CN107417790A - 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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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2430/00—Assays, e.g. immunoassays or enzyme assays, involving synthetic organic compounds as analytes
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Abstract
The invention discloses the ELISA kit based on nano antibody detection Triazophos residue and its application, the kit includes box body, the ELISA Plate and reagent being located in box body；Wherein, each hole of ELISA Plate is coated with Hostathion envelope antigen, and the reagent includes secondary antibody, buffer solution PBS, cleaning solution PBST, substrate solution, nitrite ion and reaction terminating liquid of anti-Hostathion nano antibody, Hostathion standard liquid, enzyme mark etc..In detection process, the envelope antigen that is adsorbed on ELISA Plate hole wall and Hostathion to be measured is vied each other and antibody response observes result by chromogenic reaction.Detect the Hostathion of concentration known and draw standard curve, the concentration of Hostathion to be measured can be extrapolated.It is an advantage of the invention that Triazophos residue in water, vegetables accurately can be detected delicately, the pretreatment process of sample is simple, takes less, can detect substantial amounts of sample simultaneously, 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, it is related to
ELISA kit and its application based on nano antibody detection Triazophos residue.
Background technology
Hostathion (Triazophos) is a kind of widely used organophosphorus ester insecticides, to endanger grain, cotton,
The insect of the staple crops such as oil, fruits and vegetables, tealeaves has good prevention effect.Hostathion to fish, honeybee, silkworm toxicity compared with
Greatly, medium to the toxicity of mammal, it can cause a large amount of acetylcholines to be accumulated in neural effector junction after entering human body,
Generation muscarinic, nicotine sample and central nervous system symptom.
After the high poison such as acephatemet, parathion organophosphorus insecticide is limited or prohibitted the use of, the usage amount of Hostathion
Increase sharply in recent years, because half-life period is longer, easy residual, residual excessive problem gradually causes concern in environment and food.Cause
This, strengthens the detection of Triazophos residue, for scientifically and rationally using Hostathion, ensures food safety and human health, reduces
Environmental pollution, strengthen the international competitiveness of China's agricultural byproducts, keep the sustainable development of China's agricultural etc. significant.
The Triazophos residue detection method reported at present mainly has high performance liquid chromatography (HPLC), gas chromatography
And chromatograph-mass spectrometer coupling (GC/LC-MS) (GC).Specialized laboratory, specialized instrument and equipment and specially are needed with these methods
Industry operating personnel, 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
Record 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 steady using ELISA adsorption analysis method, its heat based on anti-Hostathion nano antibody
It is qualitative to be superior to polyclonal antibody analysis method.
The content of the invention
The purpose of the present invention be for current residues of pesticides instrument analytical method cost height, complex pretreatment, poor specificity,
The deficiencies of sensitivity is low and is difficult to experimental field detection, there is provided one kind has high specific, high sensitivity, high accuracy, high-precision
Exactness, operating method are simple, and can be used for batch samples quick detection, analyze the ELISA detection reagents of Triazophos residue
Box.The quick measure of Triazophos residue suitable for the samples such as water, vegetables.
In order to realize the object of the invention, resist present invention firstly provides one for building cameloid phage display nanometer
The general PCR primer of C-terminal in body library, the nucleotide sequence such as SEQ ID NO of the primer:Shown in 7.
Then the present invention provides a kind of anti-Hostathion nano antibody, the amino acid sequence such as SEQ ID NO of the antibody:1
It is shown, or the sequence is through replacing, lacking or adding one or several amino acids formed amino acid sequences with equal function.
For example, by SEQ ID NO:Sequence shown in 1 remove end 6 it is histidine-tagged after amino acid sequence.
The anti-Hostathion nano antibody can be prepared as follows：Utilize the half of chemical reactive synthesis Hostathion
Antigen O- ethyls-O-3- (1- phenyl -1,2,4- triazolyls)-N- (3- carboxymethyls) thioate, by haptens and keyhole
Be used as immunogene after hemocyanin KLH couplings, immunization experiment animal camel, the total serum IgE of PBLC is extracted, 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, is saved through helper phage, and structure obtains bacteriophage nano antibody storehouse, filters out
Special Hostathion bacteriophage nano antibody, by its expression and purification, obtain high sensitivity, the anti-Hostathion nanometer of high specificity resists
Body.The nano antibody molecule of preparation is small, soluble strong, high temperature resistant, easy purification, easily expression.
