CN104327186A - Anti-bifenthrin monoclonal antibody and application thereof - Google Patents

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

Anti-bifenthrin monoclonal antibody and uses thereof

Technical field

The present invention relates to a kind of anti-bifenthrin monoclonal antibody, especially a kind of can the monoclonal antibody of specific recognition bifenthrin, produce the hybridoma of described monoclonal antibody, and the purposes of this monoclonal antibody, belong to biology techniques field.

Background technology

Pyrethroid (Pyrethroids) sterilant is chemical structure according to natural pyrethrum and the class ultra-high efficiency sterilant copied, because its insecticidal spectrum is wide, effective, low residue, without advantages such as cumulative effects, applies more prevalent over nearly 30 years.Pyrethoid insecticides is mainly used in killing the insect on the farm crop such as cotton, vegetables, fruit tree, tealeaves, is a kind of sterilant of broad-spectrum high efficacy.Bifenthrin esterification formal name used at school is called (1R, S)-cis-(Z)-3-(2-chloro-3,3, the fluoro-1-propenyl of 3-tri-)-2,2-dimethyl-cyclopropane-carboxylic acid-2-methyl-3-phenyl benzyl ester, bifenthrin (Bifenthrin) as a kind of strongly drive termite medicament, there is prevention, go out and control double effects, be classified as one of 5 kinds of termite-proof in advance medicaments of urban house in calendar year 2001 by Chinese Wu Xie termite control Professional Committee.While ensureing bifenthrin formulation rate as soil chemistry barrier, the potential risk that its application also will be prevented to exist, therefore need to set up a kind of for bifenthrin sensitive, fast, optionally method for detecting residue.

The method for detecting residue of current bifenthrin adopts instrument analytical method mostly, as gas chromatography.These instrument analytical methods are sensitive and accurate, but detecting instrument is expensive, sample pre-treatments is more loaded down with trivial details, detect the needs of the fast and convenient detection of satisfied a large amount of sample of wasting time and energy, be difficult to.

Immunologic detection method has fast, cheap, easy, sensitive, special advantage, in a large amount of sample rapid screening and field monitoring, demonstrate unique advantage.Enzyme immunoassay (ELISA) is a kind of immune labeled determination techniques enzymic catalytic reaction and immune response combined, and has the highly sensitive of enzymic catalytic reaction and the high specific of antigen antibody reaction, has very large advantage in sensitivity.ELISA, as a kind of detection method of rapid sensitive, has obtained more ripe application in many small molecule immune analyses.By the specific antibody of preparation for bifenthrin, set up bifenthrin immunological detection method.Completing of this inventive method, prepares gordian technique by solution bifenthrin antibody, sets up the ELISA Fast Detection Technique of bifenthrin.This invention provides effective technique means and detection method for the soil chemistry barrier setting up control termites, has important practical significance and important society, economic worth.

Summary of the invention

The object of the invention is to overcome the deficiencies in the prior art, a kind of anti-bifenthrin monoclonal antibody is provided and the bifenthrin in environment or agricultural-food is carried out to the purposes of immunodetection.

According to technical scheme provided by the invention, described anti-bifenthrin monoclonal antibody, the hybridoma cell strain 3D4 being CCTCC No.C2014135 by deposit number produces, depositary institution is China typical culture collection center, address is Wuhan, China Wuhan University, and 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-bifenthrin monoclonal antibody, is characterized in that, comprise the following steps:

(1) the immune hapten conjugation BSA shown in formula I is prepared immunogen;

(2) use immunogen immune BALB/c mouse, immunizing dose is 100 μ L immunogens/only, immunity five times altogether; First immunisation is by isopyknic immunogen and Freund's complete adjuvant emulsification, and abdominal injection, uses equal-volume immunizing antigen and Freund's incomplete adjuvant emulsification afterwards; From third time immunity, tail vein blood after each immunity, mensuration is tired;

(3) after titer plateaus, myeloma cell SP2/0 and above-mentioned best Mouse spleen cells of tiring are carried out cytogamy, and the ratio of spleen cell and myeloma cell SP2/0 is 10:l, prepares hybridoma;

(4) hybridoma cell strain of the anti-bifenthrin monoclonal antibody of energy stably excreting is obtained by series screening and subclone; Again by obtained ascites in this hybridoma cell strain injection mouse abdomen;

(5) adopt caprylic acid-ammonium purified mouse ascites, obtain described monoclonal antibody.

The application of described monoclonal antibody in the testing tool for the preparation of detection bifenthrin.Described testing tool is test kit, chip or test paper.

The present invention has following beneficial effect: (1) is novel: anti-bifenthrin monoclonal antibody is domestic and international reported first; (2) practical: the ic-ELISA utilizing anti-bifenthrin monoclonal antibody provided by the invention to realize have (only needing 2-3 hour) fast simple to operate, analysis cost low, analyze large, safe and reliable, the easy penetration and promotion of capacity, be specially adapted to batch samples detect and field monitoring, can complement one another with traditional instrument analytical procedure; (3) high specificity: the antibodies specific identification bifenthrin of generation, does not have obvious cross reaction with other pyrethroid insecticideses; (4) accuracy is high: the TIANZHU XINGNAO Capsul of ic-ELISA in pedotheque utilizing anti-bifenthrin monoclonal antibody provided by the invention to realize is 83.5-104.7%, and the variation coefficient, lower than 15.0%, meets retention analysis standard; (5) highly sensitive: in the suppression of the ic-ELISA utilizing anti-bifenthrin monoclonal antibody provided by the invention to realize, concentration (IC50) is 0.05mg/L, detectability (IC10, LOD) be 0.004mg/L, linearity range is 0.004-0.7mg/L.

Accompanying drawing explanation

Fig. 1 is that ELISA detects bifenthrin, the curve of combination rate and bifenthrin concentration; X-coordinate is the concentration of biphenyl, and unit is mg/L; Ordinate zou is combination rate, and unit is %.

Embodiment

Below in conjunction with specific embodiment, the invention will be further described.

Embodiment one: the preparation of anti-bifenthrin monoclonal antibody

1, mouse immune:

(1) the immune hapten conjugation BSA (bovine serum albumin) shown in formula I is prepared immunogen;

(2) initial immunity: get healthy BALB/c female mice in 6 ~ 8 week age, mix with equal-volume Freund's complete adjuvant (FCA) after the immunogen PBS damping fluid prepared is configured to 1mg/mL, after fully emulsified, every mouse is through abdominal injection 200 μ L (i.e. 100 μ g immunogens);

(3) booster immunization: initial immunity is after three weeks, get immunogen (with dosage such as first immunisation) and isopyknic Freund's incomplete adjuvant (FIA) mixes, after fully emulsified, every mouse is through abdominal injection 200 μ L, later every two weeks, booster immunization once, is total to immunity 4 times; Before carrying out cytogamy, 3d is to injected in mice immunogen, and injected dose identical with first immunisation dosage (not containing any adjuvant), concrete immunization protocol is in table 1.

Table 1

2, the selection of sero-fast preparation and fusion mouse: BALB/c mouse is from third time immunity, mouse tail vein blood sampling is carried out after each immune 7 ~ 10 days, place the centrifugal 10min of 30min, 12000rpm in 4 DEG C of refrigerators after 37 DEG C of placement 45min, the supernatant liquor obtained is antiserum(antisera).The BALB/c mouse of separately getting without immunity obtains negative serum in contrast.With indirect non-competing Enzyme-Linked Immunosorbent Assay sero-fast tire (extension rate of serum is and tires), after the 5th immunity, best mouse of tiring is selected to carry out cytogamy.

3, cytogamy: after immune antiserum titre is stable, carries out cytogamy by myeloma cell SP2/0 good for growth conditions and the best Mouse spleen cells of above-mentioned antiserum titre and prepares hybridoma.Screened and subclone by series, finally obtain the hybridoma cell strain of the anti-bifenthrin monoclonal antibody of a strain energy stably excreting, then by obtained ascites in this hybridoma cell strain injection mouse abdomen, after obtaining ascites, adopt caprylic acid-ammonium purification antibody.

3.1, the detailed process of cytogamy is as follows:

L () is by about 1 × 10 ⁸individual splenocyte and 1 × 10 ⁷individual myeloma cell SP2/0 (ratio is about 10:l) is mixed in 50mL centrifuge tube, and the centrifugal l0min of 1000rpm, abandons supernatant liquor, then washs once with DMEM basic culture solution;

(2) abandon supernatant, remain in the nutrient solution of the centrifugal mouth of pipe with dropper exhaustion, in order to avoid affect the concentration of PEG (polyoxyethylene glycol);

(3) play the sedimentation cell group bottom centrifuge tube gently with finger, make precipitation evenly loose, centrifuge tube is placed in 37 DEG C of water-baths subsequently;

(4) accurately draw the 50%PEG1500 of 1mL37 DEG C of preheating with syringe, be added drop-wise in centrifuge tube in 60s by 1mLPEG, adopt first slow rear fast principle, limit edged rocks gently, adds standing lmin;

(5) then dropwise add the DMEM basic culture solution of 37 DEG C of preheatings, stop PEG effect, drop to 30mL in accordance with first slow rear fast principle, 37 DEG C of centrifugal 8min of standing 10min, 800rpm, abandon supernatant;

(6) with 5mL HAT nutrient solution, cell is suspended gently, make sure to keep in mind firmly to blow and beat, in order to avoid make the cell dispersal merged open;

(7) add HAT nutrient solution to 60mL, be sub-packed in and be covered with in advance in feeder cell 96 porocyte culture plate.Every hole adds 100 μ L, and medical adhesive tape edge sealing marks, and is placed in 37 DEG C, 5%CO ₂incubator in cultivate.

The culturing process of the hybridoma that above-mentioned steps obtains is: the cell after merging is placed in 37 DEG C, 5%CO ₂cultivate in incubator, inspection in second day has pollution-free.After fusion, 5 ~ 7d changes liquid first, and according to circumstances every 3 ~ 4d changes nutrient solution once, sucks 1/2 ~ 2/3 nutrient solution, add the nutrient solution that equivalent is fresh when changing liquid.Nutrient solution used should be different according to the difference of incubation time.5th ~ 7d HAT nutrient solution after fusion, 7th ~ 14d HT nutrient solution swaps out HAT nutrient solution, uses common cell complete culture solution after three weeks instead.

3.2, the detailed process of screening is as follows:

(1) non-competing indirect elisa method primary dcreening operation: with certain density coating antigen (immune hapten conjugation ovalbumin) coated elisa plate, 3% skim-milk is closed, get the culture supernatant of hybridoma, (wherein primary antibodie is hybridoma supernatant to adopt non-competing indirect elisa method tentatively to carry out screening, with the serum of immune mouse for positive control, with myeloma cell's supernatant for negative control), choose the cell hole be positive, cell conditioned medium is retained and is used for next step detection, if the hole of being negative repeats to be once still feminine gender, can give up.

(2) indirect competitive ELISA method screening: the cell hole be positive for previous step then adopts indirect competitive ELISA method further to confirm.The positive hole producing anti-bifenthrin antibody is to the hole that bifenthrin has obvious competitive inhibition to react, should enlarged culturing carry out cloning immediately.Then can eliminate still for identical result is once repeated in hole for uncompetitive inhibited reaction.

3.3, the mono-clonal of positive hybridoma cell:

(1) getting repeatedly blowing and beating until the hybridoma pipettor of cloning evenly in a little cell suspension to 24 porocyte culture plate, then using nutrient solution doubling dilution 5 ~ 6 holes;

(2) when after cell attachment growth, the cell in several hole is counted;

(3) according to cell counts, from the hole of appropriate density, take out appropriate cell suspension (about containing 100 hybridomas) in 50mL centrifuge tube, nutrient solution is mended to 20mL.Every hole 200 μ l is inoculated in 96 porocyte culture plates, notes constantly to mix cell suspension in operating process, to prevent cell distribution irregular, makes every Kong Yuehan 1 hybridoma in theory;

(4) culture plate is placed in 37 DEG C, saturated humidity, 5%CO ₂incubator in cultivate.Within general about 7 days, can, at observation of cell clonal growth under inverted microscope, note recording each porocyte growing state and the quantity recording every porocyte group;

(5) to there being the hole of Growth of Cells to detect in good time, method should be got mono-clonal hole with 1.3.6.2 as far as possible for the cell hole be positive and be carried out cloning again, repeats 2-3 time, until screening positive rate reaches 100%.

