CN107607703B - Based on the remaining ELISA kit of nano antibody detection Fipronil and its application - Google Patents

Abstract

The invention discloses included box body, be located at the intracorporal ELISA Plate of box and reagent based on the remaining ELISA kit of nano antibody detection Fipronil and its application, the kit；Wherein, each hole of ELISA Plate is coated with Fipronil envelope antigen, and the reagent includes anti-Fipronil nano antibody, Fipronil standard solution, secondary antibody, buffer PBS, cleaning solution PBST, substrate solution, developing solution and the reaction terminating liquid of enzyme label etc..In detection process, the envelope antigen that adsorbs on ELISA Plate hole wall and Fipronil to be measured is vied each other and antibody response observes result by chromogenic reaction.It detects the Fipronil of known concentration and draws standard curve, the concentration of Fipronil to be measured can be extrapolated.It is an advantage of the invention that water, Fipronil residual in vegetables accurately can be detected delicately, the pretreatment process of sample is simple, and time-consuming is few, can detect a large amount of sample simultaneously, and sample detection cost is far below traditional instrument detection method.

Description

Based on the remaining ELISA kit of nano antibody detection Fipronil and its application

Technical field

The present invention relates to genetic engineering, display technique of bacteriophage and ELISA detection technique fields, specifically, being related to Based on the remaining ELISA kit of nano antibody detection Fipronil and its application.

Background technique

Fipronil (Fipronil) also known as fipronil, Frontline, Fipronil, which are that one kind is widely used, has three The N- Phenylpyrazole insecticides of methyl fluoride sulfonyl group, to the pest for endangering the staple crops such as grain, cotton, oil, fruits and vegetables, tealeaves There is good control efficiency.In the use process of pesticide, many pests are to machine phosphorus insecticide, carbamate chemicals for agriculture and intend Pyrethrins insecticide generates resistance.For such resistant insects, Fipronil also has preferable effect.Fipronil to aquatic products, Honeybee, silkworm, rodent, birds are toxic larger, medium to the toxicity of mammal, and Fipronil can damage mankind β 3GABAA receptor subunits lead to human nerve's developmental disorder, such as self-closing disease, angelman syndrome, epilepsy.Due to fluorine worm Nitrile is usually used in pet, equally exists risk during the mankind and pet contact.Due to the exposure in toxic environment, It is possible that the development of the brain and nervous system of meeting children and fetus.

After the high poisons such as acephatemet, parathion organophosphorus insecticide is limited or is forbidden to use, from 2009, agricultural Portion provides that Fipronil is removed as health and coating agent for seed etc., and limitation field uses.Although Fipronil is limited the use of, but still is had Violations of rules and regulations occur.Some pesticide sales companys publicize the use that Fipronil is used for crops, or it is illegally made an addition to it His pesticide has reached good vermins-proof effect.Since Fipronil half-life period is longer, easy residual, exceeded ask is remained in environment and food Topic gradually causes to pay close attention to.Therefore, reinforce the remaining detection of Fipronil, for scientifically and rationally using Fipronil, ensure food peace Complete and human health, reduces environmental pollution, and enhances the international competitiveness of China's agricultural and sideline product, keeps the sustainable development of China's agricultural Exhibition etc. is of great significance.

Fipronil method for detecting residue reported at present mainly has high performance liquid chromatography (HPLC), gas chromatography (GC) and chromatograph-mass spectrometer coupling (GC/LC-MS).Specialized laboratory, specialized instrument and equipment and specially are needed with these methods Industry operator, sample pre-treatments are complicated, and analysis cost is high, it is difficult to meet the needs of the fast slowdown monitoring in great amount of samples scene.20 generation It has recorded since the nineties, fast-developing immuno analytical method has been applied to pesticide residue analysis and environmental monitoring, has simplicity Quickly, the advantages that Cheap highly effective, high specificity, high sensitivity.

