CN202033368U - Reagent plate for detecting aflatoxins M1 in dairy - Google Patents
Reagent plate for detecting aflatoxins M1 in dairy Download PDFInfo
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- CN202033368U CN202033368U CN2010206957273U CN201020695727U CN202033368U CN 202033368 U CN202033368 U CN 202033368U CN 2010206957273 U CN2010206957273 U CN 2010206957273U CN 201020695727 U CN201020695727 U CN 201020695727U CN 202033368 U CN202033368 U CN 202033368U
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Abstract
The utility model relates to a reagent plate for detecting aflatoxins M1 in dairy, which consists of an upper plastic template, a lower plastic template and a back lining. A sample pad, a collaurum combined pad, a nitrocellulose film and a water absorption pad are sequentially tightly adhered onto the back lining, and a detecting line and a control line are sequentially sprayed onto the nitrocellulose film along the direction from the sample pad to the water absorption pad, so that reacted results can be shown by macroscopic colors. The reagent plate is capable of straightly detecting semi-quantitatively, and the integral operation process only takes 20 minutes and requires no expensive auxiliary experiment equipment. Besides, the reagent plate is favorable for selecting samples massively, is suitable for industry and commerce departments, inspection and quarantine organizations, milk stations and dairy producing and processing enterprises to fast and massively detect aflatoxins M1 in raw materials and products.
Description
Technical field
The utility model relates to a kind of agent plate that detects the aflatoxin M 1 in the dairy products, particularly a kind of immune colloid gold quick detection reagent plate that detects aflatoxin M 1 in the dairy products.
Background technology
Aflatoxin (Aflatoxin, AFT) belong to mycotoxin (Mycotoxin), it is produced by aspergillus flavus and aspergillus parasiticus, other aspergillus such as sheet are mould, mould, fusarium, head mold etc. also can produce AFT, AFT is common in peanut and goods thereof, corn, cottonseed, and in some nut fruits food and the feed.AFT was delimited a class carcinogenic substance by the cancer research mechanism of The World Health Organization (WHO) in 1993, was the extremely strong acute toxin material of a kind of toxicity.According to the survey, fungal effects are 40% food crop in the world, and produces toxin in metabolic process.FAO (Food and Agriculture Organization of the United Nation) estimates that 25% of whole world cereal supply is subjected to mycotoxin contamination.It is reported that the whole world has at least 2% crops scrap because of polluting aflatoxin every year.After the animal livestock ate aflatoxin-contaminated feed, finding in its lactation had aflatoxin M 1 residual.Aflatoxin harmfulness is big, exists scope wide, and the medical expert points out that the shortest carcinogenic time of aflatoxin only was 24 weeks.In order to prevent the generation of aflatoxicosis incident, safeguard human health, the strictness requirement of limiting the quantity of has been done to the content of aflatoxin in the food in existing in the world more than 70 countries and regions.
Federal Government relevant laws regulation, the content of M1 can not surpass 0.5 μ g/kg in human consumption's the milk, and the content in other animal feeds can not surpass 300 μ g/kg.According to the regulation of China standard GB 2761-2005 " mycotoxin is limited the quantity of in the food ", aflatoxin M 1 must not surpass 0.5 μ g/kg in sweet milk and the dairy products.
The method that detects aflatoxin in the dairy products at present mainly contains thin-layered chromatography (TLC), high performance liquid chromatography (HPLC) and enzyme linked immunosorbent assay (ELISA).
Thin-layered chromatography is to detect traditional common method of aflatoxin, be use the earliest, the widest analytical technology, once be one of China's national standard method of measuring aflatoxin.The advantage of this method is that used reagent, equipment are simple, and expense is cheap, grasps easily, is suitable for separation, the screening of a large amount of samples, and general laboratory all can be carried out.But sensitivity that thin-layered chromatography detects aflatoxin is low, it is poor to reappear, operation is loaded down with trivial details, the time is grown and shortcoming such as poor stability, the requirement of more and more inapplicable modern analysis.
High performance liquid chromatography is that present domestic mensuration aflatoxin uses at most, also is comparison authority's method, has national standard to put into effect at present, highly sensitive, precision is high, can analyze multiple aflatoxin type simultaneously.But the sample pre-treatments relative complex of this method, sense cycle is long, program is complicated, required reagent is various, need special technician when operating, and is difficult to satisfy the modern requirement quick, simple and direct, scene that detects.
