CN109734745A - A kind of preparation method and application of the antibody of fenifrothion - Google Patents
A kind of preparation method and application of the antibody of fenifrothion Download PDFInfo
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Abstract
The invention discloses a kind of preparation method and application of Direct Recognition fenifrothion antibody.The haptens of fenifrothion of the invention, structural formula such as formula (II) or (I) are shown:；.And then by the way that two kinds of artificial antigen is successfully prepared by hapten conjugation to carrier protein.Animal is immunized using one of artificial antigen, preparation can secrete the hybridoma of anti-fenifrothion antibody；Using one of artificial antigen as coating antigen, a kind of detection method of fenifrothion based on Indirect cELISA is further established.This detection method detection fenifrothion pesticide residue has the characteristics that easy quick, high specificity, with higher sensitivity, the IC of detection50Value is 7.13 ng/mL, and detection is limited to (IC10) 1.7 ng/mL, linear detection range is 2.9~17.9 ng/mL.
Description
Technical field
The present invention relates to technical field of immunoassay, more particularly, to a kind of antibody of Direct Recognition fenifrothion
Preparation method and application.
Background technique
China is large agricultural country, consumes a large amount of pesticides in agricultural generates every year, remaining pesticide easily causes environmental pollution
With the serious problems such as food acute poisoning.Fenifrothion belongs to organophosphorus insecticide, is one of now widely used insecticide.
It is detected for fenifrothion, instrumental method is main analysis method at present, but instrumental method needs expensive instrument, specially
People's operation and maintenance, complicated pre-treatment, and cannot achieve field quick detection.To make up instrumental method disadvantage, it is necessary to
A kind of analysis method that fast high-flux screening may be implemented is developed, field quick detection is used for.
Immunoassay is based on antigen and antibody specificity, the analytical technology of invertibity association reaction.Immune response relates to
And the comprehensive function of stereochemical structure, electrostatic, hydrogen bond and Van der Waals force that height is complementary between antigen and antibody molecule etc., have any
A kind of unapproachable selectivity of independent physico-chemical analysis technology and sensitivity, therefore with sensitivity and conventional instrument analysis one
The advantages that causing, being suitble to scene screening, is simple, quick, at low cost, sample aequum is few, it is considered to be 21 century is most competitive
With the Fast Detection Technique of challenge.Technique is recommended to many countries by the World Food Programme (FAO).American chemical
Immuno analytical method, chromatographic technique are classified as the major technique of pesticide, veterinary drug and fishery drugs residue analysis by meeting (ACS) jointly.
It there is no a kind of enzyme-linked immune detection method for fenifrothion at present.
Summary of the invention
The purpose of the invention is to overcome the deficiencies of the prior art and provide a kind of system of Direct Recognition fenifrothion antibody
Preparation Method and application.
The first purpose of the invention is to provide a kind of haptens of fenifrothion.
A second object of the present invention is to provide the preparation methods of the haptens.
Third object of the present invention is to provide a kind of comlete antigens of fenifrothion.
Fourth object of the present invention is to provide the preparation method of shown comlete antigen.
5th purpose of the invention is a kind of fenifrothion antibody.
6th purpose of the invention is that the haptens, the comlete antigen, and/or the antibody in detection kill snout moth's larva sulphur
Application in phosphorus or preparation detection fenifrothion kit.
7th purpose of the invention is a kind of detection method of fenifrothion based on Indirect cELISA.
To achieve the goals above, the present invention is achieved by the following technical programs:
A kind of haptens of fenifrothion, structural formula such as formula (II) or (I) are shown:
The preparation method of the haptens, comprising the following steps:
Dichloromethane solution, the NaOH aqueous solution and four of S1.3- methyl -4- nitrophenol and O- methyl thio-phosphoryl dichloride
12~16h is reacted in butylammonium bromide mixing, stirring, and by glue column chromatography, mobile phase is that volume ratio is petroleum ether: acetic acid
Ethyl ester=1~10:1 mixed solution, after purification up to compound shown in formula (II)；
S2. compound shown in formula (II) and 6-aminocaprolc acid are added to Isosorbide-5-Nitrae-dioxane, stir, and are added under 0 DEG C of ice bath
NaOH solution makes solution maintain pH 10~11, is stirred to react 12~16h to get compound shown in formula (I).
