CN114539170B - Hapten and artificial antigen for simultaneously detecting amantadine, olaquindox and chloramphenicol, and preparation method and application thereof - Google Patents
Hapten and artificial antigen for simultaneously detecting amantadine, olaquindox and chloramphenicol, and preparation method and application thereof Download PDFInfo
- Publication number
- CN114539170B CN114539170B CN202111679788.XA CN202111679788A CN114539170B CN 114539170 B CN114539170 B CN 114539170B CN 202111679788 A CN202111679788 A CN 202111679788A CN 114539170 B CN114539170 B CN 114539170B
- Authority
- CN
- China
- Prior art keywords
- hapten
- mmol
- chloramphenicol
- formula
- artificial antigen
- 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
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D241/00—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings
- C07D241/36—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings condensed with carbocyclic rings or ring systems
- C07D241/38—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings condensed with carbocyclic rings or ring systems with only hydrogen or carbon atoms directly attached to the ring nitrogen atoms
- C07D241/40—Benzopyrazines
- C07D241/44—Benzopyrazines with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to carbon atoms of the hetero ring
-
- 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
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
-
- 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
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
- C07K16/065—Purification, fragmentation
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2430/00—Assays, e.g. immunoassays or enzyme assays, involving synthetic organic compounds as analytes
-
- 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
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Gastroenterology & Hepatology (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Zoology (AREA)
- Hematology (AREA)
- Toxicology (AREA)
- General Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Pathology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a hapten and an artificial antigen for simultaneously detecting amantadine, olaquindox and chloramphenicol, and a preparation method and application thereof. The structural formula of the hapten is shown as a formula I, wherein 3 drug skeleton structures of Amantadine (AMD), 3-methyl-quinoxaline-2-carboxylic acid (MQCA) and chloramphenicol amine (CAPA) are respectively connected with amino acids and alkyl diacid with different carbon chain lengths in sequence to form a multi-cluster series linear hapten, and the hapten is coupled with carrier protein to prepare the artificial antigen. The polyclonal serum titer obtained by adopting the artificial antigen is between 8 and 32K, and the hapten inhibition rate is between 63 and 90 percent, which shows that the multi-cluster tandem linear hapten and the artificial antigen thereof prepared by the invention have good immunogenicity and can be used for preparing monoclonal antibodies, single-chain antibodies and nano antibodies subsequently.
Description
Technical Field
The invention relates to the technical field of antigen-antibody detection, in particular to an AMC multi-cluster tandem linear hapten and an artificial antigen simultaneously containing Amantadine (AMD), 3-methyl-quinoxaline-2-carboxylic acid (MQCA) and chloramphenicol amine (CAPA) skeleton structures, and a preparation method and application thereof.
Background
The Chinese is the largest livestock product production and consumption country in the world, and according to statistics, the total meat yield of the Chinese in 2010 reaches 7925.8 ten thousand tons, and the world is the first place. The reported animal product veterinary drug residue analysis and detection technology mainly integrates a large-scale precise instrument analysis technology and an immunoassay screening technology, wherein an instrument validation method is mainly liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). For example, chinese patent discloses a method for measuring the content of 11 illegally added medicines in veterinary medicine powder, which can detect 11 illegally added medicines including amantadine, olaquindox, chloramphenicol and the like at the same time, however, the instrumental method generally has the defects of complex sample pretreatment operation, time consumption, high cost, inapplicability to field detection and the like. The immunoassay method has the advantages of consistent sensitivity with the conventional instrument analysis, suitability for on-site screening, simplicity, rapidness, low cost, small sample requirement, simple and convenient pretreatment and the like. At present, in the safety detection of animal products, single-residue immunity detection methods are mainly adopted, for example, chinese patent CN108152499A, CN111077319A, CN108519480A and the like respectively disclose immunity detection methods aiming at amantadine, olaquindox and chloramphenicol. However, due to diversity of veterinary drug species and the universality and complexity of the compound use thereof, only developing single-residue immunodetection technology cannot meet the actual detection requirement, but related hapten, antigen and antibody for simultaneously detecting non-common structure small molecule drugs including amantadine, olaquindox and chloromycetin are lacking at present.
Disclosure of Invention
The invention aims to solve the technical problems and overcome the defects and the shortcomings in the prior art, and provides a hapten capable of realizing multi-residue immunodetection of non-common structure small molecule drugs including amantadine, olaquindox and chloramphenicol. The three small molecule drugs with different structures, namely amantadine, olaquindox and chloramphenicol, are regarded as different epitopes, and are connected in series into linear multi-cluster hapten in a proper way, so that the neutralizing and recognition capacity of the multi-epitope is improved.
A second object of the present invention is to provide a method for preparing the hapten.
It is a third object of the present invention to provide the use of said hapten.
It is a fourth object of the present invention to provide an artificial antigen.
A fifth object of the present invention is to provide a method for producing the artificial antigen.
A sixth object of the present invention is to provide the use of said artificial antigen.
A seventh object of the present invention is to provide an antigen-specific antibody.
The above object of the present invention is achieved by the following technical solutions:
the hapten for simultaneously detecting amantadine, olaquindox and chloramphenicol contains a skeleton structure of Amantadine (AMD), 3-methyl-quinoxaline-2-carboxylic acid (MQCA) and Chloramphenicol (CAPA), and the general structural formula of the hapten is shown in formula I:
wherein R is 1 And R is 2 Respectively selected from alkyl groups having 2 to 5 carbon atoms, i.e. R 1 And R is 2 Is respectively selected from any one of ethyl to amyl.
Preferably, said R 1 Selected from propyl or pentyl, R 2 Selected from ethyl or butyl.
Further preferably, the hapten is represented by the following AMC-4C or AMC-6C:
the preparation method of the sex hapten comprises the following steps:
s1, respectively preparing olaquindox metabolites, amantadine and chloramphenicol intermediates: methyl acetoacetate is used as a raw material to obtain a olaquindox metabolite intermediate shown in a formula II through NBS bromination, 4-nitroo-phenylenediamine cyclization, nitro reduction, amino protecting group feeding and ester bond hydrolysis; the method comprises the steps of taking adamantane as a raw material, connecting an amino acid arm protected by tert-butoxycarbonyl on 1 amino group after nitration, nitroreduction, aminoacylation and hydrolysis, then connecting fluorenylmethoxycarbonyl on the other 1 amino group, and finally removing Boc protecting group to obtain an amantadine intermediate shown in a formula III; hydrolyzing and removing dichloroacetyl of chloramphenicol to obtain chloramphenicol intermediate chloramphenicol amine shown in formula IV;
wherein R is 1 And R is 2 Respectively selected from alkyl groups having 2 to 5 carbon atoms, i.e. R 1 And R is 2 Respectively selected from any one of ethyl to amyl;
s2, synthesis of hapten: and (3) condensing and coupling an amantadine intermediate shown in a formula III and a olaquindox metabolite intermediate shown in a formula II through primary amino groups and carboxyl groups on molecules, removing DMB protecting groups of aromatic ring amino groups of the olaquindox metabolite intermediate, coupling through an acylation condensation reaction to obtain an intermediate V, performing acylation condensation on the carboxyl groups of the intermediate V and amino groups of a chloramphenicol intermediate shown in a formula IV to obtain an intermediate VI, removing Fmoc protecting groups, condensing and reducing with 4-oxybutyrate methyl ester to obtain an intermediate VII, and finally obtaining hapten shown in the formula I through water Jie Zhiji.
Preferably, said R 1 Selected from propyl or pentyl, R 2 Selected from ethyl or butyl.
The invention also provides application of the hapten in preparation of artificial antigens.
An artificial antigen is obtained by coupling hapten shown in formula I and carrier protein, and has a structure shown in formula VIII:
wherein R is 1 And R is 2 Respectively selected from alkyl groups with 2-5 carbon atoms, namely R 1 And R is 2 Selected from any one of ethyl to pentyl respectively.
Preferably, said R 1 Selected from propyl or pentyl, R 2 Selected from ethyl or butyl.
Further preferably, the R 1 And R is 2 Selected from propyl and ethyl groups, and pentyl and butyl groups, respectively, and the substituted structural formula is shown as the following AMC-4C-carrier protein or AMC-6C-carrier protein:
preferably, the carrier protein is Lactoferrin (LF), keyhole Limpet Hemocyanin (KLH) or Ovalbumin (OVA). LF, KLH are used to prepare immunogens and OVA is used to prepare coating precursors.
The preparation method of the artificial antigen is to couple carrier protein on carboxyl of hapten shown in formula I.
Preferably, the coupling is by the active ester method.
As a preferred embodiment, the method for preparing the artificial antigen comprises the following steps: hapten shown in formula I, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) are dissolved in N, N-Dimethylformamide (DMF), and the mixture is stirred and reacted overnight at 4 ℃ to obtain solution A. The carrier protein is weighed and dissolved in phosphate buffer solution (PBS, 0.01M,pH 7.4), and is stirred and dissolved to prepare solution B. Under magnetic stirring, absorbing the solution A, dropwise adding the solution A into the solution B, magnetically stirring the solution at 4 ℃ for reaction, dialyzing the reaction solution with PBS at 4 ℃ for 3 days, and changing the dialyzate for 2 times a day to obtain the artificial antigen.
The invention also provides application of the artificial antigen in preparation of monoclonal/polyclonal antibodies, single-chain antibodies or nano antibodies.
A polyclonal antibody is prepared by immunizing mice with artificial antigen shown in formula VIII.
Preferably, the carrier protein is used as Lactoferrin (LF) or artificial antigen of Keyhole Limpet Hemocyanin (KLH) as immunogen, female Balb/C mice of proper age are selected for immunization, the titer and inhibition rate of antiserum are measured after the 4 th immunization and the 5 th immunization, and the serum of the mice is collected after the performance is stable, so that the polyclonal antibody is obtained.
The invention also provides a rapid immunoassay method for detecting the artificial antigen, which is used for evaluating the performance of antisera based on an ELISA method, takes the artificial antigen with carrier protein as Ovalbumin (OVA) as a coating antigen, and uses the antigen-specific antibody as a detection antibody for detection. Preferably, the antigen-specific antibody is a murine polyclonal antibody.
Compared with the prior art, the invention has the following beneficial effects:
the invention firstly provides a hapten for simultaneously detecting amantadine, olaquindox and chloramphenicol, which has a structural general formula shown in formula I, and is characterized in that intermediates of 3 drugs including amantadine, olaquindox metabolite and chloramphenicol are connected with aminocarboxylic acid molecules with different spacing arms, and mixed chemistry is carried out to synthesize a multi-cluster tandem linear hapten in a tandem way. The artificial antigen is prepared based on the hapten, and then the murine polyclonal antiserum prepared by immunizing animals with the artificial antigen has the titer of 8-32K and the inhibition rate of 63-90% on multiple clusters of serially connected linear hapten. The artificial antigen prepared by the invention has good immunogenicity, and can be used for preparing multi-cluster tandem linear antigens or multi-epitope specific monoclonal antibodies, single-chain antibodies and nano antibodies in the follow-up process and establishing a corresponding immunoassay method. The invention lays a foundation for the preparation of the small molecule multispecific antibody and the multi-residue analysis method, and has good application prospect.
Drawings
FIG. 1 is an ultraviolet scan of a plurality of clusters of tandem linear haptens, artificial antigens, and carrier proteins.
Detailed Description
The present invention will be further described in detail with reference to the drawings and examples, wherein the reagents, methods and apparatus employed in the present invention are those conventional in the art unless otherwise specified.
EXAMPLE 1 preparation of AMC Multi-cluster tandem Linear hapten
1. A preparation method of AMC-4C hapten comprises the following steps:
s1, preparation of intermediate 3
Methyl acetoacetate, compound 1 (50 g,431 mmol) was added to water (1L), N-bromosuccinimide (i.e., NBS) (88 g,494 mmol) was added at 70℃and stirred at room temperature for half an hour until the system was transparent. 4-nitroo-phenylenediamine, compound 2 (63 g,412 mmol), was then added and stirring continued at 70℃for 8 hours, after completion of the reaction monitored by liquid-mass spectrometry (i.e., LC-MS), cooled to room temperature and extracted with organic solvent (500 mL. Times.6) with PE: EA=1:1. The organic phases were combined, dried and spun-dried. The crude product was isolated and purified by column chromatography (PE: ea=10:1) to give intermediate 3 (14 g, yield: 13.8%,57 mmol) as a yellow solid which turned to a purple-black solid after long spin-drying. MS (ESI) M/z (M+H) + 248.1。
S2, preparation of intermediate 4
Intermediate 3 (14 g,57 mmol) was dissolved in tetrahydrofuran (i.e. THF) (100 mL) and methanol (100 mL) and Zn (40 g,634 mmol) was added. Ammonium chloride (38 g,710 mmol) was added in portions at 0deg.C and the reaction was continued to stir at 0deg.C for half an hour, after completion of the reaction monitored by liquid-mass spectrometry (i.e., LC-MS), the reaction solution was filtered through celite, and the filter cake was washed with a mixed solvent of dichloromethane and methanol. Concentrating the filtrate. The residue was purified by column chromatography (PE: ea=100:0-50:50) to give intermediate 4 (7 g, yield: 56.14%,32 mmol) as a yellow solid. MS (ESI) M/z (M+H) + 218.1。 1 H NMR(400MHz, DMSO)δ7.72(d,J＝9.0Hz,1H),7.32(dd,J＝9.0,2.5Hz,1H),6.90(d,J＝2.4Hz, 1H),6.11(s,2H),3.93(s,3H),2.68(s,3H)。
S3, preparation of intermediate 5
Intermediate 4 (7 g,32 mmol) and 2, 4-dimethoxybenzaldehyde (i.e., DMB) (10.2 g,61.4 mmol) were dissolved in dichloromethane (i.e., DCM) (100 mL) at room temperature, sodium triacetoxyborohydride (i.e., STAB) (20.5 g,96.7 mmol) was added and the reaction was stirred at room temperature overnight. After completion of the reaction was monitored by liquid-mass spectrometry (i.e., LC-MS), water (100 mL) was added to the reaction solution, extracted with EA (100 ml×5), and the organic layer was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography (PE: ea=100:0-65:35) to give intermediate 5 (8.5 g, yield: 72.2%, 23.1 mmol) as a yellow solid. MS (ESI) M/z (M+H) + 368.0。 1 H NMR(400MHz,DMSO) δ7.74(t,J＝11.0Hz,1H),7.43(dt,J＝27.5,13.8Hz,1H),7.17(d,J＝8.3Hz,1H), 7.05(t,J＝5.6Hz,1H),6.63(dd,J＝16.4,2.4Hz,2H),6.47(dd,J＝8.3,2.4Hz,1H), 4.28(d,J＝5.6Hz,2H),3.91(s,3H),3.85(s,3H),3.74(s,3H),2.69(s,3H)。
S4, preparation of intermediate 6
Intermediate 5 (8.5 g,23.1 mmol) and NaOH (1.85 g,46.2 mmol) were dissolved in THF (20 mL) and water (10 mL). The reaction solution was stirred at room temperature for 1 hour. After completion of the reaction by liquid-mass spectrometry (i.e., LC-MS), the pH was adjusted to weak acidity with concentrated hydrochloric acid, followed by extraction with DCM (100 ml×8), and the organic layer was washed with saturated brine (100 mL) and dried over anhydrous sodium sulfate. After filtration, intermediate 6 (7.4 g, yield: 90.5%,20.9 mmol) was concentrated to give an orange solid, namely, a olaquindox metabolite (3-methyl-quinoxaline-2-carboxylic acid) intermediate. MS (ESI) M/z (M+H) + 354.1。 1 H NMR(400MHz, DMSO)δ7.70(d,J＝9.1Hz,1H),7.40(dd,J＝9.1,2.4Hz,1H),7.17(d,J＝8.3Hz, 1H),7.00(s,1H),6.62(dd,J＝16.2,2.4Hz,2H),6.47(dd,J＝8.4,2.3Hz,1H),4.27 (d,J＝5.6Hz,2H),3.85(s,3H),3.73(s,4H),2.66(s,3H)。
S5, preparation of intermediate 8
Amantadine, compound 7 (75 g, 553mmol), was slowly added to stirred fuming nitric acid (345 g) at 0deg.C and the reaction was continued with stirring at 0deg.C for 1 hour. Acetonitrile (55.5 g,1.37 mol) was then added dropwise at 0℃and stirring was continued at 0℃for 1 hour. Finally concentrated sulfuric acid (705 g) was slowly added at 0℃followed by stirring overnight at room temperature. After completion of the reaction, which was monitored by liquid-mass spectrometry (i.e., LC-MS), the reaction solution was poured into ice (about 1 kg), pH was adjusted to be alkaline with sodium carbonate solid, and a yellow viscous substance was precipitated. The water was decanted, EA (about 100 mL) was added to agglomerate the yellow viscous material, and filtered. The yellow precipitate was washed with EA and then with water to give intermediate 8 (80 g) as a white solid. MS (ESI) M/z (M+H) + 251.1。 1 H NMR(400MHz,DMSO)δ7.36(s,2H),2.11(d,J＝10.5Hz,2H),2.10– 1.98(m,2H),1.91–1.74(m,8H),1.73(s,6H),1.49(s,2H)。
S6, preparation of intermediate 9
Intermediate 8 (70 g) was reacted in HCl (10%, 500 mL) with stirring at 110℃for 2 days, after completion of the reaction monitored by liquid-mass spectrometry (i.e., LC-MS), the reaction mixture was concentrated under reduced pressure to give intermediate 9 (63 g,264 mmol) as a white solid. MS (ESI) M/z (M+H) + 167.1。 1 H NMR(400MHz, DMSO)δ8.40(s,6H),2.33–2.20(m,2H),2.02(s,2H),1.84–1.66(m,8H),1.52(s, 2H).
S7, preparation of intermediate 11
Boc-protected 4-aminobutyric acid, compound 10 (5.66 g,27.8 mmol), HATU (12.7 g,33.4 mmol) and DIEA (10.8 g,83.6 mmol) were dissolved in DMSO (50 mL) and the reaction stirred at room temperature for 1 h. The above liquid was slowly added dropwise to stirring intermediate 9 (10 g, 4)1.8 mmol) and DIEA (10.8 g,83.6 mmol) in DMSO (100 mL) and continuing to stir at room temperature for half an hour, fmocCl (21.63 g,83.6 mmol) and DIEA (10.8 g,83.6 mmol) were added directly to the reaction solution after completion of the reaction monitored by a liquid-mass spectrometer (i.e., LC-MS), and stirring was continued for 1 hour, water (300 mL) was added after completion of the reaction monitored by a liquid-mass spectrometer (i.e., LC-MS), extracted with EA (100 mL. Times.5), and the organic layer was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography (PE: ea=100:0-35:65) to give intermediate 11 (6.9 g, yield: 43.2%,12 mmol) as a yellow solid. MS (ESI) M/z (M+H) + 574.3。 1 H NMR(400MHz,DMSO)δ7.88(d,J ＝7.5Hz,1H),7.71(d,J＝7.4Hz,1H),7.48–7.36(m,1H),7.33(td,J＝7.4,1.1Hz, 1H),7.11(s,1H),6.76(s,1H),4.18(s,1H),2.94–2.79(m,1H),2.11(s,2H),2.03– 1.93(m,3H),1.92–1.61(m,4H),1.54(dt,J＝14.3,7.2Hz,2H),1.37(s,5H)。
S8 preparation of intermediate 12
Intermediate 11 (6.9 g,12 mmol) was dissolved in 4M dioxane hydrochloride solution (50 mL) and the reaction stirred at room temperature for 1 hour. TLC showed the reaction was complete. The reaction solution was concentrated to obtain intermediate 12 (7 g) as a pale yellow oily liquid, intermediate 12.MS (ESI) M/z (M+H) + 474.2。 1 H NMR(400MHz,DMSO)δ7.89(d,J＝7.5Hz,6H),7.71(d,J＝7.4Hz,2H),7.52(s, 1H),7.41(t,J＝7.2Hz,3H),7.33(td,J＝7.4,1.1Hz,2H),7.12(s,1H),4.20(d,J＝ 14.9Hz,3H),2.74(dd,J＝14.1,6.4Hz,2H),2.17–2.03(m,6H),1.78(ddd,J＝25.2, 22.1,15.0Hz,12H),1.51(s,2H)。
S9, preparation of intermediate 13
Intermediate 6 (3.5 g,9.9 mmol), 2- (7-azabenzotriazol) -N, N' -tetramethyluronium hexafluorophosphate (i.e., HATU) (4.5 g,11.8 mmol) and N, N-diisopropylethylamine (i.e., DIEA) (3.8 g,30 mmol) were dissolved in 20mL of dichloromethane and the reaction stirred at room temperature for 1 hour. The above liquid was added to a stirring solution of intermediate 12 (6 g) in DCM (100 mL) and the reaction was continued at room temperature for half an hour, after completion of the reaction by liquid-mass spectrometry (i.e., LC-MS), water (100 mL) was added, extracted with DCM (100 mL. Times.3), and the organic layer was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography (PE: ea=100:0-0:100) to give intermediate 13 (6.9 g, yield: 85.9%,8.5 mmol) as an orange solid. MS (ESI) M/z (M+H) + 809.2。 1 H NMR(400MHz,CDCl3)δ8.24(t,J＝6.1Hz,1H),7.81–7.70(m,3H), 7.56(t,J＝12.1Hz,2H),7.39(t,J＝7.3Hz,2H),7.34–7.28(m,2H),7.24(d,J＝8.3 Hz,1H),7.18(dd,J＝9.1,2.6Hz,1H),6.96(d,J＝2.5Hz,1H),6.51(d,J＝2.3Hz, 1H),6.46(dd,J＝8.2,2.3Hz,1H),4.38(d,J＝5.5Hz,2H),4.19(t,J＝6.1Hz,1H), 3.84(d,J＝11.5Hz,3H),3.80(s,3H),3.54(q,J＝6.5Hz,2H),3.14(dt,J＝12.8,6.4 Hz,1H),3.04(d,J＝8.3Hz,3H),2.80(s,6H),2.21(t,J＝6.9Hz,4H),1.95(dt,J＝ 30.2,8.9Hz,9H),1.45(t,J＝5.6Hz,6H)。
S10, preparation of intermediate 14
Intermediate 13 (3 g,3.7 mmol) was dissolved in DCM (20 mL), trifluoroacetic acid, TFA (2 mL) was added and the reaction was continued with stirring at room temperature for 5 min, after completion of the reaction by liquid-mass spectrometry (i.e., LC-MS), the reaction was concentrated and dried 3 times with chloroform to give 14 (3.5 g) as intermediate 14.MS (ESI) M/z (M+H) + 659.3。 1 H NMR(400MHz,DMSO)δ8.69(t,J ＝5.9Hz,1H),7.88(d,J＝7.4Hz,2H),7.70(t,J＝7.7Hz,3H),7.41(t,J＝7.4Hz, 2H),7.33(dd,J＝7.4,6.4Hz,2H),7.26(dt,J＝8.5,4.3Hz,1H),6.94(d,J＝2.4Hz, 1H),4.18(s,2H),3.16–3.08(m,3H),2.68(d,J＝7.1Hz,3H),2.17–2.03(m,5H), 2.03–1.90(m,3H),1.77(dd,J＝27.2,19.9Hz,9H)。
S11 preparation of intermediate 16
Intermediate 14 (3.5 g,3.7 mmol), succinic anhydride, i.e., compound 15 (3.7 g,37 mmol) and DIEA (2.4 g,18.5 mmol) were taken up in DCM (50 mL). The reaction was stirred at room temperature for 6 hours, and after completion of the reaction, which was monitored by liquid-mass spectrometry (i.e., LC-MS), the reaction solution was filtered, and after washing the cake with methanol, the filtrate was concentrated. The residue was purified by reverse phase column chromatography (FA, water: acetonitrile=100:0-40:60) to give intermediate 16 (1.4 g, yield: 50%,1.85 mmol) as a pale yellow solid. MS (ESI) M/z (M+H) + 759.3。 1 H NMR(400MHz,DMSO)δ10.50(s,1H),8.86(t,J＝5.8Hz,1H),8.57(d, J＝2.2Hz,1H),8.14(s,1H),7.98(d,J＝9.1Hz,1H),7.88(dd,J＝9.1,2.3Hz,3H), 7.71(d,J＝7.4Hz,2H),7.41(t,J＝7.3Hz,3H),7.33(td,J＝7.4,1.1Hz,2H),7.12(s, 1H),4.20(d,J＝15.4Hz,3H),2.79(s,3H),2.71–2.63(m,2H),2.58(t,J＝6.8Hz, 2H),2.20–2.05(m,6H),1.91–1.71(m,10H),1.57–1.41(m,3H)。
S12, preparation of intermediate 17
Chloramphenicol CAP (25 g,77.6 mmol) was dissolved in HCl (1M, 500 mL) and hydrolyzed overnight with stirring at 100deg.C, after completion of the reaction monitored by liquid-mass spectrometry (LC-MS), the reaction solution was cooled to room temperature and extracted with ethyl acetate EA (500 mL. Times.3) to remove dichloroacetic acid. The remaining aqueous phase was dried by spinning to give intermediate 17 (15 g, yield: 91.1%,70.7 mmol) as a white solid.
S13, preparation of intermediate 18
Intermediate 16 (1.4 g,1.85 mmol), intermediate 17 (3.92 g,18.5 mmol) and DIEA (1.2 g,9.25 mmol) were dissolved in DMF (20 mL), and 1-propyltricycloanhydride of phosphoric acid, T3P (2.35 g, 7.4 mmol), was added and the reaction was continued with stirring at room temperature for 1 hour, viaAfter completion of the reaction, the liquid-mass spectrometer (i.e., LC-MS) was used to monitor the reaction, water (50 mL) was added, EA (50 ml×3) was used to extract, and the organic layer was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by reverse phase column (FA, water: acetonitrile=100:0-50:50) to give intermediate 18 (950 mg, yield: 54%,1.0 mmol) as a pale yellow solid. MS (ESI) M/z (M+H) + 953.5。 1 H NMR(400MHz,DMSO)δ10.38(s, 0H),8.84(t,J＝5.8Hz,0H),8.53(d,J＝2.2Hz,0H),8.12(d,J＝8.8Hz,1H),7.96 (d,J＝9.1Hz,0H),7.86(dd,J＝13.6,4.6Hz,1H),7.71(d,J＝7.5Hz,1H),7.60(s, 1H),7.59(d,J＝8.7Hz,1H),7.40(t,J＝7.4Hz,1H),7.35–7.27(m,1H),5.80(s, 0H),5.00(s,0H),4.18(s,1H),3.98(d,J＝8.3Hz,0H),3.61–3.53(m,1H),3.17(s, 2H),2.79(s,1H),2.44–2.35(m,1H),2.10(dd,J＝13.6,6.2Hz,2H),2.02–1.95(m, 1H),1.92–1.69(m,4H),1.49(s,1H)。
S14 preparation of intermediate 19
Intermediate 18 (950 mg,1.0 mmol) was dissolved in ethanol, etOH (20 mL), diethylamine (5 mL) was added and the reaction was continued to stir at room temperature for 6 hours, after which the reaction was monitored to completion by liquid-mass spectrometry (i.e. LC-MS), the reaction was concentrated and dried twice with EtOH. The residue was dissolved in water (5 mL), and washed with petroleum ether, PE, to remove impurities and lyophilized to give intermediate 19 (720 mg, yield: 98.6%,0.98 mmol) as a pale yellow solid. MS (ESI) M/z (M+H) + 731.1。 1 H NMR(400MHz,DMSO)δ10.43 (s,1H),8.83(t,J＝5.8Hz,1H),8.54(d,J＝2.1Hz,1H),8.12(d,J＝8.7Hz,2H), 7.96(d,J＝9.0Hz,1H),7.85(dd,J＝9.1,2.2Hz,1H),7.67(d,J＝9.2Hz,1H),7.59 (d,J＝8.7Hz,2H),7.37(s,1H),5.00(d,J＝2.2Hz,1H),3.98(d,J＝8.4Hz,1H), 3.58–3.48(m,6H),3.17(s,1H),2.80(s,4H),2.46–2.38(m,3H),2.15–1.92(m, 7H),1.79(t,J＝21.7Hz,11H),1.46(s,8H),1.21(d,J＝19.9Hz,6H)。
S15 preparation of intermediate 21
Intermediate 19 (720 mg,0.98 mmol), methyl 4-oxobutyrate, compound 20 (220 mg, 1.88 mmol) and STAB (1.2 g,5.64 mmol) were stirred in DCM (20 mL) and methanol (20 mL) at room temperature overnight, after completion of the reaction monitored by liquid-mass spectrometry (i.e., LC-MS), the reaction was dried by spin-drying and the residue was isolated and purified by reverse phase column (ammonia, water: acetonitrile=100:0-70:30) to afford intermediate 21 (690 mg, yield: 84.7%,0.83 mmol) as a pale yellow solid. MS (ESI) M/z (M+H) + 831.2。 1 H NMR(400MHz,DMSO)δ10.42(s,1H),8.83(d,J＝5.5Hz,1H),8.55(d,J＝2.2 Hz,1H),8.33(s,2H),8.13(d,J＝8.8Hz,2H),7.97(d,J＝9.1Hz,1H),7.86(dd,J＝ 9.1,2.3Hz,1H),7.66(d,J＝9.1Hz,1H),7.59(d,J＝8.7Hz,2H),7.46(s,1H),5.33 (t,J＝4.8Hz,1H),5.01(s,1H),3.97(s,3H),3.57(d,J＝2.2Hz,4H),2.80(s,3H), 2.62(d,J＝7.1Hz,2H),2.38(t,J＝7.4Hz,4H),2.11(d,J＝7.1Hz,4H),2.00(dd,J ＝15.1,7.3Hz,4H),1.91(s,2H),1.84(s,2H),1.80–1.72(m,5H),1.72–1.62(m, 3H),1.58(s,5H),1.49(d,J＝10.0Hz,4H),0.86(t,J＝6.8Hz,2H).
Preparation of S16.AMC-4C
Intermediate 21 (460 mg,0.83 mmol) and lithium hydroxide, liOH (68 mg, 1.66 mmol), were dissolved in water (5 mL) and THF (10 mL) at room temperature with stirring for 6h, and after completion of the reaction monitored by liquid-mass spectrometry (i.e., LC-MS), the reaction mixture was isolated and purified by high-efficiency reverse phase column preparation to give pale yellow solid, AMC-4C (269 mg, yield: 39.8%,0.33 mmol). MS (ESI) M/z (M+H) + 817.4。 1 H NMR(400MHz,DMSO)δ10.41(s,1H),8.84(s,1H),8.55(d,J＝2.4Hz, 1H),8.12(d,J＝8.8Hz,2H),7.96(d,J＝9.1Hz,1H),7.84(dd,J＝9.1,2.3Hz,1H), 7.67(d,J＝9.0Hz,1H),7.59(d,J＝8.7Hz,2H),7.53(s,1H),5.01(s,1H),3.97(s,1H),3.60–3.52(m,1H),3.29-3.28(m,3H),2.80(s,3H),2.76(s,2H),2.48–2.44 (m,2H),2.41(d,J＝5.2Hz,2H),2.25(s,2H),2.18–2.09(m,4H),1.98(s,2H),1.88 (d,J＝12.8Hz,2H),1.83–1.72(m,4H),1.64(s,6H),1.51(s,2H)。
2. A preparation method of AMC-6C hapten comprises the following steps:
S1-S6 are the same as the AMC-4C hapten preparation process;
s7, preparation of intermediate 23
Boc-protected 6-aminocaproic acid, compound 22 (5.66 g,27.8 mmol), HATU (12.7 g,33.4 mmol) and DIEA (10.8 g,83.6 mmol) were dissolved in DMSO (50 mL) and the reaction stirred at room temperature for 1 h. The above mixture solution was slowly dropped into a DMSO (100 mL) solution of intermediate 9 (10 g,41.8 mmol) and DIEA (10.8 g,83.6 mmol) which were being stirred, and after the reaction was continued to be stirred at room temperature for half an hour, 9-fluorenylmethylchloride (FmocCl) (21.63 g,83.6 mmol) and DIEA (10.8 g,83.6 mmol) were directly added to the reaction solution after the reaction was monitored by a liquid-mass spectrometer (LC-MS) to be complete, and after the reaction was continued to be stirred for 1 hour, water (300 mL) was added, extracted by ethyl acetate (EA (100 ml×5) was added, and the organic layer was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was separated and purified by column chromatography (PE: ea=100:0-35:65) to give intermediate 23 (8.2 g, yield: 48.9%, 13.6 mmol) as pale yellow solid, i.e., intermediate 23.MS (ESI) M/z (M+H-100) + 502.1。1H NMR (400MHz,DMSO)δ7.88(d,J＝7.5Hz,2H),7.70(s,2H),7.41(s,2H),7.34(dd,J＝ 7.4,1.0Hz,3H),7.10(s,1H),6.74(t,J＝5.3Hz,1H),4.18(s,3H),4.03(q,J＝7.1 Hz,1H),3.00–2.79(m,2H),2.11(s,4H),1.90–1.70(m,8H),1.36(s,16H),1.18(t, J＝7.1Hz,4H).
S8, preparation of intermediate 24
Intermediate 23 (8.2 g,13.6 mmol) was dissolved in 4M dioxane hydrochloride (50 mL) and the reaction was continued with stirring at room temperature for 1 hour, TLC showed complete reaction, the reaction solution was purifiedConcentrated to give intermediate 24 (8.12 g) as a pale yellow oily liquid. MS (ESI) M/z (M+H) + 502.3。1H NMR(400MHz, DMSO)δ7.37(dt,J＝32.7,7.4Hz,1H),7.37(dt,J＝32.7,7.4Hz,1H),4.18(s,1H), 3.57(s,2H),2.75–2.71(m,0H),2.69(s,1H),2.52–2.48(m,1H),2.12(s,1H),2.01(t,J＝7.3Hz,0H),1.83(dd,J＝36.3,23.0Hz,1H),1.50(dtd,J＝22.5,15.0,7.5Hz, 1H),1.33–1.17(m,1H)。
S9 preparation of intermediate 25
Intermediate 6 (1.8 g,5.1 mmol), HATU (2.3 g,6.1 mmol) and DIEA (1.9 g,15.3 mmol) were dissolved in DCM (20 mL) and the reaction stirred at room temperature for an additional 1 h. The above liquid was added to a stirring solution of intermediate 24 (2.6 g,5.1 mmol) in DCM (100 mL) and the reaction was continued under stirring at room temperature for half an hour, after completion of the reaction by liquid-mass spectrometry (i.e., LC-MS), water (100 mL) was added, extracted with DCM (100 mL. Times.3), and the organic layer was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography (PE: ea=100:0-0:100) to give an orange solid, 15 (3.4 g, yield: 80.4%,4.1 mmol). MS (ESI) M/z (M+H) + 837.2。 1 H NMR(400MHz,DMSO)δ8.65(s,1H),7.88(d,J＝7.4Hz,2H),7.70 (dd,J＝8.2,4.0Hz,3H),7.40(dd,J＝13.7,4.9Hz,3H),7.32(td,J＝7.4,1.0Hz, 3H),7.15(d,J＝8.3Hz,1H),6.98(s,1H),6.63(dd,J＝20.1,2.4Hz,2H),6.46(dd,J ＝8.4,2.4Hz,1H),5.76(s,1H),4.27(d,J＝5.6Hz,2H),4.18(s,3H),3.84(s,3H), 3.73(s,3H),3.28–3.21(m,2H),2.69(d,J＝1.4Hz,4H),2.08(d,J＝17.2Hz,3H), 1.79(d,J＝20.5Hz,7H),1.58–1.41(m,7H),1.28(dd,J＝19.1,12.1Hz,7H).
S10 preparation of intermediate 26
Intermediate 25 (6 g,7.17 mmol) was dissolved in DCM (100 mL), TFA (10 mL) was added and the reaction stirred at room temperature for 5 min via liquid-mass combinationAfter the completion of the reaction, which was monitored by a spectrometer (i.e., LC-MS), the reaction mixture was concentrated and dried 3 times with chloroform to give 16 (7 g) of a red viscous liquid as intermediate 26. MS (ESI) M/z (M+H) + 687.2。
S11 preparation of intermediate 28
Monomethyl adipate, compound 27 (11.5 g,71.7 mmol) was dissolved in chloroform (100 mL) followed by the addition of thionyl chloride (25.7 g,215 mmol) and the reaction stirred at 60℃for 1 hour. The above solution was dried by spin-drying and once with toluene, dissolved in DCM (50 mL), then added to a stirred solution of intermediate 26 (7 g) and DIEA (3.37 g,26.2 mmol) in DCM (100 mL), stirred at room temperature for half an hour, and after completion of the reaction by monitoring the LC-MS, the reaction solution was added with water (100 mL), the aqueous phase was extracted with EA (100 mL. Times.3), and the organic layer was washed with saturated brine (100 mL) and dried over anhydrous sodium sulfate, followed by filtration and concentration. The residue was purified by column chromatography on silica gel (PE: EA: meoh=100:0:0-0:100:0-0:0:100) to give intermediate 28 (5 g, yield: 84.1%,6.03 mmol) as a pale yellow solid. MS (ESI) M/z (M+H) + 829.4。
S12 preparation of intermediate 29
Intermediate 28 (5 g,6.03 mmol) was reacted in 2M dilute hydrochloric acid (120 mL) and THF (100 mL) and stirring was continued at room temperature for 12 hours, after completion of the reaction monitored by liquid-mass spectrometry (i.e., LC-MS), extracted with EA (150 mL. Times.3), and the organic layer was washed with saturated brine (150 mL), dried over anhydrous sodium sulfate, filtered and concentrated to give a pale yellow solid, intermediate 29 (3 g, yield: 61.02%, 3.68 mmol). MS (ESI) M/z (M+H) + 815.4。
S13, preparation of intermediate 30
Intermediate 29 (3 g,3.68 mmol), intermediate 17 (1.56 g,7.36 mmol), HATU (2.1 g,5.52 mmol) and TEA (1.86 g,18.4 mmol) were stirred at room temperature overnight in DMF (100 mL) and after completion of the reaction by liquid-mass spectrometry (i.e., LC-MS), water (100 mL) was added to the reaction, the aqueous phase was extracted with EA (100 mL. Times.3), and the organic layer was washed with saturated brine (100 mL) and dried over anhydrous sodium sulfate, followed by filtration and concentration. The residue was separated and purified by reverse phase column chromatography (ammonia water: acetonitrile=100:0-30:70) to give intermediate 30 (2 g, yield: 53.8%,1.98 mmol) as a pale yellow solid. MS (ESI) M/z (M+H) + 1009.5。
S14, preparation of intermediate 31
Intermediate 30 (2 g,1.98 mmol) was dissolved in EtOH (20 mL), diethylamine (5 mL) was added and the reaction was continued to stir at room temperature for 12 hours, after which the reaction was complete as monitored by liquid-mass spectrometry (i.e., LC-MS), the reaction was concentrated and taken dry twice with EtOH. The residue was dissolved in water (20 mL), and after washing off impurities with EA, the intermediate 31 (1.5 g, yield: 96.2%,1.90 mmol) was obtained as a pale yellow solid by lyophilization. MS (ESI) M/z (M+H) +787.4.
S15 preparation of intermediate 33
Intermediate 31 (1.5 g,1.9 mmol), methyl 4-oxobutyrate, compound 20 (2 g,17.2 mmol) and STAB (4 g,9.5 mmol) were stirred in DCM (100 mL) and methanol (10 mL) overnight at room temperature and after completion of the reaction as monitored by liquid-mass spectrometry (i.e., LC-MS), the reaction mixture was spun dry. The residue was separated and purified by reverse phase column (ammonia, water: acetonitrile=100:0-70:30) to give intermediate 33 (1 g, yield: 58.9%,1.12 mmol) as a pale yellow solid. MS (ESI) M/z (M+H) + 887.4。1H NMR(400MHz, DMSO)δ10.35(s,1H),8.81(t,J＝5.8Hz,1H),8.58(d,J＝2.2Hz,1H),8.14(d,J＝ 8.8Hz,2H),7.97(d,J＝9.1Hz,1H),7.86(dd,J＝9.1,2.2Hz,1H),7.57(t,J＝8.3 Hz,2H),7.52(d,J＝9.2Hz,1H),5.82(d,J＝4.8Hz,1H),5.02(s,1H),4.83(s,1H), 4.17–3.96(m,3H),3.58(s,3H),3.17(s,6H),2.78(s,4H),2.40–2.20(m,5H),2.01 (dt,J＝20.2,11.2Hz,8H),1.88–1.68(m,6H),1.68–1.23(m,23H).
Preparation of S16.AMC-6C
Intermediate 33 (1 g,1.12 mmol) and LiOH (90 mg,2.8 mmol) were dissolved in water (10 mL) and THF (20 mL) at room temperature and stirred for 6h, after completion of the reaction monitored by liquid-mass spectrometry (i.e., LC-MS), the reaction mixture was isolated and purified by high-efficiency reverse phase column preparation to give pale yellow solid AMC-6C (400 mg, yield: 40.9%,0.458 mmol). MS (ESI) M/z (M+H) +873.7.1H NMR (400 MHz, DMSO). Delta.10.62 (s, 1H), 8.81 (t, J=5.8 Hz, 1H), 8.59 (d, J=2.0 Hz, 1H), 8.11 (d, J=8.8 Hz, 2H), 8.04 (s, 1H), 7.93 (dt, J=9.1, 5.6Hz, 2H), 7.60 (d, J=8.7 Hz, 2H), 7.34 (s, 1H), 5.76 (s, 2H), 5.03 (d, J=2.2 Hz, 1H), 4.06-3.96 (m, 1H), 3.58 (dd, J=10.3, 8.4Hz, 2H), 3.35-3.24 (m, 4H), 2.79 (s, 3H), 2.57 (s, 2H), 2.32 (s, 2H), 2.10 (dd, J=13.7 Hz, 1.7H), 4.06 (m, 1H), 3.58 (d, J=2.7 Hz, 1H), 4.8.9-3.24 (m, 1H), 3.9.7.7H (m, 3H).
Example 2 preparation of Multi-Cluster tandem Linear Artificial antigen
The embodiment provides a preparation method of a multi-cluster tandem linear artificial antigen, which mainly comprises the synthesis of an immunogen and a coating antigen. The immunogen is different from the coating antigen in the type of carrier, namely carrier bovine Lactoferrin (LF) and Keyhole Limpet Hemocyanin (KLH) are adopted by the immunogen; the coating antigen adopts Ovalbumin (OVA) as carrier protein. A method for preparing immunogen. The method for synthesizing immunogen/coating antigen adopted by the invention is an active ester method. The method comprises the following specific steps:
24.69mg of AMC-4℃/26.38mg of AMC-6C, 8.69mg of EDC and 5.22mg of NHS were dissolved in 600. Mu.L of DMF and the reaction was stirred overnight at 4℃and designated as solution A. 10mg of carrier protein was weighed and dissolved in 4mL of PBS buffer (0.01M, pH 7.4), and the solution was stirred and dissolved to prepare solution B. Under the magnetic stirring, the solution A is sucked and added into the solution B drop by drop, and the reaction is carried out for 12 hours under the magnetic stirring at the temperature of 4 ℃. The reaction was dialyzed against PBS at 4℃for 3 days, with 2 changes of dialysate per day. The immunogen can be obtained and frozen in a refrigerator at the temperature of minus 20 ℃ for standby. The hapten prepared in example 1 and the artificial antigen prepared in example 2 are respectively identified by ultraviolet wavelength scanning (150-400 nm), and the results are shown in figure 1, compared with the carrier protein and the hapten, the characteristic absorption peak and peak shape are changed after coupling, and the results indicate that antigen coupling is successful.
EXAMPLE 3 preparation of Multi-Cluster tandem Linear antigen-specific polyclonal antibodies
Animal immunization: using healthy 6-week-old Balb/c female mice as experimental animals, the two immunogens prepared in example 2 were each mixed and emulsified with equal amounts of adjuvant (first complete Freund's adjuvant followed by incomplete Freund's adjuvant), and subcutaneously injected into the neck, back and abdominal cavities of the mice at an immunization dose of 0.5mL (containing 0.5mg immunogen). The first immunization is carried out by emulsifying 0.5mL complete Freund's adjuvant and antigen, then carrying out booster immunization after emulsifying 4 weeks by using 0.5mL incomplete Freund's adjuvant and antigen, then carrying out antibody quality identification by taking a small amount of blood from tail vein every 2 weeks, after the antibody is stable, collecting serum from mice with the best performance, carrying out warm bath at 37 ℃ for 30min, centrifuging at room temperature for 20min, taking supernatant, sub-packaging in a centrifuge tube, and storing at-20 ℃ for use.
Evaluation of antiserum effects: the multi-cluster serial linear coating antigen prepared in example 2 is used, the collected mouse serum is taken as a detection antibody, the antiserum titer and the inhibition rate of the mouse serum are measured by adopting an indirect competition ELISA method, and the titers and the inhibition rates of the antiserum are comprehensively considered to evaluate the antiserum. The specific operation steps are as follows:
1) And (3) wrapping the plate: multiple clusters of tandem linear coated antigen were diluted to 1000ng/mL with 0.05M carbonate buffer (pH 9.6) and coated at 100. Mu.L/well overnight at 4 ℃; removing the coating liquid, washing with PBST for 2 times, adding 120 μl of sealing liquid (5% skimmed milk) into each hole, and sealing at 37deg.C for 3 hr; discarding the sealing liquid, drying at 37 ℃ for 60min, and sealing the sealing bag at 4 ℃ for standby to obtain the packaged ELISA plate.
2) SerumPotency and inhibition assay: and (3) the ELISA plate packaged in the step (1) is provided with the titer column: 50 mu L of PBS and 50 mu L of serum diluted according to gradient multiple are added into each hole respectively; inhibition column: mu.L of diluted 1000ng/mL drug (multi-cluster tandem linear hapten) and 50. Mu.L of serum diluted in a gradient fold were added to each well and 2 groups were made in parallel. Incubating at 37deg.C for 40min, washing with PBST for five times, drying the liquid in the hole, adding enzyme-labeled secondary antibody (goat anti-mouse IgG-HRP) diluted at 1:5000, incubating at 37deg.C for 30min, washing with PBST for five times, drying the liquid in the hole, adding 100 μl TMB substrate solution, and developing at 37deg.C in dark for 10min; mu.L of stop solution (10% H) was added 2 SO 4 ) Terminating the reaction; the absorbance at 450nm was read with a microplate reader.
3) Experimental results
The results of the measurement of the antiserum titer and the inhibition rate of the mouse serum based on the indirect competition ELISA method are shown in Table 1, the titer is between 8 and 32K, and the inhibition rate of the multi-cluster tandem linear hapten is between 63 and 90 percent.
TABLE 1 mouse antiserum Effect
The results show that the multi-cluster tandem linear hapten and the artificial antigen thereof prepared by the invention have good immunogenicity and can be used for preparing monoclonal antibodies, single-chain antibodies and nano antibodies subsequently.
The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (9)
1. The hapten for simultaneously detecting amantadine, olaquindox and chloramphenicol is characterized by having a structural general formula shown in formula I:
wherein R is 1 And R is 2 Respectively selected from alkylene groups with 2-5 carbon atoms.
2. The hapten of claim 1, wherein R is selected from the group consisting of 1 Selected from propylene or pentylene, R 2 Selected from ethylene or butylene.
3. The method for preparing hapten according to claim 1, comprising the steps of:
s1, respectively preparing a olaquindox metabolite intermediate, an amantadine intermediate and a chloramphenicol intermediate: methyl acetoacetate is used as a raw material to obtain a olaquindox metabolite intermediate shown in a formula II through NBS bromination, 4-nitroo-phenylenediamine cyclization, nitro reduction, amino protecting group feeding and ester bond hydrolysis; the method comprises the steps of taking adamantane as a raw material, connecting an amino acid arm protected by tert-butoxycarbonyl on 1 amino group after nitration, nitroreduction, aminoacylation and hydrolysis, then connecting fluorenylmethoxycarbonyl on the other 1 amino group, and finally removing Boc protecting group to obtain an amantadine intermediate shown in a formula III; hydrolyzing and removing dichloroacetyl of chloramphenicol to obtain chloramphenicol intermediate chloramphenicol amine shown in formula IV;
wherein R is 1 And R is 2 Respectively selected from alkylene with 2-5 carbon atoms;
s2, synthesis of hapten: and (3) condensing and coupling an amantadine intermediate shown in a formula III and a olaquindox metabolite intermediate shown in a formula II through primary amino groups and carboxyl groups on molecules, removing DMB protecting groups of aromatic ring amino groups of the olaquindox metabolite intermediate, coupling through an acylation condensation reaction to obtain an intermediate V, performing acylation condensation on the carboxyl groups of the intermediate V and amino groups of a chloramphenicol intermediate shown in a formula IV to obtain an intermediate VI, further removing Fmoc protecting groups, condensing and reducing with 4-oxybutyrate methyl ester to be aminated into an intermediate VII, and finally hydrolyzing ester groups to obtain the hapten shown in the formula I.
4. Use of the hapten according to claim 1 or 2 for the preparation of artificial antigens.
5. An artificial antigen, which is obtained by coupling the hapten and carrier protein of claim 1, and has a structure shown in a formula VIII:
wherein R is 1 And R is 2 Respectively selected from alkylene groups with 2-5 carbon atoms.
6. The artificial antigen of claim 5, wherein the carrier protein is selected from lactoferrin, keyhole limpet hemocyanin, and ovalbumin.
7. The method for preparing artificial antigen according to claim 5 or 6, wherein the antigen is obtained by coupling a carrier protein to the carboxyl group of the hapten according to claim 1.
8. The process of claim 7, wherein the coupling is by an active ester process.
9. Use of the artificial antigen according to claim 5 or 6 for the preparation of monoclonal/polyclonal antibodies, single chain antibodies or nanobodies.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111679788.XA CN114539170B (en) | 2021-12-31 | 2021-12-31 | Hapten and artificial antigen for simultaneously detecting amantadine, olaquindox and chloramphenicol, and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111679788.XA CN114539170B (en) | 2021-12-31 | 2021-12-31 | Hapten and artificial antigen for simultaneously detecting amantadine, olaquindox and chloramphenicol, and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114539170A CN114539170A (en) | 2022-05-27 |
| CN114539170B true CN114539170B (en) | 2023-05-16 |
Family
ID=81670405
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111679788.XA Active CN114539170B (en) | 2021-12-31 | 2021-12-31 | Hapten and artificial antigen for simultaneously detecting amantadine, olaquindox and chloramphenicol, and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114539170B (en) |
Family Cites Families (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3767694A (en) * | 1971-01-06 | 1973-10-23 | Squibb & Sons Inc | Adamantane carboxylic acid esters of chloramphenicol |
| CN1554649A (en) * | 1997-11-21 | 2004-12-15 | NPSҩ������˾ | Metabotropic glutamate receptor antagonists for treating central nervous system diseases |
| US9016221B2 (en) * | 2004-02-17 | 2015-04-28 | University Of Florida Research Foundation, Inc. | Surface topographies for non-toxic bioadhesion control |
| CN103304495B (en) * | 2012-03-13 | 2016-09-21 | 北京勤邦生物技术有限公司 | A kind of olaquindox metabolite hapten preparation method and applications |
| CN104950105B (en) * | 2014-03-26 | 2017-10-24 | 北京维德维康生物技术有限公司 | The preparation method and its application in chemiluminescence immunoassay kit of chloramphenicol haptens and antigen |
| CN104402753B (en) * | 2014-11-17 | 2016-04-06 | 浙江省农业科学院 | A kind of amantadine artificial semiantigen, artificial antigen and its preparation method and application |
| CN105203654B (en) * | 2015-09-11 | 2017-08-11 | 山东省分析测试中心 | It is a kind of to be used to determine the method for 11 kinds of illegal addition medicament contgs in herbal medicine powder |
| CN110057929A (en) * | 2019-03-18 | 2019-07-26 | 山东润达检测技术有限公司 | The high-flux detection method of forbidden drug in a kind of feed and feed addictive |
| CN110105232A (en) * | 2019-05-10 | 2019-08-09 | 中国农业大学 | A kind of amantadine haptens and its preparation method and application |
| CN111077319A (en) * | 2019-12-18 | 2020-04-28 | 华南农业大学 | Micro-fluidic chip immunodetection kit and detection method thereof |
| CN114539171B (en) * | 2021-12-31 | 2023-05-05 | 华南农业大学 | MAC type multi-cluster tandem linear hapten and artificial antigen as well as preparation methods and applications thereof |
-
2021
- 2021-12-31 CN CN202111679788.XA patent/CN114539170B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN114539170A (en) | 2022-05-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP0959355B1 (en) | Reagents for the detection and quantification of thryoxine in fluid samples | |
| EP0649533B1 (en) | Reagents and methods for the detection and quantification of thyroxine in fluid samples | |
| CN114539171B (en) | MAC type multi-cluster tandem linear hapten and artificial antigen as well as preparation methods and applications thereof | |
| EP3470416B1 (en) | 6-acetylmorphine analogs, and methods for their synthesis and use | |
| CN114539170B (en) | Hapten and artificial antigen for simultaneously detecting amantadine, olaquindox and chloramphenicol, and preparation method and application thereof | |
| CN114315722B (en) | Preparation and application of tolfenpyrad artificial hapten and antibody thereof | |
| CN109575123B (en) | Preparation method and application of fluoroacetamide hapten and monoclonal antibody | |
| CN108424880A (en) | A kind of hybridoma cell strain and preparation method of secretion cyproheptadine monoclonal antibody | |
| CN109824604B (en) | Sulfonamide hapten and artificial antigen as well as preparation method and application thereof | |
| CN109438424B (en) | Ribavirin hapten and artificial antigen as well as preparation method and application thereof | |
| CN101747429A (en) | Specific antibody against pesticide meta-tolyl-N-methylcarbamate | |
| CN110938007A (en) | Dicofol hapten, artificial antigen, antibody, synthetic method and application thereof | |
| CN110642743A (en) | Nifuroxazide hapten and artificial antigen as well as preparation methods and application thereof | |
| CN114031528B (en) | Florfenicol hapten, artificial antigen, antibody and synthetic method and application thereof | |
| CN111499637B (en) | Yohimbine hapten YHA, artificial antigen and antibody thereof, and preparation and application thereof | |
| EP0819254A1 (en) | Reagents and methods for the detection and quantification of vancomycin in biological fluids | |
| CN113150162A (en) | Preparation method and application of antibody of carbamate pesticide | |
| CN112939875B (en) | Trimethoprim hapten TMPO, artificial antigen, antibody and preparation method and application thereof | |
| CN111848437A (en) | Baclofen artificial hapten, artificial antigen, preparation method and application thereof | |
| CN112979457B (en) | Bisphenol diglycidyl ether compound hapten and synthesis method thereof | |
| US20240174600A1 (en) | Pregabalin artificial hapten, artificial antigen and preparation method therefor and application thereof | |
| CN113214109B (en) | Nifurosol hapten, artificial antigen, preparation methods and applications thereof | |
| CN116693443A (en) | Di-primary amine artificial hapten and artificial antigen as well as preparation methods and application thereof | |
| CN113105430B (en) | Non-natural chiral amino acid compound containing imine sulfone cycloheptyne and salt compound thereof, and preparation method and application thereof | |
| CN108191934B (en) | Tildipirosin hapten derivative and preparation method and detection kit 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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |