CN101825632B - Anti-thiram monoclonal antibody, test paper for fast testing thiram and application thereof - Google Patents
Anti-thiram monoclonal antibody, test paper for fast testing thiram and application thereof Download PDFInfo
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- CN101825632B CN101825632B CN2010101396953A CN201010139695A CN101825632B CN 101825632 B CN101825632 B CN 101825632B CN 2010101396953 A CN2010101396953 A CN 2010101396953A CN 201010139695 A CN201010139695 A CN 201010139695A CN 101825632 B CN101825632 B CN 101825632B
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Abstract
The invention belongs to the field of pesticide detection and can fast detect the residual pesticide with an immunochemical technology. More particularly, the invention relates to a hybridoma cell line with the preservation number of CGMCC No.3580, and an anti-thiram monoclonal antibody generated by the hybridoma cell line. The invention further relates to a composite for detecting the thiram, a test paper for detecting the thiram, a preparation method of the test paper, an enzyme-coupled immunization agent for detecting the thiram, and application of the anti-thiram monoclonal antibody in detecting the thiram. The monoclonal antibody has high sensitivity and specificity. The test paper has the obvious advantages of high sensitivity, high specificity, convenient operation, fast and exact detection, etc.
Description
Technical field
The invention belongs to the Pesticides Testing field, utilize the residual agricultural chemicals of immunochemical technique fast detecting, particularly, the present invention relates to a kind of hybridoma cell strain, and the anti-thiram monoclonal antibody that produces of described hybridoma cell strain.The invention still further relates to a kind of composition that detects thiram, a kind ofly detect the preparation method of the test paper of thiram, described test paper, a kind of ELISA reagent and the application of described anti-thiram monoclonal antibody in detecting thiram that detects thiram.
Background technology
Thiram (Thiram); the CAS registration number is that the 137-26-8 chemical name is tetramethyl thiuram disulfide; belong to the thiocarbamates agricultural chemicals; it is a kind of germifuge with protective effect; its has a broad antifungal spectrum can be processed seed and soil, the seedling blight of control Cereal smut and various crop; can prevent and treat some fruit trees, vegetable disease after sprinkling.
At present the residual detection method commonly used of thiram in food is mainly chromatogram analysis method, as high performance liquid chromatography (HPLC), vapor-phase chromatography (GC) and look MS (HPLC-MS-MS and GC-MS).
It is very effective, accurate and sensitive that chromatographic technique detects thiram, but also has following shortcoming: (1) sample pretreatment needs derivatization, and program is loaded down with trivial details time-consuming; (2) need the technical professional to operate, operating personnel will have abundant correlation experience, must understand the various disturbing factors that affect stratographic analysis, understand the relative merits of the preprocess method that uses, and could obtain reliable analysis result; (3) need expensive instrument and equipment auxiliary, be difficult in small and medium-sized cities and medium-sized and small enterprises universal; (4) technical requirement of instrument maintenance and maintenance is high.Maintain whether proper, accuracy that can direct impact analysis result; (5) testing cost is high.
The present invention has prepared anti-thiram monoclonal antibody, and uses this antibody and prepare a kind of high specificity, highly sensitive, easy and simple to handle, the test paper that can detect rapidly and accurately thiram, the defective that exists to overcome prior art.
Summary of the invention
One aspect of the present invention relates to a kind of hybridoma cell strain, its deposit number is CGMCCNo.3580, preservation date on January 13rd, 2010, depositary institution is China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC, the address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica), the Classification And Nomenclature of this hybridoma cell strain suggestion is: the little mouse-anti of BALA/C-thiram monoclonal antibody myeloma hybrid cell.
Another aspect of the present invention relates to a kind of anti-thiram monoclonal antibody, and its hybridoma cell strain that is CGMCC No.3580 by deposit number produces.This anti-thiram monoclonal antibody has good specificity.
Also aspect of the present invention relates to a kind of composition that detects thiram, wherein contains above-mentioned anti-thiram monoclonal antibody.
