GB/T 40223-2021.Determination of free gossypol of plant metabolites-Enzyme-linked immunosorbent assay.
1 Scope
GB/T 40223 specifies the enzyme-linked immunosorbent assay method for free gossypol of plant metabolites.
GB/T 40223 is applicable to the determination of free gossypol in cottonseed meal and fermented feed.
2 Normative references
The contents of the following documents constitute the essential clauses of this document through normative references in the text. For dated references, only the version corresponding to that date applies to this document; for undated references, the latest version (including all amendments) applies to this document.
GB/T 6682 Specifications and test methods for water for analytical laboratories
3 Terms and definitions
There are no terms and definitions that need to be defined in this document.
4 Principle
Free cottonpol in the sample solution after treatment. The cottonpol antigen fixed on the microplate competes with the added cottonpol antibody. The plate is washed to remove other unbound components and then combined with the horseradish peroxidase-labeled antibody. The enzyme-labeled substrate is added for color development. The absorbance value is measured at a wavelength of 450 nm using an enzyme reader to calculate the content of free cottonpol.
5 Reagents and materials
Unless otherwise specified, the chemical reagents used in this document are analytically pure, and the water is secondary water specified in GB/T 6682.
5.1 Detection kit for free cottonpol: See Appendix A.
5.2 Cottonpol: purity ≥95%, CAS number 652-78-8.
5.3 The required solution should be prepared according to the instructions of the kit.
6 Instruments and equipment
6.1 Small grinder.
6.2 Sieve: pore size 0.9 mm.
6.3 Homogenizer.
6.4 Vortex mixer.
6.5 Electronic balance: sensitivity 0.01 g.
6.6 Centrifuge tube with stopper: 15 mL.
The enzyme-linked immunosorbent assay method for free cottonpol, a plant metabolite, is specified.

ICS07.080
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National Standard of the People's Republic of China
GB/T40223—2021
Determination of free gossypol of plant metabolites-Enzyme-linked immunosorbent assay2021-05-21Release
State Administration for Market Regulation
National Standardization Administration
2021-12-01Implementation
People's Republic of China
National Standard
Determination of free gossypol in plant metabolites
Enzyme-linked immunosorbent assay
GB/T40223-2021
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GB/T40223—2021
This document is drafted in accordance with the provisions of GB/T1.1—2020 "Guidelines for standardization work Part 1: Structure and drafting rules of standardization documents".
Please note that some of the contents of this document may involve patents. The issuing organization of this document does not assume the responsibility for identifying patents. This document is proposed and managed by the National Technical Committee for Standardization of Biochemical Testing (SAC/TC387). The drafting units of this document: Jiangnan University, Beijing Technology and Business University, Hebei University of Science and Technology, Beijing Sambo Technology Co., Ltd. The main drafters of this document are Guo Lingling, Ma Aijin, Hao Shi, Xu Liguang, Sun Maozhong, and Song Shanshan. -riKaeerkAca
1 Scope
Determination of free gossypol in plant metabolites
Enzyme-linked immunosorbent assay
This document specifies the enzyme-linked immunosorbent assay method for free gossypol in plant metabolites. This document is applicable to the determination of high gossypol in cotton sticky and fermented feed. 2 Normative references
GB/T40223—2021
The contents of the following documents constitute the essential clauses of this document through normative references in the text. For dated references, only the version corresponding to that date applies to this document. For undated references, the latest version (including all amendments) applies to this document.
GB/T6682
3 Terms and definitions
Specifications and test methods for water for life analysis experiments
There are no terms and definitions that need to be defined in this document. 4 Principle
After the sample is treated, the free gossypol in the amniotic fluid and the cotton antigen fixed on the microplate compete with the added gossypol antibody for binding. The plate is washed to remove other unbound components and then the absorbance value is measured with the bulb peroxide meal to calculate the content of free gossypol. 5 Reagents and materials
Color using microplate reader at 50nm wavelength
Unless otherwise specified, the chemical reagents used in this document are analytically pure, and the water is secondary water specified in GB/T6682. Detection kit for free gossypol: See Appendix 5.1
Gossypol: purity ≥95%. CAS number is 652-78-8. The required solution should be prepared according to the instructions of the kit. Instruments and equipment
Small pulverizer.