Preferably, silica gel column chromatography is 400 mesh silicon.
Preferably, 3- methyl -4- nitrophenol and the dichloromethane solution and NaOH of O- methyl thio-phosphoryl dichloride are water-soluble
The volume ratio of liquid is 1~2:1~2.
Preferably, the volume ratio of ethyl acetate and petroleum ether is 10:1.
Preferably, 3- methyl -4- nitrophenol and the dichloromethane solution and NaOH of O- methyl thio-phosphoryl dichloride are water-soluble
The volume ratio of liquid is 1:1.
Preferably, the mass ratio of 3- methyl -4- nitrophenol, O- methyl thio-phosphoryl dichloride and tetrabutylammonium bromide is 1
~3:4~6:1~5 (g:g:g).
It is highly preferred that the mass ratio of 3- methyl -4- nitrophenol, O- methyl thio-phosphoryl dichloride and tetrabutylammonium bromide is
3:6:1 (g:g:g).
Preferably, the concentration of NaOH aqueous solution is 0.01~0.1g/mL in step S1.
It is highly preferred that the concentration of NaOH aqueous solution is 0.05g/mL in step S1.
Preferably, 12h is stirred to react in step S1.
Preferably, compound shown in formula (II), 6-aminocaprolc acid and Isosorbide-5-Nitrae-dioxane mass volume ratio are as follows: 1~2:
2~5:1~5 (g:g:ml).
It is highly preferred that compound shown in formula (II), 6-aminocaprolc acid and Isosorbide-5-Nitrae-dioxane mass volume ratio are as follows: 1:5:
5 (g:g:ml).
Preferably, the concentration of NaOH aqueous solution is 1~1.1g/mL in step S2.
It is highly preferred that NaOH aqueous solution is saturation NaOH solution in step S2.
Preferably, 12h is stirred to react in step S2.
Preferably, the volume of NaOH aqueous solution is 1~2 times of Isosorbide-5-Nitrae-dioxane in step S2.
It is highly preferred that the volume of NaOH aqueous solution is 2 times of Isosorbide-5-Nitrae-dioxane in step S2.
A kind of comlete antigen of fenifrothion, structural formula such as formula (III) or (IV) are shown:
Preferably, the carrier protein is one in bovine lactoferrin, bovine serum albumin(BSA), hemocyanin or ovalbumin
Kind.
The preparation method of structural formula comlete antigen as shown in formula (III), comprising the following steps:
S11. activate haptens: under stiring the n,N-Dimethylformamide solution of structural formula haptens as shown in formula (I),
1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride and n-hydroxysuccinimide mixing, are protected from light stirring 4 at room temperature
~6h obtains A liquid；
S12. carrier protein is dissolved in carbonic acid buffer, obtains B liquid, and carrier protein concentration is 5~10mg/mL；
S13. under ice bath stirring, the A drop of S11 is added in the B liquid of S12, it will with NaOH solution or carbonic acid buffer
PH to 9.5~9.6 is adjusted, 12~16h is protected from light, obtains structural formula comlete antigen as shown in formula (III) after dialysis purification；
Wherein, structural formula haptens as shown in formula (I), 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride
Molar ratio with n-hydroxysuccinimide is 1:1.2~1.5:1.2~1.5.
Preferably, stirring 4h is protected from light in step S11.
Preferably, 12h is protected from light in step S13.
Preferably, structural formula haptens as shown in formula (I), 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide hydrochloride
The molar ratio of salt and n-hydroxysuccinimide is 1:1.5:1.5.
Preferably, the carbonic acid buffer pH is 9.6.
Preferably, carrier protein is bovine lactoferrin, the n,N-Dimethylformamide of structural formula haptens as shown in formula (I)
The concentration of solution is 10~20mg/ μ L.
It is highly preferred that carrier protein is bovine lactoferrin, the N of structural formula haptens as shown in formula (I), N- dimethyl methyl
The concentration of amide solution is 47/3mg/ μ L.
Preferably, carrier protein is bovine serum albumin, the n,N-Dimethylformamide of structural formula haptens as shown in formula (I)
The concentration of solution is 20~100mg/ μ L.
It is highly preferred that carrier protein is bovine serum albumin, the N of structural formula haptens as shown in formula (I), N- dimethyl methyl
The concentration of amide solution is 170/3mg/ μ L.
Preferably, the NaOH solution concentration is 1~3M.
It is highly preferred that the NaOH solution concentration is 3M.
Preferably, the carrier protein in structural formula haptens as shown in formula (I) in the A liquid and the B liquid
Molar ratio is 30~40:1.
It is highly preferred that structural formula haptens as shown in formula (I) in the A liquid and the carrier protein in the B liquid
Molar ratio be 30:1.
The preparation method of structural formula comlete antigen as shown in formula (IV), comprising the following steps:
S21. Isosorbide-5-Nitrae-dioxane solution of structural formula haptens as shown in formula (II), referred to as A liquid；
S22. carrier protein is dissolved in 9.6 carbonic acid buffer of pH, and carrier protein concentration is 5~10mg/mL, referred to as B liquid；
S23. the A liquid of S21 is added drop-wise in the B liquid of S22 under ice bath stirring, pH to 9.5 will be adjusted with NaOH solution
~9.6, it is protected from light 12~16h, obtains structural formula comlete antigen as shown in formula (IV) after dialysis purification.
Preferably, it is protected from light 12h.
Preferably, the carbonic acid buffer pH is 9.6.
Preferably, carrier protein is bovine serum albumin, and Isosorbide-5-Nitrae-dioxane of structural formula haptens as shown in formula (II) is molten
The concentration of liquid is 0.01~0.05mg/ μ L.
It is highly preferred that carrier protein is bovine serum albumin, Isosorbide-5-Nitrae-dioxane of structural formula haptens as shown in formula (II)
The concentration of solution is 0.026mg/mL.
Preferably, the concentration of carrier protein is 10mg/mL in B liquid.
Preferably, the NaOH solution concentration is 1~3M
It is highly preferred that the NaOH solution concentration is 3M.
Preferably, the carrier protein in structural formula haptens as shown in formula (II) in the A liquid and the B liquid
Molar ratio is 30~40:1.
It is highly preferred that structural formula haptens as shown in formula (II) in the A liquid and the carrier protein in the B liquid
Molar ratio be 30:1.
A kind of fenifrothion antibody, it is completely anti-using one or both of haptens described above, and/or the above
One or both of original is prepared.
Preferably, the antibody is polyclonal antibody, monoclonal antibody or genetic engineering antibody.
A kind of detection method of the fenifrothion based on Indirect cELISA is with compound shown in formula (III)
Immunogene prepares antibody, using compound shown in formula (IV) as coating antigen,
Preferably, the carrier protein of compound shown in the formula (III) of the immunogene is bovine lactoferrin (LF).
Preferably, the carrier protein of compound shown in coating former formula (IV) is bovine serum albumin(BSA) (BSA).
Preferably, the working concentration of coating antigen is 500~2000ng/mL, the working concentration of fenifrothion antibody is 10~
100ng/mL。
It is highly preferred that the working concentration of coating antigen is 1000ng/mL, the working concentration of antibody is 10ng/mL.
Most preferably, a kind of detection method of the fenifrothion based on Indirect cELISA, with formula (III) institute
Show that compound is that immunogene prepares antibody, using compound shown in formula (IV) as coating antigen,
Wherein, the carrier protein of compound shown in the formula (III) of the immunogene is bovine lactoferrin (LF)；
Preferably, the carrier protein of compound shown in coating former formula (IV) is bovine serum albumin(BSA) (BSA)；
It is highly preferred that the working concentration of coating antigen is 1000ng/mL, the working concentration of antibody is 10ng/mL.
A kind of detection kit of the fenifrothion based on Indirect cELISA, including it is coated with formula (IV) institute
The antibody for showing the enzyme mark version of compound and being prepared using compound shown in formula (III) as immunogene,
Preferably, the carrier protein of compound shown in the formula (III) of the immunogene is bovine lactoferrin (LF).
Preferably, the carrier protein of compound shown in coating former formula (IV) is bovine serum albumin(BSA) (BSA).
Preferably, the working concentration of coating antigen is 500~2000ng/mL, the working concentration of fenifrothion antibody is 10~
100ng/mL。
It is highly preferred that the working concentration of coating antigen is 1000ng/mL, the working concentration of antibody is 10ng/mL.