Of the present inventionly also relate in one aspect to a kind of test paper that detects thiram, it contains the above-mentioned anti-thiram monoclonal antibody of effective dose.Described antibody is mark in many ways, for example fluorescence labeling.Preferably, described anti-thiram monoclonal antibody colloid gold label.
In one embodiment of the invention, described test paper has the detection line that thiram-carrier protein couplet thing consists of, and preferably, described test paper also has the nature controlling line that dynamics consists of.
In the present invention, described detection line is the band that thiram-carrier protein couplet thing forms, and described nature controlling line is the coated band that forms of anti-mouse IgG, and the width of these two bands is not particularly limited.The distance of detection line and nature controlling line is not particularly limited, preferred 5-8mm.
In one embodiment of the invention, described test paper comprises:
Sample pad (1), pad (2), protein combination film (3), adsorptive pads (4) and backing (7), and described sample pad (1), pad (2), protein combination film (3), adsorptive pads (4) along continuous straight runs stick to backing (7) upper (as shown in Figure 2) successively;
It is characterized in that,
Described pad is coated with above-mentioned anti-thiram monoclonal antibody on (2), described anti-thiram monoclonal antibody colloid gold label;
Be coated with respectively the detection line (5) of thiram-carrier protein couplet thing formation and the nature controlling line (6) that dynamics consists of on described protein combination film (3).Preferably, described detection line (5) is 5-8mm with the spacing of nature controlling line (6).
In the present invention, in conjunction with coated albumen (for example acting as of described protein combination film, thiram-carrier protein couplet thing or antibody), then the albumen and the substance reaction to be detected that are coated with, therefore can select any material with this effect, include but not limited to: nitrocellulose filter, pvdf membrane etc.Preferably, the protein combination film is nitrocellulose filter.
In the present invention, described carrier protein be can with any albumen of thiram coupling, include, but are not limited to: human serum albumins, bovine serum albumin(BSA) or ovalbumin.Preferably, carrier protein is bovine serum albumin(BSA).
In the present invention, described backing can be selected the material of multiple supporting function, and for example, it can be the PVC backing.
The present invention selects thiram-carrier protein couplet thing as detection line, anti-thiram monoclonal antibody specific is as the antibody of colloid gold label, utilize competition law to detect and whether contain thiram in testing sample, by the thiram in testing sample be coated in thiram on the protein combination film-carrier protein couplet thing and jointly compete anti-thiram monoclonal antibody-colloid gold label thing, accumulation in various degree due to colloid gold particle, the red stripes that occurs being of different shades at the detection line place or band do not occur, red stripes appears in the nature controlling line place.If band does not appear in the testing sample detection paper, simultaneously red stripes occurs on nature controlling line and be judged as positive, namely in testing sample the concentration of thiram higher than 500ppb; If red stripes has appearred in the testing sample detection paper, red stripes occurs on nature controlling line simultaneously and judge that the concentration of thiram in testing sample is lower than 500ppb; If do not occur red stripes on nature controlling line, this test paper invalid (as shown in Figure 3).
General technical route map of the present invention as shown in Figure 1.
The preparation method of above-mentioned test paper comprises the steps:
1) with thiram and carrier protein couplet, form thiram-carrier protein couplet thing;
2) prepare above-mentioned anti-thiram monoclonal antibody with thiram-carrier protein couplet thing as antigen;
3) preparation dynamics;
4) with trisodium citrate and gold chloride reaction preparation collaurum;
5) with step 2) in the preparation anti-thiram monoclonal antibody add step 4) preparation collaurum in, obtain anti-thiram monoclonal antibody-colloid gold label thing;
6) anti-thiram monoclonal antibody-colloid gold label thing is coated on pad (2);
7) thiram-carrier protein couplet thing is coated on the upper formation of protein combination film (3) detection line (5); And will resist mouse IgG to be coated on the upper formation of protein combination film (3) nature controlling line (6);
8) adhere to successively sample pad (1), pad (2), protein combination film (3), adsorptive pads (4) at the upper along continuous straight runs of described PVC backing (7).
