CN105372416A - Preparation method of reagent plate for detecting sterigmatocystin - Google Patents
Preparation method of reagent plate for detecting sterigmatocystin Download PDFInfo
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- CN105372416A CN105372416A CN201510776029.3A CN201510776029A CN105372416A CN 105372416 A CN105372416 A CN 105372416A CN 201510776029 A CN201510776029 A CN 201510776029A CN 105372416 A CN105372416 A CN 105372416A
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- sterigmatocystin
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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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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Abstract
The invention discloses a preparation method of a reagent plate for detecting sterigmatocystin. A colloidal gold binding pad of a nitrocellulose film is coated with an anti-sterigmatocystin monoclonal antibody-colloidal gold mark, a detection line and a quality control line are orderly arranged from a sample pad to a water absorption pad and are respectively coated with a sterigmatocystin-carrier protein conjugate and goat anti-mouse IgG, and the carrier protein is a rabbit serum protein. The nitrocellulose film is spray-coated with the sterigmatocystin-carrier protein conjugate and goat anti-mouse IgG through a film formation machine so that the detection line and the quality control line are obtained, the nitrocellulose film is dried by an oven at a temperature of 38 DEG C for 8h, the colloidal gold binding pad is spray-coated with the anti-sterigmatocystin monoclonal antibody-colloidal gold mark through the film formation machine, and the colloidal gold binding pad is dried by the oven at a temperature of 38 DEG C for 8h. The reagent plate has the advantages of fast detection rate, sensitivity, accuracy, quantification and simple operation. The preparation method has simple processes, realizes detection of a single sample, has a low production cost and greatly reduces a detection cost.
Description
Technical field
The present invention relates to biological technical field, specifically a kind of preparation method detecting the agent plate of sterigmatocystin.
Background technology
The final metabolic product of sterigmatocystin (Sterigmatocystin) mainly aspergillus versicolor (Aspergillusversicolor) and aspergillus nidulans (Aspergillusnidulans) is again the intermediate product that aspergillus flavus (Aspergillusflavus) and aspergillus parasiticus (AspergillusparasiticusSpeare) synthesize the yellow blood mould toxin process later stage simultaneously.It is reported, infected the corn of aspergillus versicolor under the environment of 27 DEG C, within 21 days, more than sterigmatocystin 12g/kg can be produced.
The rat oral LD50 of sterigmatocystin with mg/kg weighing scale, male be 166, female be 120, mouse is more than 800.Monkey is 32 through lumbar injection LDso.Can be found out by LDso value, primate (monkey) susceptibility to sterigmatocystin is higher than rodent.The lethal pathology that sterigmatocystin causes is mainly liver, kidney organa parenchymatosum is downright bad.Sterigmatocystin has carcinogenicity, and hay bacillus and mouse cell also can be made to undergo mutation reaction.ST is widespread in nature, and because structure is similar to aflatoxin, therefore its toxicity and carcinogenicity are subject to the great attention of countries in the world.The characteristics of lesion of sterigmatocystin acute poisoning is liver, renal necrosis.But the chronic toxic effect of sterigmatocystin is comparatively strong, and main manifestations is to hepatic and/or renal toxicity.Once there is equus and lamb and search for food after the feed that polluted by sterigmatocystin and occur toxicity symptom in China Ningxia, China, pathology cuts open inspection and is characterized as cirrhosis, skin and internal organs height xanthochromia.Sterigmatocystin also can make rat occur the tumour such as angiosarcoma, back of the body Tissue Blood tuberculation.About the carcinogenic mechanism of sterigmatocystin, have scholar to think relevant with the double bond of its two furan nucleus ends, this double bond can form adduct with the uracil of DNA molecular, DNA structure is changed, copy error.Also do not have at present to detect fast, accurately kit it is detected.
Summary of the invention
The object of the present invention is to provide the preparation method of agent plate of a kind of quick detection, sensitive, accurate, detection sterigmatocystin that can be quantitative, easy and simple to handle, to solve the problem proposed in above-mentioned background technology.
