CN114560834B - Spirodiclofen hapten, antigen and antibody as well as preparation methods and applications thereof - Google Patents

Spirodiclofen hapten, antigen and antibody as well as preparation methods and applications thereof Download PDF

Info

Publication number
CN114560834B
CN114560834B CN202210229226.3A CN202210229226A CN114560834B CN 114560834 B CN114560834 B CN 114560834B CN 202210229226 A CN202210229226 A CN 202210229226A CN 114560834 B CN114560834 B CN 114560834B
Authority
CN
China
Prior art keywords
spirodiclofen
formula
antigen
hapten
coupling reaction
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202210229226.3A
Other languages
Chinese (zh)
Other versions
CN114560834A (en
Inventor
崔永亮
金雅琪
彭怡霖
焦必宁
张耀海
赵其阳
何悦
李晶
王成秋
陈爱华
李志霞
赵希娟
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Southwest University
Original Assignee
Southwest University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Southwest University filed Critical Southwest University
Priority to CN202210229226.3A priority Critical patent/CN114560834B/en
Publication of CN114560834A publication Critical patent/CN114560834A/en
Application granted granted Critical
Publication of CN114560834B publication Critical patent/CN114560834B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/94—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom spiro-condensed with carbocyclic rings or ring systems, e.g. griseofulvins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C07K14/765—Serum albumin, e.g. HSA
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C07K14/77—Ovalbumin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/795—Porphyrin- or corrin-ring-containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • 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
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Peptides Or Proteins (AREA)
  • Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)

Abstract

The invention discloses a spirodiclofen hapten, antigen and antibody as well as preparation methods and applications thereof, wherein the hapten and the antigen respectively have structural formulas shown in formulas A1 and A2, and the preparation method comprises the following steps: 3- (2, 4-dichlorophenyl) -4-hydroxy-1-oxaspiro [4.5]]Reacting dec-3-en-2-one with a compound containing an ester group to obtain a hapten compound A1 containing a carboxyl group, performing a first coupling reaction on the hapten compound A1 containing the carboxyl group and N-hydroxysuccinimide to obtain a precursor compound, and performing a second coupling reaction on the precursor compound and carrier protein to obtain the antigen A2. The spirodiclofen antigen prepared by the preparation method of the invention can further obtain the spirodiclofen antibody with good specificity and high sensitivity, realizes the rapid, accurate and low-cost detection of the spirodiclofen,