In the present invention, in conjunction with coated albumen (for example acting as of described protein combination film, thiram-carrier protein couplet thing or antibody), then allow coated albumen and substance reaction to be detected, therefore can select any material with this effect, include but not limited to: nitrocellulose filter, pvdf membrane etc.Preferably, the protein combination film is nitrocellulose filter.
In the present invention, described carrier protein be can with any albumen of thiram coupling, include, but are not limited to: human serum albumins, bovine serum albumin(BSA) or ovalbumin.Preferably, carrier protein is bovine serum albumin(BSA).
In the present invention, described backing can be selected the material of multiple supporting function, and for example, it can be the PVC backing.
In one embodiment of the invention, the described dynamics step 3) is rabbit anti-mouse igg antibody.
Of the present inventionly also relate in one aspect to a kind of ELISA reagent that detects thiram, its anti-thiram monoclonal antibody of the present invention is as first antibody.
Of the present inventionly also relate in one aspect to the application of anti-thiram monoclonal antibody in detecting thiram.
The application that also relates in one aspect in the thiram of anti-thiram monoclonal antibody in detecting vegetables or fruit of the present invention.
The beneficial effect of the invention
Compared with prior art, the present invention has following outstanding advantages:
1. anti-thiram monoclonal antibody of the present invention has good specificity and sensitivity.
2. the immune colloid gold test paper of detection thiram of the present invention, high specificity, highly sensitive, particularly detection time short, be approximately 20 minutes, and existing chromatogram detects and needs more than 2 hours at least.
3. Test paper of the present invention is without any need for specific apparatus, equipment, and testing cost is low.
4. Test paper of the present invention is easy and simple to handle, does not need to be operated by the professional.
5. Test paper of the present invention stores conveniently, stablizes, and is not high to temperature requirement, can reach 1 year at 4-8 ℃ of lower shelf life; At room temperature can preserve six months.
Description of drawings
Fig. 1: the technology of the present invention route map.
Fig. 2: the schematic diagram of Test paper of the present invention.Wherein: 1 is sample pad, and 2 is pad, and 3 is the protein combination film, and 4 is absorption pad, and 5 is detection line, and 6 is nature controlling line, and 7 is backing.
Fig. 3: the schematic diagram that Test paper result of the present invention is judged.The negative result of A, the line of top are nature controlling line, and the line of below is detection line, and red stripes all appears in two lines; The positive result of B only has nature controlling line red stripes to occur; C, D are test paper and lost efficacy, and red stripes does not all appear in nature controlling line.
Embodiment
Below in conjunction with embodiment, embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example only is used for explanation the present invention, and should not be considered as limiting scope of the present invention.Unreceipted actual conditions person in embodiment carries out according to the condition of normal condition or manufacturer's suggestion.The unreceipted person of production firm of agents useful for same or instrument, being can be by the conventional products of commercial acquisition.
Embodiment 1: detect the preparation of the immune colloid gold test paper of thiram
1, the preparation of thiram-carrier protein couplet thing
1) take the 40mg thiram and be dissolved in the 20mM PBS solution that contains 0.02%Tween-20, be mixed with thiram solution.
2) under ice-water bath and magnetic agitation, thiram solution dropwise is added in 2.4ml 1mol/lHCl, carries out diazotising (Itaru Y, Kohji I.Enzyme immunoassay forclenbuteol, an β
2-adrnergicstimulant[J] .Journal ofimmunoassay, 1982,3 (2): 155-171) reaction adds rear continuation reaction 5min.
3) under magnetic agitation, the diazotising product dropwise is added to respectively in human serum albumins (HSA), bovine serum albumin(BSA) (BSA) and ovalbumin (OVA) solution, transfer pH value to 9.5 with 11mol/1NaOH, 4 ℃ of slow stirrings are spent the night, obtain three kinds and connect product, i.e. thiram-HSA, thiram-BSA, thiram-OVA.
