CN107817341B - ELISA kit and its application based on nano antibody detection Triazophos residue - Google Patents
ELISA kit and its application based on nano antibody detection Triazophos residue Download PDFInfo
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Abstract
The invention discloses ELISA kit and its application based on nano antibody detection Triazophos residue, the kit includes box body, is located at the intracorporal ELISA Plate of box and reagent；Wherein, each hole of ELISA Plate is coated with Hostathion envelope antigen, and the reagent includes anti-Hostathion nano antibody, Hostathion 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 Hostathion to be measured is vied each other and antibody response observes result by chromogenic reaction.In detection process, the envelope antigen that adsorbs on ELISA Plate hole wall and Hostathion to be measured is vied each other and antibody response observes result by chromogenic reaction.It is an advantage of the invention that water accurately can delicately be detected, Triazophos residue in vegetables, 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
Technical field
The present invention relates to genetic engineering, display technique of bacteriophage and ELISA detection technique fields, specifically, being related to
ELISA kit and its application based on nano antibody detection Triazophos residue.
Background technique
Hostathion (Triazophos) is a kind of widely used organophosphorus ester insecticides, to endanger grain, cotton,
The pest of the staple crops such as oil, fruits and vegetables, tealeaves has good control efficiency.Hostathion to fish, honeybee, silkworm toxicity compared with
Greatly, medium to the toxicity of mammal, it will cause a large amount of acetylcholines after entering human body and be accumulated in neural effector junction,
Muscarinic, nicotine sample and central nervous system symptom occurs.
After the high poisons such as acephatemet, parathion organophosphorus insecticide is limited or is forbidden to use, the usage amount of Hostathion
It increases sharply in recent years, since half-life period is longer, easy residual, residual excessive problem gradually causes to pay close attention in environment and food.Cause
This, reinforces the detection of Triazophos residue, for scientifically and rationally using Hostathion, ensures food safety and human health, reduces
Environmental pollution enhances the international competitiveness of China's agricultural and sideline product, and the sustainable development etc. of China's agricultural is kept to be of great significance.
Triazophos residue detection method 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.
Triazophos residue ELISA adsorption analysis method based on conventional antibody (polyclonal antibody and monoclonal antibody)
Stability is relatively low, and the present invention is based on anti-Hostathion 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 ELISA detection reagent of Triazophos residue
Box.The quick measurement of Triazophos residue 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-Hostathion nano antibody, the amino acid sequence of the antibody such as SEQ ID
Shown in NO:1 or the sequence is through replacement, missing or one or several amino acids formed amino acid with same function of addition
Sequence.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-Hostathion nano antibody can be prepared as follows: utilize the half of chemical reactive synthesis Hostathion
Antigen O- ethyl-O-3- (1- phenyl -1,2,4- triazolyl)-N- (3- carboxymethyl) thioate, by haptens and keyhole
It is used as immunogene after hemocyanin KLH coupling, immunization experiment animal camel extracts the total serum IgE of peripheral blood lymphocytes, through inverting
Record and nest-type PRC, clone nano antibody heavy chain (VHH) genetic fragment, are connected by digestion, by gene fragment clone to phagocytosis
Grain carrier, highly effective iodine to Escherichia coli are saved through helper phage, and building obtains bacteriophage nano antibody library, are filtered out
Its expression and purification it is anti-to be obtained high sensitivity, the anti-Hostathion nanometer of high specificity by special Hostathion bacteriophage nano antibody
Body.The nano antibody molecule of preparation is small, soluble strong, and high temperature resistant, easy purification is easily expressed.
The ELISA detection kit of analysis Triazophos residue 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 Hostathion envelope antigen, described
Reagent includes secondary antibody, the buffer PBS, cleaning solution of the anti-Hostathion nano antibody, Hostathion standard solution, enzyme label
PBST, developing solution (A liquid), developing solution (B liquid) and reaction terminating liquid etc..
The Hostathion envelope antigen is haptens O- ethyl-O-3- (1- phenyl -1,2,4- triazolyl)-N- (3- carboxylic first
Base) thioate and bovine serum albumin coupled complex, the envelope antigen the preparation method is as follows:
(1) claim 7.4mg (0.02mmol) O- ethyl-O-3- (1- phenyl -1,2,4- triazolyl)-N- (3- carboxymethyl) thio
Phosphoramidate (MW=370), 2.65mg (0.024mmol) NHS (n-hydroxysuccinimide, MW=115), 4.8mg
(0.023mmol) DCC (dicyclohexylcarbodiimide, MW=206) is dissolved in 200 μ L anhydrous DMFs (N- N-formyl morpholine N), at room temperature
It is stirred to react overnight.Reaction solution is centrifuged (5000rpm, 10min), abandons precipitating, supernatant is active ester.
(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 20~30min (preferably 20min or so) is added.
Then continue to be stirred to react 4~6h (preferably 4h or so) at room temperature.
(3) reaction solution is packed into bag filter, is dialysed with PBS (0.01mol/L pH7.4).Every 6~8h (preferably 6h or so)
It changes the liquid once, changes 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, the peridium concentration of envelope antigen are 250ng/mL.
The concentration of the nano antibody is about 126ng/mL.The secondary antibody of the enzyme label is horseradish peroxidase-labeled
Anti- HA tag antibody, concentration be 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 Hostathion ELISA detection reagent, effective component is the anti-Hostathion nano antibody.
The application of the Triazophos residue 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 Hostathion envelope antigen Hostathion sample to be measured and
The anti-Hostathion nano antibody, solid-phase coating antigen and Hostathion 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 triazole phosphorus 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 triazole phosphorus 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 Hostathion standard solution of known concentration, can be pushed away
Calculate the concentration of Hostathion to be measured.
The present invention accurately can delicately detect Triazophos residue in water and vegetables, 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 Triazophos residue of batch sample detects, and realizes that on-site supervision has important practical significance.
Detailed description of the invention
Fig. 1 is the standard suppression curve of the Hostathion based on nano antibody in the embodiment of the present invention 5.Regression Equations are
Y=1.0013+1.0757/ [1+ (x/8.4244) ^0.0047] (R2=0.9994) concentration IC in inhibiting50=8.6ng/mL, most
Low detection limits IC10=1.15ng/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 Hostathion 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 7.4mg O- ethyl-O-3- (1- phenyl -1,2,4- triazolyl)-N- (3- carboxymethyl) thioate
(MW=370) (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.By reaction solution centrifugation (5000rpm,
10min), precipitating is abandoned, 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 20min is added.Then continue to be stirred to react 4h at room temperature.
(3) reaction solution is packed into bag filter, is dialysed with PBS (0.01mol/L pH7.4).Every 6h is changed the liquid once, Zong Gonghuan
Liquid 5~6 times.It is centrifuged after dialysis, abandons precipitating, take supernatant, -20 DEG C of preservations.
The building in 2 Hostathion 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 59.7mg O- ethyl-O-3- (1- phenyl -1,2,4- triazolyl)-N- (5- carboxylic amyl) thioate
(MW=398) (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.By reaction solution centrifugation (5000rpm,
10min), precipitating is abandoned, 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
20min is added.Then continue to be stirred to react 4h at room temperature.
(3) reaction solution is packed into bag filter, is dialysed with PBS (0.01mol/L pH7.4).Every 6h is changed the liquid once, and changes liquid 5 altogether
~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 Hostathion 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 (5 ' -3 '):
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 Hostathion 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 Hostathion 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 Hostathion
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 Hostathion 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-Hostathion of high-purity
Nano antibody is analyzed through amino acid sequencing, and the amino acid sequence of gained nano antibody is as shown in SEQ ID NO:1.
ELISA kit and its application of the embodiment 5 based on nano antibody detection Triazophos residue
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 Hostathion envelope antigen of embodiment 1, and the reagent includes the anti-of embodiment 4
Hostathion nano antibody, Hostathion 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 triazole of 1000ng/mL
50 μ L standard specimens or the sample handled well are added into each hole in phosphorus 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 126ng/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 Hostathion 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 Hostathion 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 250ng/mL) that is adsorbed on ELISA Plate hole wall and to
Survey Hostathion is vied each other and antibody response, competition results are come out by chromogenic reaction.It detects the Hostathion of known concentration and draws
Standard curve processed can extrapolate the concentration of Hostathion to be measured.It is an advantage of the invention that water and vegetables accurately can be detected delicately
Middle Triazophos residue, 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-Hostathion 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 ELISA detection kit of Triazophos residue, 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 Hostathion envelope antigen, and the reagent includes
Secondary antibody, the buffer PBS, cleaning solution PBST, developing solution that nano antibody described in claim 1, Hostathion standard solution, enzyme mark
And reaction terminating liquid.
3. kit according to claim 2, which is characterized in that the Hostathion envelope antigen is haptens O- ethyl-
The coupled complex of O-3- (1- phenyl -1,2,4- triazolyl)-N- (3- carboxymethyl) thioate and bovine serum albumin, institute
State envelope antigen the preparation method is as follows:
(1) claim 7.4mg O- ethyl-O-3- (1- phenyl -1,2,4- triazolyl)-N- (3- carboxymethyl) thioate,
2.65mg NHS, 4.8mg DCC are dissolved in 200 μ L anhydrous DMFs, are stirred to react at room temperature overnight；Reaction solution is centrifuged, it is heavy to abandon
It forms sediment, 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 250ng/mL.
5. kit according to claim 2, which is characterized in that the concentration of the nano antibody is 126ng/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. Hostathion ELISA detection reagent, effective component is nano antibody described in claim 1.
9. the Hostathion in ELISA method detection sample of reagent described in any one of the claim 2-7 kit or claim 8
Remaining application.
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