6.2 Sampling sieve: aperture 0.9mm.
6.3 Homogenizer.
6.4 Vortex mixer.
6.5 Electronic dog balance: sensitivity 0.01g.
6.6 Centrifuge tube with stopper: 15mL
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GB/T40223—2021
6.7 Microplate reader: with 150Ⅱm filter. 6.8 Instruments required by the test kit.
Sample preparation and preservation
Sample preparation
Weigh 100g sample and crush it with a small crusher. The crushed sample passes through a 0.9mm sieve. Put it into a clean sealed container and mark it for testing.
7.2 Sample preservation
After sampling, the sample should be tested immediately. If it cannot be tested in time, it should be frozen in a refrigerator at 18℃ and stored for no more than 2 weeks. 8 Test steps
8.1 Sample pretreatment
Weigh 1.00g of the sieved sample, place it in a 15mL stoppered centrifuge tube, add the extract required by the kit, and dilute and test it according to the method described in the kit manual.
8.2 Determination
Perform quantitative detection of the sample (liquid) to be tested according to the operating steps described in the enzyme-linked immunosorbent assay kit. 8.3 Parallel test
According to the above steps, parallel tests should be carried out on each standard solution and each sample solution. 8.4 Blank test
Except for not weighing the sample, all the above steps are carried out. 8.5 Positive quality control
According to the content of free cottonpol in the sample to be tested, select an appropriate quality control standard to determine the accuracy of the test process. Test data processing
Drawing the standard working curve of quantitative detection of enzyme-linked immunosorbent assay kit 9.1
Draw the standard working curve according to the relationship between the mass concentration of the standard and the change in absorbance according to the calculation method or computer software provided in the kit manual.
9.2 Calculation of mass concentration of test solution
Draw the standard working curve according to the calculation method and computer software provided in the kit manual according to the absorbance of the test solution into the standard working curve obtained in 9.1 to calculate the mass concentration (p) of the test solution. 9.3 Calculation of results
The content of free cottonpol in the sample is calculated according to formula (1): 2
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Wherein:
X-pxVxax1000
mx1000
GB/T40223-2021
X—the content of free cottonpol in the sample, in milligrams per kilogram (mg/kg); β—the mass concentration of free cottonpol in the sample obtained from the standard working curve, in micrograms per milliliter (μug/mL); V—the volume of sample extract, in milliliters (mL); the dilution multiple during the pretreatment process;
m—the weight of the sample, in grams (g). The calculation result is expressed as the arithmetic mean of two independent determination results obtained under repeatability conditions, retaining 3 significant figures. 10
Precision
The absolute difference between two independent determination results obtained under repeatability conditions shall not exceed 20% of the arithmetic mean. 11Accuracy
When the spiked concentration of the free cottonpol solution tested by the ELISA kit is 5μg/mL, the average recovery rate is 77.3%~111.6%, when the spiked concentration of the free cottonpol solution tested by the ELISA kit is 10μg/mL, the average recovery rate is 80.1%~110.5%, when the spiked concentration of the free cottonpol solution tested by the ELISA kit is 50g/m, the average recovery rate is 86.4%~105.4%
12Others
When the sample weight is 1.00g, the detection limit of free cottonpol is 2mg/kg and the quantification limit is 3mg/kg. rrKaeerKAca-
GB/T40223—2021
Enzyme labeling plate coated with free gossypol. AI
Sample extract: methanol water (95+5).
Appendix A
(Informative)
Free gossypol detection kit
Sample diluent: 0.01mol/LpH7.4 phosphate buffer solution containing 0.01% Tween-20. Standard solution of free gossypol: 0μg/mL, 2μg/mL, 5μg/mL10μg/mL.50ug/mL and 100μg/mL. Horseradish peroxidase labeled antibody
Washing solution concentrate (10 times concentrated): can be used after dilution with water. Substrate solution.
Reaction termination solution.
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GB/T40223-2021
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