4) with step 3) connection product thiram-HSA, the thiram-BSA, the thiram-OVA decibel that the obtain bag filter of packing into, with the phosphate buffer (formula: NaCl 8g, KCl 0.2g, Na of 0.01MPH 7.4
2HPO
412H
2O 2.9g, KH
2PO
40.2g, be dissolved to 1000ml with distilled water) dialysed 3 days, after removing the free little molecule of thiram ,-20 ℃ save backup.
2, the preparation of anti-thiram monoclonal antibody:
Utilize the prepared thiram-human serum albumins of the inventor (HSA) conjugate immunity Balb/C mouse, immune programme for children is to get solution and isopyknic Freund's complete adjuvant (available from the Sigma company) emulsification that contains thiram-human serum albumins (HSA) conjugate 100 μ g, the injection mouse peritoneal, strengthened once at interval of ten days later on, use incomplete Freunds adjuvant emulsification (available from Sigma company) instead.At last in merging first three day, (preferably and immunity last time be separated by more than 4 weeks), the abdominal cavity reinforced immunological, the antigen amount doubles (200 μ g), does not add adjuvant.During fusion, one of the Balb/C mouse of the last reinforced immunological of learning from else's experience, eye socket sacrificed by exsanguination (collect serum, be positive serum) is soaked the 5min sterilization in 75% alcohol.Aseptic taking-up mouse spleen is isolated splenocyte.
The splenocyte of separation and the SP2/0 myeloma cell of fresh preparation are pressed 1-2 * 10
7(1: 10-1: ratio 15) is mixing in the 50ml centrifuge tube, 1500rpm, centrifugal 10min for individual SP2/0 and 108 immunocytes.Evacuation supernatant (filter paper of available sterilization blots) knocks gently and manages at the end, makes cell precipitation loosening slightly.The centrifuge tube that cell mixture is housed is put in 37 ℃ of water-baths.Then slowly splash into 50% polyglycol (PEG) 0.8ml (available from Sigma company) of pre-temperature to 37 ℃ in 1min, the limit edged stirs with pipette tip gently, continues to stir 1min.Then 1640 (available from the Sigma company commodity) basal liquid (formula: 164010.4g, NaHCO that slowly adds 37 ℃ of pre-temperature
32g is settled to 1000ml with distilled water, transfers PH to 7.2) 10ml.Concrete grammar is: dropwise splashed into 1ml in first minute.Added 1ml in second minute, added 3ml in 3-4 minute, added remaining 5ml on the 5th minute, each added-time need slowly add, and not only gently stirring.Add at last 30ml 1640 liquid, also need slowly add.The centrifugal 5min of 800rpm removes supernatant, places 5-8min in 37 ℃.With HAT (available from Sigma company commodity, its composition is 100% content) the nutrient culture media suspension, simultaneously also with the HAT nutrient culture media suspend the raising splenocyte for preparing and with fusion after mixing with cells, add as required the appropriate HAT nutrient culture media (composition of nutrient culture media: 80ml 1640 basal liquids, the 20ml calf serum of sterilizing, the HAT of 1ml100%, 1ml is two anti-), minute kind cultivate in version in 96 holes, approximately 250 μ l/ holes.
Single cell fusion can be inoculated 4-8 piece 96 orifice plates.Also can plant less as required, generally press the cell number of SP2/0 and calculate, every hole inoculum concentration approximately contains 104 a left and right SP2/0 cell.In 37 ℃, 5%CO
2Cultivate in incubator.
After merging, second day begins to observe had pollution-freely, added 1 HAT nutrient culture media in the 4th day, sucked 100 μ l nutrient culture media and changed HT (available from Sigma company) nutrient culture media 100 μ l in 8-10 days.Treat that the fused cell colony grows to culture hole 1/4, when nutrient culture media omits flavescence, carry out antibody test.
Adopt thiram-ovalbumin (OVA) as screening antigen, utilize the ELISA method to filter out the positive hole of the anti-thiram of secretion.Use at once limiting dilution assay (with reference to Xue Qingshan " philosophy and technique of in vitro culture " Science Press calendar year 2001 version) to clone, screen to the positive hole that screens.Through the 3-4 time cloning, finishing screen is selected the monoclonal hybridoma strain of the anti-thiram of secretion, this cell line was deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), deposit number: CGMCC No.3580 on 01 13rd, 2010.
In addition, this cell line has been carried out chromosome counting, the result demonstration, the chromosomal average of SP2/0 is 70, splenocyte chromosome is 40, and the chromosome number of hybridoma is between 80-94.All higher than the chromosome number of two parent's cells, the cell of SP2/0 really of fused cell and the hybridization product of splenocyte are described, the chromosome number of hybridoma is obviously more than the chromosome of myeloma cell SP2/0.
With this cell line injection Balb/C mouse peritoneal, manufacture order clonal antibody.
3, the purifying of anti-thiram monoclonal antibody
with reference to Zhu Li equality, " immunology common experimental method ", the method for the 2000 editions instructions in People's Medical Officer Press: the mouse ascites 5ml that gets gained mixes with appropriate silicon dioxide, adds isopyknic bar than damping fluid (formula: sodium chloride 85.00g, barbital sodium 3.75g, Sodium azide 2.00g is settled to 2000ml with distilled water), after room temperature vibration 1H, the standing 30min of room temperature, get in supernatant and clean centrifuge tube, 4 ℃, the centrifugal 10min of 3000rpm, get supernatant 8ml, add 16ml 0.06mol sodium-acetate buffer, transfer PH to 4.5 with HCL, after slowly adding sad 132 μ l under fully stirring, stirring at room 30min, then change 4 ℃ of refrigerators over to and fully precipitate 2h, 4 ℃, the centrifugal 30min of 15000rpm, get supernatant 22ml, add 2.2ml PH7.2, 0.1m phosphate buffer (PB), transfer pH value to 7.6 with NaOH, slowly adding ammonium sulfate to final concentration under stirring is 0.277g/ml, 4 ℃, the centrifugal 30min of 12000rpm, abandon supernatant, precipitation is resuspended with 5mlM pH7.2 phosphate buffer (PBS), the bag filter of packing into, after 5000ml0.01M pH7.2 phosphate buffer is fully dialysed, again to the 2000ml distill water dialysis, at last 3000ml three is boiled off the ionized water dialysis, the protein solution that fully dialysis is good is concentrated into 3ml with PEG-400 (PEG-20000), then 4 ℃, the centrifugal 30min of 12000rpm, abandon precipitation, collect supernatant, recording protein concentration is 1.6mg/ml.Be accredited as the monoclonal antibody of purifying through sds page sex change electrophoresis (SDS-PAGE), purity is greater than 95%.This monoclonal antibody can be used for preparing immune colloid gold.
4, the preparation of rabbit anti-mouse igg antibody:
Utilize the Balb/C mouse IgG immune health rabbit of inventor place microorganism and immunization experiment chamber extraction, the preparation high specific, the rabbit anti-mouse igg hyper-immune serum of high-titer, adopt the saturated ammonium sulphate method slightly to carry to hyper-immune serum, obtain highly purified rabbit anti-mouse igg antibody after DEAE crosses post.Utilize this antibody as nature controlling line.
5, the preparation of anti-thiram monoclonal antibody-colloid gold label thing
(1) preparation of collaurum:
With two ionized waters that boil off, 1% gold chloride is diluted to 0.01%, put to stir on the magnetic force heating stirrer and boil, add 2ml 1% trisodium citrate by every 100ml 0.01% gold chloride, continue to boil, be stopped heating until liquid is orange red, supply dehydration after being cooled to room temperature.The collaurum outward appearance for preparing should be pure, bright, without precipitation and floating thing, one week of the term of validity.