Enzyme linked immunosorbent assay is the immune analysis method that the efficient catalytic of using antigen and antibody specific reaction and enzyme is used for measuring aflatoxin content, has specificity height, advantage such as highly sensitive, quick, easy.But the activity of enzyme is subject to the reaction conditions influence in the ELISA method, easily causes measurement result repeatability relatively poor thus.In addition the ELISA reagent life-span short, therefore need low-temperature preservation, and this method need touch the aflatoxin standard items when the preparation typical curve, operate dangerous.
The utility model content
The utility model provides a kind of immune colloid gold quick detection reagent plate that is used for on-the-spot rapid screening aflatoxin positive sample at 1 residual the conducting a research of the aflatoxin M in the dairy products.The utlity model has simple, fast, characteristics such as sensitivity height, need not instrument and equipment cooperate and measure, detect and both can in the laboratory, carry out, also can measure on the spot, 5～10min finishes the qualitative and semiquantitative determination to aflatoxin M in the sample 1.
The utility model agent plate is made up of up and down two plastic formworks and backing, is closely pasting sample pad, collaurum pad, nitrocellulose filter and adsorptive pads on the backing successively.Have between sample pad, collaurum pad, the adjacent each several part of nitrocellulose filter wherein that 1-2mm's is overlapping with adsorptive pads, its purpose is that the effect of assurance chromatography is carried out to the adsorptive pads position smoothly from sample pad on the one hand, be in order to make sample solution and sample be lined with sufficient reaction time on the other hand, buffer system on the sample pad can in and the potential of hydrogen of sample solution, guarantee that effective constituent in the sample solution and the golden labeling antibody on the collaurum pad react smoothly.
Be coated with the bond of aspergillus flavus resisting toxin M1 monoclonal antibody and collaurum on the collaurum pad; Be coated with aflatoxin M 1-carrier protein couplet thing and sheep anti-mouse igg successively from sample pad to the adsorptive pads direction on the nitrocellulose filter, respectively as detection line and control line.The carrier protein of coupling aflatoxin M 1 can be bovine serum albumin(BSA) (BSA), ovalbumin (OVA), hemocyanin (KLH).
The utility model agent plate is used chromatography type antibody mediated immunity competition principle, and by antigen and golden labeling antibody reaction solution, the aflatoxin M 1 in the specific detection dairy products is residual.If it is residual that sample solution contains aflatoxin M 1, antibody response on aflatoxin M 1 elder generation and the colloid gold particle, therefore when colloid gold particle diffused to detection line with sample solution, the avtive spot of antibody can't combine with 1 specific antigen of aflatoxin M on the detection line because of being occupied by the aflatoxin M in the sample solution 1 on the colloid gold particle; When 1 content of the aflatoxin M in the sample surpassed the agent plate detection limit, the detection line colour developing on the agent plate was shallow even do not have colour developing than control line, is judged to be the positive.Otherwise when 1 content of aflatoxin M in the sample below the agent plate detection limit or during noresidue, the detection line colour developing on the agent plate is close with control line or partially deeply, is judged to be feminine gender.
The each several part constituent and the function of the utility model agent plate are as follows:
Plastic formwork plays fixedly test strips and referential function district (well, detection zone, control zone).
Backing as the PVC plate, plays fixing other ingredients of test paper of supporting for simultaneously scribbling the toughness material that does not absorb water of adhesive sticker.
Sample pad is made by glass fibre, works to absorb sample solution and buffering sample solution pH value.
The collaurum pad is made by polyester film, is marked with the bond of aspergillus flavus resisting toxin M1 monoclonal antibody and collaurum on it, is the place that effective constituent in the sample solution and golden labeling antibody react.
It is that reaction result is come out with macroscopic characterization that the cellulose nitrate membrane portions mainly acts on.
Adsorptive pads is a filter paper, and its effect is that the excessive solution that will move up absorbs as the suction part.
The utility model agent plate has following beneficial effect:
(1) specificity is good.The aspergillus flavus resisting toxin M1 monoclonal antibody of the utility model agent plate is 100% to the cross reacting rate of aflatoxin M 1, comprises that with the microbiotic of other kinds medicines such as zearalenone, volt horse mycin, ochracin, deoxynivalenol do not have cross reaction.
(2) highly sensitive.According to the regulation of China standard GB 2761-2005 " mycotoxin is limited the quantity of in the food ", aflatoxin M 1 allowance standard is in China's food: aflatoxin M 1 must not limit the quantity of above 0.5 μ g/kg in sweet milk and the dairy products.The sensitivity of the utility model agent plate can reach 0.5 μ g/kg, can satisfy the detection demand in market fully.