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 the reagent that is located in box body.Wherein, each hole of the ELISA Plate is coated with Hostathion envelope antigen, described
Reagent includes the anti-Hostathion nano antibody, Hostathion standard liquid, secondary antibody, buffer solution PBS, the cleaning solution of enzyme mark
PBST, nitrite ion (A liquid), nitrite ion (B liquid) and reaction terminating liquid etc..
The Hostathion envelope antigen is haptens O- ethyls-O-3- (1- phenyl -1,2,4- triazolyls)-N- (3- carboxylic first
Base) thioate and bovine serum albumin coupled complex, the preparation method of the envelope antigen is as follows：
(1) claim 7.4mg (0.02mmol) O- ethyls-O-3- (1- phenyl -1,2,4- triazolyls)-N- (3- carboxymethyls) 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 dry DMFs (N- N-formyl morpholine Ns), at room temperature
Stirring reaction is stayed overnight.Reaction solution is centrifuged into (5000rpm, 10min), abandons precipitation, supernatant is active ester.
(2) claim 20mg BSA (bovine serum albumin, MW=67000) be dissolved in 2mL carbonate buffer solutions (0.05mol/mL,
PH9.5 150 μ L active ester liquid are added dropwise in), under stirring, slow during dropwise addition, 20~30min (preferably 20min or so) is added.
Then 4~6h of stirring reaction (preferably 4h or so) is continued at room temperature.
(3) reaction solution loads in bag filter, is dialysed with PBS (0.01mol/L pH7.4).Every 6~8h (preferably 6h or so)
Change liquid once, change liquid altogether 5~6 times.Centrifuged after dialysis, abandon precipitation, take supernatant, as antigen coat liquid.The ELISA Plate is
96 hole elisa Plates, the coating concentration of envelope antigen is 285ng/mL.
The concentration of the nano antibody is about 142ng/mL.The secondary antibody of the enzyme mark is horseradish peroxidase-labeled
Anti- HA tag antibodies, concentration is 0.1 μ g/mL.Purchased from Abcam companies, goods number:ab1265.
The nitrite ion A liquid is by urea peroxide 1g, citric acid 10.3g, Na2HPO4·12H2O 35.8g, Tween-20 100
μ L and distilled water 1000mL are formulated, pH value 5.
The nitrite ion 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 2M sulfuric acid liquid.
The present invention also provides a kind of Hostathion ELISA detection reagents, and its active ingredient 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.
Analysis detection when, 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 to react 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 it is few by sessile antibody binding capacity, be eventually adding substrate solution and colour developing
Liquid, chromogenic reaction is shallow, and the OD values detected with ELIASA are low, show inhibiting rate height；Conversely, when triazole phosphorus concentration to be measured is low, then
The OD values surveyed are high, and inhibiting rate is low.Standard curve is drawn according to the Hostathion standard liquid detection of concentration known, you can is 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, 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 consumes
When it is few, substantial amounts 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 detection of batch sample, realizes that on-site supervision has important practical significance.
Brief description of the drawings
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=-0.1591+1.1293/ [1+ (x/106.4572) ^0.8473] (R2=0.989) concentration IC in suppressing50=71.46ng/
ML, minimum detection limit IC10=4.33ng/mL.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.Unless otherwise specified, embodiment
According to conventional laboratory conditions, as Sambrook equimoleculars Cloning: A Laboratory Manual (Sambrook J＆Russell DW,
Molecular Cloning:A Laboratory Manual, 2001), or the condition according to manufacturer's specification suggestion.
The preparation of the Hostathion envelope antigen of embodiment 1
Coupled complex is prepared with haptens and bovine serum albumin, as envelope antigen.Preparation method is as follows：
(1) 7.4mg O- ethyls-O-3- (1- phenyl -1,2,4- triazolyls)-N- (3- carboxymethyls) thioate is claimed
(MW=370) (0.02mmol), 2.65mg NHS (MW=115) (0.024mmol), 4.8mg DCC (MW=206)
(0.023mmol) is dissolved in 200 μ L dry DMFs, stirs anti-reaction overnight at room temperature.Reaction solution is centrifuged (5000rpm,
10min), precipitation is abandoned, supernatant is active ester.
(2) claim 20mg BSA (MW=67000) to be dissolved in 2mL carbonate buffer solutions (0.05mol/mL, pH9.5), stir
Mix down and 150 μ L active ester liquid are added dropwise, slow during dropwise addition, about 20min is added.Then stirring reaction 4h is continued at room temperature.
