CN110981907A - Malathion hapten, artificial antigen and preparation method thereof - Google Patents
Malathion hapten, artificial antigen and preparation method thereof Download PDFInfo
- Publication number
- CN110981907A CN110981907A CN201911072717.6A CN201911072717A CN110981907A CN 110981907 A CN110981907 A CN 110981907A CN 201911072717 A CN201911072717 A CN 201911072717A CN 110981907 A CN110981907 A CN 110981907A
- Authority
- CN
- China
- Prior art keywords
- malathion
- hapten
- artificial antigen
- reacting
- carrier protein
- 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.)
- Pending
Links
- JXSJBGJIGXNWCI-UHFFFAOYSA-N diethyl 2-[(dimethoxyphosphorothioyl)thio]succinate Chemical compound CCOC(=O)CC(SP(=S)(OC)OC)C(=O)OCC JXSJBGJIGXNWCI-UHFFFAOYSA-N 0.000 title claims abstract description 84
- 239000005949 Malathion Substances 0.000 title claims abstract description 80
- 229960000453 malathion Drugs 0.000 title claims abstract description 80
- 239000000427 antigen Substances 0.000 title claims abstract description 35
- 102000036639 antigens Human genes 0.000 title claims abstract description 35
- 108091007433 antigens Proteins 0.000 title claims abstract description 35
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 238000000034 method Methods 0.000 claims abstract description 18
- CYQAYERJWZKYML-UHFFFAOYSA-N phosphorus pentasulfide Chemical compound S1P(S2)(=S)SP3(=S)SP1(=S)SP2(=S)S3 CYQAYERJWZKYML-UHFFFAOYSA-N 0.000 claims abstract description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 24
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 24
- 102000014914 Carrier Proteins Human genes 0.000 claims description 16
- 108010078791 Carrier Proteins Proteins 0.000 claims description 16
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 15
- 239000013067 intermediate product Substances 0.000 claims description 12
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 claims description 10
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 9
- UCKMPCXJQFINFW-UHFFFAOYSA-N Sulphide Chemical compound [S-2] UCKMPCXJQFINFW-UHFFFAOYSA-N 0.000 claims description 9
- 239000006228 supernatant Substances 0.000 claims description 9
- 235000001014 amino acid Nutrition 0.000 claims description 8
- 150000001413 amino acids Chemical class 0.000 claims description 8
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 6
- SLXKOJJOQWFEFD-UHFFFAOYSA-N 6-aminohexanoic acid Chemical compound NCCCCCC(O)=O SLXKOJJOQWFEFD-UHFFFAOYSA-N 0.000 claims description 5
- XLYMOEINVGRTEX-ARJAWSKDSA-N Ethyl hydrogen fumarate Chemical compound CCOC(=O)\C=C/C(O)=O XLYMOEINVGRTEX-ARJAWSKDSA-N 0.000 claims description 5
- 229960002684 aminocaproic acid Drugs 0.000 claims description 5
- XLYMOEINVGRTEX-UHFFFAOYSA-N fumaric acid monoethyl ester Natural products CCOC(=O)C=CC(O)=O XLYMOEINVGRTEX-UHFFFAOYSA-N 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 4
- 229940098773 bovine serum albumin Drugs 0.000 claims description 4
- 238000005406 washing Methods 0.000 claims description 4
- 238000001816 cooling Methods 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- SNDPXSYFESPGGJ-UHFFFAOYSA-N 2-aminopentanoic acid Chemical compound CCCC(N)C(O)=O SNDPXSYFESPGGJ-UHFFFAOYSA-N 0.000 claims description 2
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 claims description 2
- 108010088751 Albumins Proteins 0.000 claims description 2
- 102000009027 Albumins Human genes 0.000 claims description 2
- 102000002322 Egg Proteins Human genes 0.000 claims description 2
- 108010000912 Egg Proteins Proteins 0.000 claims description 2
- 239000004471 Glycine Substances 0.000 claims description 2
- 108091006905 Human Serum Albumin Proteins 0.000 claims description 2
- 102000008100 Human Serum Albumin Human genes 0.000 claims description 2
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims description 2
- 108010039918 Polylysine Proteins 0.000 claims description 2
- 235000004279 alanine Nutrition 0.000 claims description 2
- 229940124277 aminobutyric acid Drugs 0.000 claims description 2
- 210000000991 chicken egg Anatomy 0.000 claims description 2
- 230000008878 coupling Effects 0.000 claims description 2
- 238000010168 coupling process Methods 0.000 claims description 2
- 238000005859 coupling reaction Methods 0.000 claims description 2
- 238000001035 drying Methods 0.000 claims description 2
- 235000014103 egg white Nutrition 0.000 claims description 2
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 claims description 2
- 108060003552 hemocyanin Proteins 0.000 claims description 2
- 229920000656 polylysine Polymers 0.000 claims description 2
- 238000003556 assay Methods 0.000 claims 2
- 238000001514 detection method Methods 0.000 abstract description 22
- 238000006243 chemical reaction Methods 0.000 abstract description 15
- 230000003053 immunization Effects 0.000 abstract description 10
- 230000015572 biosynthetic process Effects 0.000 abstract description 5
- 238000003786 synthesis reaction Methods 0.000 abstract description 5
- 238000003018 immunoassay Methods 0.000 abstract description 4
- 230000002163 immunogen Effects 0.000 abstract description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 241001465754 Metazoa Species 0.000 abstract description 2
- 239000002994 raw material Substances 0.000 abstract description 2
- 239000007858 starting material Substances 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 18
- 239000008055 phosphate buffer solution Substances 0.000 description 10
- 238000005119 centrifugation Methods 0.000 description 8
- 238000004458 analytical method Methods 0.000 description 7
- 238000002649 immunization Methods 0.000 description 7
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 6
- 239000002671 adjuvant Substances 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 210000002966 serum Anatomy 0.000 description 6
- 241000699666 Mus <mouse, genus> Species 0.000 description 5
- 239000007853 buffer solution Substances 0.000 description 5
- 230000010355 oscillation Effects 0.000 description 5
- 230000035484 reaction time Effects 0.000 description 5
- 241000699670 Mus sp. Species 0.000 description 4
- 230000004913 activation Effects 0.000 description 4
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 4
- 238000000502 dialysis Methods 0.000 description 4
- 239000011259 mixed solution Substances 0.000 description 4
- 239000000575 pesticide Substances 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 3
- 150000002148 esters Chemical class 0.000 description 3
- 230000036039 immunity Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 125000004185 ester group Chemical group 0.000 description 2
- 239000003337 fertilizer Substances 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000000447 pesticide residue Substances 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 239000012460 protein solution Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 238000004566 IR spectroscopy Methods 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 230000003187 abdominal effect Effects 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 244000245420 ail Species 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 150000008064 anhydrides Chemical class 0.000 description 1
- 150000004945 aromatic hydrocarbons Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010241 blood sampling Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 150000001718 carbodiimides Chemical class 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 238000002967 competitive immunoassay Methods 0.000 description 1
- 239000012043 crude product Substances 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000001804 emulsifying effect Effects 0.000 description 1
- 125000003754 ethoxycarbonyl group Chemical group C(=O)(OCC)* 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000004186 food analysis Methods 0.000 description 1
- 235000004611 garlic Nutrition 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 231100000086 high toxicity Toxicity 0.000 description 1
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 238000002329 infrared spectrum Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 239000003987 organophosphate pesticide Substances 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/06—Phosphorus compounds without P—C bonds
- C07F9/16—Esters of thiophosphoric acids or thiophosphorous acids
- C07F9/165—Esters of thiophosphoric acids
- C07F9/17—Esters of thiophosphoric acids with hydroxyalkyl compounds without further substituents on alkyl
-
- 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
- G01N33/18—Water
- G01N33/1826—Organic contamination in water
- G01N33/184—Herbicides, pesticides, fungicides, insecticides or the like
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Analytical Chemistry (AREA)
- Biomedical Technology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Hematology (AREA)
- Zoology (AREA)
- Urology & Nephrology (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a malathion hapten, an artificial antigen and a preparation method thereof. The malathion hapten has a structure shown in a formula (I), wherein n is 1-9. The invention takes phosphorus pentasulfide as a starting material to synthesize a malathion hapten; further preparing artificial antigen of malathion, and immunizing animals with the prepared artificial immunogen can induce and generate specific antibody aiming at the malathion. The method has the advantages of simple and convenient synthesis steps, readily available raw materials, mild reaction conditions, suitability for production in laboratories and factories, application in establishing a rapid detection method for malathion and developing an immunoassay kit, satisfaction of the detection requirement for malathion residues, wide application prospect and great popularization value.
Description
Technical Field
The invention belongs to the technical field of food analysis. More particularly, relates to a malathion hapten, an artificial antigen and a preparation method thereof.
Background
Malathion (malathion), also known as malathion, 4049, is a colorless or yellow oily liquid with a garlic odor and pungent odor. The molecular weight is 330, and the product is easily soluble in organic solvents such as alcohols, acetone, esters and aromatic hydrocarbons, and slightly soluble in water and petroleum ether. Malathion is an ideal pesticide for replacing high-toxicity variety as a novel broad-spectrum organophosphorus pesticide with the characteristics of low toxicity and high efficiency. At present, the fertilizer is widely applied to agriculture and grain storage production, but the residue of the fertilizer in the environment and food poses a potential threat to the health of people to a certain extent. In part of fruits, vegetables and grains, malathion is one of pesticides with higher detection amount and is a necessary detection variety for a plurality of pesticide residue detection items.
At present, the quantitative detection of pesticide residues is mainly carried out in China by adopting instrument methods such as gas chromatography, gas chromatography-mass spectrometry, high performance liquid chromatography, liquid chromatography-mass spectrometry and the like. Although the instrumental detection method is accurate and credible, the method also has the defects of complicated sample pretreatment, time consumption, high cost, unsuitability for field detection and the like. In comparison, the immunoassay method represented by the enzyme-linked immunosorbent assay has the advantages of convenient and rapid detection and suitability for rapid detection of large-scale samples, and plays an increasingly important role in the field of rapid detection.
However, the basis for the establishment of immunological detection methods is to have high quality and strong specificity of antibodies, and the design of haptens and the synthesis of artificial antigens are the key points for obtaining high quality antibodies. At present, malathion hapten, artificial antigen, antibody and immunoassay based on the malathion hapten are not mature, the structure of the existing hapten is unreasonable, the synthesis steps are complex, the affinity and the specificity of the antibody are low, and the development of an immunological detection method of malathion is seriously hindered.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects and shortcomings of the prior art and provide a malathion hapten, an artificial antigen and a preparation method thereof. The malathion hapten has the advantages of reasonable structure, simple synthesis steps, high affinity of the prepared malathion antiserum and good sensitivity.
The first purpose of the invention is to provide a hapten of malathion.
The second object of the present invention is to provide a method for preparing the above hapten.
The third purpose of the invention is to provide an artificial antigen of malathion.
The fourth purpose of the invention is to provide an antibody of malathion.
The fifth purpose of the invention is to provide the application of the hapten, the artificial antigen and/or the antibody in establishing a malathion analysis and detection method and/or a malathion analysis and detection kit.
The sixth purpose of the invention is to provide a malathion detection kit.
The above purpose of the invention is realized by the following technical scheme:
a hapten of malathion has a structure shown in a formula (I), wherein n is 1-9,
formula (I)
The haptens have a carboxyl structure and arms of different lengths.
The preparation method of the hapten comprises the following steps:
s11, dissolving phosphorus pentasulfide in toluene, dropwise adding methanol at 35-45 ℃, reacting for 1.5-2.5 h at 50-60 ℃, and cooling to obtain a crude sulfide a, wherein the reaction formula is as follows:
s12, mixing the crude sulfide a and the ethyl hydrogen maleate in the step S11, reacting at 50-60 ℃ for 1-2 h and at 70-75 ℃ for 6-10 h to obtain an intermediate product b, wherein the reaction formula is as follows:
s13, dissolving the intermediate product b in the step S12 in a Dimethylformamide (DMF) solution, adding Dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS), reacting for 10-12 h at 0-4 ℃, centrifuging, taking the supernatant, dropwise adding the supernatant into amino acid solutions with different carbon chain lengths at the speed of 0.2-0.5 mL/min, reacting for 8-10 h under stirring at 0-4 ℃, washing and drying to obtain the hapten, wherein the reaction formula is as follows:
the amino acid solutions with different carbon chain lengths comprise glycine, alanine, aminobutyric acid, aminopentanoic acid and aminocaproic acid.
Preferably, the mass volume ratio of the phosphorus pentasulfide to the toluene in the step S11 is 66-100%.
More preferably, the mass volume ratio of the phosphorus pentasulfide to the toluene in the step S11 is 83%.
Preferably, methanol is added dropwise when the temperature is raised to 45 ℃ in step S11.
Preferably, the dropping speed of the methanol in the step S11 is 0.1-0.3 mL/min.
More preferably, the methanol dropping rate in step S11 is 0.2 mL/min.
Preferably, the mass-to-volume ratio of phosphorus pentasulfide to methanol in step S11 is 1: 1 to 1.5.
More preferably, the mass-to-volume ratio of phosphorus pentasulfide to methanol in step S11 is 1: 1.
preferably, the temperature of the reaction in step S11 is 55 ℃.
Preferably, the reaction time in step S11 is 2 h.
Preferably, when the temperature is cooled to below 40 ℃ in the step S11, crude sulfide a is obtained.
Preferably, the molar ratio of the crude sulfide a to the ethyl hydrogen maleate in the step S12 is 1-1.2: 1 to 1.5.
More preferably, the molar ratio of the crude sulfide a to the ethyl hydrogen maleate in the step S12 is 1: 1.
preferably, the reaction conditions in step S12 are 55 ℃ for 1 h.
Preferably, the reaction condition in the step S12 is 70-75 ℃ for 8 h.
Preferably, the mass-to-volume ratio of the intermediate product b to the DMF solution in the step S13 is 1-3: 1.
more preferably, the mass-to-volume ratio of the intermediate product b to the DMF solution in step S13 is 1.5: 1.
preferably, the molar ratio of the intermediate product b to DCC and NHS in the step S13 is 0.8-1.2: 1.2-2: 1.2 to 2.
More preferably, the molar ratio of the intermediate product b to DCC, NHS in step S13 is 1: 1.2: 1.2.
preferably, the intermediate product b, DCC, NHS in step S13 is reacted for 12 h.
Preferably, the centrifugation condition in the step S13 is 10000-15000 rpm centrifugation for 10-15 min.
More preferably, the centrifugation conditions in step S13 are 12000rpm centrifugation for 10 min.
Preferably, the dropping speed in step S13 is 0.2 mL/min.
Preferably, the amino acid solution with different carbon chain lengths in step S13 is aminocaproic acid.
Preferably, the supernatant droplets are reacted with an amino acid solution at 4 ℃ for 10h in step S13.
Preferably, the reagent required for the washing in step S13 is ethyl acetate.
Preferably, the molar ratio of the intermediate product b to the amino acid in step S13 is 1: 1 to 2.
More preferably, the molar ratio of intermediate b to amino acid in step S13 is 1: 1.2.
an artificial antigen of malathion, which is obtained by coupling the hapten and carrier protein and has a structure shown in a formula (II):
formula (II).
Preferably, the artificial antigen is prepared by an active ester method, a carbodiimide method or a mixed anhydride method.
Preferably, the artificial antigen is prepared by an active ester method, and comprises the following steps:
s21, mixing the hapten, the DCC and the NHS in a molar ratio of 0.8-1.2: 1.2-2: dissolving 1.2-2 in DMF, and carrying out vibration activation reaction for 10-15 h at the temperature of 4 ℃ in the dark to obtain an activated malathion derivative;
s22, centrifuging the derivative obtained in the step S21, and collecting supernatant to obtain a malathion hapten derivative which is marked as solution A; dissolving appropriate amount of carrier protein BSA, KLH, OVA or PLL in PBS buffer solution, and recording as solution B;
and S23.4 ℃, slowly dripping the solution A in the step S22 into the solution B to ensure that the molar ratio of the hapten to the carrier protein is 200-600: 1, carrying out oscillation reaction for 8-12 h to obtain a malathion artificial antigen mixed solution;
s24, transferring the mixed solution obtained in the step S23 into a dialysis bag, dialyzing by using a PBS solution to obtain an artificial antigen, subpackaging, and storing at-20 ℃ for later use.
Preferably, the molar ratio of the hapten, the DCC and the NHS in the step S21 is 1: 1.5: 1.5.
preferably, the final concentration of the hapten in the step S21 is 0.5-1.5 mmoL/100 muL.
More preferably, the final concentration of the hapten in step S21 is 1 mmoL/100. mu.L.
Preferably, the light-shielding oscillation activation reaction time in the step S21 is 10-15 h.
More preferably, the light-shielding shaking activation reaction time in the step S21 is 12 h.
Preferably, the centrifugation condition in the step S22 is 10000-15000 rpm centrifugation for 10-15 min.
More preferably, the centrifugation conditions in step S22 are 12000rpm centrifugation for 10 min.
Preferably, the pH of the PBS buffer solution in the step S22 is 6-8.
More preferably, the pH of the PBS buffer in step S22 is 7.4.
Preferably, the final concentration of the carrier protein solution in the step S22 is 4-6 mg/mL.
More preferably, the final concentration of the carrier protein solution in step S22 is 5 mg/mL.
Preferably, the molar ratio of the hapten to the carrier protein in the step S23 is 200-600: 1.
more preferably, the molar ratio of hapten to carrier protein in step S23 is 400: 1.
preferably, the oscillation reaction time in the step S23 is 8-12 h.
More preferably, the oscillation reaction time in step S23 is 10 h.
Preferably, the pH of the PBS buffer solution in the step S24 is 6-8.
More preferably, the pH of the PBS solution in step S24 is 7.4.
Preferably, the dialysis conditions in step S24 are 3 days of dialysis, with 3 dialysate changes per day.
Preferably, the carrier protein is bovine serum albumin, chicken egg white albumin, hemocyanin, polylysine, human serum albumin.
More preferably, the carrier protein is bovine serum albumin.
In addition, the malathion antibody prepared by the artificial antigen is also within the protection scope of the invention.
Preferably, the antibody specifically comprises the following steps:
s31, selecting a mouse as an experimental animal, mixing the artificial antigen as an immunogen with a complete adjuvant with the same volume, and injecting the mixture into the abdomen, the back and the sole of a foot to perform primary immunization;
s32, carrying out second immunization, replacing a complete adjuvant with an incomplete adjuvant after three weeks, and carrying out the same operation method as the step S31;
s33, enhancing immunity for the third time, wherein the enhancing immunity is performed once every two weeks, and the operation method is the same as that of the step S32;
s34, blood is taken, the mixture is kept stand at 37 ℃ for 30min, then the mixture is centrifuged at 12000rpm at 4 ℃ for 15min, supernatant is taken, and the titer of the mouse serum is detected by adopting an indirect competitive immunoassay method.
Preferably, the mice in step S31 are 6-8 weeks old mice.
Preferably, the primary immunization in step S31 is carried out at a dose of 40-75 μ g (calculated as protein content) per mouse.
More preferably, the primary immunization in step S31 is performed at a dose of 50. mu.g (in terms of protein content) per mouse.
The application of the hapten, the artificial antigen and/or the polyclonal antibody in establishing a malathion analysis detection method and/or preparing a malathion analysis detection kit is also within the protection scope of the invention.
A malathion analysis and detection method is prepared by using the hapten, the artificial antigen and/or the antibody.
The invention has the following beneficial effects:
the invention provides a malathion hapten, which well maintains the chemical structure and chemical characteristics of a raw pesticide, maintains the characteristic structures of a malathion dimethoxy and an carbethoxy, simultaneously introduces an arm containing a plurality of carbon, exposes a carboxyl end active group, synthesizes an artificial antigen by using the malathion hapten coupled with a carrier protein, is used for immunizing animals, and obtains a polyclonal antibody with high sensitivity. The method has the advantages of simple and convenient synthesis steps, readily available raw materials, mild reaction conditions, suitability for production in laboratories and factories, application in establishing a rapid detection method for malathion and developing an immunoassay kit for rapidly detecting the residues of the malathion, satisfaction of the detection requirements of the malathion residues, wide application prospect and great popularization value.
Drawings
FIG. 1 is the ultraviolet scanning map of malathion hapten MLH (n ═ 5) and its artificial antigen, carrier protein.
Fig. 2 is a graph showing the performance of malathion antiserum prepared from malathion hapten MLH (n-5).
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
The following examples illustrate the preparation of malathion hapten with 6-aminocaproic acid (n ═ 5).
Example 1 preparation of malathion hapten MLH (n ═ 5)
1. Experimental procedure
The malathion hapten MLH (n-5) is prepared by the following synthetic route:
the method comprises the following specific steps:
1.1 get 1g P2S5Putting into a reaction bottle, adding 1.2mL of toluene, stirring, heating to 45 ℃, starting to dropwise add 1mL of methanol according to 0.2mL/min, slowly dropwise adding into the reaction bottle through a dropper, keeping the temperature at 55 +/-5 ℃, reacting for 2h, and cooling to below 40 ℃, and taking out colorless crude sulfide with pungent smell;
1.2 taking 0.948g of the crude sulfide in the step 1.1, adding 6mmoL of ethyl hydrogen maleate, carrying out heat preservation reaction at 55 ℃ for 1h after the addition is finished, and carrying out heat preservation reaction at 70-75 ℃ for 8h to obtain an intermediate product b;
1.3 taking 0.302mg (0.83 mmoL) of the intermediate product b in the step 1.2, dissolving in 0.2mL of DMF solution, respectively adding 1mmoL of DCC and NHS solution, reacting for 12h at 4 ℃, centrifuging the reacted mixed solution for 10min at 12000rpm, dropwise adding the supernatant into 6-aminocaproic acid solution containing 1mmoL at 0.2mL/min, reacting for 10h at 0-4 ℃, washing with ethyl acetate, evaporating the solvent, and obtaining a crude product which is a light yellow solid.
2. Results of the experiment
Regulating by TLC (thin layer chromatography), and purifying by column chromatography to obtain malathion hapten with structural formula shown in (III).
2.1 identification of malathion hapten
2.1.1 Fourier Infrared Spectroscopy: fourier infrared spectrum analysis (FTIR) analysis is carried out on the malathion hapten (III) obtained by preparation, and the characteristic frequency range of carbonyl (C ═ O) stretching vibration is 1850-1600 cm-1The malathion has an ester group at 1725-1750 cm-1The prepared malathion hapten also has a strong absorption peak at the position, which indicates that the malathion hapten retains an ester group and has a wave number of 1600cm-1The structure is preliminarily considered to have carboxyl, and the structure of the malathion original medicine has no carboxyl group;
2.1.2 identification by electrospray ionization mass spectrometry (ESI): identifying carboxylated malathion (III) by utilizing an electric spray separation mass spectrum, wherein the molecular weight of the prepared malathion hapten (III) is 415, and the ESI [ M-1] -molecular ion peak in a mass spectrum analysis chart is 414, which is consistent with the molecular weight of the prepared malathion hapten.
2.1.3 nuclear magnetic resonance hydrogen spectrum (1H NMR) identification:1H NMR(600MHz,DMSO)δ8.55(s, 1H),8.34(s,2H),8.22(s,1H),7.95(s,1H),6.99(d,J＝15.5Hz,1H),6.55(d,J＝ 15.5Hz,1H),4.18(dd,J＝14.2,7.1Hz,3H),3.73(ddd,J＝15.3,9.9,3.9Hz,2H), 3.65(s,1H),3.40(dd,J＝14.8,4.0Hz,2H),2.89(s,3H),2.73(s,3H),2.50(s,7H), 2.17(s,1H),2.09(s,1H),1.45(ddd,J＝21.8,14.3,7.0Hz,2H),1.38–1.31(m, 1H),1.24(d,J＝7.1Hz,2H),1.15(t,J＝6.9Hz,1H),1.06(t,J＝7.0Hz,1H)。
EXAMPLE 2 preparation of malathion Artificial antigen MLH-BSA
1. Experimental procedure
The preparation method of the malathion artificial antigen comprises the following specific steps:
1.1 dissolving malathion hapten (III) prepared in example 1 in 2mmoL of DMF (200 muL), adding 3mmoL of DCC and 3mmoL of NHS, and carrying out oscillation activation reaction at 4 ℃ in dark place for 12h to obtain an activated malathion derivative;
1.2 centrifuging the malathion derivative in the step 1.1 at 12000rpm for 10min, and collecting supernatant to obtain the malathion hapten derivative. Preparing a solution containing 5mg/mL carrier protein BSA by using a PBS buffer solution with the pH value of 7.4;
1.3 dropwise adding the hapten derivative in the step 1.2 into a PBS buffer solution containing carrier protein, so that the molar ratio of malathion hapten to carrier protein is 400: oscillating and reacting for 10 hours at the temperature of 1 and 4 ℃ to obtain malathion artificial antigen mixed liquor;
and 1.4, transferring the mixed solution obtained in the step 1.3 into a dialysis bag, dialyzing for 3d by using PBS (phosphate buffer solution) with the pH value of 7.4, replacing the dialyzate for 3 times every day to obtain malathion artificial antigen MLH-BSA, subpackaging, and storing at-20 ℃ for later use.
2. Results of the experiment
And respectively preparing a 1mg/mL solution of malathion hapten MLH (n ═ 5), carrier protein (BSA) and malathion artificial antigen (MLH-BSA) for ultraviolet scanning (200-400 nm).
The result is shown in figure 1, and the ultraviolet scanning images of malathion hapten MLH, malathion artificial antigen MLH-BSA and carrier protein BSA are compared, so that the malathion MLH-BSA is deviated, and the three curves are not overlapped, which indicates that the malathion artificial antigen is successfully coupled.
EXAMPLE 3 preparation of polyclonal Malathion antibody
1. Experimental procedure
The preparation method of the malathion polyclonal antibody comprises the following specific steps:
1.1 mixing and emulsifying the prepared malathion artificial antigen MLH-BSA with an equivalent amount of complete adjuvant to be used as immunogen, selecting mice as experimental animals, adopting an abdominal, back and sole subcutaneous injection mode, immunizing the mice of 6 weeks old according to the dose of 50 mu g (calculated by protein content) of each mouse, and carrying out primary immunization;
1.2 second immunization: after three weeks, the incomplete adjuvant replaces the complete adjuvant, the boosting immunization is carried out once, and the operation method is the same as the step 1.1;
1.3 third booster: boosting the immunity once every two weeks, wherein the operation method is the same as the step 1.2;
1.4 blood sampling: collecting blood from tail, standing at 37 deg.C for 30min, centrifuging at 4 deg.C and 12000rpm for 15min, and collecting upper layer serum.
1.5, detection: the prepared serum is diluted in a gradient way, the corresponding serum dilution multiple of which the absorbance value is about 1.0 is determined according to an indirect competitive enzyme-linked immunoassay method, the dilution multiple is taken as the serum effect value, and the inhibition effect of the serum on the pesticide malathion is determined at the same time.
2. Results of the experiment
The result is shown in fig. 2, and the result shows that the antiserum titer corresponding to the malathion hapten MLH (n-5) designed by the invention is 13000-16000, and the malathion hapten MLH has good identification capability and high sensitivity.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (8)
1. A malathion hapten is characterized by having a structure shown in a formula (I), wherein n is 1-9,
2. a method for preparing the hapten of claim 1, comprising the steps of:
s11, dissolving phosphorus pentasulfide in toluene, dropwise adding methanol at 35-45 ℃, reacting at 50-60 ℃ for 1.5-2.5 h, and cooling to obtain a crude sulfide a;
s12, mixing the crude sulfide a obtained in the step S11 with ethyl hydrogen maleate, reacting at 50-60 ℃ for 1-2 h, and reacting at 70-75 ℃ for 6-10 h to obtain an intermediate product b;
s13, dissolving the intermediate product b in the step S12 in a dimethylformamide solution, adding dicyclohexylcarbodiimide and N-hydroxysuccinimide, reacting for 10-12 hours at 0-4 ℃, centrifuging, taking the supernatant, dropwise adding the supernatant into amino acid solutions with different carbon chain lengths at a speed of 0.2-0.5 mL/min, reacting for 8-10 hours under stirring at 0-4 ℃, washing and drying to obtain a hapten; the amino acid solutions with different carbon chain lengths comprise glycine, alanine, aminobutyric acid, aminopentanoic acid and aminocaproic acid.
3. An artificial antigen of malathion, which is obtained by coupling the hapten of claim 1 with a carrier protein and has the structure shown in formula (II):
4. the artificial antigen of claim 3, wherein the carrier protein is bovine serum albumin, chicken egg white albumin, hemocyanin, polylysine, or human serum albumin.
5. The artificial antigen of claim 4, wherein the carrier protein is bovine serum albumin.
6. An antibody against malathion, which is produced from the artificial antigen according to any one of claims 3 to 5.
7. Use of a hapten according to claim 1, an artificial antigen according to claim 3 and/or an antibody according to claim 6 for the establishment of a malathion assay and/or for the preparation of a kit for the malathion assay.
8. A kit for assaying malathion, characterized in that it is prepared from the hapten of claim 1, the artificial antigen of claim 3 and/or the antibody of claim 6.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911072717.6A CN110981907A (en) | 2019-11-05 | 2019-11-05 | Malathion hapten, artificial antigen and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911072717.6A CN110981907A (en) | 2019-11-05 | 2019-11-05 | Malathion hapten, artificial antigen and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110981907A true CN110981907A (en) | 2020-04-10 |
Family
ID=70083247
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911072717.6A Pending CN110981907A (en) | 2019-11-05 | 2019-11-05 | Malathion hapten, artificial antigen and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110981907A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994027149A1 (en) * | 1993-05-18 | 1994-11-24 | The Minister Of Agriculture Fisheries And Food In Her Britannic Majesty's Government Of The United Kingdom Of Great Britain And Northern Ireland | Methods, agents and kits for detection of organophosphorus compounds |
| JP2000236875A (en) * | 1999-02-23 | 2000-09-05 | Kankyo Meneki Gijutsu Kenkyusho:Kk | Malathion haptenic compound, antibody and measurement |
| WO2019179798A1 (en) * | 2018-03-20 | 2019-09-26 | Radiometer Turku Oy | Luminescent lanthanide chelates and their use |
-
2019
- 2019-11-05 CN CN201911072717.6A patent/CN110981907A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994027149A1 (en) * | 1993-05-18 | 1994-11-24 | The Minister Of Agriculture Fisheries And Food In Her Britannic Majesty's Government Of The United Kingdom Of Great Britain And Northern Ireland | Methods, agents and kits for detection of organophosphorus compounds |
| JP2000236875A (en) * | 1999-02-23 | 2000-09-05 | Kankyo Meneki Gijutsu Kenkyusho:Kk | Malathion haptenic compound, antibody and measurement |
| WO2019179798A1 (en) * | 2018-03-20 | 2019-09-26 | Radiometer Turku Oy | Luminescent lanthanide chelates and their use |
Non-Patent Citations (1)
| Title |
|---|
| N. LEE WOLFE等: "Synthesis and Carbon-13 Studies of Malathion Acid Derivatives", 《J. AGRIE. FOOD CHEM.》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111187346B (en) | Colloidal gold test strip for detecting fipronil and its metabolites and preparation method thereof | |
| CN114315722B (en) | Preparation and application of tolfenpyrad artificial hapten and antibody thereof | |
| CN110441512B (en) | Colloidal gold immunochromatography detection device for ethyl maltol hapten and ethyl maltol | |
| CN101100456A (en) | Fipronil artificial hapten and synthesis method, antigen and antibody and application | |
| CN109265401B (en) | Preparation method and application of iprodione hapten and antigen | |
| CN109307761B (en) | An indirect competitive ELISA method for the detection of furosine | |
| CN101100486A (en) | Fipronil artificial antigen, antibody and use thereof | |
| CN113264834A (en) | Abienol hapten, artificial antigen and antibody as well as preparation method and application thereof | |
| CN111269262B (en) | Profenofos hapten, artificial antigen and antibody, and preparation method and application thereof | |
| CN114716535B (en) | Synthetic method of patulin artificial antigen and preparation and application of monoclonal antibody thereof | |
| CN110872344B (en) | A kind of chloramphenicol complete antigen and its preparation method and application | |
| CN110981907A (en) | Malathion hapten, artificial antigen and preparation method thereof | |
| CN115215811B (en) | Prothioconazole hapten, antigen, antibody, detection device, preparation and application thereof | |
| CN111978271A (en) | Hexythiazole hapten and preparation method thereof, hexythiazole antigen, antibody and application thereof | |
| CN102942519B (en) | Nitenpyram hapten, artificial antigen and antibody, their preparation methods and application thereof | |
| CN115340986B (en) | A hybridoma cell line secreting phorate monoclonal antibody and its application | |
| CN102295698B (en) | Cyclosporine A immunogen, cyclosporine A specific antibody, detection reagent, and detection kit | |
| CN109942624B (en) | Glufosinate hapten, artificial antigen, antibody, preparation method and detection device thereof | |
| CN108132348B (en) | Sudan red III hapten, coupling antigen, antibody and colloidal gold rapid detection device and application thereof | |
| CN113637642A (en) | Hybridoma cell strain capable of secreting monoclonal antibody of dicofol and application of hybridoma cell strain | |
| CN115322089B (en) | A raspberry ketone hapten, artificial antigen and preparation method, antibody and application thereof | |
| CN112358549A (en) | Preparation of dimethyl isotrichum monoclonal antibody and enzyme-linked immunosorbent assay method thereof | |
| CN112480167A (en) | Isocarbophos hapten, artificial antigen and antibody, and preparation method and application thereof | |
| CN115925740B (en) | Methylisosalphos hapten, complete antigen, antibody, preparation method and application | |
| CN113480474B (en) | Sulfoxaflor hapten, preparation method thereof, antigen, antibody and application thereof |
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 | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200410 |