(3) easy and simple to handle quick.Most of raw material that the utility model agent plate is reacted immunochromatography required has been incorporated in the reagent strip, and antigen-antibody reaction is carried out on immobilon-p fast behind the sample, has shortened the sample time greatly, promptly can read the result in 5～10 minutes behind the sample.The operation of the utility model agent plate does not need any professional training, and the ordinary person all can operate, and only needs to get final product by interpretation with the naked eye behind the explanation sample.
(4) do not rely on experimental facilities.The utility model agent plate is after directly dripping sample race plate, the shade of judging detection line and control line on the nitrocellulose filter by naked eyes is to sentence read result recently, whole testing process need not to use any experimental facilities, really accomplished experimental facilities zero requirement, be particularly suitable for field and execute-in-place, be easy to promote.
(5) cost is low, and is profitable.The utility model agent plate production technology is simple, can realize single pattern detection, and production cost is low, greatly reduces testing cost.
Description of drawings
Fig. 1 is aflatoxin M 1 an immune colloid gold quick detection reagent plate structure synoptic diagram, and wherein 1 is sample pad, and 2 is the collaurum pad, and 3 is nitrocellulose filter, and 4 is detection line, and 5 is control line, and 6 is adsorptive pads, and 7 is adhesive sticker, and 8 is the PVC plate.
Fig. 2 is aflatoxin M 1 an immune colloid gold quick detection reagent plate operation chart, and wherein S is a well, and C is the control zone, and T is a detection zone.
Fig. 3 really judges synoptic diagram for aflatoxin M 1 immune colloid gold quick detection reagent hardens, and wherein C is the control zone, and T is a detection zone.
Embodiment
The preparation of the utility model agent plate comprises the assembling of preparation, aspergillus flavus resisting toxin M1 MONOCLONAL ANTIBODIES SPECIFIC FOR, colloidal gold solution, colloid gold label aspergillus flavus resisting toxin M1 MONOCLONAL ANTIBODIES SPECIFIC FOR and the aflatoxins toxin immunity collaurum quick detection reagent plate of aflatoxin M 1 carrier protein couplet thing.
1. the coupling of aflatoxin M 1 and carrier protein
Adopt carbodlimide method (EDC method) that aflatoxin M 1 and carrier protein (bovine serum albumin BSA and ovalbumin OVA) coupling are prepared immunizing antigen and envelope antigen.
Take by weighing 2mg aflatoxin M 1 and 4mg CMO, be dissolved in the 400 μ L pyridines, 25 ℃ of lucifuge jolting reactions.Until reacting completely.With the reaction product freeze drying, residue obtained with the dissolving of 3mL ultrapure water, with 0.1mol/L sodium hydroxide solution adjust pH to 8.0.Divide 3 times and add 10mL benzene (4mL, 3mL, 3mL), unreacted aflatoxin M 1 in the oscillation extraction organic phase.Drip 0.1mol/L watery hydrochloric acid adjust pH to 3.0 at aqueous phase, must precipitate, with ethyl acetate extracting sediment, vacuum drying gets intermediate product aflatoxin M 1 oxime.
Take by weighing 0.2mg aflatoxin M 1 oxime, be dissolved in 100 μ L DMF-water (6: 9, V/V) in, add 2mg EDC, the lucifuge mixing.Add 0.5%C-BSA solution again, 25 ℃ of lucifuges, 100r/min react 4h, add EDC 2mg, continue reaction.With the coupled product bag filter of packing into, at the mid-4 ℃ of dialysis 3d of 0.01mol/L PBS (pH 7.4), during change dislysate 3 times (every 24h changes once).
Substitute BSA with OVA, adopt to prepare aflatoxin M 1-ovalbumin conjugate with quadrat method.
2. aspergillus flavus resisting toxin M1 MONOCLONAL ANTIBODIES SPECIFIC FOR
Get female Balb/c mouse in 6～8 ages in week, will be as immunogenic aflatoxin M 1-BSA conjugate and isopyknic Freund's complete adjuvant emulsification, press 100 a μ g/ dosage hypodermic injection, every 3 all booster immunizations 1 time, full adjuvant lumbar injection toos many or too much for use afterwards.The 3d reinforced immunological is 1 time before merging, and without adjuvant, dosage doubles.Fusion of Cells is carried out according to a conventional method: the Sp2/0 multiple myeloma cells is mixed in 1: 10 ratio with immune spleen cell, merge under 50% polyglycol effect, the HAT nutrient culture media suspends, and divide and plant in 96 well culture plates, 37 ℃, 5%CO
2Cultivate in the incubator.
After the fusion, treat that cell grows into 1/4 o'clock of culture hole area, adopt and divide step screening method screening hybridoma.Primary dcreening operation selects 10mg/L aflatoxin M 1-BSA bag by elisa plate, and measured hole adds culture supernatant, after hatching, cleaning, adds sheep anti-mouse igg-HRP (1: 1000), the OPD colour developing.The elisa plate of the positive Kong Zaiyong aflatoxin M 1-ovalbumin conjugate bag quilt that filters out is blocked indirect ELISA.With cells and supernatant and 2 * 10
-3Mol/L aflatoxin M 1 solution mixed in equal amounts, 1h is made in 37 ℃ of senses, adds to have wrapped in the ELISA Plate of quilt.Use PBS (0.01mol/L, pH7.4) to substitute aflatoxin M 1 solution in addition and compare, all the other steps are the same.If the OD value after aflatoxin M 1 blocking-up is reduced to below 50% of control wells, then be judged to positive hole.Through the hole that 2～3 detections all are positive, carry out cloning with limiting dilution assay immediately.
In vitro culture: with the cell line enlarged culture of cloning, cell concentration reaches 5 * 10
5Stop to change liquid during/mL, nutrient solution is collected in all dead back of cell.Induce ascites in the body: give the mouse peritoneal injection cloning cell line 10 of lumbar injection whiteruss after 10 days
7Individual cell extracted ascites after 7 days.
3. the preparation of colloidal gold solution
The mean size of colloid gold particle is 30nm, and its preparation method is to add 1mL 1% trisodium citrate in the 100mL deionized water, boils the back and adds 1mL 1% gold chloride rapidly, continues to boil 10min, after the cooling, preserves standby down for 4 ℃.
4. colloid gold label aspergillus flavus resisting toxin M1 MONOCLONAL ANTIBODIES SPECIFIC FOR
Get the colloidal gold solution 100mL that has prepared, transfer pH to 8.0 with the 0.1mol/L solution of potassium carbonate.Add 2mg aspergillus flavus resisting toxin M1 monoclonal antibody while stirring, stir 20min, dropwise add 2mL 25mol/L Macrogol 2000 0 (PEG20000), stir 15min.20, the centrifugal 15min of 000rpm abandons supernatant, and the PBS damping fluid (containing 0.4mol/L PEG) that adds 10mL pH 7.4 cleans 2 times.Precipitation is dissolved with the PBS damping fluid (pH 7.4) that 10mL contains 2%BSA, and after filtering with 0.22 μ m sterilizing filter, 4 ℃ of preservations are standby.
5. the assembling of aflatoxin M 1 immune colloid gold quick detection reagent plate
With a film machine aflatoxin M 1-carrier protein couplet thing and the sheep anti-mouse igg of debita spissitudo are sprayed on the nitrocellulose filter, respectively as detection line and control line, 37 ℃ of oven drying 8h.In kind, golden mark aflatoxin M 1 monoclonal antibody for preparing is coated on the collaurum pad.
Detectable consists of a PVC backing, is stained with sample pad, collaurum pad, nitrocellulose filter and adsorptive pads thereon in order.With cutting machine the kilocalorie that posts is cut into the wide bar of 4mm, make the detectable plate in the plastic formwork of packing into, put into the airtight storage of aluminium foil bag of band drying agent again.
6. aflatoxin M 1 immune colloid gold quick detection reagent plate detects the implementation and operation method
6.1 specimen preparation
Open warm bath power supply and heater switch, preheating, the loading slot actual temperature is maintained about 50 ℃, with fill former milk centrifuge tube and agent plate be put on the test tube rack preheating 5 minutes, the former milk that pipettor is got 100 μ L preheatings adds in the enzyme mark hole, with dropper Kong Zhongjin is marked abundant mixing, reacted 1 minute, leave standstill to be checked.
6.2 detection step
Draw sample solution 100 μ L to be checked and be added drop-wise in the well, pick up counting behind the application of sample; The result should read at 3～5min, and the other times interpretation is invalid.During observation, agent plate is placed horizontally at the observer front.
6.3 the result judges
When reading as a result, the agent plate level is placed the observer front.
Negative (-): T line colour developing (detection line is near well one end) than C line (control line) deeply or equally dark, aflatoxin M 1 residual content is lower than detectability or does not contain aflatoxin M 1 residual in the expression sample.
Positive (+): the colour developing of T line is more shallow than C line, or the T line do not have colour developing, and aflatoxin M 1 residual content is higher than detectability in the expression sample, and the colour developing of T line is more shallow more than C line, and aflatoxin M 1 content is high more in the expression sample.
Invalid: the C line not occurring, may be that misoperation or agent plate lost efficacy.Should read instructions once more, and test again with new agent plate.
Claims (3)
1. agent plate that detects aflatoxin M 1 in the dairy products, by up and down two plastic formworks and backing are formed, closely pasting sample pad on the backing successively, the collaurum pad, nitrocellulose filter and adsorptive pads, it is characterized in that sample pad, the collaurum pad, have between nitrocellulose filter and the adsorptive pads each several part that 1-2mm's is overlapping, be coated with the bond of aspergillus flavus resisting toxin M1 monoclonal antibody and collaurum on the collaurum pad, be coated with aflatoxin M 1 carrier protein couplet thing and sheep anti-mouse igg successively from sample pad to the adsorptive pads direction on the nitrocellulose filter, respectively as detection line and control line.
2. agent plate according to claim 1 is characterized in that the carrier protein of coupling aflatoxin M 1 can be bovine serum albumin(BSA), ovalbumin, hemocyanin.
3. agent plate according to claim 1 is characterized in that backing is the PVC plate that one side scribbles adhesive sticker, and sample pad is made by glass fibre, and the collaurum pad is made by polyester film, and adsorptive pads is a filter paper.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010206957273U CN202033368U (en) | 2010-12-29 | 2010-12-29 | Reagent plate for detecting aflatoxins M1 in dairy |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010206957273U CN202033368U (en) | 2010-12-29 | 2010-12-29 | Reagent plate for detecting aflatoxins M1 in dairy |
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| Publication Number | Publication Date |
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| CN202033368U true CN202033368U (en) | 2011-11-09 |
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| CN2010206957273U Expired - Lifetime CN202033368U (en) | 2010-12-29 | 2010-12-29 | Reagent plate for detecting aflatoxins M1 in dairy |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103091494A (en) * | 2013-01-14 | 2013-05-08 | 华南农业大学 | Chemiluminescence enzyme-linked immune detection kit of aflatoxin M1 and using method |
| CN103412124A (en) * | 2013-07-25 | 2013-11-27 | 北京陆桥技术有限责任公司 | Aflatoxin M1 gold label quick detectiontest card and preparation method and application thereof |
| CN103513035A (en) * | 2012-06-28 | 2014-01-15 | 北京勤邦生物技术有限公司 | Test strip and method for detecting aflatoxin M1 |
| CN108663505A (en) * | 2018-06-08 | 2018-10-16 | 中国科学院上海生命科学研究院湖州营养与健康产业创新中心 | The remaining kits of aflatoxins M1 and preparation method in a kind of detection dairy produce |
| CN109061077A (en) * | 2018-06-29 | 2018-12-21 | 中山出入境检验检疫局检验检疫技术中心 | It is a kind of for detecting the device of Aflatoxins M1 in milk |
-
2010
- 2010-12-29 CN CN2010206957273U patent/CN202033368U/en not_active Expired - Lifetime
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103513035A (en) * | 2012-06-28 | 2014-01-15 | 北京勤邦生物技术有限公司 | Test strip and method for detecting aflatoxin M1 |
| CN103513035B (en) * | 2012-06-28 | 2016-09-21 | 北京勤邦生物技术有限公司 | A kind of test strips detecting Aflatoxins M1 and method |
| CN103091494A (en) * | 2013-01-14 | 2013-05-08 | 华南农业大学 | Chemiluminescence enzyme-linked immune detection kit of aflatoxin M1 and using method |
| CN103091494B (en) * | 2013-01-14 | 2015-07-29 | 华南农业大学 | Aflatoxin M 1chemical luminescence ELISA detection kit and using method |
| CN103412124A (en) * | 2013-07-25 | 2013-11-27 | 北京陆桥技术有限责任公司 | Aflatoxin M1 gold label quick detectiontest card and preparation method and application thereof |
| CN108663505A (en) * | 2018-06-08 | 2018-10-16 | 中国科学院上海生命科学研究院湖州营养与健康产业创新中心 | The remaining kits of aflatoxins M1 and preparation method in a kind of detection dairy produce |
| CN109061077A (en) * | 2018-06-29 | 2018-12-21 | 中山出入境检验检疫局检验检疫技术中心 | It is a kind of for detecting the device of Aflatoxins M1 in milk |
| CN109061077B (en) * | 2018-06-29 | 2021-04-30 | 中山出入境检验检疫局检验检疫技术中心 | Device for detecting aflatoxin M1 in milk |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CX01 | Expiry of patent term |
Granted publication date: 20111109 |
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| CX01 | Expiry of patent term | ||
| DD01 | Delivery of document by public notice |
Addressee: Patent director of Hangzhou Nankai Rixin Biotechnology Co.,Ltd. Document name: Notice of expiration of patent right |
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| DD01 | Delivery of document by public notice |