Compared with prior art, the invention has the following beneficial effects:
Two kinds of fenifrothion haptens are synthesized, by being successfully prepared two kinds on hapten conjugation to carrier protein
Artificial antigen.Animal is immunized using one of artificial antigen, preparation can secrete the hybridoma of anti-fenifrothion antibody；
Using one of artificial antigen as coating antigen, a kind of fenifrothion based on Indirect cELISA is further established
Detection method.This detection method, which detects fenifrothion pesticide residue, has easy quick, high specificity, with higher sensitivity spy
Point, the IC of detection50Value is 7.13ng/mL, and detection is limited to (IC10) 1.7ng/mL, linear detection range is 2.9~17.9ng/
mL。
Detailed description of the invention
Fig. 1 is the flow chart of the preparation method of fenifrothion haptens.
Fig. 2 is to kill snout moth's larva sulphur comlete antigen III-1 qualification figure.
Fig. 3 is to kill snout moth's larva sulphur comlete antigen III-2 qualification figure.
Fig. 4 is to kill snout moth's larva sulphur comlete antigen IV-1 qualification figure.
Fig. 5 is the ELISA competition test curve graph of fenifrothion antibody.
Specific embodiment
The present invention is made with specific embodiment with reference to the accompanying drawings of the specification and further being elaborated, the embodiment
It is served only for explaining the present invention, be not intended to limit the scope of the present invention.Test method as used in the following examples is such as without spy
Different explanation, is conventional method；Used material, reagent etc., unless otherwise specified, for the reagent commercially obtained
And material.
1 fenifrothion hapten synthesis of embodiment
One, experimental method
(1) fenifrothion haptens shown in formula (II)
3g 3- methyl -4- nitrophenol and 6g O- methyl thio-phosphoryl dichloride are dissolved in 20mLDCM (methylene chloride), add
20mLNaOH (0.05g/mL) aqueous solution and 1g tetrabutylammonium bromide (TBAB), vigorous stirring overnight, crude product are pure through silicagel column
Change, mobile phase is petroleum ether, obtains compound shown in formula (II).
(2) fenifrothion haptens shown in formula (I)
It is added to 5mL Isosorbide-5-Nitrae-dioxane under compound shown in 1g formula (II) and 5g 6-aminocaprolc acid low temperature, is stirred.Ice
It is gradually added into saturation NaOH solution 10mL under bath, so that solution is maintained alkalinity, is stirred overnight, obtains compound shown in formula (I).
Specific synthesis flow is shown in Fig. 1.
Two, experimental result
It is as shown in the table for haptens I and II mass spectrum and nuclear-magnetism identification.
The synthesis of 2 fenifrothion comlete antigen of embodiment
One, experimental method
(1) fenifrothion comlete antigen shown in formula (III)
(i) 9.4mg fenifrothion haptens shown in formula (I) is dissolved in 600 μ LN, in dinethylformamide, be followed by stirring for
Lower addition 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride and n-hydroxysuccinimide, are protected from light stir at room temperature
4h is mixed, the haptens after being activated, referred to as A liquid.Wherein, fenifrothion haptens shown in (I), the 1- (3- dimethylamino
Propyl) molar ratio of -3- ethyl-carbodiimide hydrochloride and the n-hydroxysuccinimide is 1:1.5:1.5.By 50mg ox
Lactoferrin (LF) is dissolved in 9.6 carbonic acid buffer of pH, and bovine lactoferrin concentration is 5~10mg/mL, referred to as B liquid.?
Above-mentioned A liquid is added drop-wise in B liquid under ice bath stirring, in the haptens as shown in formula (I) of the structural formula in A liquid and the B liquid
The molar ratio of the carrier protein is 30:1.After dropwise addition, pH is adjusted to 9.5~9.6 with 3M NaOH.It is protected from light overnight, and
The fenifrothion comlete antigen as shown in formula (III-1) is obtained after dialysis purification:
(ii) 34mg fenifrothion haptens shown in formula (I) is dissolved in 600 μ LN, in dinethylformamide, be followed by stirring for
Lower addition 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride and n-hydroxysuccinimide, are protected from light stir at room temperature
4h is mixed, the haptens after being activated, referred to as A liquid.Wherein, fenifrothion haptens shown in (I), the 1- (3- dimethylamino
Propyl) molar ratio of -3- ethyl-carbodiimide hydrochloride and the N- HOSu NHS is 1:1.5:1.5.By 200mg
Bovine serum albumin (BSA) is dissolved in 9.6 carbonic acid buffer of pH, and bovine serum albumin concentration is 5~10mg/mL, referred to as B
Liquid.Above-mentioned A liquid is added drop-wise in B liquid under ice bath stirring, the haptens as shown in formula (I) of the structural formula in A liquid and the B
The molar ratio of the carrier protein in liquid is 30:1.After dropwise addition, pH is adjusted to 9.5~9.6 with 3M NaOH.It is protected from light
Overnight, and after dialysis purification the fenifrothion comlete antigen as shown in formula (III-2) is obtained:
(2) fenifrothion comlete antigen shown in formula (IV)
26mg fenifrothion haptens shown in formula (II) is dissolved in 1mL Isosorbide-5-Nitrae-dioxane, referred to as A liquid.Wherein, will
Carrier protein is dissolved in 9.6 carbonic acid buffer of pH, and carrier protein concentration is 5~10mg/mL, referred to as B liquid.It is stirred in ice bath
Mix it is lower above-mentioned A liquid is added drop-wise in B liquid, the haptens as shown in formula (II) of the structural formula in A liquid with it is described in the B liquid
The molar ratio of carrier protein is 30:1.After dropwise addition, pH is adjusted to 9.5~9.6 with 3M NaOH.It is protected from light overnight, and passes through
The fenifrothion comlete antigen as shown in formula (IV) is obtained after crossing dialysis purification.
When carrier protein is bovine serum albumin(BSA) (BSA), shown in the comlete antigen being prepared such as formula (IV-1):
Two, experimental result
By scanning haptens, conjugate and carrier protein solution respectively in the absorption light of ultra-violet (UV) band (200~400nm).
As shown in figs. 2 to 4, the characteristic ultraviolet absorption peak of conjugate all has red shift to a certain degree or blue shift relative to body protein published originally,
Prove that artificial antigen is successfully prepared.
The preparation of the anti-fenifrothion monoclonal antibody of embodiment 3
One, experimental method
6~8 week old Bal b/c mouse (Guangdong Province's Experimental Animal Center) are taken, are 1 mg/mL formula by the concentration of preparation
(III-1) immunizing antigen of fenifrothion comlete antigen shown in and Freund's complete adjuvant mixed in equal amounts inject mouse completely after emulsification
Abdomen and back, every mouse inject 100 μ L.Immune for the first time to use Freund's complete adjuvant, rear booster immunization uses Freund not
Freund's complete adjuvant, altogether booster immunization 4 time immune primary every 3 weeks.Second of booster immunization latter week takes blood from tail vein
Examine potency and inhibition.After 4th booster immunization, chooses all higher mouse of potency and inhibiting rate and carry out cell fusion, before fusion
Doubling dose reinforced immunological is primary within 3 days.
Mouse myeloma SP2/0 cell and splenocyte are mixed with the ratio of 5:1, merged at 50%PEG, washing, from
With the suspension of HAT culture medium after the heart, it is inoculated in 96 well culture plates containing feeder cells, in 37 DEG C 5% of CO2It is trained in incubator
Liquid is changed with HAT culture medium after supporting 3 days, changes HT culture medium within the 10th day.When the 1/3 of the long extremely culture hole area of plate inner cell,
ELISA method screening cell positive hole is connect, with structural formula is that the comlete antigen as shown in formula (IV-1) and (III-1) is distinguished when screening
As envelope antigen.Indirect ELISA evaluation and screening is further used in positive hole, and limiting dilution assay is cloned into thin every about hole < 1
Born of the same parents, test positive and the preferable monoclonal hole gained cell strain of competition are the cell strain of secrete monoclonal antibody after 10 days.
After hybridoma expands culture, prepared for monoclonal antibody.
Two, experimental result
The results are shown in Table 1, when using comlete antigen IV-1 as coating antigen, inhibiting rate highest, therefore select comlete antigen
IV-1 is as coating antigen.
1 mouse resisting anteserum of table characterization:
The foundation and specific detection of the anti-fenifrothion detection method of embodiment 4
One, experimental method
The monoclonal antibody that Example 3 is prepared establishes standard curve, and the working concentration of coating antigen is 1000 ng/
ML, using three groups of parallel tests (n=3).