(2) preparation of anti-thiram monoclonal antibody-colloid gold label thing:
Under magnetic agitation, transfer the pH value to 8.2 of collaurum with 0.1M sal tartari, add anti-thiram monoclonal antibody by 1-2 μ g antibody/ml collaurum, continue to stir and evenly mix 30min, adding 10%BSA is 1% to final concentration, standing 30min.12000rp, 4 ℃ of centrifugal 30min, abandon supernatant, precipitation (is filled a prescription: boric acid 0.1237g, PEG-20000 1g with the borate buffer solution of 0.02m PH9.0, heat up in a steamer water with three and be settled to 1000ml, transfer PH to 9.0) washed twice, (fill a prescription: boric acid 0.1237g, PEG-20000 1g with the borate buffer solution of the 0.02M PH9.0 of 1/20th initial collaurum volume, heat up in a steamer water with three and be settled to 1000ml, transfer PH to 9.0) will precipitate resuspended, put 4 ℃ standby, the term of validity 60 days.
6, pad is coated
The resuspended liquid of resuspended anti-thiram monoclonal antibody-collaurum is adopted 0.01M PH 7.4 phosphate buffers (formula and preparation: BSA 20g, sucrose 25g, pvp k-303g, NaN
30.2g NaCl 8g, KCl 0.2g, Na
2HPO
412H
2O 2.9g, KH
2PO
40.2g, be settled to 1000ml with distilled water) dilute acquisition anti-thiram monoclonal antibody-colloid gold label thing, evenly drip on pad, every 100 square centimeters of pads drip 20ml anti-thiram monoclonal antibody-colloid gold label thing dilution, vacuum drying, Vacuum Package, put 4 ℃ standby.
7, the processing of sample pad
Sample pad is soaked in 0.01M PH7.4 phosphate buffer (formula and preparation: BSA20g, sucrose 25g, pvp k-30 3g, NaN
30.2g, NaCl 8g, KCl 0.2g, Na
2HPO
412H
2O2.9g, KH
2PO
40.2g, be settled to 1000ml with distilled water) in after 30min, in 37 ℃ of oven dry, Vacuum Package, put 4 ℃ standby.
8, the protein combination film is coated
With 0.01M PH7.4PBS damping fluid (containing 3% methyl alcohol), thiram-bovine serum albumin(BSA) (BSA) conjugate is diluted to 65 μ g/ml, with Biodot point film instrument, it is coated in the protein combination film as detection line, package amount is 0.6 μ l/cm, nature controlling line is near adsorptive pads, apart from the about 8mm of absorption pad, two linear distance 5-8mm.37 ℃ of oven dry encapsulate standby.
9, the assembling of test paper
Sample pad (1), pad (2), protein combination film (3), adsorptive pads (4) are sticked on PVC backing (7) successively by order shown in Figure 2, be cut into wide little of 3mm, Vacuum Package.4-8 ℃ of preservation is valid for one year; Normal temperature is preserved, the term of validity 6 months.
Described sample pad, pad, protein combination film, adsorptive pads, PVC backing are all available from Millipore company.
Embodiment 2: the sensitivity of anti-thiram monoclonal antibody and specific detection
Coated thiram-bovine serum albumin(BSA) conjugate on microwell plate, package amount is the 10ng/ hole; Carry out mark with described anti-thiram monoclonal antibody and HRP enzyme and obtain the anti-thiram monoclonal antibody enzyme marking reagent, use at 1: 1000; Adopt PBST (0.5%Tween-20, the phosphate buffer of 0.02MpH 7.4) as washing lotion; The TMB Color Appearance System is adopted in colour developing; Colour developing stops adopting the 0.2M concentrated sulphuric acid.Detect as the thiram detection system with this, result adopts microplate reader to read.
Select damping fluid (phosphate buffer of 0.01MpH7.4, formula: NaCl 8g, KCl0.2g, Na
2HPO
412H
2O 2.9g, KH
2PO
40.2g, be settled to 1000ml with distilled water), adding therein thiram and the concentration of different content is 3000ppb ziram, fervam, bis methylarsine, Propineb, ambam, Mancozeb, dithiocyano-methane, Bravo, iprodione, S-Ethyl ethylthio sulfonate, prepares dissimilar sample.Adopt the ELISA reagent of using anti-thiram monoclonal antibody of the present invention to detect, the results are shown in Table 1.
Testing result shows that anti-thiram monoclonal antibody of the present invention has outstanding sensitivity and splendid specificity.
Table 1: use the detection (wavelength 450nm) that monoclonal antibody of the present invention is carried out
Embodiment 3: the specificity of immune colloid gold test paper and sensitivity detect
1, specific detection
Ziram, fervam, bis methylarsine, Propineb, ambam, Mancozeb, dithiocyano-methane, Bravo, iprodione, S-Ethyl ethylthio sulfonate are mixed with the sample that concentration is 3000ppb.Directly draw the immune colloid gold test paper that 80 μ l add detection thiram of the present invention.Test findings (seeing Table 2) shows, when detecting ziram, fervam, bis methylarsine, Propineb, ambam, Mancozeb, dithiocyano-methane, Bravo, iprodione, S-Ethyl ethylthio sulfonate sample, red stripes appears in the detection paper line, on nature controlling line, red stripes appears simultaneously, this shows ziram, fervam, bis methylarsine, Propineb, ambam, Mancozeb, dithiocyano-methane, Bravo, iprodione, S-Ethyl ethylthio sulfonate and test paper no cross reaction of the present invention, shows that test paper of the present invention has good specificity.
Table 2: the result of test paper specific detection of the present invention
| Ziram | Fervam | Bis methylarsine | Propineb | Ambam | Mancozeb | Dithiocyano-methane | Bravo | Iprodione | S-Ethyl ethylthio sulfonate | |
| Testing result | - | - | - | - | - | - | - | - | - | - |
Annotate: "+" expression is positive, and "-" expression is negative.
2, sensitivity test
With Test paper of the present invention respectively detectable concentration be 0,5,50,500,1000,3000ppb thiram standard items, repeat 8 times, detect and have repeatability, the results are shown in Table 3.As seen Test paper sensitivity of the present invention can reach 500ppb, can satisfy the demand fully.
Table 3: test paper sensitivity of the present invention (susceptibility) test findings
| Thiram concentration | 0ppb | 5ppb | 50ppb | 500ppb | 1000ppb | 3000ppb |
| Testing result | - | - | - | + | + | + |
Annotate: "+" expression is positive, and "-" expression is negative.
Embodiment 4: use the thiram in immune colloid gold test paper detection vegetables
1, the pre-service of sample
Get sample that 5.0g smashs to pieces in the 50mL centrifuge tube, add phosphate buffer (1: 4) and the 0.50g Graphon (GCB) of 10mL acetone: 0.01MpH7.4, mechanical shaking extraction 5-10min is with the centrifugal 15min of 3000r/min, get supernatant, as test specimens liquid.
2, detect
0.01MpH7.4 phosphate buffer (formula: NaCl 8g, KCl 0.2g, Na
2HPO
412H
2O 2.9g, KH
2PO
40.2g, be settled to 1000ml with distilled water) and sample to be checked respectively get 80 μ l and add on colloid gold test paper, observations after 20 minutes.
3, result is judged
As shown in Figure 3, if band does not appear in testing sample detection paper line, simultaneously red stripes occurs on nature controlling line and be judged as positive findings, namely in testing sample the concentration of thiram higher than 500ppb (Fig. 3 B); If red stripes has appearred in testing sample detection paper line, simultaneously red stripes occurs on nature controlling line and be judged as negative findings (Fig. 3 A), namely in testing sample the concentration of thiram lower than 500ppb; If do not occur red stripes on nature controlling line, this test paper invalid (Fig. 3 C, Fig. 3 D).
Embodiment 5: add the analog detection test of thiram in the tomato sample
Add respectively the thiram standard solution in the tomato sample, make that in the tomato sample, thiram concentration is respectively 0ppb, 1000ppb.Press the described method of embodiment 4, respectively 0ppb and 1000ppb thiram tomato sample are detected with Test paper of the present invention, repeat 3 times, the result demonstration, red stripes appears in 0ppb sample detection line, the negative sample of interpretation; And red stripes does not appear in 1000ppb sample detection line, is judged to positive.Illustrate that Test paper of the present invention can be used for the thiram that detects in vegetables (for example tomato) residual.
Although the specific embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that.According to disclosed all instructions, can carry out various modifications and replacement to those details, these change all within protection scope of the present invention.Four corner of the present invention is provided by claims and any equivalent thereof.
Claims (18)
1. hybridoma cell strain, its deposit number is CGMCC No.3580, preservation date on 01 13rd, 2010, depositary institution is China Committee for Culture Collection of Microorganisms's common micro-organisms center.
2. anti-thiram monoclonal antibody, its hybridoma cell strain by claim 1 produces.
3. composition that detects thiram, it contains anti-thiram monoclonal antibody claimed in claim 2.
4. test paper that detects thiram, it contains the anti-thiram monoclonal antibody claimed in claim 2 of effective dose, described anti-thiram monoclonal antibody colloid gold label; Described test paper has the detection line that thiram-carrier protein couplet thing consists of; Described test paper also has the nature controlling line that dynamics consists of; And described test paper comprises:
Sample pad (1), pad (2), protein combination film (3), adsorptive pads (4) and backing (7), and described sample pad (1), pad (2), protein combination film (3), adsorptive pads (4) along continuous straight runs stick on backing (7) successively;
It is characterized in that,
Described pad is coated with anti-thiram monoclonal antibody claimed in claim 2 on (2), described anti-thiram monoclonal antibody colloid gold label;
Be coated with respectively the detection line (5) of thiram-carrier protein couplet thing formation and the nature controlling line (6) that dynamics consists of on described protein combination film (3), described detection line (5) is 5-8mm with the spacing of nature controlling line (6).
5. test paper according to claim 4, wherein, described protein combination film is selected from nitrocellulose filter or pvdf membrane.
6. test paper according to claim 4, wherein, described protein combination film is nitrocellulose filter.
7. test paper according to claim 4, wherein, described backing is the PVC backing.
8. test paper according to claim 4, wherein, described carrier protein is selected from human serum albumins, bovine serum albumin(BSA) or ovalbumin.
9. test paper according to claim 4, wherein, described carrier protein is bovine serum albumin(BSA).
10. the preparation method of test paper claimed in claim 4, comprise the steps:
1) with thiram and carrier protein couplet, form thiram-carrier protein couplet thing;
2) prepare anti-thiram monoclonal antibody claimed in claim 2 with hybridoma cell strain claimed in claim 1;
3) preparation dynamics;
4) with trisodium citrate and gold chloride reaction preparation collaurum;
5) with step 2) in the preparation anti-thiram monoclonal antibody add step 4) preparation collaurum in, obtain anti-thiram monoclonal antibody-colloid gold label thing;
6) anti-thiram monoclonal antibody-colloid gold label thing is coated on pad (2);
7) thiram-carrier protein couplet thing is coated on the upper formation of protein combination film (3) detection line (5); And will resist mouse IgG to be coated on the upper formation of protein combination film (3) nature controlling line (6);
8) adhere to successively sample pad (1), pad (2), protein combination film (3), adsorptive pads (4) at the upper along continuous straight runs of described backing (7).
11. method according to claim 10, wherein, described carrier protein is selected from human serum albumins, bovine serum albumin(BSA) or ovalbumin.
12. method according to claim 10, wherein, described carrier protein is bovine serum albumin(BSA).
13. method according to claim 10, wherein, step 3) the described dynamics in is rabbit anti-mouse igg antibody; Described protein combination film (3) is selected from nitrocellulose filter or pvdf membrane.
14. method according to claim 10, wherein, described protein combination film (3) is nitrocellulose filter.
15. method according to claim 10, wherein, described backing (7) is the PVC backing.
16. an ELISA reagent that detects thiram, its with antibody claimed in claim 2 as first antibody.
17. the application of antibody claimed in claim 2 in detecting thiram.
18. the application in the thiram of antibody claimed in claim 2 in detecting vegetables or fruit.
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