(3) reaction solution loads in bag filter, is dialysed with PBS (0.01mol/L pH7.4).Liquid is changed per 6h once, Zong Gonghuan
Liquid 5~6 times.Centrifuged after dialysis, abandon precipitation, take supernatant, -20 DEG C of preservations.
The structure in the Hostathion phage display nano antibody storehouse of embodiment 2
Haptens and keyhole limpet hemocyanin are coupled using active ester method, specific method is as follows：
(1) 59.7mg O- ethyls-O-3- (1- phenyl -1,2,4- triazolyls)-N- (5- carboxylics amyl group) thioate is claimed
(MW=398) (0.15mmol), 17.825mg NHS (MW=115) (0.155mmol), 31.518mg DCC (MW=206)
(0.153mmol) is dissolved in 1500 μ L dry DMFs, and stirring reaction is stayed overnight at room temperature.Reaction solution is centrifuged (5000rpm,
10min), precipitation 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, it is slow during dropwise addition, about
20min is added.Then stirring reaction 4h is continued at room temperature.
(3) reaction solution loads in bag filter, is dialysed with PBS (0.01mol/L pH7.4).Liquid is changed per 6h once, changes liquid 5 altogether
~6 times.Centrifuged after dialysis, abandon precipitation, receive supernatant, -20 DEG C of preservations.
Take 1mg conjugates to be dissolved in 1mL physiological saline, mixed with 1mL complete Freund's adjuvants, white horse with a black mane is injected after fully emulsified
Camel, change incomplete Freund's adjuvant into and mixed with immunogene booster immunization every two weeks once later, the subcutaneous multiple spot of nape part is exempted from
Epidemic disease, it 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
Go out VHH genetic fragments, modify cohesive end with restriction enzyme SfiI, be connected to VHH genetic fragments by T4 ligases
Phasmid pComb3x, highly effective iodine to Escherichia coli ER2738, build the bacteriophage nano antibody storehouse of Hostathion.After measured,
Primary storage capacity is up to 108Cfu, add helper phage (infection multiplicity 20:1) M13KO7 is saved, and is obtained bacteriophage and is received
Rice antibody library, storage capacity 1014Pfu/mL, the diversity in storehouse are good.
Reverse transcription PCR：
Reverse transcription reagent box uses PrimeScriptTMRT-PCR Kit, purchased from TaKaRa companies, 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, carries out the synthesis of the chains of cDNA first.
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 pre-degeneration 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 pre-degeneration 3min；94 DEG C of 30s, 62 DEG C of 40s, 72 DEG C of 40s, 25 circulations；72℃
5min。
Following (the SEQ ID NO of nest-type PRC primer sequence: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 represents that base A/G, W represent that base A/T, K represent bases G/T.
The screening of the specific Hostathion phage display nano antibody of embodiment 3
The envelope antigen of embodiment 2 is coated with the 1st hole of 96 hole elisa Plates, coating concentration is 100 μ g/mL, 4 DEG C of mistakes
Night；Next day, coating buffer is poured out, washed 3 times with PBST, the 1st, 2 two hole of ELISA Plate is closed with BSA, 37 DEG C of incubation 1h；Pour out
Confining liquid, washed 3 times with PBST；The phage antibody library of embodiment 2 is added into the 1st hole, reacts 2h；Liquid is poured out, clean
Pat dry on net blotting paper, washed 5 times with PBST；Add 100 μ L Hostathions standard items into the 1st hole, react 1h；Suction out the 1st
Liquid in individual hole, the 2nd hole is added, react 1h, remove the bacteriophage combined with BSA；Eluent is collected, takes 5 μ L to be used to drip
Degree measure, remaining is used to expand.
Bacteriophage elution liquid is added to fresh Escherichia coli ER2738 bacterium solutions, 37 DEG C of standing 15min；Add carboxylic benzyl mould
Plain (working concentration 50mg/L) and SB culture mediums, 37 DEG C of 220rpm shaken cultivations 2h；Adding helper phage M13KO7, (infection is multiple
Number MOI=20:1) and kanamycins, overnight incubation；Next day, centrifuging and taking supernatant, add PEG-NaCl solution deposition and purification phagocytosis
Body.