Antiserum indirect competitive ELISA detecting step is as follows:
S1. it is coated with: being diluted coating antigen (fenifrothion comlete antigen shown in formula (IV-1)) with 9.6 carbonic acid buffer of pH
It to 1 μ g/mL, is added in ELISA Plate hole, 100 holes μ L/, in 37 DEG C of water baths overnight.
S2. wash: liquid in hole of inclining, board-washing are machine-washed plate 2 times, and every hole adds 250 μ L of cleaning solution, dry liquid in hole.
S3. close: 120 μ L confining liquids are added in every hole, and 37 DEG C of closing 3h dry liquid in hole, are inverted in 37 DEG C of baking ovens
1h is spare.
S4. it is loaded and is incubated for: fenifrothion and its analogue being diluted to graded series titer, are diluted to respectively
5000,1000,200,40,8,1.6,0.32ng/mL, every hole adds 50 μ L, and 10ng/mL antibody (3 Chinese style of embodiment is then added
(III-1) fenifrothion comlete antigen shown in is immune to be prepared) 50 μ L of dilution, react 40min in 37 DEG C of water baths
Afterwards, board-washing is machine-washed plate 5 times, and 250 μ L of cleaning solution is added in every hole, dries liquid in hole.
Plus secondary antibody S5.: the HRP- sheep anti mouse that 100 μ L dilute 5000 times is added in every hole, reacts 30min in 37 DEG C of water baths
Afterwards, the same S4 of board-washing.
S6. develop the color: tmb substrate liquid and substrate buffer solution mix in equal volume, and 100 μ L of mixed liquor is added in every hole, are placed in 37 DEG C
It is developed the color after 10min in water bath, 50 μ L 10%H are added in every hole2SO4Terminate liquid.
S7. it measures: measuring each hole A with enzyme-linked immunosorbent assay instrument450nmLight absorption value.
S8. it calculates: calculating the IC of suppression curve with the four parameter fitting modules of Origin8.510、IC20、IC50、 IC80Value.
Cross reacting rate R (%) is as follows:
R (%)=IC50(fenifrothion)/IC50(fenifrothion analogue) × 100%.
Two, experimental result
Standard curve is shown in Fig. 5.Gained standard curve I C50Value is 7.13ng/mL, and detection is limited to (IC10) 1.7 ng/mL, line
Property detection range be 2.9~17.9ng/mL.
Specific detection is as shown in table 2.
Table 2:
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
Limitations on the scope of the patent of the present invention therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to guarantor of the invention
Protect range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (10)
1. a kind of haptens of fenifrothion, which is characterized in that its structural formula such as formula (II) or (I) are shown:
；。
2. the preparation method of haptens described in claim 1, which is characterized in that 3- methyl -4- nitrophenol and O- methyl sulphur
For dichloromethane solution, NaOH aqueous solution and the tetrabutylammonium bromide mixing of phosphinylidyne dichloro, stirring is reacted 12~16 h, is passed through
Silica gel column chromatography column purification, mobile phase are that volume ratio is petroleum ether: ethyl acetate=1~10:1 mixed solution, after purification to obtain the final product
Compound shown in formula (II)；Compound, 6-aminocaprolc acid shown in formula (II) are mixed with Isosorbide-5-Nitrae-dioxane, stirring, under ice bath with
The mixing of NaOH solution, makes solution maintain pH 10~11, stirs 12~16 h to get compound shown in formula (I).
3. a kind of comlete antigen of fenifrothion, which is characterized in that its structural formula such as formula (III) or (IV) are shown:
；。
4. comlete antigen according to claim 3, which is characterized in that the carrier protein is bovine lactoferrin, cow's serum
One of albumin, hemocyanin or ovalbumin.
5. the preparation method of structural formula comlete antigen as shown in the formula (III), which comprises the following steps:
S11. haptens is activated: n,N-Dimethylformamide solution, the 1- of structural formula haptens as shown in the formula (I) under stiring
(3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride and n-hydroxysuccinimide mixing, under be protected from light 4~6h of stirring,
Obtain A liquid；
S12. carrier protein is dissolved in carbonic acid buffer, obtains B liquid, and carrier protein concentration is 5~10 mg/mL；
S13. it will be mixed in the A liquid of S11 and the B liquid of S12, adjust pH to 9.5~9.6 with NaOH solution or carbonic acid buffer, keep away
12~16 h of light reaction obtains structural formula comlete antigen as shown in the formula (III) after dialysis purification；
Wherein, structural formula haptens as shown in the formula (I), 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride and N-
The molar ratio of HOSu NHS is 1:1.2~1.5:1.2~1.5.