The residual enzyme-linked immunosorbent assay of Fipronil based on conventional antibody (polyclonal antibody and monoclonal antibody) Stability is relatively low, and the present invention is based on anti-Fipronil nano antibody, and using ELISA adsorption analysis method, heat is steady It is qualitative to be superior to polyclonal antibody analysis method.

Summary of the invention

The purpose of the present invention is for current pesticide residue instrument analytical method is at high cost, complex pretreatment, poor specificity, The deficiencies of sensitivity is low and is difficult to experimental field detection, providing a kind of has high specific, high sensitivity, high accuracy, high-precision Exactness, operating method are simple, and can be used for what batch samples quickly detected, analyze the remaining ELISA detection reagent of Fipronil Box.The remaining quick measurement of Fipronil suitable for the samples such as water, vegetables.

In order to achieve the object of the present invention, it is anti-for constructing cameloid phage display nanometer that present invention firstly provides one The general PCR primer of the C-terminal in body library, the nucleotide sequence of the primer is as shown in SEQ ID NO:7.

Then the present invention provides a kind of anti-Fipronil nano antibody, the amino acid sequence of the antibody such as SEQ ID NO:1 The shown or sequence is through replacement, missing or one or several amino acids formed amino acid sequences with same function of addition. For example, sequence shown in SEQ ID NO:1 to be removed to the amino acid sequence after the histidine tag of end 6.

The anti-Fipronil nano antibody can be prepared as follows: utilize the half of chemical reactive synthesis Fipronil Antigen 6- (3- cyano -5- (2,6- bis- chloro- 4- (trifluoromethyl) phenyl) the amyl- 1,3- diene -1- base of ring) amino) -6- oxo oneself Acid will be used as immunogene after haptens and keyhole limpet hemocyanin KLH coupling, and immunization experiment animal camel extracts periphery hemolymph The total serum IgE of cell clones nano antibody heavy chain (VHH) genetic fragment, is connected by digestion through reverse transcription and nest-type PRC, will Gene fragment clone to phagemid vector, highly effective iodine to Escherichia coli is saved through helper phage, and building obtains bacteriophage Nano antibody library filters out special Fipronil bacteriophage nano antibody and its expression and purification is obtained high sensitivity, specificity Strong anti-Fipronil nano antibody.The nano antibody molecule of preparation is small, soluble strong, and high temperature resistant, easy purification is easily expressed.

The remaining ELISA detection kit of analysis Fipronil provided by the invention, including box body, be located in box body it is detachable ELISA Plate and be located at the intracorporal reagent of box.Wherein, each hole of the ELISA Plate is coated with Fipronil envelope antigen, described Reagent includes secondary antibody, the buffer PBS, cleaning solution of the anti-Fipronil nano antibody, Fipronil standard solution, enzyme label PBST, developing solution (A liquid), developing solution (B liquid) and reaction terminating liquid etc..

The Fipronil envelope antigen is haptens 6- (3- cyano -5- (2,6- bis- chloro- 4- (trifluoromethyl) phenyl) ring Amyl- 1,3- diene -1- base) amino) -6- oxo caproic acid and bovine serum albumin coupled complex, the preparation of the envelope antigen Method is as follows:

(1) claim 8.8mg (40.15mmol) 6- (3- cyano -5- (2,6- bis- chloro- 4- (trifluoromethyl) phenyl) amyl- 1,3- of ring Diene -1- base) amino) -6- oxo caproic acid (MW=440.15), 2.65mg (0.024mmol) NHS (n-hydroxysuccinimide, MW=115), 4.8mg (0.023mmol) DCC (dicyclohexylcarbodiimide, MW=206) is dissolved in 200 μ L anhydrous DMF (N- formyls Morpholine) in, it is stirred to react at room temperature overnight.Reaction solution is centrifuged (5000rpm, 10min), abandons precipitating, supernatant is active ester, That is 6- (3- cyano -5- (2,6- bis- chloro- 4- (trifluoromethyl) phenyl) the amyl- 1,3- diene -1- base of ring) amino) -6- oxo caproic acid.

(2) claim 20mg BSA (bovine serum albumin, MW=67000) be dissolved in 2mL carbonate buffer solution (0.05mol/mL, PH9.5 150 μ L active ester liquid are added dropwise in), under stirring, when dropwise addition is slow, and about 20~30min is added.Then at room temperature after It is continuous to be stirred to react 4~6h.

(3) reaction solution is packed into bag filter, is dialysed with PBS (0.01mol/L pH7.4).Every 6~8h is changed the liquid once, and is changed altogether Liquid 5~6 times.It is centrifuged after dialysis, abandons precipitating, supernatant is taken, as antigen coat liquid.The ELISA Plate is 96 hole elisa Plates, packet It is 180ng/mL by the peridium concentration of antigen.

The concentration of the nano antibody is about 124ng/mL.The secondary antibody of the enzyme label is horseradish peroxidase-labeled Anti- HA tag antibody, concentration are 0.1 μ g/mL.Purchased from Abcam company, goods number: ab1265.

The developing solution A liquid is by urea peroxide 1g, citric acid 10.3g, Na₂HPO₄·12H₂O35.8g, 100 μ of Tween-20 L and distilled water 1000mL are formulated, pH value 5.

The developing solution B liquid is by tetramethyl benzidine 700mg, DMSO 40mL, citric acid 10.3g and distilled water 1000mL It is formulated, pH value 2.4.

The reaction terminating liquid is the sulfuric acid liquid of 2M.

The present invention also provides a kind of Fipronil ELISA detection reagent, effective component is the anti-Fipronil nano antibody.

The remaining application of Fipronil in ELISA method detection sample the present invention further provides the kit or reagent. When analysis detection, sequentially added into each hole for the ELISA Plate for being coated with the Fipronil envelope antigen Fipronil sample to be measured and The anti-Fipronil nano antibody, solid-phase coating antigen and Fipronil to be measured are vied each other and are reacted with nano antibody, due to each The nano antibody uniform content of solid phase antigen and addition in hole causes, therefore when Fipronil concentration to be measured is high, is then combined Antibody on solid phase antigen is few, the ELIAS secondary antibody of addition with to be fixed antibody binding capacity few, be eventually adding substrate solution and colour developing Liquid, chromogenic reaction is shallow, and the OD value detected with microplate reader is low, shows inhibiting rate height；Conversely, when Fipronil concentration to be measured is low, then The OD value surveyed is high, and inhibiting rate is low.Drawn standard curve is detected according to the Fipronil standard solution of known concentration, can be pushed away Calculate the concentration of Fipronil to be measured.

The present invention accurately can delicately detect Fipronil in water and vegetables and remain, and the pretreatment process of sample is simple.For Water sample is detected after need to only being filtered；Pre-treatment for vegetable sample, referring to " Triazophos residue detects direct competitive The development and application of ELISA kit, beam red week etc., Chinese food journal, the 6th phase of volume 8, in December, 2008 ".This method consumption When it is few, a large amount of sample can be detected simultaneously, sample detection cost is far below traditional instrument detection method.The present invention is big to solving The Fipronil residue detection of batch sample realizes that on-site supervision has important practical significance.

Detailed description of the invention

Fig. 1 is the standard suppression curve of the Fipronil based on nano antibody F1 in the embodiment of the present invention 5.Regression Equations For y=-0.2244+1.1454/ [1+ (x/111.7956) ^0.7704] (R²=0.9933) concentration IC in inhibiting₅₀= 55.27ng/mL minimum detection limit IC₁₀=0.64ng/mL.

Specific embodiment

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment According to conventional laboratory conditions, such as Sambrook molecular cloning experiment handbook (Sambrook J&Russell DW, Molecular Cloning:a Laboratory Manual, 2001), or according to the condition of manufacturer's specification suggestion.

The preparation of 1 Fipronil envelope antigen of embodiment

Coupled complex is prepared with haptens and bovine serum albumin, as envelope antigen.The preparation method is as follows:

(1) claim 8.8mg 6- (3- cyano -5- (2,6- bis- chloro- 4- (trifluoromethyl) phenyl) the amyl- 1,3- diene -1- base of ring) Amino) -6- oxo caproic acid (MW=440.15) (0.02mmol), 2.65mg NHS (MW=115) (0.024mmol), 4.8mg DCC (MW=206) (0.023mmol) is dissolved in 200 μ L anhydrous DMFs, stirs anti-reaction overnight at room temperature.Reaction solution is centrifuged (5000rpm, 10min) abandons precipitating, and supernatant is active ester.

(2) claim 20mg BSA (MW=67000) to be dissolved in 2mL carbonate buffer solution (0.05mol/mL, pH9.5), stir It mixes down and 150 μ L active ester liquid is added dropwise, when dropwise addition is slow, and about 20~30min is added.Then continue to be stirred to react 4 at room temperature ~6h.

(3) reaction solution is packed into bag filter, is dialysed with PBS (0.01mol/L pH7.4).Every 6~8h is changed the liquid once, in total It changes liquid 5~6 times.It is centrifuged after dialysis, abandons precipitating, take supernatant, -20 DEG C of preservations.

The building in 2 Fipronil phage display nano antibody library of embodiment

Haptens and keyhole limpet hemocyanin are coupled using active ester method, the specific method is as follows:

(1) claim 66.02mg 6- (3- cyano -5- (2,6- bis- chloro- 4- (trifluoromethyl) phenyl) amyl- 1,3- diene -1- of ring Base) amino) -6- oxo caproic acid (MW=440.15) (0.15mmol), 17.825mg NHS (MW=115) (0.155mmol), 31.518mg DCC (MW=206) (0.153mmol) is dissolved in 1500 μ L anhydrous DMFs, is stirred to react at room temperature overnight.It will reaction Liquid is centrifuged (5000rpm, 10min), abandons precipitating, supernatant is active ester.

(2) 6mL KLH solution (6.8mg/mL) is taken, is added dropwise 1200 μ L active ester liquid under stirring, when dropwise addition is slow, about 20~30min is added.Then continue to be stirred to react 4~6h at room temperature.

(3) reaction solution is packed into bag filter, is dialysed with PBS (0.01mol/L pH7.4).Every 6~8h is changed the liquid once, and is changed altogether Liquid 5~6 times.It is centrifuged after dialysis, abandons precipitating, receive supernatant, -20 DEG C of preservations.

It takes 1mg conjugate to be dissolved in 1mL physiological saline, is mixed with 1mL complete Freund's adjuvant, inject white horse with a black mane after fully emulsified Camel, booster immunization is primary every two weeks later, changes incomplete Freund's adjuvant into and mixes with immunogene, and the subcutaneous multiple spot of the nape of the neck is exempted from Epidemic disease is immunized 5 times altogether.Since third time is immune, latter week is immunized every time from neck lock venous blood collection detection serum titer.

Leucocyte is separated from peripheral blood of the 5th after immune, extracts total serum IgE, through reverse transcription PCR and nest-type PRC, clone VHH genetic fragment (including IgG2, IgG3a and IgG3b) out, modifies cohesive end with restriction enzyme SfiI, is connected by T4 It connects enzyme and VHH genetic fragment is connected to phasmid pComb3x, highly effective iodine constructs Fipronil to Escherichia coli ER2738 Bacteriophage nano antibody library.After measured, primary storage capacity is up to 10⁸Cfu is added helper phage (infection multiplicity 20:1) M13KO7 is saved, and bacteriophage nano antibody library, storage capacity 10 are obtained¹⁴The diversity of pfu/mL, library are good.

Reverse transcription PCR:

Reverse transcription reagent box uses PrimeScriptTMRT-PCR Kit, is purchased from TaKaRa company, goods number: AK2701。

Reverse transcription system is as follows:

65 DEG C of reaction 5min.Taking-up is placed on ice, is loaded by following system, and the synthesis of the first chain of cDNA is carried out.

30℃10min；42℃1h；72℃5min.

Nest-type PRC:

First round PCR:

Reaction system is as follows:

Response procedures are as follows: 94 DEG C of initial denaturation 3min；94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 50s, 25 circulations；72℃ 5min。

Second wheel PCR:

Reaction system is as follows:

Response procedures are as follows: 94 DEG C of initial denaturation 3min；94 DEG C of 30s, 62 DEG C of 40s, 72 DEG C of 40s, 25 circulations；72℃ 5min。

Nest-type PRC primer sequence is following (SEQ ID NO:2-7):

GSP-RT:5 '-CGCCATCAATRTACCAGTTGA-3 '

LP-leader:5 '-GTGGTCCTGGCTGCTCTW-3 '

F:5 '-CATGCCATGACTGTGGCCCAGGCGGCCCAGKTGCAGCTCGTGGAGTC-3 '

R1:5 '-CATGCCATGACTCGCGGCCGGCCTGGCCATGGGGGTCTTCGCTGTGGTGCG-3 '

R2:5 '-CATGCCATGACTCGCGGCCGGCCTGGCCGTCTTGTGGTTTTGGTGTCTTGGG-3 '

R5:5 '-CATGCCATGACTCGCGGCCGGCCTGGCCCTTGCATACTTCATTCGTTCCTG-3 '

Wherein, R indicates that base A/G, W indicate that base A/T, K indicate bases G/T.

The screening of the specific Fipronil phage display nano antibody of embodiment 3

In the envelope antigen of the 1st hole of 96 hole elisa Plates coating embodiment 2, peridium concentration is 100 μ g/mL, 4 DEG C of mistakes Night；Next day pours out coating buffer, is washed 3 times with PBST, the 1st, 2 two hole of ELISA Plate is closed with BSA, 37 DEG C of incubation 1h；It pours out Confining liquid is washed 3 times with PBST；The 1st hole is added in the phage antibody library of embodiment 2, reacts 2h；Liquid is poured out, clean It pats dry on net blotting paper, is washed 5 times with PBST；Add 100 μ L Fipronil standard items into the 1st hole, reacts 1h；It is sucked out the 1st The 2nd hole is added in liquid in a hole, reacts 1h, removes the bacteriophage in conjunction with BSA；Eluent is collected, takes 5 μ L for dripping Degree measurement, remaining is for expanding.

Bacteriophage elution liquid is added to fresh Escherichia coli ER2738 bacterium solution, 37 DEG C of standing 15min；Carboxylic benzyl mould is added Plain (working concentration 50mg/L) and SB culture medium, 37 DEG C of 220rpm shaken cultivation 2h；Helper phage M13KO7 is added, and (infection is multiple Number MOI=20:1) and kanamycins, overnight incubation；PEG-NaCl solution deposition and purification phagocytosis is added in next day, centrifuging and taking supernatant Body.

Amplified production is subjected to next round screening, guarantees that every wheel screening additional amount is identical, antigen coat concentration and Fipronil Standard items competitive elution concentration is successively decreased by 2 times, calculates the titre of every wheel, and picking monoclonal carries out amplification and ELISA identification.Through 3 Wheel elutriation obtains positive monoclonal.

The expression of the specific Fipronil nano antibody of embodiment 4

Positive monoclonal plasmid is extracted, change goes to Escherichia coli TOP10F ' competent cell, and solid training is coated on after recovery Feeding base is incubated overnight.Next day, picking are individually cloned in training in SB- carboxylic benzyl culture medium (carbenicillin working concentration is 50mg/L) It supports, the expression of IPTG overnight induction is added；Next day is used ni-sepharose purification, that is, is utilized with Ultrasonic Cell Disruptor lytic cell after membrane filtration Histidine tag and the affinity chromatography of nickel chloride in nickel column isolate and purify nano antibody, obtain the anti-Fipronil of high-purity Nano antibody is analyzed through amino acid sequencing, and the amino acid sequence of gained nano antibody F1 is as shown in SEQ ID NO:2.

Embodiment 5 analyzes the remaining ELISA detection kit of Fipronil and its application

The kit includes box body, is located at dismountable 96 hole elisa Plates in box body and is located at the intracorporal reagent of box. Wherein, each hole of the ELISA Plate is coated with the Fipronil envelope antigen of embodiment 1, and the reagent includes the anti-of embodiment 4 Fipronil nano antibody, Fipronil standard solution, enzyme label secondary antibody, buffer PBS, cleaning solution PBST, substrate solution (A liquid), Developing solution (B liquid) and reaction terminating liquid etc..

The secondary antibody of enzyme label is the anti-HA tag antibody of horseradish peroxidase-labeled, and concentration is 0.1 μ g/mL.It is purchased from Abcam company, goods number: ab1265.

A liquid is by urea peroxide 1g, citric acid 10.3g, Na₂HPO₄·12H₂O 35.8g, 100 μ L of Tween-20 and distilled water 1000mL is formulated, pH value 5.

B liquid is formulated by tetramethyl benzidine 700mg, DMSO 40mL, citric acid 10.3g and distilled water 1000mL, pH Value 2.4.

Reaction terminating liquid is the sulfuric acid liquid of 2M.

Envelope antigen is coated in 96 hole elisa Plates, each hole peridium concentration is 100 μ g/mL, 4 DEG C of reaction overnights；It is secondary Day, the liquid in hole is thrown away, is washed 3 times with the PBST containing 0.05% tween, ELISA Plate is upside down on blotting paper and is patted dry；Envelope is added Liquid is closed, 37 DEG C are incubated for 30 minutes, throw away the liquid in hole, are washed 3 times with 0.05%PBST, ELISA Plate is upside down on blotting paper and is clapped It is dry；Prepare 0ng/mL, 1ng/mL, 4ng/mL, 12ng/mL, 37ng/mL, 111ng/mL, 333ng/mL, the fluorine worm of 1000ng/mL 50 μ L standard specimens or the sample handled well are added into each hole in nitrile titer.For water sample, detected after need to only being filtered； Pre-treatment for vegetable sample, referring to " Triazophos residue detects the development and application of direct competive ELISA kit, Liang Chi Week etc., Chinese food journal, the 6th phase of volume 8, in December, 2008, the 2nd section of P103 right column " carries out, can be into dilution dissolution Row detection.Standard specimen and sample do 2-4 repetition, are added the 50 diluted antibody of μ L (concentration is about 124ng/mL after dilution), and 37 DEG C It is incubated for 30 minutes；The liquid in hole is thrown away, is washed 3 times with PBST, ELISA Plate is upside down on blotting paper and is patted dry；Enzyme mark two is added Anti-, 37 DEG C are incubated for 30 minutes；The liquid in hole is thrown away, with PBST board-washing 3 times, is patted dry；A liquid and B liquid is taken to mix in equal volume, every hole Add 100 μ L, be protected from light colour developing 10~15 minutes, terminate liquid is added and terminates reaction, it is at 450nm that each hole is measured in microplate reader in wavelength OD value.

The OD value that the OD value of the sample wells of standard containing 0ng/mL subtracts the sample wells of standard containing maximum concentration is set to B₀, remaining Kong Jingtong OD value after quadrat method correction is set to B；With B/B₀Value is ordinate, and respective standard product concentration is abscissa, draws Fipronil mark Quasi- suppression curve (Fig. 1).The concentration of counter sample can be found out according to the regression equation of curve, can also find out Fipronil inhibition Middle concentration IC₅₀(B/B₀=50%) and minimum detectable level IC₁₀(B/B₀=90%).

In actual sample detection process, the envelope antigen (peridium concentration 180ng/mL) that is adsorbed on ELISA Plate hole wall and to Survey Fipronil is vied each other and antibody response, competition results are come out by chromogenic reaction.It detects the Fipronil of known concentration and draws Standard curve processed can extrapolate the concentration of Fipronil to be measured.It is an advantage of the invention that water and vegetables accurately can be detected delicately Middle Fipronil residual, the pretreatment process of sample simply for water sample, are detected after need to only being filtered；For vegetable sample Pre-treatment, referring to " Triazophos residue detect direct competive ELISA kit development and application, beam red week etc., Chinese food Journal, the 6th phase of volume 8, in December, 2008 ".This method time-consuming is few, can detect a large amount of sample simultaneously, and sample detection cost is remote Lower than traditional instrument detection method.

Although above the present invention is described in detail with a general description of the specific embodiments, On the basis of the present invention, it can be modified or is improved, this will be apparent to those skilled in the art.Cause This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.

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Claims (9)

1. anti-Fipronil nano antibody, which is characterized in that the amino acid sequence of the antibody is as shown in SEQ ID NO:1.

2. analyze the remaining ELISA detection kit of Fipronil, including box body, it is located in box body dismountable ELISA Plate and sets In the intracorporal reagent of box, which is characterized in that each hole of the ELISA Plate is coated with Fipronil envelope antigen, and the reagent includes Secondary antibody, the buffer PBS, cleaning solution PBST, developing solution that nano antibody described in claim 1, Fipronil standard solution, enzyme mark And reaction terminating liquid.

3. kit according to claim 2, which is characterized in that the Fipronil envelope antigen is haptens 6- (3- cyanogen Base -5- (2,6- bis- chloro- 4- (trifluoromethyl) phenyl) amyl- 1,3- diene -1- base of ring) amino) -6- oxo caproic acid and cow's serum egg White coupled complex, the envelope antigen the preparation method is as follows:

(1) claim 8.8mg 6- (3- cyano -5- (2,6- bis- chloro- 4- (trifluoromethyl) phenyl) the amyl- 1,3- diene -1- base of ring) ammonia Base) -6- oxo caproic acid, 2.65mg NHS, 4.8mg DCC be dissolved in 200 μ L anhydrous DMFs, be stirred to react at room temperature overnight；It will be anti- It answers liquid to be centrifuged, abandons precipitating, supernatant is active ester；

(2) claim 20mg BSA to be dissolved in the carbonate buffer solution 2mL of 0.05mol/mL pH9.5,150 μ are added dropwise under stirring The above-mentioned active ester of L, 20~30min are added；Then continue to be stirred to react 4~6h at room temperature；

(3) reaction solution is packed into bag filter, is dialysed with the PBS of 0.01mol/L pH7.4；Every 6~8h is changed the liquid once, and changes liquid 5 altogether ~6 times；It is centrifuged after dialysis, abandons precipitating, take supernatant as antigen coat liquid.

4. kit according to claim 2, which is characterized in that the ELISA Plate is 96 hole elisa Plates, envelope antigen Peridium concentration is 180ng/mL.

5. kit according to claim 2, which is characterized in that the concentration of the nano antibody is 124ng/mL.

6. kit according to claim 2, which is characterized in that the developing solution includes A liquid and B liquid, and A liquid is by peroxidating Urea 1g, citric acid 10.3g, Na₂HPO₄·12H₂O 35.8g, Tween-20 100 μ L and distilled water 1000mL are formulated, pH value 5；B liquid is formulated by tetramethyl benzidine 700mg, DMSO 40mL, citric acid 10.3g and distilled water 1000mL, pH value 2.4.

7. according to the described in any item kits of claim 2-6, which is characterized in that the reaction terminating liquid is the sulfuric acid of 2M Liquid.

8. Fipronil ELISA detection reagent, effective component is nano antibody described in claim 1.

9. the Fipronil in ELISA method detection sample of reagent described in any one of the claim 2-7 kit or claim 8 Remaining application.