CN108226512A - The enzyme linked immunological kit of aflatoxin B1 in a kind of detection bottled rice wine - Google Patents
The enzyme linked immunological kit of aflatoxin B1 in a kind of detection bottled rice wine Download PDFInfo
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- CN108226512A CN108226512A CN201611159443.0A CN201611159443A CN108226512A CN 108226512 A CN108226512 A CN 108226512A CN 201611159443 A CN201611159443 A CN 201611159443A CN 108226512 A CN108226512 A CN 108226512A
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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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
- G01N33/531—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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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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Abstract
The invention discloses a kind of enzyme linked immunological kits for detecting aflatoxin B1 in bottled rice wine.The enzyme linked immunological kit of aflatoxin B1 in detection bottled rice wine provided by the present invention, including coated aflatoxin b1 antibody and enzyme-labelled antigen.The enzyme linked immunological kit of aflatoxin B1 is multi-residue determination kit in the detection bottled rice wine of the present invention, low to the pre-treatment requirement of sample, and sample pretreatment process is simple, quickly can detect gross sample simultaneously；Minimum detection limit is up to 0.05 μ gkg‑1, can directly judgement sample it is whether exceeded, there is high specific, high sensitivity, pinpoint accuracy, high accuracy, can play a significant role in aflatoxin B1 residue detection in food bottled rice wine.
Description
Technical field
Aflatoxin is mainly generated by aspergillus flavus, aspergillus parasiticus, aspergillus fumigatus and a small number of Aspergillus wentti bacterial strains, is one group of change
Learn two similar furocoumarin derivatives of structure.At present, it is B and G to detach the natural aflatoxin main Types identified
Toxin, including aflatoxin B1 (AFB1), aflatoxin B 2 (AFB2), aflatoxin G 1 (AFG1), Huang Qu
Mould toxin G2 (AFG2), wherein, it is most strong with aflatoxin B1 toxicity.1993, international cancer research institution's definition was yellow bent
Mould toxin B1 is a kind of carcinogenic substance, and toxicity is equivalent to 10 times of potassium cyanide, 68 times of arsenic, especially to humans and animals liver
With extremely strong carcinogenicity.Toxicologic study, which shows that the harm of aflatoxin further includes, causes immunity of organisms low, teratogenesis and
Mutagenicity.According to the food national standards of GB2761-2011《Mycotoxin is limited the quantity in food》Regulation, paddy, brown rice, rice
And its aflatoxin B1 limitation is 10 μ gkg in product-1, wheat, barley, other Grain and its products limitation be 5 μ
g·kg-1, dispensed food for baby and accesary foods limitation are 0.5 μ gkg-1.European Union member countries' regulation is stringenter, it is desirable that
The content of aflatoxin B1 in cereal preparation and baby food is processed no more than 0. 1 μ gkg-1, but at present, GB
2761-2011 provide the limitation of aflatoxin B1 in yellow rice wine not yet.Yellow rice wine is a kind of distinctive brewed wine in China,
It is also one of three great Gu wine of the world (beer, yellow rice wine and grape wine).
Yellow rice wine is mainly made with rice, milled glutinous broomcorn millet, black rice, wheat, corn, glutinous rice etc. for raw material, because it is with antioxygen
Change, anti-aging, blood pressure lowering, norcholesterol, metabolism and immunological regulation and other effects, it is deep to be liked by compatriots.Yellow rice wine raw material (such as grain in storage for years)
, brewing process, storage link in be likely to occur aflatoxin contamination, therefore detect yellow rice wine in aflatoxin content have
Its necessity.
At present, in food using more extensive Determination Methods of Aflatoxins have high performance liquid chromatography (HPLC),
Thin-layered chromatography (TLC), enzymoimmunoassay (ELISA), liquid chromatography mass Tandem analysis technology (LC-MS/
MS) etc..Although HPLC and LC-MS/MS methods are quantitative accurate, sample pre-treatments generally need specific decontaminating column,
Decontaminating column price is relatively high, while needs to configure expensive detecting instrument, and operating personnel need to be specifically targeted with instrument training,
And enzyme-linked immunization only needs full scan microplate reader, easy to operate, quick, high sensitivity, the easy left-hand seat of testing staff, in many
Enterprise and small-sized testing agency are widely applied.This research and utilization enzyme linked immunosorbent detection technology (ELISA), using yellow rice wine as
Sample has detected the content of AFB1 in the bottled rice wine randomly selected in local market, and detection method is effectively commented
Estimate.
Innovation and creation content
The object of the present invention is to provide a kind of detection, the object of the present invention is to provide aspergillus flavus in a kind of detection bottled rice wine
The enzyme linked immunological kit of toxin B1.