For achieving the above object, the invention provides following technical scheme:
A kind of preparation method detecting the agent plate of sterigmatocystin, gold conjugation pad on nitrocellulose filter is coated with anti-sterigmatocystin monoclonal antibody-colloid gold label thing, detection line and nature controlling line successively from sample pad to adsorptive pads direction, be coated with sterigmatocystin-carrier protein couplet thing and sheep anti-mouse igg, wherein carrier protein adopts rabbit serum proteins; By a film machine, sterigmatocystin-carrier protein couplet thing and sheep anti-mouse igg are sprayed on nitrocellulose filter, respectively as detection line and nature controlling line, 38 DEG C of oven drying 8h, by a film machine, anti-sterigmatocystin monoclonal antibody-colloid gold label thing is sprayed on gold conjugation pad, after 38 DEG C of oven drying 8h and get final product.
As the further scheme of the present invention: collaurum and anti-sterigmatocystin monoclonal antibody are mixed, collaurum and anti-sterigmatocystin monoclonal antibody is made to form stable colloid gold particle, by the anti-sterigmatocystin monoclonal antibody-colloid gold label thing of concentrated formation.
As the further scheme of the present invention: when the sterigmatocystin content in sample exceedes agent plate detection limit, the detection line colour developing in agent plate is shallow compared with control line even without colour developing, is judged to be the positive; Otherwise, when sterigmatocystin content in sample below agent plate detection limit or noresidue time, the detection line colour developing in agent plate is close with control line or partially deeply, is judged to be feminine gender.
Compared with prior art, the invention has the beneficial effects as follows:
The present invention prepares a kind of immune colloid gold quick detection reagent plate, and agent plate application chromatography type antibody mediated immunity competition principle, by antigen and golden labeling antibody reaction solution, the sterigmatocystin in specific detection corn remains.Remain if sample solution contains sterigmatocystin, the antibody response of sterigmatocystin first and on colloid gold particle, therefore, when colloid gold particle diffuses to detection line with sample solution, on colloid gold particle, the avtive spot of antibody cannot be combined by sterigmatocystin specific antigen because being occupied by the sterigmatocystin in sample solution on detection line; When the sterigmatocystin content in sample exceedes agent plate detection limit, the detection line colour developing in agent plate is shallow compared with control line even without colour developing, is judged to be the positive.Otherwise, when sterigmatocystin content in sample below agent plate detection limit or noresidue time, the detection line colour developing in agent plate is close with control line or partially deeply, is judged to be feminine gender.The present invention have quick detection, sensitive, accurate, can be quantitative, advantage easy and simple to handle, agent plate production technology of the present invention is simple, and can realize single pattern detection, production cost is low, greatly reduces testing cost.
Embodiment
Below in conjunction with the embodiment of the present invention, be clearly and completely described the technical scheme in the embodiment of the present invention, obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.
Embodiment 1
In the embodiment of the present invention, a kind of preparation method detecting the agent plate of sterigmatocystin, concrete steps are as described below.
1. the coupling of sterigmatocystin and carrier protein
Adopt carbodlimide method (EDC method) that sterigmatocystin and carrier protein (rabbit serum proteins) coupling are prepared immunizing antigen and envelope antigen.
Take 2mg sterigmatocystin and 3mgCMO, be dissolved in 375 μ L pyridines, 28 DEG C of lucifuge jolting reactions.Until react completely.By reaction product freeze drying, residue obtained 4mL ultrapure water dissolves, with 0.1mol/L sodium hydroxide solution adjust pH to 8.2.Divide and add 8mL benzene (4mL, 2mL, 2mL) for 3 times, unreacted sterigmatocystin in oscillation extraction organic phase.In aqueous phase, drip 0.1mol/L watery hydrochloric acid adjust pH to 3.5, obtain precipitation, with ethyl acetate extracting sediment, vacuum drying obtains intermediate product sterigmatocystin oxime.
Take 0.3mg sterigmatocystin oxime, be dissolved in 100 μ LDMF-water (6: 9, V/V), add 2.5mgEDC, lucifuge mixes.Add 0.5%C-rabbit serum proteins solution again, 28 DEG C of lucifuges, 100r/min react 4h, add EDC2mg, continue reaction.Coupled product is loaded bag filter, and at the mid-5 DEG C of dialysis 3d of 0.01mol/LPBS (pH7.4), period changes dislysate 3 times (every 24h changes once).
