CN104327186B - Anti-bifenthrin monoclonal antibody and application thereof - Google Patents
Anti-bifenthrin monoclonal antibody and application thereof Download PDFInfo
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- CN104327186B CN104327186B CN201410359926.XA CN201410359926A CN104327186B CN 104327186 B CN104327186 B CN 104327186B CN 201410359926 A CN201410359926 A CN 201410359926A CN 104327186 B CN104327186 B CN 104327186B
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Abstract
The invention relates to an anti-bifenthrin monoclonal antibody and an application thereof and especially provides a monoclonal antibody capable of specifically recognizing bifenthrin, a hybrid tumor generating the monoclonal antibody and the application of the monoclonal antibody. The invention belongs to the field of biology. A method includes following steps: immunizing BALB/c female mice by an artificial antigen; performing cell fusion with myeloma cell SP2/0 and mice spleen cells to prepare a hybridoma; obtaining a hybridoma strain which can stably secret the anti-bifenthrin monoclonal antibody; injecting the hybridoma strain into abdomen of the mice to prepare ascites; purifying the ascites in an octanoic acid-ammonium sulfate manner to obtain the specific anti-bifenthrin specifically monoclonal antibody. The monoclonal antibody does not cross-react with other compounds. ELISA method established by the antibody can be used for quickly, sensitively, conveniently and cheaply detecting residual bifenthrin in environment and agricultural products.
Description
Technical field
The present invention relates to a kind of anti-Biphenthrin monoclonal antibody, especially a kind of can specific recognition Biphenthrin list
Clonal antibody, produces the hybridoma of described monoclonal antibody, and the purposes of this monoclonal antibody, belongs to biology techniques neck
Domain.
Background technology
Pyrethroid (pyrethroids) insecticide is to be copied according to the chemical constitution of natural pyrethrum
One class ultra high efficiency insecticide, because its insecticidal spectrum is wide, effect is good, low-residual, no cumulative action the advantages of, apply day over nearly 30 years
Benefit is universal.Pyrethoid insecticides is mainly used in killing the insect on the crops such as Cotton Gossypii, vegetable, fruit tree, Folium Camelliae sinensis, is one
Plant the insecticide of broad-spectrum high efficacy.Biphenthrin esterification scientific name is referred to as (1r, s)-cis-(z) -3- (chloro- 3,3, the 3- tri- fluoro- 1- of 2-
Acrylic) -2,2- dimethyl-cyclopropane-carboxylic acid -2- methyl -3- phenyl benzyl ester, Biphenthrin (bifenthrin) is as a kind of
Strong drive Coptotermes formosanus Shtrari. medicament, has prevention, goes out and control double effectses, is arranged by Chinese Wu Xie termite control Professional Committee in calendar year 2001
For one of pre- 5 kinds of termite-proof medicaments of urban house.While ensureing the Biphenthrin formulation rate as soil chemistry barrier,
Also the potential risk that its application exists to be prevented it is therefore desirable to set up a kind of sensitive, quick, selective for Biphenthrin
Method for detecting residue.
The method for detecting residue of Biphenthrin adopts instrument analytical method, such as gas chromatography mostly at present.These
Instrument analytical method is sensitive and accurate, but detecting instrument is expensive, sample pre-treatments are cumbersome, detection is wasted time and energy, it is big to be difficult to satisfaction
The needs of the amount fast and convenient detection of sample.
Immunologic detection method has the advantages that quick, cheap, easy, sensitive, special, in a large amount of sample rapid screening with now
Unique advantage is shown in the monitoring of field.Enzyme immunoassay (elisa) is that enzymic catalytic reaction and immunoreation are combined by one kind
Immune labeled determination techniques, have the high sensitivity of enzymic catalytic reaction and the high specific of antigen antibody reaction, in sensitivity side
There is very big advantage in face.Elisa, as a kind of detection method of rapid sensitive, has obtained relatively in the analysis of many small molecule immune
Ripe application.By preparation for the specific antibody of Biphenthrin, set up Biphenthrin immunological detection method.This invention
Completing of method, will solve Biphenthrin Antibody preparation key technology, set up the elisa Fast Detection Technique of Biphenthrin.Should
Invent and provide effective technological means and detection method for the soil chemistry barrier setting up preventing and treating Coptotermes formosanus Shtrari., there is important reality meaning
Adopted and important society, economic worth.
Content of the invention
The purpose of the present invention is to overcome the deficiencies in the prior art, provide a kind of anti-Biphenthrin monoclonal antibody and
It carries out the purposes of immune detection to the Biphenthrin in environment or agricultural product.
The technical scheme providing according to the present invention, described anti-Biphenthrin monoclonal antibody, is cctcc by deposit number
The hybridoma cell strain 3d4 of no.c2014135 produces, and depositary institution is China typical culture collection center, and address is China
Wuhan Wuhan University, preservation date is on July 11st, 2014.
Described hybridoma cell strain 3d4, its deposit number is cctcc no.c2014135.
The preparation method of described anti-Biphenthrin monoclonal antibody, is characterized in that, comprise the following steps:
(1) the immune hapten conjugation bsa shown in formula () is prepared immunogen；
(2) use immunogen immune balb/c mice, immunizing dose is 100 μ l immunogens/only, immunity five times altogether；Exempt from first
Epidemic disease, by isopyknic immunogen and Freund's complete adjuvant emulsifying, lumbar injection, uses equal-volume immunizing antigen and Freund not complete afterwards
Full adjuvant emulsion；From the beginning of third time immunity, tail vein blood after immunity every time, measure potency；
(3) after titer plateaus, Mouse spleen cells best with above-mentioned potency for myeloma cell sp2/0 are carried out cell
Merge, the ratio of spleen cell and myeloma cell sp2/0 is 10:l, prepares hybridoma；
(4) hybridoma of energy stably excreting anti-Biphenthrin monoclonal antibody is obtained by series screening and sub-clone
Strain；Again this hybridoma cell strain is injected and ascites in mice abdomen, is obtained；
(5) adopt caprylic acid-ammonium purified mouse ascites, obtain described monoclonal antibody.
Described monoclonal antibody is used for the application in the detection instrument detecting Biphenthrin in preparation.Described detection instrument
For test kit, chip or reagent paper.
The method have the advantages that (1) is novel: anti-Biphenthrin monoclonal antibody is domestic and international reported first；
(2) practical: using the ic-elisa that the anti-Biphenthrin monoclonal antibody that the present invention provides is realized have simple to operate quick (only
Need 2-3 hour), analysis cost is low, capacity is big, safe and reliable for analysis, easy penetration and promotion, is particularly well-suited to batch samples
Detection and field monitoring, can be complemented one another with traditional instrument analysis method；(3) high specificity: the antibody specificity of generation is known
Other Biphenthrin, does not have obvious cross reaction with other pyrethroid insecticideses；(4) accuracy is high: utilizes the present invention
TIANZHU XINGNAO Capsul in pedotheque for the ic-elisa that the anti-Biphenthrin monoclonal antibody providing is realized is 83.5-
104.7%, the coefficient of variation is less than 15.0%, meets retention analysiss standard；(5) sensitivity is high: anti-using present invention offer
In the suppression of ic-elisa that benzene chrysanthemum ester monoclonal antibody is realized, concentration (ic50) is 0.05mg/l, and test limit (ic10, lod) is
0.004mg/l, the range of linearity is 0.004-0.7mg/l.
Brief description
Fig. 1 detects the curve of Biphenthrin, combination rate and bifenthrin concentration for elisa；Abscissa is the concentration of biphenyl,
Unit is mg/l；Vertical coordinate is combination rate, and unit is %.
Specific embodiment
With reference to specific embodiment, the invention will be further described.
Embodiment one: the preparation of anti-Biphenthrin monoclonal antibody
1st, mouse immune:
(1) the immune hapten conjugation bsa (bovine serum albumin) shown in formula () is prepared immunogen；
(2) initial immunity: take 6～8 week old health balb/c female mices, the immunogen preparing is buffered with pbs
Liquid is mixed with equal-volume Freund's complete adjuvant (fca) after being configured to 1mg/ml, fully emulsified after, every mice is through lumbar injection
200 μ l (i.e. 100 μ g immunogen)；
(3) booster immunization: initial immunity, after three weeks, takes immunogen (with dosage such as first immunisation) and isopyknic Freund
Freund's incomplete adjuvant (fia) mix, fully emulsified after, every mice is through lumbar injection 200 μ l, later every two weeks, booster immunization
Once, immunity 4 times altogether；Before carrying out cell fusion, to injected in mice immunogen, injection dosage is identical with first immunisation dosage for 3d
(without any adjuvant), concrete immunization protocol is shown in Table 1.
Table 1
2nd, sero-fast preparation and merge mice selection: balb/c mice from the beginning of third time immunity, every time immunity 7～
Carry out mouse tail vein blood sampling after 10 days, place 45min for 37 DEG C and place 30min after 4 DEG C of refrigerators, 12000rpm is centrifuged 10min,
The supernatant obtaining is antiserum.Non-immunized balb/c mice is separately taken to obtain negative serum as comparison.With indirectly non-
Competitive enzyme-linked immune adsorption analyses method measures sero-fast potency (extension rate of serum is potency), after the 5th immunity,
The mice selecting potency best carries out cell fusion.
3rd, cell fusion: after immune antiserum titre is stable, by myeloma cell sp2/0 good for growth conditions with upper
State the best Mouse spleen cells of antiserum titre and carry out cell fusion and prepare hybridoma.By series screening and sub- gram
Grand, finally obtain one plant can stably excreting anti-Biphenthrin monoclonal antibody hybridoma cell strain, then by this hybridoma
Ascites is obtained in strain injection mice abdomen, after obtaining ascites, antibody is purified using caprylic acid-ammonium.
3.1st, the detailed process of cell fusion is as follows:
L () will about 1 × 108Individual splenocyte and 1 × 107Individual myeloma cell sp2/0 (ratio is about 10:l) is mixed in 50ml
In centrifuge tube, 1000rpm is centrifuged l0min, abandons supernatant, then be washed once with dmem basic culture solution；
(2) abandon supernatant, remain in the culture fluid of the centrifugation mouth of pipe with dropper exhaustion, in order to avoid impact peg (Polyethylene Glycol) is dense
Degree；
(3) gently bounce off the sedimentation cell group of heart bottom of the tube with finger, make precipitation loose uniformly, subsequently centrifuge tube is placed in
In 37 DEG C of water-baths；
(4) accurately draw the 50%peg1500 of 1ml37 DEG C of preheating with syringe, in 60s, 1mlpeg is added drop-wise to centrifugation
Guan Zhong, using first rear slowly fast principle, side edged gently rocks, and adds standing lmin；
(5) and then be added dropwise over the dmem basic culture solutions of 37 DEG C of preheatings, terminate peg effect, in accordance with first slowly after fast former
Then drop to 30ml, 37 DEG C of standing 10min, 800rpm are centrifuged 8min, abandon supernatant；
(6) with 5ml hat culture fluid, cell is gently suspended, make sure to keep in mind firmly to blow and beat, in order to avoid make to merge
Cell scatter；
(7) add hat culture fluid to 60ml, be sub-packed in and be covered with advance in feeder cells 96 porocyte culture plates.Every hole adds
Enter 100 μ l, medical adhesive tape edge sealing labelling, be placed in 37 DEG C, 5%co2Incubator in cultivate.
The incubation of the hybridoma that above-mentioned steps obtain is: the cell after merging is placed in 37 DEG C, 5%co2Training
Cultivate in foster case, check within second day polluting.After fusion, 5～7d changes liquid first, and according to circumstances every 3～4d changes culture
Liquid once, changes and sucks 1/2～2/3 culture fluid during liquid, adds the fresh culture fluid of equivalent.Culture fluid used should be according to culture
The difference of time and different.5～7d hat culture fluid after fusion, 7～14d ht culture fluid swap out hat culture
Liquid, uses common cell complete culture solution instead after three weeks.
3.2nd, the detailed process of screening is as follows:
(1) non-competing Indirect Elisa primary dcreening operation: with certain density coating antigen (immune hapten conjugation ovalbumin) bag
By ELISA Plate, 3% defatted milk powder closing, take the culture supernatant of hybridoma, tentatively carried out using non-competing Indirect Elisa
(wherein one resists for hybridoma supernatant, with the serum of immune mouse as positive control, with myeloma cell's supernatant as the moon for screening
Property comparison), choose the cell hole that is positive, cell conditioned medium is retained for next step detection, after the Kong Ruo being negative is repeated once
Still then can give up for feminine gender.
(2) indirect competition elisa method screening: indirect competition elisa method is then adopted for the cell hole that previous step is positive
Further confirmed.The hole having obvious competitive inhibition reaction to Biphenthrin as produces anti-Biphenthrin antibody
Positive hole, should amplification culture carry out cloning immediately.The hole reacted for uncompetitive suppression is still identical after being repeated once
Then can the eliminating of result.
3.3rd, the monoclonal of positive hybridoma cell:
(1) a little cell suspension will be taken thin to 24 holes after the hybridoma pipettor of cloning is blown and beaten uniformly repeatedly
In born of the same parents' culture plate, then use 5～6 holes of culture fluid doubling dilution；
(2) after cell attachment grows, count the cell in several holes；
(3) according to cell counts, take out appropriate cell suspension from the hole of appropriate density and (hybridize containing about 100
Oncocyte) to 50ml centrifuge tube, culture fluid is mended to 20ml.Every hole 200 μ l is inoculated in 96 porocyte culture plates, notes grasping
Cell suspension to constantly be mixed during work, to prevent cell distribution irregular, make every hole in theory containing about 1 hybridoma；
(4) by culture plate be placed in 37 DEG C, saturated humidity, 5%co2Incubator in cultivate.General 7 days about can fall
Put the growth of basis of microscopic observation cell clone, note the quantity recording each hole cell growth status and recording Mei Kong population of cells；
(5) hole having cell growth to be detected in good time, method should for the cell hole being positive with 1.3.6.2
Take monoclonal hole to carry out cloning again as far as possible, repeat 2-3 time, until screening positive rate reaches 100%.
3.4th, the preparation of mouse ascites: balb/c female mice lumbar injection 0.5ml liquid paraffin sensitization, after 7～14 days
Injection 1～2 × 106Individual hybridoma.7～10 days about after inoculation, mouse peritoneal substantially expands, and is extracted with syringe abdominal cavity
Ascites, took every 1～3 day 1 time, and 4000rpm is centrifuged 15min, collected supernatant, and -20 DEG C of preservations are to be clean.
4th, the purification of monoclonal antibody: using caprylic acid-ammonium purified mouse ascites, specific operating procedure is as follows:
(1) mouse ascites dilute (0.06mm, ph 4.0) with 4 times of volume sodium-acetate buffers, adjust ph with 0.1mnaoh
To 4.5；
(2) under room temperature, Deca is sad (33ul/ml serum dilution) while stirring, continues stirring 30min, standing after adding
2h (4 DEG C), centrifugation 30min (4 DEG C, 10000r/min), collect supernatant；
(3) use pbs (0.1m, ph7.4) to dilute supernatant with 1:10, adjust ph to 7.4, pre-cooling 15min (4 with 1m naoh
DEG C), calculate overall solution volume；
(4) it is slowly added to (nh4)2so40.277g/ml, stirring while adding, it is further continued for stirring reaction 30min, stand 2h (4
℃)；
(5) centrifugation 30min (4 DEG C, 12000r/min), abandons supernatant, precipitate is molten with a small amount of pbs (0.01m, ph 7.4)
Solution, with dialysing 3 days in 4 DEG C containing normal saline, changes liquid 3～5 times daily；
(6) by dialysis after antibody subpackage, frozen in -20 DEG C.
The purified antibody titer made is 9.8 × 105.
5th, the mensure of antibody variable region amino acid sequence:
5.1 extracting mrna:
(1) collect the hybridoma in cell bottle, abandon supernatant, plus trizol reagent 1ml, piping and druming (observation: liquid immediately
Become viscous, cell takes off wall).
(2) cell pyrolysis liquid that each in the hole has been digested be drawn onto the 1.5ml that a depc (pyrocarbonic acid diethyl ester) processed from
In heart pipe, the chloroform 0.2ml of Jia Xinkai, jog 20 seconds.
(3) after room temperature stands 5 minutes, 12000rpm, 15min, 4 DEG C, centrifugation.Then take supernatant colourless aqueous phase to centrifuge tube
(depc was processed), plus the isopropanol that equal-volume is newly opened, reverse centrifuge tube for several times, stands 10 minutes under room temperature after mixing.
(4) 12000rpm, 10 minutes, 4 DEG C, centrifugation.Observe the white precipitate in ttom of pipe for total rna, supernatant discarded, 75% second
After alcohol 1.0ml washing (newly being prepared with depc water), 7500rpm, 5min, 4 DEG C of centrifugations, it is repeated twice washing.
(5) remove supernatant, put from blotting liquid with little suction pipe.Air dry precipitates 5～10 minutes, and depc processes water 20～30ul
Add, middle rifle beats, total rna is dissolved in 55～60 DEG C of water-baths for 10 minutes, survey od value.
(6) 1.2% sepharose electrophoresis, 155v, 30min.
5.2 reverse transcriptions: with primescripttm rt reagent kit with gdna eraser test kit
(takara) reverse transcription obtains cdna, and -20 DEG C frozen.
5.3dna fragment amplification and sequencing: with trans-tap enzyme reagent kit (the full formula in Beijing gold biology company limited), with
Cdna is template, carries out pcr amplification, obtains purpose dna fragment, being sequenced, (Beijing six directions Hua Da Gene science share is limited
Shanghai branch company of company).
Pcr condition: light chain: 95 DEG C, 5min---94 DEG C, 30s---49 DEG C, 30s---72 DEG C, 30s--- repetitive cycling 30
Secondary --- 72 DEG C, 8min---12 DEG C, terminate；
Heavy chain: 95 DEG C, 5min---94 DEG C, 30s---60 DEG C, 30s---72 DEG C, 30s--- repetitive cycling 30 times --- 72
DEG C, 8min---12 DEG C, terminate.
The determination of 5.4 antibody variable region amino acid sequences:
(1) heavy chain of antibody variable region amino acid sequence is the sequence shown in seq id no.1:
qvklqqsgpglvapsqslsiactvsglslsssgvswvrqppgkslewlgviwsdgrtnyhsalisrltiskdnsksq
vflnlnslqiddsatyycvqrrwggynmdywsqgttvtvss；
(2) light chain of antibody variable region amino acid sequence is the sequence shown in seq id no.2:
dieltqspssmyaslgervtltckashginsylswfqqkpgkspktliyranglidgvpsrfsgggfgqdfsltiss
leyedlgiyyclqyyefpwtfgggtkleikr.
6th, the foundation of Biphenthrin ic-elisa
6.1 Method And Principles:
Using indirect competition immune analysis method.It is coupled being coated hapten shown in formula () with ova (chicken ovalbumin)
Prepared complex is adsorbed on solid phase carrier (96 hole elisa Plates) as envelope antigen, is prepared into solid phase antigen, is subsequently adding
Pesticide to be measured and corresponding antibodies.Solid phase antigen, pesticide to be measured, it is at war with association reaction with antibody, pesticide concentration to be measured is many, quilt
Just few in conjunction with the antibody on solid phase antigen, on the contrary the antibody combining in solid phase antigen is many, and after reaction, addition ELIAS secondary antibody (can only
Combine with combining the antibody on solid phase antigen), finally carry out developing the color being measured with substrate, when amount of antibody one timing, plus
The pesticide volume to be measured entering is more, and the antibody being combined with solid phase antigen is fewer, and colour developing just weakens, and combination rate reduces, conversely, then showing
Color strengthens, and combination rate raises, thus can extrapolate to be measured according to the combination rate of the standard curve of known quantity pesticide and measuring samples
The concentration of pesticide.
6.2 antigen-antibody working concentrations:
The determination square formation titrimetry of ic-elisa antigen-antibody working concentration, selection od value is antigen-antibody when 1.0
Diluted concentration.Through experiment, envelope antigen concentration 0.25 μ g/ml, antibody concentration 1.02 μ g/ml is as the most suitable working concentration.
6.3 indirect competition immunoreation programs:
(1) it is coated: with cbs buffer (0.05mol/l, ph 9.6), the envelope antigen as shown in formula () is diluted to
Suitable concentration, 100 μ l/ holes add 96 hole elisa Plates (maxisorptm transparent polyethylene plate), and 37 DEG C are coated 2h；
(2) close: take out the ELISA Plate being coated, discard and be coated liquid, with the phosphate buffered solution of 0.5% tween 20
(pbst) after washing, add pbs (0.01m, the ph 7.4) confining liquid of 3.0% defatted milk powder with the dilution of pbs buffer solution
230 μ l/ holes, incubate 1h in 37 DEG C of incubators；
(3) it is loaded: the ELISA Plate after learn from else's experience closing and washing, add the Biphenthrin titer of series concentration or treat test sample
Product extracting solution 50 μ l/ hole, adds antibody diluent 50 μ l/ hole, simultaneously setting blank and negative control, 37 DEG C of incubations
1h；
(4) add ELIAS secondary antibody: discard in the hole liquid, washed with pbst solution.Add the sheep of horseradish peroxidase-labeled
Anti- 5000 times of diluents of Mus 100 μ l/ hole, 37 DEG C of incubation 1h, discard in the hole liquid, washed with pbst solution；
(5) chromogenic reaction: add tetramethyl benzidine (tmb)-h2o2Substrate solution 100 μ l/ hole, incubates in 37 DEG C of incubators
15min, with 50 μ l/ hole 2mol/l h2so4Terminating reaction.In imarktmLight absorption value under 490nm wavelength is measured on microplate reader
(a).
6.4 standard curves and sensitivity: the relation curve of combination rate and bifenthrin concentration is as shown in figure 1, according to combination
Rate obtains standard curve with the logarithm mapping of bifenthrin concentration, calculates concentration (ic in suppression50) and lowest detectable limit
(ic10, lod).Combination rate (b/bo, %) is calculated as follows: b/bo (%)=[(ax amin)/(amax amin)] × 100, its
In: ax is light absorption value during not dosing, and amax is the light absorption value of negative control, and amin is the light absorption value of blank.
The linear equation of standard curve is: b/bo (%)=- 0.3510logc+0.0454.
Bifenthrin concentration is in the range of 0.004～0.7mg/l, linear, and correlation coefficient is r2=0.9903,
ic50For 0.05mg/l, lod is 0.004mg/l.
The specificity of 6.5 antibody: the specificity of antibody refers to the ability of its homospecificity antigen binding and with this Antigens
Comparison like thing binding ability.Conventional cross reaction is as the major criterion evaluated.Cross reaction is less, and the specificity of antibody is got over
Good.
As shown in table 2, the antibody of preparation and other pyrethrin series bactericidal agents do not have cross reaction (cr% < 0.7%).From
And understand, prepared antibody specificity is strong, can be used for the analysis of Biphenthrin.
Table 2
| Pesticide | ic50(mg/l) | Cross reacting rate (%) |
| Biphenthrin | 0.05 | 100 |
| Grenade (ICI). | >7.5 | <0.7 |
| Time acid | >7.5 | <0.7 |
| Cypermethrin | >7.5 | <0.7 |
| Deltamethrin | >7.5 | <0.7 |
| Fenpropathrin | >7.5 | <0.7 |
| Blue or green penta chrysanthemum fat | >7.5 | <0.7 |
| Kresoxim-methyl | >7.5 | <0.7 |
| Tetramethrin | >7.5 | <0.7 |
The immune analysis method of the Biphenthrin residual that the present invention sets up meets pesticide residue analysis standard.The method can use
The residue detection of Biphenthrin in pedotheque, and pre-treating method is simple compared with instrument analytical method, suitable mass detection
And field monitoring.
<160> 2
<210>seq id no:1
<211> 118
<212> prt
<213>
<400> 1
gln val lys leu gln gln ser gly pro gly leu val ala pro ser gln
1 5 10 15
ser leu ser ile ala cys thr val ser gly leu ser leu ser ser ser
20 25 30
gly val ser trp val arg gln pro pro gly lys ser leu glu trp leu
35 40 45
gly val ile trp ser asp gly arg thr asn tyr his ser ala leu ile
50 55 60
ser arg leu thr ile ser lys asp asn ser lys ser gln val phe leu
65 70 75 80
asn leu asn ser leu gln ile asp asp ser ala thr tyr tyr cys val
85 90 95
gln arg arg trp gly gly tyr asn met asp tyr trp ser gln gly thr
100 105 110
thr val thr val ser ser
115
<210> seq id no: 2
<211> 108
<212> prt
<213>
<400> 2
asp ile glu leu thr gln ser pro ser ser met tyr ala ser leu gly
1 5 10 15
glu arg val thr leu thr cys lys ala ser his gly ile asn ser tyr
20 25 30
leu ser trp phe gln gln lys pro gly lys ser pro lys thr leu ile
35 40 45
tyr arg ala asn gly leu ile asp gly val pro ser arg phe ser gly
50 55 60
gly gly phe gly gln asp phe ser leu thr ile ser ser leu glu tyr
65 70 75 80
glu asp leu gly ile tyr tyr cys leu gln tyr tyr glu phe pro trp
85 90 95
thr phe gly gly gly thr lys leu glu ile lys arg
100 105
Claims (4)
1. a kind of anti-Biphenthrin monoclonal antibody, is produced by the hybridoma cell strain for cctcc no.c2014135 for the deposit number
Raw.
2. a kind of hybridoma cell strain, its deposit number is cctcc no.c2014135.
3. monoclonal antibody as claimed in claim 1 is used for the application in the detection instrument detecting Biphenthrin in preparation.
4. apply as claimed in claim 3, described detection instrument is test kit, chip or reagent paper.
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| CN105628922A (en) * | 2016-01-07 | 2016-06-01 | 无锡市白蚁防治中心 | Rapid gold-labeled test strip of bifenthrin and preparation method thereof |
| CN106754737A (en) * | 2016-12-20 | 2017-05-31 | 郑州伊美诺生物技术有限公司 | The method that pollution hybridoma is processed using mouse model |
| CN107356748A (en) * | 2017-07-17 | 2017-11-17 | 广东志道医药科技有限公司 | Biphenthrin colloidal gold immuno-chromatography test paper strip and its preparation and application |
| CN107505467A (en) * | 2017-07-18 | 2017-12-22 | 宁夏医科大学 | ELISA detection reagents, kit, application and the detection method of pyrethroid residual |
| CN108840933A (en) * | 2018-07-16 | 2018-11-20 | 夏泉 | A kind of preparation method of Lamotrigine monoclonal antibody |
| CN109942710B (en) * | 2018-12-25 | 2021-06-18 | 苏州快捷康生物技术有限公司 | Biphenthrin monoclonal antibody and preparation method and application thereof |
| CN112779225B (en) * | 2021-03-17 | 2022-10-18 | 江南大学 | Hybridoma cell strain capable of secreting kresoxim-methyl monoclonal antibody and application thereof |
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| CN101135683A (en) * | 2007-10-16 | 2008-03-05 | 南京农业大学 | Bifenthrin antigen, antibody and uses thereof |
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