CN102087284A - Preparation method of reagent board for rapidly detecting aflatoxin B1 - Google Patents
Preparation method of reagent board for rapidly detecting aflatoxin B1 Download PDFInfo
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- CN102087284A CN102087284A CN201010619519XA CN201010619519A CN102087284A CN 102087284 A CN102087284 A CN 102087284A CN 201010619519X A CN201010619519X A CN 201010619519XA CN 201010619519 A CN201010619519 A CN 201010619519A CN 102087284 A CN102087284 A CN 102087284A
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Abstract
The invention discloses a preparation method of a reagent board for rapidly detecting aflatoxin B1, belonging to the field of mycotoxin detection and particularly relating to a preparation method of a reagent board for rapidly detecting aflatoxin B1 in grains and edible oil. The reagent board comprises an upper plastic template, a lower plastic template and a back lining; a sample pad, a colloidal gold combining pad, a nitrocellulose membrane and a water sucking pad are sequentially and tightly attached on the back lining; a detection line and a quality control line are sequentially sprayed on the nitrocellulose membrane in the direction of from the sample pad to the water sucking pad, and a reaction result can be represented by using a color visible to the unaided eye. The reagent board can be used for performing semi-quantitative and intuitive detection, the whole operation process only takes 40 minutes, no any expensive experimental device is needed for assistance, thereby the invention is good for sample screening on a large scale and suitable for industry and business departments, inspection and quarantine organizations and grain and oil production and processing enterprises to carry out rapid detection on the aflatoxin in the grains and the edible oil on a large scale.
Description
Technical field
The present invention relates to a kind of preparation method who detects the quick detection reagent plate of aflatoxin B1, particularly a kind of preparation method who detects the immune colloid gold quick detection reagent plate of aflatoxin B1 in cereal and the edible oil.
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 comprises kinds more than 10 such as B1, B2, G1 and G2, and wherein common with aflatoxin B1, toxicity is the strongest.B1 has a very wide distribution, and can cause the acute poisoning death of the mankind (especially children), various letting animals feeds, can also teratogenesis, carcinogenic, mutagenesis, even the aflatoxin B1 that every kilogram of material contains tens micrograms also has very big toxicity.Therefore very big to the health threat of people, poultry, domestic animal.
Aflatoxin harmfulness is big, exists scope wide, " waste oil " incident that caused a stir in recent years, and what cause numerous common people's fear is exactly that it contains aflatoxin, and its toxicity is 100 times of arsenic; In addition, " the malicious rice " of the ripe title of people, the carcinogen that wherein contains also is an aflatoxin, 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, aflatoxin content in human consumption's food and the milk cow forage (total amount that refers to B1+B2+G1+G2) can not surpass 20 μ g/kg, 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.The ECNo.1525/98 rules and regulations that European Union began to carry out on January 1st, 1999, the content that directly offers human edible food and form aflatoxin B1 in the component of food can not surpass 2 μ g/kg, and the total amount of aflatoxin B1, B2, G1, G2 must not surpass 4 μ g/kg.Japan's regulation, the content of aflatoxin B1 can not surpass 10 μ g/kg in the food.According to the regulation of China standard GB 2761-2005 " mycotoxin is limited the quantity of in the food ", aflatoxin B1 allowance standard is in China's food: must not surpass 20 μ g/kg in corn, shelled peanut, the peanut oil; Must not surpass 20 μ g/kg in corn and the shelled peanut goods (pressing the raw material conversion); 10 μ g/kg must not be surpassed in rice, other edible oils, 5 μ g/kg must not be surpassed in other grains, beans, the fermented food.
The method that detects aflatoxin B1 in cereal and the edible oil 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 B1, be use the earliest, the widest analytical technology, once be one of China's national standard method of measuring AFB1 in food and the feed.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, 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.
Summary of the invention
The present invention is directed to residual the conducting a research of aflatoxin B1 in cereal and the edible oil, a kind of detection technique that is used for on-the-spot rapid screening aflatoxin positive sample is provided, prepares the on-the-spot immune colloid gold quick detection reagent plate that detects aflatoxin B1 in cereal and the edible oil.The present invention has simply, fast, characteristics such as sensitivity height, need not cooperate mensuration by instrument and equipment, detect and both can in the laboratory, carry out, also can be on the farm, cereal compound department, eating and drinking establishment etc. measure on the spot, 5～10min finishes the qualitative and semiquantitative determination to aflatoxin B1 in the sample.
The present invention prepares a kind of immune colloid gold quick detection reagent plate, and agent plate is made up of up and down two plastic formworks and backing, is closely pasting sample pad, the golden pad of glue, 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 B1 monoclonal antibody and collaurum on the collaurum pad; Be coated with aflatoxin B1-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 nature controlling line.The carrier protein of coupling aflatoxin B1 can be bovine serum albumin(BSA) (BSA), ovalbumin (OVA), hemocyanin (KLH).
The present invention prepares a kind of immune colloid gold quick detection reagent plate, and agent plate is used chromatography type antibody mediated immunity competition principle, and by antigen and golden labeling antibody reaction solution, the aflatoxin B1 in specific detection cereal and the edible oil is residual.If it is residual that sample solution contains aflatoxin B1, antibody response on aflatoxin B1 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 aflatoxin B1 specific antigen on the detection line because of being occupied by the aflatoxin B1 in the sample solution on the colloid gold particle; When the aflatoxin B1 content 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 aflatoxin B1 content 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 present invention prepares a kind of immune colloid gold quick detection reagent plate, and the each several part constituent and the function of 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 B1 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.
Agent plate of the present invention has following beneficial effect:
(1) specificity is good.The aspergillus flavus resisting toxin B1 monoclonal antibody of agent plate of the present invention is 100% to the cross reacting rate of aflatoxin B1, 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 B1 allowance standard is in China's food: must not surpass 20 μ g/kg in corn, shelled peanut, the peanut oil; Must not surpass 20 μ g/kg in corn and the shelled peanut goods (pressing the raw material conversion); 10 μ g/kg must not be surpassed in rice, other edible oils, 5 μ g/kg must not be surpassed in other grains, beans, the fermented food.The sensitivity of agent plate of the present invention can reach 5 μ g/kg, can satisfy the detection demand in market fully.
(3) easy and simple to handle quick.Most of raw material that agent plate of the present invention 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 5～8 minutes behind the sample.The operation of agent plate of the present invention 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.Agent plate of the present invention 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.Agent plate production technology of the present invention is simple, can realize single pattern detection, and production cost is low, greatly reduces testing cost.
Description of drawings
Fig. 1 is an aflatoxin B1 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 an aflatoxin B1 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 B1 immune colloid gold quick detection reagent hardens, and wherein C is the control zone, and T is a detection zone.
Specific implementation method
The preparation of agent plate of the present invention comprises the assembling of preparation, aspergillus flavus resisting toxin B1 MONOCLONAL ANTIBODIES SPECIFIC FOR, colloidal gold solution, colloid gold label aspergillus flavus resisting toxin B1 MONOCLONAL ANTIBODIES SPECIFIC FOR and the aflatoxins toxin immunity collaurum quick detection reagent plate of aflatoxin B1 carrier protein couplet thing.
1. the coupling of aflatoxin B1 and carrier protein
Adopt carbodlimide method (EDC method) that aflatoxin B1 and carrier protein (bovine serum albumin BSA and ovalbumin OVA) coupling are prepared immunizing antigen and envelope antigen.
Take by weighing 2mg aflatoxin B1 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 B1 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 B1 oxime.
Take by weighing 0.2mg aflatoxin B1 oxime, be dissolved in 100 μ LDMF-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 B1-ovalbumin conjugate with quadrat method.
2. aspergillus flavus resisting toxin B1 MONOCLONAL ANTIBODIES SPECIFIC FOR
Get female Balb/c mouse in 6～8 ages in week, will be as immunogenic aflatoxin B1-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 B1-BSA to wrap 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 B1 that filters out-ovalbumin conjugate bag quilt is blocked indirect ELISA.With cells and supernatant and 2 * 10
-3Mol/L aflatoxin B1 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 B1 solution in addition and compare, all the other steps are the same.If the OD value after the aflatoxin B1 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 B1 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 B1 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 B1 immune colloid gold quick detection reagent plate
With a film machine aflatoxin B1 of debita spissitudo-carrier protein couplet thing and sheep anti-mouse igg are sprayed on the nitrocellulose filter, respectively as detection line and nature controlling line, 37 ℃ of oven drying 8h.In kind, the golden mark aflatoxin B1 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 B1 immune colloid gold quick detection reagent plate detects the implementation and operation method
6.1 specimen preparation
Rice: get the 20g rice in the 50mL centrifuge tube, add the 20mL damping fluid, concuss 1-2min leaves standstill 1min, draws at least 100 μ L lower floor solution, and is to be checked.
Edible oil: get the 1g edible oil in the 50mL centrifuge tube, add the 1mL normal hexane, concuss 1min leaves standstill a moment, adds the 0.5mL damping fluid in pipe, concuss 1-2min, and standing demix is drawn at least 100 μ L lower floor solution, and is 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 5～8min, 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, the aflatoxin B1 residual content is lower than detectability or does not contain aflatoxin B1 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 the aflatoxin B1 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 B1 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 (5)
1. preparation method who detects the quick detection reagent plate of aflatoxin B1 in cereal and the edible oil, it is characterized in that being coated with on the collaurum pad on the cellulose acetate film aspergillus flavus resisting toxin B1 monoclonal antibody-colloid gold label thing, is detection line and nature controlling line from sample pad successively to the adsorptive pads direction, is coated with aflatoxin B1-carrier protein couplet thing and sheep anti-mouse igg.
2. as the preparation method of the said agent plate of claim 1, it is characterized in that collaurum and aspergillus flavus resisting toxin B1 monoclonal antibody mixing by a certain percentage, make collaurum and aspergillus flavus resisting toxin B1 monoclonal antibody form stable colloid gold particle, form aspergillus flavus resisting toxin B1 monoclonal antibody-colloid gold label thing by concentrating.
3. as the preparation method of claims 1 said agent plate, it is characterized in that the carrier protein of coupling aflatoxin B1 on the detection line can be bovine serum albumin(BSA) (BSA), ovalbumin (OVA), hemocyanin (KLH).
4. as the preparation method of the said agent plate of claim 1, when it is characterized in that aflatoxin B1 content in the sample surpasses the agent plate detection limit, the detection line colour developing on the agent plate is shallow even do not have colour developing than control line, is judged to be the positive; Otherwise when aflatoxin B1 content 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.
5. as the preparation method of the said agent plate of claim 1, it is characterized in that technological process comprises preparation aflatoxin B1-BSA conjugate, preparation aspergillus flavus resisting toxin B1 monoclonal antibody, preparation colloidal gold solution, preparation colloid gold label aspergillus flavus resisting toxin B1 monoclonal antibody and assembling aflatoxin B1 immune colloid gold quick detection reagent plate.
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