2. the preparation of anti-sterigmatocystin monoclonal antibody
Get female Balb/c mouse in 6 ~ 8 week age, will as immunogenic sterigmatocystin-rabbit serum proteins conjugate and isopyknic Freund's complete adjuvant emulsification, by a 100 μ g/ injected sc, afterwards every 3 weeks booster immunizations 1 time, cannot be used up full adjuvant lumbar injection.3d reinforced immunological 1 time before merging, without adjuvant, dosage doubles.Fusion of Cells is carried out according to a conventional method: mixed with the ratio of immune spleen cell in 1:9 by Sp2/0 multiple myeloma cells, merges under 50% polyglycol effect, and HAT nutrient culture media suspends, and divides and plants in 96 well culture plates, 38 DEG C, 5%CO
2cultivate in incubator.
After fusion, when Growth of Cells arrives 1/4 of culture hole area, adopt a point step screening method screening hybridoma.Primary dcreening operation selects 10mg/L sterigmatocystin-rabbit serum proteins bag by elisa plate, and measured hole adds culture supernatant, and after hatching, cleaning, add sheep anti-mouse igg-HRP (1: 1000), OPD develops the color.The elisa plate of the positive Kong Zaiyong sterigmatocystin filtered out-rabbit serum proteins conjugate bag quilt carries out blocking-up indirect ELISA.By cells and supernatant and 2 × 10
-3mol/L sterigmatocystin solution even mixes, and 1h is made in 38 DEG C of senses, adds and wraps in the ELISA Plate of quilt.Use PBS (0.01mol/L, pH7.4) to substitute sterigmatocystin solution in addition to compare, all the other steps are the same.If the OD value after sterigmatocystin blocks is down to less than 50% of control wells, be then judged to positive hole.Detect the hole of being all positive through 2 ~ 3 times, carry out cloning with limiting dilution assay immediately.
In vitro culture: expanded by the cell line of cloning and cultivate, cell concentration reaches 5.5 × 10
5stop during/mL changing liquid, after complete cell death, collect nutrient solution.Ascites is induced: to the mouse peritoneal injection cloning cell line 10 of lumbar injection whiteruss after 10 days in body
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, for add 1mL1% trisodium citrate in 100mL deionized water, boils and adds rapidly 1mL1% gold chloride afterwards, continue to boil 10min, after cooling, save backup at 4 DEG C.
4. the preparation of the anti-sterigmatocystin monoclonal antibody of colloid gold label
Get the colloidal gold solution 100mL prepared, adjust pH to 8.0 with 0.1mol/L solution of potassium carbonate.Add the anti-sterigmatocystin monoclonal antibody of 2mg while stirring, stir 20min, dropwise add 2mL25mol/L PEG 20000 (PEG20000), stir 15min.The centrifugal 15min of 20,000rpm, abandons supernatant, and the PBS damping fluid (containing 0.4mol/LPEG) adding 10mLpH7.4 cleans 2 times.Precipitation dissolved containing the PBS damping fluid (pH7.4) of 2% rabbit serum proteins with 10mL, after 0.22 μm of sterile filter, 4 DEG C save backup.
5. the assembling of sterigmatocystin immune colloid gold quick detection reagent plate
With a film machine, the sterigmatocystin of debita spissitudo-carrier protein couplet thing and sheep anti-mouse igg are sprayed on nitrocellulose filter, respectively as detection line and nature controlling line, 38 DEG C of oven drying 8h.In kind, the gold prepared mark sterigmatocystin monoclonal antibody is coated on gold conjugation pad.
Detect reagent set and become a PVC backing, be stained with sample pad, gold conjugation pad, nitrocellulose filter and adsorptive pads in order thereon.With cutting machine, the kilocalorie posted is cut into the wide bar of 4.5mm, load in plastic formwork and make detection agent plate, then put into the aluminium foil bag sealed storage of band drying agent.
6. sterigmatocystin immune colloid gold quick detection reagent plate detects implementation and operation method
6.1 sample preparation
Corn: get 20g corn in 50mL centrifuge tube, add 20mL damping fluid, concuss 1-2min, leaves standstill 1min, draws at least 100 μ L lower floor solution, to be checked.
6.2 detecting step
Drawing measuring samples solution 100 μ L is added drop-wise in well, starts timing after application of sample; Result should read at 5 ~ 8min, and other times interpretation is invalid.During observation, agent plate is placed horizontally at observer front.
6.3 results judge
When reading result, agent plate level is placed in observer front.