Description

Spirodiclofen hapten, antigen and antibody as well as preparation methods and applications thereof
Technical Field
The invention relates to the technical field of spirodiclofen detection reagents.
Background
The spirodiclofen is also called as fenugreek, fenugreek and quaternary ketoester, is a high-efficiency novel non-systemic acaricide, is a white powdery pure product, has no special smell, and mainly comprises 24% of 240 g/L of suspending agent and 15% of aqueous emulsion, and has the chemical name of 3- (2, 4-dichlorophenyl) -2-oxo-1-oxaspiro [4,5] -dec-3-en-4-yl-2, 2-dimethylbutyrate, and has the following structural formula:
the spirodiclofen has a brand-new action mechanism, has a touch-killing effect and no systemic property, can mainly inhibit fat synthesis of mites, blocks energy metabolism of the mites, has good killing effect on eggs, young mites and if mites of mites, has the effect of inhibiting spawning hatchability of female mites, and has wide mite killing spectrum and strong adaptability. In addition, the spirodiclofen has good control effects on red mites, yellow mites, tea yellow mites, tetranychus cinnabarinus, tetranychus urticae, and the like, has control effects on pests such as pear psyllids, elm scale and leafhoppers, and can be used for crops such as oranges, pomes, drupes, grapes, strawberries, hops, nuts, and the like; can be mixed with other quick-acting acaricides (pyridaben, abamectin, etc.), and can improve the drug effect and reduce the risk of resistance of harmful mites.
On the other hand, part of the existing researches consider that spirodiclofen is a pesticide which may have cancerogenic action on human beings, and the research on spirodiclofen at home and abroad is mainly focused on the analysis of raw medicines and preparations and the field efficacy, and the research on the residual degradation of spirodiclofen on crops is less.
At present, the residual detection method of spirodiclofen in agricultural products mainly comprises a gas chromatography method, a liquid chromatography method, a gas chromatography-mass spectrometry method and the like, and the detection sensitivity of an instrument method is high, accurate and reliable, but the requirement on a sample pretreatment process is high, the detection time is long, and the required instrument is huge and expensive and is not suitable for mass sample measurement.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a novel spirodiclofen hapten, antigen and antibody as well as preparation and application methods thereof, and the detection method based on the antigen realizes quantitative detection of a target spirodiclofen through specific combination between the antibody and the antigen, and has high sensitivity and selectivity, and simultaneously is simple, quick and low in cost.
The invention firstly provides a spirodiclofen hapten which has a structural formula shown in a formula A1:
wherein,
r is selected from- (CH) 2 ) m -or-X- (CH) 2 ) a X is selected from any one of aromatic ring and aromatic heterocycle;
n is selected from integers from 0 to 6, m is selected from integers from 0 to 6, and a is selected from integers from 0 to 6;
the invention further provides a spirodiclofen antigen, which has a structural formula shown in a formula A2:
wherein,
r is selected from- (CH) 2 ) m -or-X- (CH) 2 ) a X is selected from aromatic ring and aromatic heterocycleAny of (2) to (3);
n is selected from integers from 0 to 6, m is selected from integers from 0 to 6, and a is selected from integers from 0 to 6;
protein represents a carrier Protein.
According to some embodiments of the invention, the carrier protein is selected from one or more of bovine serum albumin, ovalbumin and hemocyanin.
Further, as exemplified in some embodiments of the present invention, spirodiclofen antibodies can be obtained according to the above hapten compounds and/or antigens.
The invention further provides a preparation method of the spirodiclofen hapten or the spirodiclofen antigen, which comprises the following steps:
and (3- (2, 4-dichlorophenyl) -4-hydroxy-1-oxaspiro [4.5] dec-3-en-2-one) shown in the formula B and a compound containing an ester group shown in the formula C are mixed in an organic solvent to react, so that a compound containing a carboxyl group shown in the formula D, namely a hapten shown in the formula A1 is obtained.
Further, the preparation method further comprises the following steps:
carrying out a first coupling reaction on a compound shown in a formula D and N-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide or 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride to obtain a compound shown in a formula E;