Amplified production is subjected to next round screening, ensures that often wheel screening addition is identical, antigen coat concentration and Hostathion
Standard items competitive elution concentration is successively decreased by 2 times, calculates the titre often taken turns, and picking monoclonal is expanded and ELISA identifications.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 cells, and solid training is coated on after recovery
Foster base is incubated overnight.Next day, single be cloned in SB- carboxylic benzyls culture medium (carbenicillin working concentration is 50mg/L) of picking are trained
Support, add the expression of IPTG overnight inductions；Next day, with Ultrasonic Cell Disruptor cell lysis, ni-sepharose purification is used after membrane filtration, that is, is utilized
The affinity chromatography of nickel chloride isolates and purifies to nano antibody in the histidine-tagged post with nickel, obtains the anti-Hostathion of high-purity
Nano antibody, analyzed through amino acid sequencing, the amino acid sequence such as SEQ ID NO of gained nano antibody:Shown in 1.
Embodiment 5 analyzes ELISA detection kit and its application of Triazophos residue
The kit includes box body, is located at dismountable 96 hole elisa Plates and the reagent being located in box body in box body.
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 liquid, enzyme mark secondary antibody, buffer solution PBS, cleaning solution PBST, substrate solution (A liquid),
Nitrite ion (B liquid) and reaction terminating liquid etc..
The secondary antibody of enzyme mark is the anti-HA tag antibodies of horseradish peroxidase-labeled, and concentration is 0.1 μ g/mL.It is purchased from
Abcam companies, goods number:ab1265.
A liquid is by urea peroxide 1g, citric acid 10.3g, Na2HPO4·12H2O 35.8g, the μ L of Tween-20 100 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 2M sulfuric acid liquid.
Envelope antigen is coated in 96 hole elisa Plates, each hole coating 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 patted dry；Add envelope
Liquid is closed, 37 DEG C are incubated 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 clapped
It is dry；Prepare 0ng/mL, 1ng/mL, 4ng/mL, 12ng/mL, 37ng/mL, 111ng/mL, 333ng/mL, 1000ng/mL triazole
Phosphorus titer, 50 μ L standard specimens or the sample handled well are added into each hole.For water sample, detected after need to only being filtered；
Pre-treatment for vegetable sample, with reference 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 columns " is carried out, and can be entered with dilution dissolving
Row detection.Standard specimen and sample are 2-4 and repeated, the antibody (concentration is about 142ng/mL after dilution) of addition 50 μ L dilutions, 37 DEG C
It is incubated 30 minutes；The liquid in hole is thrown away, is washed 3 times with PBST, ELISA Plate is upside down on blotting paper and patted dry；Add enzyme mark two
Anti-, 37 DEG C are incubated 30 minutes；The liquid in hole is thrown away, with PBST board-washings 3 times, is patted dry；A liquid and B liquid is taken to mix in equal volume, per hole
Add 100 μ L, lucifuge develops the color 10~15 minutes, adds terminate liquid terminating reaction, and it is at 450nm that each hole is determined on ELIASA in wavelength
OD values.
The OD values that the OD values of the sample wells of standard containing 0ng/mL are subtracted to the sample wells of standard containing Cmax are set to B0, remaining Kong Jingtong
OD values after quadrat method correction are set to B；With B/B0It is abscissa to be worth for ordinate, respective standard product concentration, draws Hostathion mark
Quasi- suppression curve (Fig. 1).The concentration of counter sample can be obtained according to the regression equation of curve, Hostathion suppression can also be obtained
Middle concentration IC50(B/B0=50%) and minimum detectable level IC10(B/B0=90%).
In actual sample detection process, the envelope antigen (coating concentration is 285ng/mL) that is adsorbed on ELISA Plate hole wall and treat
Survey Hostathion is vied each other and antibody response, competition results are come out by chromogenic reaction.Detect the Hostathion of concentration known and paint
Standard curve processed, the concentration of Hostathion to be measured can be extrapolated.It is an advantage of the invention that it accurately can delicately detect water and vegetables
Middle Triazophos residue, the pretreatment process of sample are detected simply for water sample 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 is time-consuming few, can detect substantial amounts of sample simultaneously, sample detection cost is remote
Less 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 improved, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Sequence table
<110>China Agricultural University
<120>ELISA kit and its application based on nano antibody detection Triazophos residue
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35 40 45
Ala Ser Ile Asp Tyr Ser Gly Trp Ala Asn His Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Gln Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala
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Claims (10)
1. anti-Hostathion nano antibody, it is characterised in that the amino acid sequence of the antibody such as SEQ ID NO:Shown in 1, or should
Sequence is through replacing, lacking or adding one or several amino acids formed amino acid sequences with equal function.
2. analyze the ELISA detection kit of Triazophos residue, including box body, it is located in box body dismountable ELISA Plate and sets
Reagent in box body, it is characterised in that each hole of the ELISA Plate is coated with Hostathion envelope antigen, and the reagent includes
Nano antibody, Hostathion standard liquid, secondary antibody, buffer solution PBS, cleaning solution PBST, the nitrite ion of enzyme mark described in claim 1
And reaction terminating liquid.
3. kit according to claim 2, it is characterised in that the Hostathion envelope antigen be haptens O- ethyls-
The coupled complex of O-3- (1- phenyl -1,2,4- triazolyls)-N- (3- carboxymethyls) thioates and bovine serum albumin, institute
The preparation method for stating envelope antigen is as follows：
(1) claim 7.4mg O- ethyls-O-3- (1- phenyl -1,2,4- triazolyls)-N- (3- carboxymethyls) thioate,
2.65mg NHS, 4.8mg DCC are dissolved in 200 μ L dry DMFs, and stirring reaction is stayed overnight at room temperature；Reaction solution is centrifuged, it is heavy to abandon
Form sediment, supernatant is active ester；
(2) claim 20mg BSA to be dissolved in 0.05mol/mL pH9.5 carbonate buffer solution 2mL, 150 μ are added dropwise under stirring
The above-mentioned active esters of L, 20~30min are added；Then 4~6h of stirring reaction is continued at room temperature；
(3) reaction solution loads in bag filter, with 0.01mol/L pH7.4 PBS；Every 6~8h changes liquid once, changes liquid 5 altogether
~6 times；Centrifuged after dialysis, abandon precipitation, take supernatant as antigen coat liquid.
4. kit according to claim 3, it is characterised in that the ELISA Plate is 96 hole elisa Plates, envelope antigen
Coating concentration is 285ng/mL.
5. kit according to claim 2, it is characterised in that the concentration of the nano antibody is 142ng/mL.
6. kit according to claim 2, it is characterised in that the secondary antibody of the enzyme mark is horseradish peroxidase mark
The anti-HA tag antibodies of note, concentration is 0.1 μ g/mL.
7. kit according to claim 2, it is characterised in that the nitrite ion includes A liquid and B liquid, and A liquid is by peroxidating
Urea 1g, citric acid 10.3g, Na2HPO4·12H2O 35.8g, the μ L of Tween-20 100 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.
8. according to the kit described in claim any one of 2-7, it is characterised in that the reaction terminating liquid is 2M sulfuric acid
Liquid.
9. Hostathion ELISA detection reagents, its active ingredient is nano antibody described in claim 1.
10. reagent triazole in ELISA method detection sample described in any one of the claim 2-8 kits or claim 9
The application of phosphorus residual.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110684080A (en) * | 2019-08-26 | 2020-01-14 | 中国农业大学 | scFv-ELISA kit suitable for thiamethoxam residue analysis |
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|---|---|---|---|---|
| CN1624481A (en) * | 2004-10-20 | 2005-06-08 | 扬州大学 | Triazole phospho direct competition joint immune absorption analysis technology and its kit |
| CN106680484A (en) * | 2016-11-15 | 2017-05-17 | 中国农业大学 | ELISA (enzyme-linked immuno sorbent assay) detection kit for analyzing residual carbaryl and application thereof |
| CN107817341A (en) * | 2017-07-31 | 2018-03-20 | 中国农业大学 | ELISA kit and its application based on nano antibody detection Triazophos residue |
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2017
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|---|---|---|---|---|
| CN1624481A (en) * | 2004-10-20 | 2005-06-08 | 扬州大学 | Triazole phospho direct competition joint immune absorption analysis technology and its kit |
| CN106680484A (en) * | 2016-11-15 | 2017-05-17 | 中国农业大学 | ELISA (enzyme-linked immuno sorbent assay) detection kit for analyzing residual carbaryl and application thereof |
| CN107817341A (en) * | 2017-07-31 | 2018-03-20 | 中国农业大学 | ELISA kit and its application based on nano antibody detection Triazophos residue |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110684080A (en) * | 2019-08-26 | 2020-01-14 | 中国农业大学 | scFv-ELISA kit suitable for thiamethoxam residue analysis |
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