6. the preparation method of structural formula comlete antigen as shown in formula (IV), which comprises the following steps:
S21. Isosorbide-5-Nitrae-dioxane solution of structural formula haptens as shown in the formula (II), referred to as A liquid；
S22. carrier protein is dissolved in carbonic acid buffer, and carrier protein concentration is 5~10 mg/mL, referred to as B liquid；
S23. the A liquid of S21 is mixed with the B liquid of S22, adjusts pH to 9.5~9.6 with NaOH solution, is protected from light 12~16
H obtains structural formula comlete antigen as shown in formula (IV) after dialysis purification.
7. according to any preparation method of claim 5 or 6, which is characterized in that the structural formula such as formula (I) in the A liquid
Or the molar ratio of the carrier protein in haptens shown in (II) and the B liquid is 30~40:1.
8. a kind of antibody of fenifrothion, which is characterized in that using one or both of haptens described in claim 1, and/
Or it is prepared using one or both of comlete antigen described in claim 3.
9. antibody described in comlete antigen described in haptens, claim 3 described in claim 1, and/or claim 8 is detecting
Application in fenifrothion or preparation detection fenifrothion kit.
10. a kind of detection method of the fenifrothion based on Indirect cELISA, which is characterized in that with formula (III) institute
Show that compound is immunogene, using compound shown in formula (IV) as coating antigen.
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| CN201811647747.0A CN109734745B (en) | 2018-12-29 | 2018-12-29 | Preparation method and application of fenitrothion antibody |
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| CN201811647747.0A CN109734745B (en) | 2018-12-29 | 2018-12-29 | Preparation method and application of fenitrothion antibody |
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| CN109734745B CN109734745B (en) | 2021-05-04 |
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| CN201811647747.0A Active CN109734745B (en) | 2018-12-29 | 2018-12-29 | Preparation method and application of fenitrothion antibody |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113092749A (en) * | 2021-04-07 | 2021-07-09 | 华南农业大学 | Fluorescence ratio type immunoassay method for detecting fenitrothion |
| CN113214109A (en) * | 2021-04-15 | 2021-08-06 | 中国农业大学 | Nifurosol hapten, artificial antigen, preparation methods and applications thereof |
| CN113307877A (en) * | 2021-04-14 | 2021-08-27 | 华南农业大学 | Preparation and application of nano antibody capable of simultaneously recognizing fenitrothion and methyl parathion |
| CN113388037A (en) * | 2021-04-14 | 2021-09-14 | 华南农业大学 | Preparation and application of specific recognition fenitrothion nano antibody |
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| JP2000219695A (en) * | 1998-11-24 | 2000-08-08 | Kankyo Meneki Gijutsu Kenkyusho:Kk | Hapten compound for fenitrothion, antibody and measurement |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113092749A (en) * | 2021-04-07 | 2021-07-09 | 华南农业大学 | Fluorescence ratio type immunoassay method for detecting fenitrothion |
| CN113307877A (en) * | 2021-04-14 | 2021-08-27 | 华南农业大学 | Preparation and application of nano antibody capable of simultaneously recognizing fenitrothion and methyl parathion |
| CN113388037A (en) * | 2021-04-14 | 2021-09-14 | 华南农业大学 | Preparation and application of specific recognition fenitrothion nano antibody |
| CN113307877B (en) * | 2021-04-14 | 2022-04-29 | 华南农业大学 | Preparation and application of nano antibody capable of simultaneously recognizing fenitrothion and methyl parathion |
| CN113388037B (en) * | 2021-04-14 | 2022-04-29 | 华南农业大学 | Preparation and application of specific recognition fenitrothion nano antibody |
| CN113214109A (en) * | 2021-04-15 | 2021-08-06 | 中国农业大学 | Nifurosol hapten, artificial antigen, preparation methods and applications thereof |
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| CN109734745B (en) | 2021-05-04 |
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