carrying out a second coupling reaction on the compound shown in the formula E and carrier protein to obtain the spirodiclofen antigen;
wherein:
Y-(CH 2 )n-R-COO-(CH 2 )b-CH 3
C
wherein R is selected from- (CH) 2 ) m -or-X- (CH) 2 ) a X is selected from any one of aromatic ring and aromatic heterocycle, Y is selected from any one of Cl, br and I, n is selected from an integer of 0-6, m is selected from an integer of 0-6, a is selected from an integer of 1-6, and b is selected from an integer of 0-6.
Preferably, wherein the carrier protein is selected from one or more of bovine serum albumin, ovalbumin and hemocyanin.
In the preparation method, the spirodiclofen antigen is a conjugate formed by connecting a compound shown in a formula E and carrier protein through an amide bond, wherein the amide bond is formed by carboxyl on the compound shown in a formula D through active ester and amino on the carrier protein.
According to some preferred embodiments of the present invention, the compound represented by formula C is selected from one or more of methyl chloroformate, ethyl chloroacetate, butyl chloroacetate, methyl chloropropionate, methyl chlorobutyrate, ethyl chlorobutyrate, propyl chlorobutyrate, methyl bromoacetate, ethyl bromoacetate, butyl bromoacetate, methyl bromobutyrate, ethyl bromobutyrate, butyl bromobutyrate, ethyl iodoacetate, methyl iodobutyrate, ethyl iodobutyrate, methyl p-chlorobenzoate, ethyl o-chloroacetate, ethyl m-chloroacetate, methyl p-bromophenylacetate, ethyl p-bromomethylbenzoate, methyl m-bromomethylbenzoate, ethyl m-bromobenzoate, methyl p-iodobenzoate, ethyl p-iodophenylacetate, methyl o-iodobenzoate.
According to some preferred embodiments of the invention, the organic solvent is selected from one or more of pyridine, N-dimethylformamide, dimethylsulfoxide and tetrahydrofuran.
According to some preferred embodiments of the invention, the molar ratio of the compound of formula C to the compound of formula B is (0.1-1): 01.
According to some preferred embodiments of the invention, the molar ratio of the compound of formula D to the N-hydroxysuccinimide and the dicyclohexylcarbodiimide is 1: (1-5): (1-5).
According to some preferred embodiments of the present invention, the molar ratio of the compound represented by formula D to the N-hydroxysuccinimide and the 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride is 1: (1-5): (1-5).
According to some preferred embodiments of the invention, the temperature of the mixing reaction is between 0 and 100 ℃ and/or the reaction time is between 6 and 48 hours.
According to some preferred embodiments of the invention, the temperature of the first coupling reaction is between 0 and 50 ℃ and/or the reaction time is between 4 and 24 hours.
According to some preferred embodiments of the invention, the molar ratio of the compound of formula E to the carrier protein is (5-30): 1.
according to some preferred embodiments of the invention, the temperature of the second coupling reaction is between 0 and 50 ℃ and/or the reaction time is between 8 and 36 hours.
According to some preferred embodiments of the invention, the second coupling reaction is carried out at a pH of 5 to 9.
According to some preferred embodiments of the invention, the second coupling reaction is performed in a solution of the carrier protein, the solution of the carrier protein being obtained by adding the carrier protein to a buffer solution, the buffer solution being selected from one or more of a carbonate buffer, a phosphate buffer, a borate buffer and a 4-hydroxyethylpiperazine ethanesulfonic acid buffer.
More preferably, the pH of the buffer is 4 to 10.
According to some preferred embodiments of the invention, the method of preparing further comprises: and dialyzing the mixed system obtained after the second coupling reaction.
More preferably, the dialysate used in the dialysis is phosphate buffer solution having a pH of 4 to 10 and/or a concentration of 0.01 to 0.2 mol/L.
The invention further provides application of the hapten and/or the antigen and/or the antibody prepared by the preparation method in spirodiclofen detection.
According to some embodiments of the invention, the detection comprises one or more of an enzyme-linked immunosorbent assay, a luminescent immunosorbent assay, an immunoaffinity chromatography column assay.
According to some embodiments of the invention, the test sample for which the test is directed may comprise one or more of a body of water, a pharmaceutical, a food, an agricultural product, or soil.