Negative (-): T line colour developing (detection line, near well one end) than C line (control line) deeply or equally dark, to represent in sample that sterigmatocystin residual content is lower than detectability or do not remain containing sterigmatocystin.
Positive (+): the colour developing of T line is more shallow than C line, or T line is without colour developing, represent that in sample, sterigmatocystin residual content is higher than detectability, the colour developing of T line is more shallow than C line, represents that in sample, sterigmatocystin content is higher.
Invalid: not occur C line, may be that misoperation or agent plate lost efficacy.Again should read instructions, and retest by new agent plate.
To those skilled in the art, obviously the invention is not restricted to the details of above-mentioned one exemplary embodiment, and when not deviating from spirit of the present invention or essential characteristic, the present invention can be realized in other specific forms.Therefore, no matter from which point, all should embodiment be regarded as exemplary, and be nonrestrictive, scope of the present invention is limited by claims instead of above-mentioned explanation, and all changes be therefore intended in the implication of the equivalency by dropping on claim and scope are included in the present invention.
In addition, be to be understood that, although this instructions is described according to embodiment, but not each embodiment only comprises an independently technical scheme, this narrating mode of instructions is only for clarity sake, those skilled in the art should by instructions integrally, and the technical scheme in each embodiment also through appropriately combined, can form other embodiments that it will be appreciated by those skilled in the art that.
Claims (3)
1. one kind is detected the preparation method of the agent plate of sterigmatocystin, it is characterized in that, gold conjugation pad on nitrocellulose filter is coated with anti-sterigmatocystin monoclonal antibody-colloid gold label thing, detection line and nature controlling line successively from sample pad to adsorptive pads direction, be coated with sterigmatocystin-carrier protein couplet thing and sheep anti-mouse igg, wherein carrier protein adopts rabbit serum proteins; By a film machine, sterigmatocystin-carrier protein couplet thing and sheep anti-mouse igg are sprayed on nitrocellulose filter, respectively as detection line and nature controlling line, 38 DEG C of oven drying 8h, by a film machine, anti-sterigmatocystin monoclonal antibody-colloid gold label thing is sprayed on gold conjugation pad, after 38 DEG C of oven drying 8h and get final product.
2. the preparation method of the agent plate of detection sterigmatocystin according to claim 1, it is characterized in that, collaurum and anti-sterigmatocystin monoclonal antibody are mixed, collaurum and anti-sterigmatocystin monoclonal antibody is made to form stable colloid gold particle, by the anti-sterigmatocystin monoclonal antibody-colloid gold label thing of concentrated formation.
3. the preparation method of the agent plate of detection sterigmatocystin according to claim 1, it is characterized in that, when sterigmatocystin content in sample exceedes agent plate detection limit, the detection line colour developing in agent plate is shallow compared with control line even without colour developing, is judged to be the positive; Otherwise, when sterigmatocystin content in sample below agent plate detection limit or noresidue time, the detection line colour developing in agent plate is close with control line or partially deeply, is judged to be feminine gender.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201510776029.3A CN105372416A (en) | 2015-11-13 | 2015-11-13 | Preparation method of reagent plate for detecting sterigmatocystin |
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| CN201510776029.3A CN105372416A (en) | 2015-11-13 | 2015-11-13 | Preparation method of reagent plate for detecting sterigmatocystin |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107703298A (en) * | 2017-07-25 | 2018-02-16 | 广东省药品检验所（广东省药品质量研究所、广东省口岸药品检验所） | Colloid gold immune detection kit, preparation method and the detection method of a kind of aflatoxin B1 |
| WO2018161905A1 (en) * | 2017-03-07 | 2018-09-13 | 中国农业科学院油料作物研究所 | Time-resolved fluorescence immunochromatographic kit for simultaneous detection of mixed contamination of five mycotoxins such as aflatoxin b1 and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018161905A1 (en) * | 2017-03-07 | 2018-09-13 | 中国农业科学院油料作物研究所 | Time-resolved fluorescence immunochromatographic kit for simultaneous detection of mixed contamination of five mycotoxins such as aflatoxin b1 and application thereof |
| CN107703298A (en) * | 2017-07-25 | 2018-02-16 | 广东省药品检验所（广东省药品质量研究所、广东省口岸药品检验所） | Colloid gold immune detection kit, preparation method and the detection method of a kind of aflatoxin B1 |
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Application publication date: 20160302 |