3.4, the preparation of mouse ascites: BALB/c female mice abdominal injection 0.5mL whiteruss sensitization, injected 1 ~ 2 × 10 after 7 ~ 14 days ⁶individual hybridoma.Latter about 7 ~ 10 days of inoculation, mouse peritoneal obviously expands, and extracts ascites, get 1 time, the centrifugal 15min of 4000rpm every 1 ~ 3 day with syringe abdominal cavity, and collect supernatant liquor ,-20 DEG C of preservations are to be clean.

4, the purifying of monoclonal antibody: adopt caprylic acid-ammonium purified mouse ascites, concrete operation steps is as follows:

(1) mouse ascites 4 times of volume sodium-acetate buffers dilute (0.06mM, pH 4.0), regulate pH to 4.5 with 0.1MNaOH;

(2) drip while stirring under room temperature sad (33uL/mL serum dilution), add rear continuation and stir 30min, leave standstill 2h (4 DEG C), centrifugal 30min (4 DEG C, 10000r/min), collect supernatant liquor;

(3) dilute supernatant liquor with PBS (0.1M, pH7.4) with 1:10, adjust pH to 7.4, precooling 15min (4 DEG C) with 1M NaOH, calculate overall solution volume;

(4) (NH is slowly added ₄) ₂sO ₄0.277g/mL, limit edged stirs, then continues stirring reaction 30min, leaves standstill 2h (4 DEG C);

(5) centrifugal 30min (4 DEG C, 12000r/min), abandons supernatant liquor, and throw out a small amount of PBS (0.01M, pH 7.4) dissolves, and dialyses 3 days, change liquid every day 3 ~ 5 times with containing physiological saline in 4 DEG C;

(6) by the antibody packing after dialysis, frozen in-20 DEG C.

The purified antibody titer made is 9.8 × 10 ⁵.

5, the mensuration of antibody variable region amino acid sequence:

5.1 extract mRNA:

(1) hybridoma in collecting cell bottle, abandons supernatant, adds TRIZOL reagent 1ml, blows and beats immediately (observing: liquid becomes sticky thick, and cell takes off wall).

(2) digested for each hole good cell pyrolysis liquid is drawn onto in the 1.5ml centrifuge tube that a DEPC (diethylpyrocarbonate) processed, the chloroform 0.2ml of Jia Xinkai, jog 20 seconds.

(3) room temperature left standstill after 5 minutes, and 12000rpm, 15min are 4 DEG C, centrifugal.Then get the colourless aqueous phase of supernatant to centrifuge tube (DEPC process), add the Virahol that equal-volume is newly opened, put upside down centrifuge tube for several times, left at room temperature 10 minutes after mixing.

(4) 12000rpm is 10 minutes, 4 DEG C, centrifugal.Observe the white precipitate of total serum IgE at the bottom of pipe, supernatant discarded, after 75% ethanol 1.0ml washs (newly preparing with DEPC water), 7500rpm, 5min, 4 DEG C centrifugal, repeats twice washing.

(5) remove supernatant, put from, blot liquid with little suction pipe.Gas does precipitation 5 ~ 10 minutes, and DEPC process water 20 ~ 30ul adds, and middle rifle beats, and 55 ~ 60 DEG C of water-baths dissolve total serum IgE in 10 minutes, survey OD value.

(6) 1.2% agarose electrophoresiss, 155V, 30min.

5.2 reverse transcriptions: obtain cDNA with PrimeScriptTM RT reagent Kit with gDNA Eraser test kit (TaKaRa) reverse transcription ,-20 DEG C frozen.

5.3DNA fragment amplification and order-checking: with Trans-Tap enzyme reagent kit (the biological company limited of the full formula in Beijing gold), take cDNA as template, carry out pcr amplification, obtain target DNA fragment, carry out check order (Shanghai, Beijing Liuhe Huada Genomics Technology Co., Ltd branch office).

PCR condition: light chain: 95 DEG C, 5min---94 DEG C, 30s---49 DEG C, 30s---72 DEG C, 30s---recirculation 30 times---72 DEG C, 8min---12 DEG C, stops;

Heavy chain: 95 DEG C, 5min---94 DEG C, 30s---60 DEG C, 30s---72 DEG C, 30s---recirculation 30 times---72 DEG C, 8min---12 DEG C, stops.

The determination of 5.4 antibody variable region amino acid sequences:

(1) antibody heavy chain variable region aminoacid sequence is the sequence shown in SEQ ID No.1:

QVKLQQSGPGLVAPSQSLSIACTVSGLSLSSSGVSWVRQPPGKSLEWLGVIWSDGRTNYHSALISRLTISKDNSKSQVFLNLNSLQIDDSATYYCVQRRWGGYNMDYWSQGTTVTVSS；

(2) antibody chain variable region aminoacid sequence is the sequence shown in SEQ ID No.2:

DIELTQSPSSMYASLGERVTLTCKASHGINSYLSWFQQKPGKSPKTLIYRANGLIDGVPSRFSGGGFGQDFSLTISSLEYEDLGIYYCLQYYEFPWTFGGGTKLEIKR。

6, the foundation of bifenthrin ic-ELISA

6.1 Method And Principles:

Adopt indirect competition immune analysis method.The mixture wrapped shown in formula II by haptens and OVA (chicken ovalbumin) coupling obtain is adsorbed on solid phase carrier (96 hole enzyme plate) as envelope antigen, be prepared into solid phase antigen, then add agricultural chemicals to be measured and corresponding antibodies.Solid phase antigen, agricultural chemicals to be measured, to be at war with association reaction with antibody, pesticide concentration to be measured is many, the antibody be bonded on solid phase antigen is just few, otherwise the antibody being combined in solid phase antigen is many, ELIAS secondary antibody (can only combining with the antibody be combined on solid phase antigen) is added after reaction, finally carry out developing the color being measured with substrate, when antibody amount one timing, the pesticide volume to be measured added is more, the antibody be combined with solid phase antigen is fewer, colour developing just weakens, combination rate reduces, otherwise, then develop the color enhancing, combination rate raises, thus can according to the combination rate of the typical curve of known quantity agricultural chemicals and measuring samples, extrapolate the concentration of agricultural chemicals to be measured.

6.2 antigen-antibody working concentrations:

The determination square formation volumetry of ic-ELISA antigen-antibody working concentration, antigen-antibody weaker concn when selecting OD value to be 1.0.Through experiment, envelope antigen concentration 0.25 μ g/mL, antibody concentration 1.02 μ g/mL is as the suitableeest working concentration.

6.3 indirect competition immune response programs:

(1) bag quilt: with CBS damping fluid (0.05mol/L, pH 9.6) envelope antigen such as shown in formula II is diluted to optimal concentration, 100 μ L/ holes add 96 hole enzyme plates (MaxisorpTM transparent polyethylene plate), and 37 DEG C of bags are by 2h;

(2) close: take out bag by good enzyme plate, discard coating buffer, after phosphate buffer soln (PBST) washing of 0.5% tween 20, add the PBS (0.01M of the skim-milk of use PBS buffered soln to dilute 3.0%, pH 7.4) confining liquid 230 μ L/ hole, incubation 1h in 37 DEG C of incubators;

(3) application of sample: the enzyme plate of learning from else's experience after closing and washing, adds bifenthrin reference liquid or the testing sample extracting solution 50 μ L/ hole of series concentration, then add antibody diluent 50 μ L/ hole, arrange blank and negative control, 37 DEG C of incubation 1h simultaneously;

(4) add ELIAS secondary antibody: discard liquid in hole, use PBST solution washing.Add sheep anti mouse 5000 times of diluent 100 μ L/ holes of horseradish peroxidase-labeled, 37 DEG C of incubation 1h, discard liquid in hole, use PBST solution washing;

(5) color reaction: add tetramethyl benzidine (TMB)-H ₂o ₂substrate solution 100 μ L/ hole, incubation 15min in 37 DEG C of incubators, with 50 μ L/ hole 2mol/L H ₂sO ₄termination reaction.At iMark ^tMmicroplate reader measures the light absorption value (A) under 490nm wavelength.

6.4 typical curves and sensitivity: the relation curve of combination rate and bifenthrin concentration as shown in Figure 1, is mapped according to the logarithm of combination rate and bifenthrin concentration and namely obtained typical curve, calculate concentration (IC in suppressing ₅₀) and lowest detectable limit (IC ₁₀, LOD).Combination rate (B/Bo, %) calculate with following formula: B/Bo (%)=[(Ax – Amin)/(Amax – Amin)] × 100, wherein: light absorption value when Ax is not dosing, Amax is the light absorption value of negative control, and Amin is the light absorption value of blank.

The linear equation of typical curve is: B/Bo (%)=-0.3510LogC+0.0454.

Bifenthrin concentration is within the scope of 0.004 ~ 0.7mg/L, linear, and relation conefficient is R ²=0.9903, IC ₅₀for 0.05mg/L, LOD are 0.004mg/L.

The specificity of 6.5 antibody: the specificity of antibody refers to the ability that its homospecificity antigen combines and comparing with this antigen-analogues ability.Conventional cross reaction is as the major criterion evaluated.Cross reaction is less, and the specificity of antibody is better.

As shown in table 2, the antibody of preparation and other pyrethrin series bactericidal agents do not have cross reaction (CR%<0.7%).Thus known, prepared antibodies specific is strong, may be used for the analysis of bifenthrin.

Table 2

Agricultural chemicals	IC 50(mg/L)	Cross reacting rate (%)
			Bifenthrin	0.05	100
Lambda-cyhalothrin	>7.5	<0.7
			Time acid	>7.5	<0.7
Cypermethrin	>7.5	<0.7
			Deltamethrin	>7.5	<0.7
Fenvalerate	>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 that the bifenthrin that the present invention sets up remains meets pesticide residue analysis standard.The method can be used for the residue detection of bifenthrin in pedotheque, and pre-treating process comparatively instrument analytical method is simple, be applicable to 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 (5)

1. an anti-bifenthrin monoclonal antibody, the hybridoma cell strain being CCTCC No.C2014135 by deposit number produces.

2. a hybridoma cell strain, its deposit number is CCTCC No.C2014135.

3. a preparation method for anti-bifenthrin monoclonal antibody, is characterized in that, comprise the following steps:

(1) the immune hapten conjugation BSA shown in formula I is prepared immunogen;

(2) use immunogen immune BALB/c mouse, immunizing dose is 100 μ L immunogens/only, immunity five times altogether; First immunisation is by isopyknic immunogen and Freund's complete adjuvant emulsification, and abdominal injection, uses equal-volume immunizing antigen and Freund's incomplete adjuvant emulsification afterwards; From third time immunity, tail vein blood after each immunity, mensuration is tired;

(3) after titer plateaus, myeloma cell SP2/0 and above-mentioned best Mouse spleen cells of tiring are carried out cytogamy, and the ratio of spleen cell and myeloma cell SP2/0 is 10:l, prepares hybridoma;

(4) hybridoma cell strain of the anti-bifenthrin monoclonal antibody of energy stably excreting is obtained by series screening and subclone; Again by obtained ascites in this hybridoma cell strain injection mouse abdomen;

(5) adopt caprylic acid-ammonium purified mouse ascites, obtain described monoclonal antibody.

4. the application of monoclonal antibody as claimed in claim 1 in the testing tool for the preparation of detection bifenthrin.

5. applied research as claimed in claim 4, described testing tool is test kit, chip or test paper.