The preparation method can conveniently and rapidly obtain the spirodiclofen antigen, and has simple and clear synthesis steps, low synthesis cost and good synthesis effect.
The spirodiclofen antibody obtained by immunization with the spirodiclofen antigen has good specificity and low minimum detection limit.
The spirodiclofen antigen and/or antibody provided by the invention have the advantages of high sensitivity, strong selectivity and wide application prospect in the rapid immunodetection of spirodiclofen.
Drawings
FIG. 1 is a schematic representation of a specific synthetic route to the spirodiclofen antigen.
Detailed Description
The present invention will be described in detail with reference to the following examples and drawings, but it should be understood that the examples and drawings are only for illustrative purposes and are not intended to limit the scope of the present invention in any way. All reasonable variations and combinations that are included within the scope of the inventive concept fall within the scope of the present invention.
Materials, reagents, and the like used in the following examples, unless otherwise specified, are commercially available and include, for example: 3- (2, 4-dichlorophenyl) -4-hydroxy-1-oxaspiro [4.5] dec-3-en-2-one, ethyl 4-bromobutyrate, dicyclohexylcarbodiimide (DCC), 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), freund's complete adjuvant, freund's incomplete adjuvant, bovine serum albumin and ovalbumin, sheep anti-mouse IgG-HRP, single-component TMB color development solution.
Example 1 preparation of spirodiclofen-ovalbumin (spirodiclofen-OVA) antigen
The antigen is prepared by a synthetic route shown in fig. 1, and specifically comprises the following steps:
(1) Synthesis of Compound of formula I
Into a 50mL three-necked flask, 0.5g of 3- (2, 4-dichlorophenyl) -4-hydroxy-1-oxaspiro [4.5] was charged]Dec-3-en-2-one was dissolved by adding 40mL of acetonitrile, 0.374g of ethyl 4-bromobutyrate was added dropwise, 0.44g of anhydrous potassium carbonate was added, the mixture was stirred and heated to 100℃and reacted for 24 hours, and after evaporation to dryness by rotation, extracted with ethyl acetate (3X 50 mL) and dried over anhydrous magnesium sulfate. Spin desolventizing, column chromatography to obtain a compound of formula I 1 H-NMR、 13 The C-NMR and mass spectrum characterization data are as follows:
1 H NMR(400MHz,DMSO)δ7.72(s,1H),7.48(s,2H),4.03(q,2H),3.83(m,2H),2.29(t,2H),1.82(m,2H),1.76(m,10H),1.15(t,3H).
13 C NMR(101MHz,DMSO)δ178.35,172.50,170.39,135.86,134.76,134.61,129.10,129.08,127.75,98.37,83.06,71.89,60.39,33.52,32.81,30.08,24.32,21.95,21.93,14.51.
ESI-MS:m/z calcd for C 20 H 20 N 3 O 4 (M+Na + )449.09,found 449.00.
the structural formula is as follows:
(2) Synthesis of Compound of formula II
Into a 50mL three-necked flask, 0.5g of the compound of formula I, 0.3g of lithium hydroxide was charged, and the mixture was dissolved in 20mL of methanol and 5mL of water, followed by stirring at room temperature for 8 hours. After the reaction is finished, removing methanol by rotary evaporation, adding 20mL of water, adjusting the pH value to 3 by using concentrated hydrochloric acid, filtering and drying to obtain a compound shown in a formula II 1 H-NMR、 13 C-NMR and Mass Spectrometry characterization dataThe following steps:
1 H NMR(400MHz,DMSO)δ12.01(s,1H),7.71(s,1H),7.18(s,2H),3.81(m,2H),2.21(t,2H),1.87(m,2H),1.75(m,10H).
13 C NMR(101MHz,DMSO)δ178.40,174.03,170.41,135.87,134.75,134.58,129.11,129.09,127.73,98.29,83.08,72.00,33.54,32.83,30.15,24.59,24.30,21.96.
ESI-MS:m/z calcd for C 20 H 20 N 3 O 4 (M+Na + )421.06,found 421.00.
the structural formula is as follows:
(3) Coupling reaction
Weighing spirodiclofen hapten 0.03mmol,NHS 0.045mmo shown in a formula II and obtained in the step (2), dissolving DCC (digital versatile chloride) 0.033mmol in 1mL anhydrous DMF (dimethyl formamide), stirring and reacting at room temperature of 25 ℃ for 6 hours, centrifuging the reaction solution at 8000 revolutions for 5 minutes, and taking supernatant to obtain a compound shown in a formula III:
(4) Slowly and dropwise adding the compound shown in the formula III obtained in the step (3) into a carrier protein OVA solution, wherein the carrier protein solution is prepared by uniformly mixing 20mg of OVA in 5mL of Phosphate Buffer Solution (PBS) with the pH value of 7.4, the feeding molar ratio of the compound shown in the formula III to the carrier protein is 20:1, and stirring at the temperature of 4 ℃ overnight.
(5) And (3) dialysis: dialyzing the reaction solution obtained in the step (4) by using PBS solution with the pH value of 7.4 and the concentration of 0.01mol/L for three days, diluting the completely dialyzed reaction product solution (spirodiclofen-OVA) into a solution with the concentration of 1mg/mL, and putting the solution into a freeze-storage at the temperature of-40 ℃ for standby.
In the above process, the PBS solution is prepared according to the following method: naCl, KH 2 PO 4 And Na (Na) 2 HPO 4 ·12H 2 O is mixed with the following components in mass ratio of 8.0:0.2:2.96 in water byThe volume of water is fixed to 1L.
In the process, unreacted spirodiclofen hapten and other small molecules can be removed by dialysis, so that the conjugate of spirodiclofen shown in the formula IV-1 and OVA, namely spirodiclofen antigen shown in the formula A2, is obtained:
example 2 preparation of spirodiclofen-ovalbumin (spirodiclofen-OVA) antigen
The spirodiclofen hapten 0.036mmol,NHS 0.047mmol shown in the formula II and obtained in the step (1) in the example 1 and EDC 0.030mmol are weighed and dissolved in 1mL of water, and after stirring and reacting for 6 hours at the room temperature of 25 ℃, the compound shown in the formula III is obtained, and then the spirodiclofen antigen shown in the formula IV-1 and OVA conjugate can be obtained according to the steps (4) to (5) in the example 1.
Example 3 preparation of spirodiclofen-hemocyanin (spirodiclofen-KLH) antigen
(1) A spirodiclofen hapten of formula II and a compound of formula III were synthesized by the procedure of example 1 or 2.
(2) Slowly dropwise adding the compound shown in the formula III into a carrier protein solution, wherein the carrier protein solution is obtained by uniformly mixing 16.8mg of KLH in 5mL of Phosphate Buffer Solution (PBS) buffer solution with the pH value of 7.4, the feeding molar ratio of the compound shown in the formula III to the carrier protein is 800:1, and stirring at the temperature of 4 ℃ overnight.
(3) Dialyzing the reaction solution obtained in the step (2) by using PBS solution with the pH value of 7.4 and the concentration of 0.01mol/L for three days, diluting the completely dialyzed reaction product solution (spirodiclofen-KLH) into 1mg/mL solution, and placing the solution in a frozen state at the temperature of-40 ℃ for standby.
In the above process, the PBS solution is prepared according to the following method: naCl, KH 2 PO 4 And Na (Na) 2 HPO 4 ·12H 2 O is mixed with the following components in mass ratio of 8.0:0.2:2.96 in water to a volume of 1L with water.
The obtained product is a conjugate of spirodiclofen shown in a formula IV-2 and KLH, namely spirodiclofen antigen shown in a formula A2:
example 4 preparation of antibodies Using spirodiclofen-hemocyanin (spirodiclofen-KLH) antigen
(1) Bal b/c mice at 8-10 weeks of age were used as experimental animals.
(2) Basic immunization: the diluted spirodiclofen-KLH antigen solution with the concentration of 1mg/mL is obtained in the example 3, filtered by a sterile filter, added with an equal volume of Freund complete adjuvant, and fully stirred and emulsified by a magnetic stirrer until the spirodiclofen-KLH antigen solution is not diffused in dripping water; bal b/c mice were subcutaneously multi-point injected with 0.1mg emulsified antigen per mouse on the abdominal and back with emulsified complete antigen.
(3) Boosting: after basic immunization is carried out for 2 weeks, 1mL of the diluted spirodiclofen-BSA antigen solution is taken, then 1mL of Freund's incomplete adjuvant is added, and the mixture is fully stirred and emulsified by a magnetic stirrer until the mixture is not diffused in dripping water; the emulsified antigens were injected subcutaneously into Bal b/C mice intraperitoneally and subcutaneously at the back at a dose of 0.1mg of emulsified diluted antigen per mouse (8-week-old Bal b/C mice weighing about 23-25 g).
Boosting is performed once every 15 days, starting from the third boosting, taking blood from the eye sockets of a mouse on the 3 rd to 5 th days after each immunization, measuring the titer of antibodies, diluting the antigen with 1mg/mL spirodiclofen-OVA for 500 times, taking blood after the titer is more than 1:8000 (the dilution factor of serum when the titer is defined as zero Kong Xian color value is 1), taking the blood after the eyeball is removed, standing the blood for 1 hour at room temperature, standing the blood for 2 hours in a refrigerator at 4 ℃, centrifuging the blood for 5 minutes at 8000r/min in a centrifuge, and separating antiserum to obtain spirodiclofen-KLH antibodies for detection described below.
