute to 100mL, filter and sterilize with 0.22μm microporous membrane, and store at 4℃ for later use. A.4 0.1mol/L sodium carbonate stop solution Weigh 1.06g of sodium carbonate, add water to dissolve and dilute to 100mL, filter and sterilize with 0.22μm microporous membrane, and store at 4℃ for later use. A.5 Tyrode's solution (Tyrode's solution) Weigh 7.90g sodium chloride, 0.20g potassium chloride, 0.26g magnesium sulfate heptahydrate, 0.07g sodium dihydrogen phosphate dihydrate, 1.00g sodium bicarbonate, 0.20g calcium chloride and 1.00g glucose, dissolve in 980mL water, adjust the pH to 7.2 with hydrochloric acid, and then add water to make the volume to 1000mL, 0.22μm microporous filter membrane is filtered and sterilized, stored at 4℃ for later use or use similar commercial products. A.6 Cell culture medium Eagle's MinimalEssential Medium (MEM), add calf serum and penicillin-streptomyces Was prepared into a cell culture medium containing 10% calf serum and 1% penicillin-streptomycin, in which the final concentration of penicillin and streptomycin The degrees are 100U/mL and 100μg/mL, respectively. EMEM medium can choose powder medium supplied by various commodities, according to the manufacturer's proposal For data preparation and sterilization, store at 4℃ for later use. A.7 Cell Lysate Commercialized 1% TritionX-100 cell lysate, the main components are 50mmol Tris-HCl (pH7.2), 150mmol sodium chloride And 5mmol ethylenediaminetetraacetic acid, the volume fraction is 1% Triton X-100.0.22μm microporous membrane filter sterilization, 4 ℃ storage for later use. A.8 Trypsin solution Weigh 2.50g trypsin (activity 1.250), add 1000mL PBS buffer (A.2), mix well, filter through 0.22μm microporous membrane Filter out bacteria, store at 4℃ for later use or use similar commercial products. A.9 Basophils (RBL-2H3 cells) Rat basophilic leukemia cells, ATCCCRL-2256 cell line. A.10 IgE globulin Mouse source, purity >95%, mass concentration >50μg/mL, various similar commercial products can be selected. ......

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