The enzyme linked immunological kit of aflatoxin B1 in detection bottled rice wine provided by the present invention, it is anti-including being coated with
Body, enzyme marker.
In order to be more convenient on-site supervision and great amount of samples screening, the kit further includes A, B, C, D, E, F reagents
Liquid, G reagent solutions, H reagent solutions, I reagent solutions, J reagent solutions.
The A, B, C, D, E, F reagent solutions are aflatoxin B1 series concentration standard solution in bottled rice wine.
The G reagent solutions are enzyme-labelled antigen.
The H reagent solutions are cleaning solution.
The I reagent solutions are substrate solution.
The J reagent solutions are terminate liquid.
The enzyme linked immunological kit of aflatoxin B1 in the detection bottled rice wine of the present invention, including in coating bottled rice wine
Aflatoxin b1 antibody and enzyme-labelled antigen.
The testing principle of the present invention is according to sample to be tested amount, intercepts corresponding ELISA Plate hole count, sequentially adds what is be configured
50 μ L of standard solution or yellow rice wine sample extracting solution, add 50 μ L of enzyme-labelled antigen, and reaction plate is put into 37 DEG C of constant incubators
30 min of middle incubation take out reaction plate, firmly get rid of reaction solution, after patting dry, the washing of 250 μ L cleaning solutions are added in per hole, is repeated
Board-washing 4 times adds in assay chromogenic substrate solution A and B per hole, shakes up, and reaction plate is put into 37 DEG C of constant incubators and develops the color 15
After colour developing, 50 μ L terminate liquids are added in per hole, measure the suction in each hole after shaking up at 450 nm wavelength with microplate reader by min
Shading value.
It is of the invention that aflatoxin B1 in bottled rice wine is qualitatively or quantitatively mainly detected using indirect competitive ELISA method
Residual quantity；Low to the pre-treatment requirement of sample, sample pretreatment process is simple, quickly can detect gross sample simultaneously；It is minimum
Detection is limited up to 0. 05 μ gkg-1, can directly judgement sample it is whether exceeded, have high specific, high sensitivity, pinpoint accuracy,
The features such as high accuracy, can play a significant role in food bottled rice wine in aflatoxin B1 residue detection.
Description of the drawings
Fig. 1 enzyme-linked immunization measures the linear relationship of AFB1.
Specific embodiment
The enzyme linked immunological kit of aflatoxin B1 in embodiment 1, detection bottled rice wine
（1）Detect the structure of the enzyme linked immunological kit of aflatoxin B1 in bottled rice wine
Material and reagent：
Different brands and 26, the yellow rice wine sample in the place of production, aflatoxin AFB1 enzyme linked immunological quantitative tests box (are revived in Jiangsu Province
Pico- biological study Co., Ltd).It is limited that aflatoxin B1 standard items (purity ＞ 98%) are purchased from Beijing Thailand auspicious science and technology of pleasure
Company (Singapore Pribolab).Hplc grade methanol is purchased from Te-dia (U.S., Ohio, Fairfield).
Instrument and equipment：
1500 type all-wave length microplate reader (power ＆ light company of the U.S.), electronic balance BSA 822-cw (Sai Duolisi science instrument
Device), GNP-9160 type water isolation types constant incubator (the upper macro experimental facilities Co., Ltd of Nereid), DS-7510DTH types surpass
Sound wave washer (Shanghai Sheng Xi ultrasonic instruments Co., Ltd).
（2）The preparation of agents useful for same
The A, B, C, D, E, F reagent solutions are that aflatoxin B1 series concentration standard solution is 0. 0 in bottled rice wine,
0. 10,0. 25,0. 50,1. 0,2. 0 μ gL-1。
The G reagent solutions are enzyme-labelled antigen.
The H reagent solutions are cleaning solution.
The I reagent solutions are substrate solution.
The J reagent solutions are terminate liquid.
The pre-treatment and detection of embodiment 2, sample
（1）The pre-treatment of sample
Using the sample treatment in the AFB1 enzyme linked immunological quantitative test boxes of Su Wei microbe researches Co., Ltd production as base
Plinth, and this method is improved.5. 0 g yellow rice wine accurately are weighed in 50mL graduated centrifuge tubes, addition methanol: water (
Volume ratio 1: 1) be settled to 25 mL, and be vortexed 3 min, and then 50 DEG C of 10 min of ultrasound, finally take 1. 0 mL
0. 22 μm of film is crossed, sample liquid is to be measured.
（2）Detection
According to sample to be tested amount, corresponding ELISA Plate hole count is intercepted, sequentially adds the standard solution being configured or yellow rice wine sample extraction
50 μ L of liquid add 50 μ L of enzyme-labelled antigen, reaction plate are put into 37 DEG C of constant incubators and is incubated 30 min, take out anti-
Plate is answered, firmly gets rid of reaction solution, after patting dry, the washing of 250 μ L cleaning solutions is added in per hole, repeats board-washing 4 times, is added in per hole
Assay chromogenic substrate solution A and B, shake up, and reaction plate are put into 37 DEG C of constant incubators 15 min that develop the color, colour developing terminates
Afterwards, 50 μ L terminate liquids are added in per hole, measure the absorbance value in each hole after shaking up at 450 nm wavelength with microplate reader.
（3）Standard curve
By series A FB1 standard solution (0. 0,0. 10,0. 25,0. 50,1. 0,2. 0 μ gL-1) suction that measures
Shading value draws standard curve (Fig. 1).Abscissa is the common logarithm value (lgc) of standard curve concentration, and ordinate is
Each titer absorbance value and 0 ngmL-1The ratio of the absorbance value in standard solution hole is worth linear equation y=- 0.4679x
+ 0.3132, R2=0. 9982, aflatoxin B1 is in 0.1~2.0 μ gL-1In the range of it is linear related.
Embodiment 3, kit sensitivity, specificity, preci-sion and accuracy
1. kit sensitivity test
According to test procedure, by adding in aflatoxin B1 to sample successively, it is close to reduce light of the AFB1 in 450 nm
It spends to determine minimal detectable concentration, that is, the sensitivity detected.The results are shown in Table 1.As it can be seen from table 1 AFB1 a concentration of 0.
05 μg·kg-1When, the light absorption value under 450 nm of microplate reader is 1.264, and the difference of the light absorption value 1.424 when AFB1 is 0 is big
In 0.1, this concentration can be identified as the sensitivity of this method, therefore this law is 0.05 μ gkg to the concentration limit of AFB1-1, i.e. this method is 0. 05 μ gkg to the detection sensitivity of AFB1 in yellow rice wine-1。
2. accuracy determination
1# yellow rice wine sample is selected to add 0,2. 0,3. 5,5. 0 μ gkg respectively as addition recovery test sample-1Four
Concentration measures AFB1 contents in addition sample according to test procedure, and each concentration makees 3 reproducible results such as table 2.Work as sample-adding
Concentration is in 2.0~3.5 μ gkg-1When, the recovery of standard addition of sample is 105% or so;When loading concentrations further increase to
5.0 μg·kg-1When, the recovery of standard addition of sample is down to 90%.
3. repeatability measures
According to the content 6 times of AFB1 in testing inspection step replication yellow rice wine, its standard deviation and relative standard are calculated
Deviation, testing result are as shown in table 3.As can be seen that the average value that multiple sample measures is 1.319, and standard deviation 0.050, phase
It is 3. 5% to standard deviation, reproducibility disclosure satisfy that the requirement of analysis method.
4. the content of aflatoxin B1 in different bottled rice wines
More dry type yellow rice wine, half-dry type yellow rice wine, characteristic half-dry type yellow rice wine, semi-sweet yellow rice wine and spy are consumed in selection currently on the market
5 class yellow rice wine sample of type semi-sweet yellow rice wine, sample number into spectrum 1 to 26.The aflatoxin B1 content of measure can be seen that from result, 26
There is detection aflatoxin B1 in the bottled rice wine sample of a stochastic buying, wherein 10 sample aflatoxin B1s contain
Amount is less than 0.5 μ gkg-1, aflatoxin B1 content is in 0.5~1.0 μ gkg in 14 samples-1Between, 2 samples
Aflatoxin B1 content is more than 1.0 μ gkg-1, but less than 2.0 μ gkg-1.All in all, the bottled Huang of stochastic buying
Aflatoxin content is low in wine sample.Due to the national regulations formal currently not yet of limiting the quantity of AFB1 in yellow rice wine, with reference to food
(AFB1 limitations are 10 μ gkg to mycotoxin limit standard in 2761-2011 pairs of rice of GB in product-1, AFB1 in wheat
Limitation requirement is 5 μ gkg-1), the content of aflatoxin B1 is not exceeded in 26 yellow rice wine samples of extraction.
1 yellow rice wine sample of table adds the absorbance value that the aflatoxin B1 of various concentration is surveyed successively
2 yellow rice wine sample difference of table adds concentration recovery test result
| Add concentration/(μ gkg-1) | Background values/(μ gkg-1) | Average measurement/(μ gkg-1) | Average recovery rate/% |
| 2.0 | 0.29 | 2.4 | 105.5±0.55 |
| 3.5 | 0.29 | 4.0 | 106.0±0.57 |
| 5.0 | 0.29 | 4.8 | 90.2±0.54 |
3 yellow rice wine sample repeatability result of the test of table
| Sample | D450 |
| Parallel 1 | 1.255 |
| Parallel 2 | 1.384 |
| Parallel 3 | 1.353 |
| Parallel 4 | 1.316 |
| Parallel 5 | 1.284 |
| Parallel 6 | 1.324 |
| Average value | 1.319 |
| Standard deviation | 0.050 |
| Relative standard deviation/% | 3.5 |
Claims (3)
1. a kind of enzyme linked immunological kit for detecting aflatoxin B1 in bottled rice wine, it is characterised in that：By porous coating plate,
Buffer solution, aflatoxin B1 standard items in bottled rice wine, enzyme-labelled antigen, cleaning solution are formed.
2. a kind of detection side for detecting the enzyme linked immunological kit of aflatoxin B1 in bottled rice wine according to claim 1
Method, including Sample pretreatment, it is characterised in that：
（1）With the sample treatment in the AFB1 enzyme linked immunological quantitative test boxes of Su Wei microbe researches Co., Ltd production
Based on, and this method is improved；5.0 g yellow rice wine accurately are weighed in 50mL graduated centrifuge tubes, add in methanol: water
(volume ratio 1: 1) is settled to 25 mL, and be vortexed 3 min, and then 50 DEG C of 10 min of ultrasound, finally take 1.0 mL mistakes
0.22 μm of film, sample liquid are to be measured；（2）The determinand of step (1) is subjected to enzyme linked immunological, reference standard curve is calculated in sample
AFB1 content.
3. the detection method of the enzyme linked immunological kit of aflatoxin B1 in bottled rice wine is detected according to claim l,
It is characterized in that：The solid phase carrier is porous coating plate, is carried using more micropores coating plate in 48 or 96 holes as solid phase
Body；
The detection method that the enzyme linked immunological kit of aflatoxin B1 in bottled rice wine is detected described in seeking l is played according to right,
It is characterized in that：According to sample to be tested amount, corresponding ELISA Plate hole count is intercepted, sequentially adds the standard solution being configured or yellow rice wine sample
50 μ L of extracting solution add 50 μ L of enzyme-labelled antigen, reaction plate are put into 37 DEG C of constant incubators and is incubated 30 min, take out anti-
Plate is answered, firmly gets rid of reaction solution, after patting dry, the washing of 250 μ L cleaning solutions is added in per hole, repeats board-washing 4 times, is added in per hole
Assay chromogenic substrate solution A and B, shake up, and reaction plate are put into 37 DEG C of constant incubators the 15min that develops the color, after colour developing, often
Hole adds in 50 μ L terminate liquids, measures the absorbance value in each hole after shaking up at 450 nm wavelength with microplate reader.
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| CN201611159443.0A CN108226512A (en) | 2016-12-15 | 2016-12-15 | The enzyme linked immunological kit of aflatoxin B1 in a kind of detection bottled rice wine |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001056456A2 (en) * | 2000-02-02 | 2001-08-09 | Cygene, Inc. | Methods and compositions for detection of disease |
| CN102955031A (en) * | 2011-08-31 | 2013-03-06 | 北京勤邦生物技术有限公司 | Enzyme-linked immunosorbent assay kit for detecting aflatoxin B1-containing medicine and application for same |
| CN103134924A (en) * | 2011-11-26 | 2013-06-05 | 无锡艾科瑞思产品设计与研究有限公司 | Aflatoxin B1 detection kit |
| CN104569380A (en) * | 2015-01-23 | 2015-04-29 | 天津伯克生物科技有限公司 | Method for detecting aflatoxin B1 and enzyme-linked immunosorbent assay kit |
| CN105277708A (en) * | 2015-03-20 | 2016-01-27 | 贵州勤邦食品安全科学技术有限公司 | Enzyme linked immunosorbent assay kit for detecting aflatoxin B1 in chili |
-
2016
- 2016-12-15 CN CN201611159443.0A patent/CN108226512A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001056456A2 (en) * | 2000-02-02 | 2001-08-09 | Cygene, Inc. | Methods and compositions for detection of disease |
| CN102955031A (en) * | 2011-08-31 | 2013-03-06 | 北京勤邦生物技术有限公司 | Enzyme-linked immunosorbent assay kit for detecting aflatoxin B1-containing medicine and application for same |
| CN103134924A (en) * | 2011-11-26 | 2013-06-05 | 无锡艾科瑞思产品设计与研究有限公司 | Aflatoxin B1 detection kit |
| CN104569380A (en) * | 2015-01-23 | 2015-04-29 | 天津伯克生物科技有限公司 | Method for detecting aflatoxin B1 and enzyme-linked immunosorbent assay kit |
| CN105277708A (en) * | 2015-03-20 | 2016-01-27 | 贵州勤邦食品安全科学技术有限公司 | Enzyme linked immunosorbent assay kit for detecting aflatoxin B1 in chili |
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