Example 5 antibody Effect detection
The antigen obtained in example 4 was subjected to antibody inhibition assay using the following buffers:
coating buffer solution: carbonate buffer of 0.05M, pH 9.6.6.
Phosphate buffer PBS (ph=7.4): weigh 4.0g NaCl, 0.1g KH 2 PO 4 、1.48g Na 2 HPO 4 ·12H 2 O was fixed to 500mL with distilled water at a concentration of 0.01. 0.01M, pH in 7.4 phosphate buffer.
Sample dilution PBSTG: from 0.5mL Tween 20, 0.5g gelatin, and 500mL PBS buffer at a concentration of 0.1. 0.1M, pH of 7.4.
Stop buffer: 2.0M aqueous sulfuric acid.
Washing liquid: from NaCl, KH 2 PO 4 、Na 2 HPO 4 ·12H 2 O, tween-20 and water; the concentration of NaCl in the washing liquid is 8.0g/L, KH 2 PO 4 The concentration in the washing liquid is 0.2g/L, na 2 HPO 4 ·12H 2 The concentration of O in the washing liquid is 2.96g/L, and the volume percentage of Tween-20 in the washing liquid is 1:1000.
The detection process comprises the following steps:
(1) Preparation of spirodiclofen-OVA coated antigen solution
After the diluted 1mg/mL spirodiclofen-OVA antigen prepared in the example 1 is completely thawed, gradient dilution is carried out by using coating buffer solution according to the ratio of 1:500, 1:1000, 1:2000 and 1:4000, so as to obtain the coating antigen solutions of the spirodiclofen-OVA with different concentrations.
(2) Preparation of spirodiclofen standard solution
Weighing 10mg of spirodiclofen standard sample, and fully dissolving the sample in 10mL of absolute methanol to obtain 1.0mg/mL of spirodiclofen standard solution; the obtained 1.0mg/mL spirodiclofen standard solution is prepared into a spirodiclofen standard solution with the concentration of 1000ng/mL by using a sample diluent.
(3) Preparation of spirodiclofen-KLH antiserum diluent
The spirodiclofen-KLH antibody prepared in example 4 was diluted in a gradient with sample dilutions 1:500, 1:1000, 1:2000, 1:4000 to give a spirodiclofen-KLH antiserum dilution.
(4) Checkerboard experiment of antigen and antibody
Coating: mu.L of spirodiclofen-OVA coated antigen solution was added to each well of a 96-well ELISA plate, and the plate was coated at 37℃for 3 hours and washed 4 times with washing solution.
Closing: weighing 5g of skimmed milk powder, fully dissolving in 100mL of sample diluent to obtain 5% sealing solution, adding 200 mu L of sealing solution into each hole of a 96-hole ELISA plate, sealing for 1h in a 37 ℃ wet box, discarding the sealing solution, and washing for 3 times.
Competing: 50 μl of sample dilution was added to each well of the zero wells, and 50 μl of spirodiclofen standard solution was added to each well of the inhibition wells. The spirodiclofen-KLH antiserum dilutions were run from 1X 10 3 Multiple by 8×10 3 The double was added to the ELISA plate (50. Mu.l/well), the plate was washed 4 times at 37℃for 30min in a wet box.
Adding enzyme-labeled secondary antibodies: sheep anti-mouse enzyme-labeled secondary antibody (IgG-HRP, jackson, cat. No. 79556) was diluted 1000-fold in 0.01M PBSTG pH 7.4, 100. Mu.L per well, placed in a humidity box at 37deg.C for 30min, and washed 4 times.
Color development: the single-component TMB color developing solution is added into the ELISA plate, 100 mu l of each hole is used for color development for 15min in dark.
And (3) terminating: 50. Mu.L of stop buffer was added to each well and the OD of each well was measured at 450nm using an ELISA reader.
The results are shown in table 1, where titers are defined as serum dilution at zero well OD of 1:
TABLE 1 detection of serum titers of anti-spirodiclofen mice (Single-component TMB color development at room temperature for 15min,1000ng inhibition of standard)
Note that: i represents the inhibition wells in the ELISA plate and C represents the control wells in the ELISA plate.
The results show that the spirodiclofen-KLH obtained in the above way can be used as an immunogen to prepare antibodies for detecting spirodiclofen.
Those of ordinary skill in the art will appreciate that the embodiments described herein are intended to aid the reader in understanding the practice of the invention and that the scope of the invention is not limited to such specific statements and embodiments. Those of ordinary skill in the art can make various other specific modifications and combinations from the teachings of the present disclosure without departing from the spirit thereof, and such modifications and combinations remain within the scope of the present disclosure.

Claims (13)

1. A spirodiclofen hapten which is characterized by having a structural formula shown in a formula A1:
wherein,
r is selected from- (CH) 2 ) m -, and m+n=4.
2. A spirodiclofen antigen, characterized in that it has the structural formula shown in formula A2:
wherein,
r is selected from- (CH) 2 ) m -, and m+n=4;
protein represents a carrier Protein.
3. The spirodiclofen antigen according to claim 2, wherein the carrier protein is selected from one or more of bovine serum albumin, ovalbumin and hemocyanin.
4. A spirodiclofen hapten according to claim 1 or a spirodiclofen antibody obtained in response to a spirodiclofen antigen according to claim 2 or 3.
5. A method of preparing a spirodiclofen hapten as defined in claim 1 or a spirodiclofen antigen as defined in claim 2 or 3, comprising:
mixing a compound shown in a formula B with a compound shown in a formula C in an organic solvent to obtain a compound shown in a formula D, namely the spirodiclofen hapten shown in a formula A1;
or further comprises:
carrying out a first coupling reaction on a compound shown in a formula D and N-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide or 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride to obtain a compound shown in a formula E;
carrying out a second coupling reaction on the compound shown in the formula E and carrier protein to obtain the spirodiclofen antigen,
wherein:
Y-(CH 2 )n-R-COO-(CH 2 )b-CH 3
C
wherein R is selected from- (CH) 2 ) m And m+n=4; y is selected from any one of Cl, br and I, and b is selected from an integer of 0-6.
6. The preparation method according to claim 5, wherein the compound represented by the formula C is selected from one or more of methyl chlorobutyrate, ethyl chlorobutyrate, propyl chlorobutyrate, methyl bromobutyrate, ethyl bromobutyrate, butyl bromobutyrate, methyl iodobutyrate, ethyl iodobutyrate; the organic solvent is selected from one or more of pyridine, N-dimethylformamide, dimethyl sulfoxide and tetrahydrofuran.
7. The production method according to claim 5, wherein a molar ratio of the compound represented by the formula C to the compound represented by the formula B is 0.1 to 1:0.1, and a molar ratio of the compound represented by the formula D to the N-hydroxysuccinimide and the dicyclohexylcarbodiimide is 1:1 to 5:1 to 5; the molar ratio of the compound shown in the formula D to the N-hydroxysuccinimide and the 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride is 1:1 to 5:1 to 5; the molar ratio of the compound shown in the formula E to the carrier protein is 5-30: 1.
8. the method according to claim 5, wherein the temperature of the mixing reaction is 0 to 100 ℃, the reaction time is 6 to 48 hours, the temperature of the second coupling reaction is 0 to 50 ℃, and the reaction time is 8 to 36 hours; the second coupling reaction is carried out under the condition that the pH value is 5-9; the temperature of the first coupling reaction is 0-50 ℃ and the reaction time is 4-24 hours.
9. The method according to claim 5, wherein the second coupling reaction is performed in a solution of the carrier protein obtained by adding the carrier protein to a buffer solution selected from one or more of a carbonate buffer, a phosphate buffer, a borate buffer, and a 4-hydroxyethylpiperazine ethanesulfonic acid buffer.
10. The method according to claim 9, wherein the buffer has a pH of 4 to 10.
11. The method of manufacturing according to claim 5, further comprising: and (3) dialyzing the mixed system obtained after the second coupling reaction, wherein the dialysate used in the dialyzing is phosphate buffer solution with the pH value of 4-10.
12. Use of a spirodiclofen hapten as defined in claim 1 or a spirodiclofen antigen as defined in claim 2 or 3 or a spirodiclofen antibody as defined in claim 4 for the preparation of a spirodiclofen detection preparation.
13. The use of claim 12, wherein the detection comprises one or more of an enzyme-linked immunosorbent assay, a luminescent immunosorbent assay, an immunoaffinity chromatography column assay.
CN202210229226.3A 2022-03-10 2022-03-10 Spirodiclofen hapten, antigen and antibody as well as preparation methods and applications thereof Active CN114560834B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210229226.3A CN114560834B (en) 2022-03-10 2022-03-10 Spirodiclofen hapten, antigen and antibody as well as preparation methods and applications thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210229226.3A CN114560834B (en) 2022-03-10 2022-03-10 Spirodiclofen hapten, antigen and antibody as well as preparation methods and applications thereof

Publications (2)

Publication Number Publication Date
CN114560834A CN114560834A (en) 2022-05-31
CN114560834B true CN114560834B (en) 2024-02-09

Family

ID=81717910

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210229226.3A Active CN114560834B (en) 2022-03-10 2022-03-10 Spirodiclofen hapten, antigen and antibody as well as preparation methods and applications thereof

Country Status (1)

Country Link
CN (1) CN114560834B (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102627628A (en) * 2012-03-08 2012-08-08 中国农业大学 Chlorantraniliprole antigen, its preparation method and application
CN113980117A (en) * 2021-11-17 2022-01-28 西南大学 Fenpyroximate antigen and preparation method and application thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102627628A (en) * 2012-03-08 2012-08-08 中国农业大学 Chlorantraniliprole antigen, its preparation method and application
CN113980117A (en) * 2021-11-17 2022-01-28 西南大学 Fenpyroximate antigen and preparation method and application thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Predictive Models of Prenatal Developmental Toxicity from ToxCast High-Throughput Screening Data;Nisha S. Sipes等;《TOXICOLOGICAL SCIENCES》;第124卷(第1期);第109–127页 *

Also Published As

Publication number Publication date
CN114560834A (en) 2022-05-31

Similar Documents

Publication Publication Date Title
CN101413955B (en) ELISA test box for detecting zearalenone and preparing and detecting method thereof
CN102621326B (en) Method for detecting furaltadone metabolite content in food
CN108640850B (en) Malachite green hapten, artificial antigen and application of malachite green hapten and artificial antigen in fluorescence quantitative immunochromatography
CN101245032A (en) Leuco malachite green hapten, produced antibody and application of the antibody
CN109734621B (en) 1-naphthol hapten as well as preparation method and application thereof
CN106866568A (en) Furaxone metabolite AOZ derivatizations haptens, the preparation method and applications of artificial antigen
CN108680755B (en) Sulfonamide synergist hapten and holoantigen as well as preparation method and application thereof
CN105503632A (en) Preparation method and application of hapten and artificial antigen capable of being used for detecting crystal violet and malachite green together
CN106831498B (en) Furacilin metabolite SEM derivatizations haptens, artificial antigen preparation method and applications
CN106588805A (en) Furaltadone metabolite (AMOZ) derivatization hapten, preparation method of artificial antigen and application of artificial antigen
CN110256298B (en) 4, 4' -dinitrophenylurea hapten and artificial antigen as well as preparation methods and application thereof
CN108484751B (en) Nobiletin antigen and preparation method and application thereof
CN108558718B (en) Florfenicol, florfenicol amine antigen and antibody and simultaneous detection enzyme-linked immunoassay method thereof
CN111978271B (en) Hexythiazox hapten and preparation method thereof, hexythiazox antigen, antibody and application thereof
CN114560834B (en) Spirodiclofen hapten, antigen and antibody as well as preparation methods and applications thereof
CN110683965B (en) Nifurtida hapten and artificial antigen as well as preparation methods and application thereof
CN110642743B (en) Nifuroxazide hapten and artificial antigen as well as preparation methods and application thereof
CN101135683B (en) Bifenthrin antigen, antibody and uses thereof
CN107118159A (en) Cistofuran metabolite AHD derivatizations haptens, the preparation method and applications of artificial antigen
CN109824599B (en) Albendazole hapten as well as preparation method and application thereof
CN102627628B (en) Chlorantraniliprole antigen, its preparation method and application
CN109438424B (en) Ribavirin hapten and artificial antigen as well as preparation method and application thereof
CN108640866B (en) Flubendiamide antigen and preparation method and application thereof
CN109575123B (en) Preparation method and application of fluoroacetamide hapten and monoclonal antibody
CN104513314A (en) Diazepam monoclonal antibody screening and application

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant