CN114539170A - 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
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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, 3 drug framework structures of Amantadine (AMD), 3-methyl-quinoxaline-2-carboxylic acid (MQCA) and chloramphenicol amine (CAPA) are respectively connected in sequence by adopting amino acids and alkyl diacid with different carbon chain lengths to form a multi-cluster series linear hapten, and the hapten is coupled with carrier protein to prepare the artificial antigen. The titer of the multi-antiserum obtained by adopting the artificial antigen is between 8 and 32K, and the inhibition rate of the multi-antiserum to the hapten 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, and in particular relates to an AMC type multi-cluster tandem linear hapten and artificial antigen simultaneously containing Amantadine (AMD), 3-methyl-quinoxaline-2-carboxylic acid (MQCA) and chloramphenicol amine (CAPA) framework structures, and a preparation method and application thereof.
Background
China is the biggest livestock product producing and consuming country in the world, and according to statistics, the total yield of meat reaches 7925.8 ten thousand tons in China in 2010, and the meat is at the top of the world. The reported veterinary drug residue analysis and detection technologies of animal products mainly focus on large-scale precise instrument analysis technology and immunoassay screening technology, wherein the instrument identification method mainly adopts liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). For example, chinese patent discloses a method for determining the content of 11 illegally added drugs in veterinary drug powder, which can simultaneously detect 11 illegally added drugs including amantadine, olaquindox, chloramphenicol, etc. for detection, however, the instrumental method generally has the disadvantages of complicated sample pretreatment operation, time-consuming, high cost, unsuitability for field detection, etc. The immunoassay method has the advantages of consistent sensitivity with the analysis of a conventional instrument, suitability for field screening, simplicity, rapidness, low cost, less required sample amount, simple and convenient pretreatment and the like. Currently, the single-residue immunoassay is mainly used in the safety detection of livestock products, and for example, chinese patents CN108152499A, CN111077319A, CN108519480A and the like disclose immunoassay methods for amantadine, olaquindox and chloramphenicol, respectively. However, due to the diversity of veterinary drug types and the universality and complexity of compound use thereof, the development of only a single-residue immunoassay technology cannot meet the actual detection requirements, but related haptens, antigens and antibodies for simultaneously detecting small molecule drugs of amantadine, olaquindox and chloramphenicol with non-shared structures are lacking at present.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects in the prior art and provide a hapten for realizing multi-residue immunoassay of small molecular drugs of non-shared structures, such as amantadine, olaquindox and chloramphenicol. Three small molecule drugs with different structures, namely amantadine, olaquindox and chloramphenicol, are regarded as different epitopes and are connected in series in a proper way to form a linear multi-cluster hapten, so that the neutralizing and recognizing capability of the multi-epitope is improved.
The second purpose of the invention is to provide a preparation method of the hapten.
A third object of the invention is to provide the use of said hapten.
The fourth purpose of the invention is to provide an artificial antigen.
The fifth purpose of the invention is to provide a preparation method of the artificial antigen.
The sixth purpose of the invention is to provide the application of the artificial antigen.
It is a seventh object of the present invention to provide an antigen-specific antibody.
The above object of the present invention is achieved by the following technical solutions:
a hapten for simultaneously detecting amantadine, olaquindox and chloramphenicol, which simultaneously contains the skeleton structures of Amantadine (AMD), 3-methyl-quinoxaline-2-carboxylic acid (MQCA) and chloramphenicol amine (CAPA), and the structural general formula is shown as formula I:
wherein R is1And R2Are respectively selected from alkyl with 2-5 carbon atoms, namely R1And R2Are respectively selected from any one of ethyl to amyl.
Preferably, said R is1Selected from propyl or pentyl, R2Selected from ethyl or butyl.
Further preferably, the hapten is of the formula AMC-4C or AMC-6C as follows:
the preparation method of the sex hapten comprises the following steps:
s1, preparing olaquindox metabolite, amantadine and chloramphenicol intermediates respectively: methyl acetoacetate is taken as a raw material, and subjected to NBS bromination, 4-nitro o-phenylenediamine cyclization, nitro reduction, amino protecting group addition and ester bond hydrolysis to obtain a olaquindox metabolite intermediate shown in a formula II; taking adamantane as a raw material, carrying out nitration, nitro reduction, amino acylation and hydrolysis, connecting an amino acid arm protected by tert-butyloxycarbonyl on 1 amino group, then connecting fluorenylmethoxycarbonyl acyl on the other 1 amino group, and finally removing a Boc protecting group to obtain an adamantanamine intermediate shown in a formula III; hydrolyzing and removing dichloroacetyl of chloramphenicol to obtain chloramphenicol intermediate chloramphenicol amine shown in formula IV;
wherein R is1And R2Are respectively selected from alkyl with 2-5 carbon atoms, namely R1And R2Respectively selected from any one of ethyl to amyl;
s2, synthesis of hapten: 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 on molecules and carboxyl groups, removing DMB protecting groups of aromatic cyclic amino groups of the olaquindox metabolite intermediate, coupling through acylation condensation reaction to obtain an intermediate V, subjecting the intermediate V to acylation condensation through 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, reducing and aminating the intermediate VI with 4-oxobutyric acid methyl ester respectively to obtain an intermediate VII, and finally hydrolyzing ester groups to obtain the hapten shown in a formula I.
Preferably, said R is1Selected from propyl or pentyl, R2Selected from ethyl or butyl.
The invention also provides application of the hapten in preparing an artificial antigen.
An artificial antigen is obtained by coupling hapten shown in the formula I and carrier protein, and the structure of the artificial antigen is shown in the formula VIII:
wherein R is1And R2Are respectively selected from alkyl with 2-5 carbon atoms, namely R1And R2Each selected from any one of ethyl to pentyl.
Preferably, said R is1Selected from propyl or pentyl, R2Selected from ethyl or butyl.
Further preferably, said R1And R2Selected from the group consisting of propyl and ethyl, pentyl and butyl, respectively, substituted as shown by 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 and KLH are used for preparing immunogen, OVA is used for preparing coating antigen.
The preparation method of the artificial antigen is obtained by coupling carrier protein on carboxyl of hapten shown in a formula I.
Preferably, the coupling is by an active ester method.
As a preferable embodiment, the method for preparing the artificial antigen comprises the steps of: hapten shown as formula I, 1-ethyl- (3-dimethyl amino propyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) are dissolved in N, N-dimethyl formamide (DMF), and the mixture is stirred and reacted at 4 ℃ overnight, and is recorded as solution A. Weighing carrier protein, dissolving in phosphate buffer solution (PBS, 0.01M, pH 7.4), stirring, and dissolving to obtain solution B. And (3) under magnetic stirring, sucking the solution A, dropwise adding the solution A into the solution B, reacting under magnetic stirring at 4 ℃, dialyzing the reaction solution with PBS for 3 days at 4 ℃, and replacing the dialyzate for 2 times every day to obtain the artificial antigen.
The invention also provides application of the artificial antigen in preparation of a monoclonal/polyclonal antibody, a single-chain antibody or a nano antibody.
A polyclonal antibody is prepared by immunizing mice with artificial antigen shown in formula VIII.
Preferably, artificial antigen with carrier protein of Lactoferrin (LF) or Keyhole Limpet Hemocyanin (KLH) is used as immunogen, female Balb/C mice of the right age are selected for immunization, the titer and the inhibition rate of antiserum are measured after 4 th immunization and 5 th immunization, and after the performance is stable, mouse serum is collected to obtain the polyclonal antibody.
The invention also provides a rapid immunoassay method for detecting the artificial antigen, which is used for evaluating the performance of antiserum based on an ELISA method, takes the artificial antigen taking the carrier protein as Ovalbumin (OVA) as the coating antigen, and takes the antigen specific antibody as the 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 prepared by connecting intermediates of 3 medicines of amantadine, olaquindox metabolite and chloramphenicol with aminocarboxylic acid molecules of different spacer arms, and performing mixed chemical tandem to synthesize a multi-cluster tandem linear hapten. The artificial antigen is prepared based on the hapten, and then the mouse multi-antiserum prepared by immunizing animals with the artificial antigen has the titer of 8-32K and the inhibition rate of 63-90% on multi-cluster series linear hapten. The artificial antigen prepared by the invention has good immunogenicity, and can be used for subsequently preparing multi-cluster tandem linear antigens or multi-epitope specific monoclonal antibodies, single-chain antibodies and nano-antibodies and establishing a corresponding immunoassay method. The invention lays a foundation for the preparation of the micromolecule multispecific antibody and the multi-residue analysis method, and has good application prospect.
Drawings
FIG. 1 is an ultraviolet scan of multi-cluster tandem linear haptens, artificial antigens and carrier proteins.
Detailed Description
The present invention will be described in further detail with reference to the following figures and specific examples, wherein reagents, methods and apparatus are conventional in the art unless otherwise specified.
Example 1 preparation of AMC-type Multi-Cluster tandem Linear hapten
1. A method for preparing AMC-4C hapten comprises the following steps:
s1. preparation of intermediate 3
Methyl acetoacetate, Compound 1(50g,431mmol) was added to water (1L) at 70 deg.C with N-bromosuccinimide (NBS) (88g,494mmol) and stirred at room temperature for half an hour until the system was clear. 4-Nitrophthalene diamine, Compound 2(63g,412mmol) was then added and stirring continued at 70 ℃ for 8h, after monitoring the reaction completion by liquid-mass spectrometry (LC-MS), cooled to room temperature and extracted with an organic solvent PE: EA ═ 1:1 (500 mL. times.6). The organic phases were combined, dried and spin dried. The crude product is subjected to column chromatography(PE: EA ═ 10:1) was isolated and purified to give intermediate 3(14g, yield: 13.8%, 57mmol) as a yellow solid, which turned purple-black after long-time spin-drying. MS (ESI) M/z (M + H)+248.1。
S2. preparation of intermediate 4
Intermediate 3(14g,57mmol) was dissolved in tetrahydrofuran (i.e., THF) (100mL) and methanol (100mL) and Zn (40g,634mmol) was added. Ammonium chloride (38g,710mmol) was added portionwise at 0 ℃ and the reaction was continued for half an hour with stirring at 0 ℃ and, after completion of the reaction was monitored by a liquid-mass spectrometer (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. And (5) concentrating the filtrate. The residue was separated and purified by column chromatography (PE: EA 100:0-50:50) to give intermediate 4(7g, yield: 56.14%, 32mmol) as a yellow solid. MS (ESI) M/z (M + H)+218.1。1H 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(7g,32mmol) and 2, 4-dimethoxybenzaldehyde (i.e., DMB) (10.2 g,61.4mmol) were dissolved in dichloromethane (i.e., DCM) (100mL) at room temperature, and sodium triacetoxyborohydride (i.e., STAB) (20.5g,96.7mmol) was added and the reaction stirred at room temperature overnight. After completion of the reaction was monitored by LC-MS, water (100mL) was added to the reaction solution, and extraction was performed with EA (100 mL. times.5), and the organic layer was washed with saturated brine (100mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was separated and purified by column chromatography (PE: EA 100:0-65:35) to give intermediate 5(8.5g, yield: 72.2%, 23.1mmol) as a yellow solid. MS (ESI) M/z (M + H)+368.0。1H 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(8.5g, 23.1mmol) and NaOH (1.85g,46.2mmol) were dissolved in THF (20mL) and water (10 mL). The reaction solution was stirred at room temperature for 1 hour. After completion of the reaction was monitored by LC-MS, the pH was adjusted to weak acidity by concentrated hydrochloric acid, followed by extraction with DCM (100 mL. times.8), and the organic layer was washed with saturated brine (100mL) and dried over anhydrous sodium sulfate. After filtration, concentration was carried out to obtain intermediate 6(7.4g, yield: 90.5%, 20.9mmol) as an orange solid, i.e., a olaquindox metabolite (3-methyl-quinoxaline-2-carboxylic acid) intermediate. MS (ESI) M/z (M + H)+354.1。1H 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(75g, 551mmol), was added slowly to stirred fuming nitric acid (345g) at 0 ℃ and the reaction was stirred for an additional 1 hour at 0 ℃. Acetonitrile (55.5 g,1.37mol) was then added dropwise at 0 ℃ and stirring was continued for 1 hour at 0 ℃. Finally concentrated sulfuric acid (705g) was added slowly at 0 ℃ followed by stirring overnight at room temperature. After the reaction was monitored by a liquid-mass spectrometer (LC-MS), the reaction solution was poured into ice (about 1kg), pH was adjusted to alkaline with sodium carbonate solid, and a yellow viscous substance was precipitated. The water was decanted, EA (approximately 100mL) was added to cake the yellow viscous mass, and filtered. Washing the yellow precipitate with EA and then with waterIntermediate 8(80g) was washed as a white solid. MS (ESI) M/z (M + H)+251.1。1H 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(70g) was reacted in HCl (10%, 500mL) with stirring at 110 ℃ for 2 days, and after completion of the reaction was monitored by a mass spectrometer liquid-mass spectrometer (i.e., LC-MS), the reaction was concentrated under reduced pressure to give intermediate 9(63g, 264mmol) as a white solid. MS (ESI) M/z (M + H)+167.1。1H 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
tert-Butoxycarbonyl (Boc) protected 4-aminobutyric acid, compound 10(5.66g, 27.8mmol), HATU (12.7g,33.4mmol) and DIEA (10.8g,83.6mmol) were dissolved in DMSO (50mL) and the reaction was stirred at room temperature for 1 h. The above liquid was slowly added dropwise to a stirring DMSO (100mL) solution of intermediate 9 (10g,41.8mmol) and DIEA (10.8g,83.6mmol), the reaction was continued for half an hour at room temperature, after completion of the reaction was monitored by a liquid mass spectrometer (LC-MS), FmocCl (21.63g,83.6mmol) and DIEA (10.8g,83.6mmol) were directly added to the reaction solution, the reaction was continued for 1 hour with stirring, after completion of the reaction was monitored by a liquid mass spectrometer (LC-MS), water (300mL) was added, extraction was performed with EA (100mL × 5), the organic layer was washed with saturated brine (100mL), 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 11(6.9g, yield: 43.2%, 12mmol) as a yellow solid. MS (ESI) M/z (M + H)+574.3。1H 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(6.9g, 12mmol) was dissolved in 4M dioxane hydrochloride solution (50mL) and the reaction was stirred at room temperature for an additional 1 hour. TLC showed the reaction was complete. The reaction mixture was concentrated to give intermediate 12(7g) as a pale yellow oily liquid, intermediate 12. MS (ESI) M/z (M + H)+474.2。1H 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(3.5g,9.9mmol), 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium hexaflourophosphate (i.e. HATU) (4.5g,11.8mmol) and N, N-diisopropylethylamine (i.e. DIEA) (3.8 g,30mmol) were dissolved in 20mL of dichloromethane and the reaction was stirred at room temperature for 1 hour. The above liquid was added to a stirring solution of intermediate 12(6g) in DCM (100mL) and the reaction was continued for half an hour at room temperature, after completion of the reaction was monitored by liquid mass spectrometer (i.e. LC-MS), water (100mL) was added, DCM (100mL × 3) was extracted, the organic layer was washed with saturated brine (100mL), dried over anhydrous sodium sulfate, filtered and concentrated. The residue was separated and purified by column chromatography (PE: EA 100:0-0:100) to give intermediate 13(6.9g, yield: 85.9%, 8.5mmol) as an orange solid. MS (ESI) M/z (M + H)+809.2。1H 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(3g, 3.7mmol) was dissolved in DCM (20mL), trifluoroacetic acid i.e. TFA (2mL) was added and the reaction was stirred at room temperature for 5 minutes and after completion of the reaction was monitored by a mass spectrometer (i.e. LC-MS), the reaction was concentrated and taken up 3 times with chloroform to give 14(3.5g) of a red viscous liquid as intermediate 14. MS (ESI) M/z (M + H)+659.3。1H 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(3.5g,3.7mmol), succinic anhydride, compound 15(3.7g,37mmol) and DIEA (2.4g,18.5mmol) were dissolved in DCM (50 mL). The reaction was stirred at room temperature for 6 hours, and after completion of the reaction was monitored by a liquid mass spectrometer (i.e., LC-MS), the reaction solution was filtered, and after washing the cake with methanol, the filtrate was concentrated. The residue was separated and purified by reverse phase column chromatography (FA, water: acetonitrile 100:0-40:60) to give intermediate 16(1.4g, yield: 50%, 1.85mmol) as a pale yellow solid. MS (ESI) M/z (M +H) +759.3。1H 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 (25g, 77.6mmol) was dissolved in HCl (1M,500mL), hydrolyzed overnight with stirring at 100 deg.C, and after completion of the reaction was monitored by liquid-mass spectrometer (LC-MS), the reaction liquid was cooled to room temperature and extracted with ethyl acetate EA (500 mL. times.3) to remove dichloroacetic acid. The remaining aqueous phase was spin-dried to give intermediate 17(15g, yield: 91.1%, 70.7mmol) as a white solid.
S13. preparation of intermediate 18
Intermediate 16(1.4g,1.85mmol), intermediate 17(3.92g,18.5mmol) and DIEA (1.2g, 9.25mmol) were dissolved in DMF (20mL), 1-propylphosphoric acid tricyclo anhydride, T3P (2.35g, 7.4mmol) was added and the reaction was continued for 1 hour at room temperature, after completion of the reaction was monitored by a liquid-mass spectrometer (LC-MS), water (50mL) was added, extracted with EA (50mL × 3), the organic layer was washed with saturated brine (50mL), dried over anhydrous sodium sulfate, filtered and concentrated. The residue was separated and purified by a reverse phase column (FA, water: acetonitrile 100:0-50:50) to give intermediate 18(950mg, yield: 54%, 1.0mmol) as a pale yellow solid. MS (ESI) M/z (M + H)+953.5。1H 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(950mg, 1.0mmol) was dissolved in ethanol, EtOH (20mL), diethylamine (5mL) was added and the reaction was stirred at room temperature for an additional 6 hours, and after completion of the reaction was monitored by a mass spectrometer (LC-MS), the reaction was concentrated and taken up twice with EtOH. The residue was dissolved in water (5mL), washed with petroleum ether, PE, to remove impurities and lyophilized to give intermediate 19(720mg, yield: 98.6%, 0.98mmol) as a pale yellow solid. MS (ESI) M/z (M + H)+731.1。1H 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(720mg,0.98mmol), methyl 4-oxobutyrate, compound 20(220mg, 1.88mmol) and STAB (1.2g,5.64mmol) were stirred in DCM (20mL) and methanol (20mL) at room temperature overnight, after completion of the reaction was monitored by a liquid-mass spectrometer (LC-MS), the reaction solution was spun dry, and the residue was separated and purified by a reverse phase column (ammonia, water: acetonitrile: 100:0-70:30) to give intermediate 21(690mg, yield: 84.7%, 0.83mmol) as a pale yellow solid. MS (ESI) M/z (M + H)+831.2。 1H 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).
S16. preparation of AMC-4C
At room temperature, intermediate 21(690mg, 0.83mmol) and lithium hydroxide, LiOH (68mg, 1.66mmol) were dissolved in water (5mL) and THF (10mL) and stirred for reaction for 6h, and after completion of the reaction was monitored by a liquid-mass spectrometer (LC-MS), the reaction solution was subjected to preparative separation on a high performance reverse phase column to obtain a pale yellow solid, AMC-4C (269mg, yield: 39.8%, 0.33 mmol). MS (ESI) M/z (M + H)+817.4。1H 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 method for preparing AMC-6C hapten comprises the following steps:
S1-S6 are the same as the preparation process of the AMC-4C hapten;
s7. preparation of intermediate 23
tert-Butoxycarbonyl protected 6-aminocaproic acid, Compound 22(5.66g, 27.8 mmo)l), HATU (12.7g,33.4mmol) and DIEA (10.8g,83.6mmol) were dissolved in DMSO (50mL) and the reaction was stirred at room temperature for 1 h. The above mixture solution was slowly added dropwise to a stirring DMSO (100mL) solution of intermediate 9 (10g,41.8mmol) and DIEA (10.8g,83.6mmol), the reaction was continued for half an hour at room temperature, the completion of the reaction was monitored by a mass spectrometer for liquid-mass spectrometry (LC-MS), chloroformate-9-fluorenylmethyl ester (FmocCl (21.63g,83.6mmol) and DIEA (10.8g,83.6mmol) were directly added to the reaction solution, the reaction was continued for 1 hour with stirring, after the completion of the reaction was monitored by a mass spectrometer for liquid-mass spectrometry (LC-MS), water (300mL) was added, extraction was performed with Ethyl Acetate (EA) (100mL × 5), the organic layer was washed with a saturated saline solution (100mL), 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(8.2g, yield: 48.9%, 13.6mmol) as a pale yellow solid, 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(8.2g, 13.6mmol) was dissolved in 4M dioxane hydrochloride solution (50mL) and the reaction was stirred at room temperature for 1 hour, TLC showed completion of the reaction, and the reaction was concentrated to give intermediate 24(8.12g) as a pale yellow oil. 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(1.8g,5.1mmol), HATU (2.3g,6.1mmol) and DIEA (1.9g,15.3 mmol) were dissolved in DCM (20mL) and the reaction was stirred for an additional 1h at room temperature. The above liquid was added to a stirring solution of intermediate 24(2.6g,5.1mmol) in DCM (100mL) and the reaction was continued for half an hour at room temperature with stirring, after completion of the reaction was monitored by a liquid mass spectrometer (i.e. LC-MS), water (100mL) was added, extraction was performed with DCM (100mL × 3), and the organic layer was washed with saturated brine (100mL), dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography (PE: EA 100:0-0:100) to give 15(3.4g, yield: 80.4%, 4.1mmol) as an orange solid. MS (ESI) M/z (M + H)+837.2。1H 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(6g, 7.17mmol) was dissolved in DCM (100mL), TFA (10mL) was added and the reaction was continued for 5 minutes at room temperature with stirring, and after completion of the reaction was monitored by a mass spectrometer liquid mass spectrometer (LC-MS), the reaction was concentrated and dried with chloroform for 3 times to give 16(7g) of a red viscous liquid as intermediate 26. MS (ESI) M/z (M + H)+687.2。
S11. preparation of intermediate 28
Mixing adipic acidMonomethyl ester, compound 27(11.5g,71.7mmol) was dissolved in chloroform (100mL), followed by addition of thionyl chloride (25.7g,215mmol) and stirring of the reaction at 60 deg.C for 1 hour. After the above liquid was spin-dried and taken up once with toluene, it was dissolved in DCM (50mL), followed by addition of a stirred solution of intermediate 26(7g) and DIEA (3.37g,26.2mmol) in DCM (100mL), reaction was stirred at room temperature for half an hour, completion of reaction was monitored by LC-MS, water (100mL) was added to the reaction solution, the aqueous phase was extracted with EA (100 mL. times.3), the organic layer was washed with saturated brine (100mL), dried over anhydrous sodium sulfate, and then concentrated by filtration. The residue was separated and purified by silica gel column (PE: EA: MeOH: 100:0:0-0:100:0-0:0:100) to obtain intermediate 28(5g, yield: 84.1%, 6.03mmol) as a pale yellow solid. MS (ESI) M/z (M + H)+829.4。
S12. preparation of intermediate 29
Intermediate 28(5g,6.03mmol) was dissolved in 2M dilute hydrochloric acid (120mL) and THF (100mL) and stirred at room temperature for 12 hours, after completion of the reaction was monitored by LC-MS, extraction was performed with EA (150 mL. times.3), the organic layer was washed with saturated brine (150mL), dried over anhydrous sodium sulfate, filtered and concentrated to give intermediate 29(3g, yield: 61.02%, 3.68mmol) as a pale yellow solid. MS (ESI) M/z (M + H)+815.4。
S13. preparation of intermediate 30
Intermediate 29(3g,3.68mmol), intermediate 17(1.56g,7.36mmol), HATU (2.1 g,5.52mmol) and TEA (1.86g,18.4mmol) were stirred in DMF (100mL) at room temperature overnight, after completion of the reaction was monitored by a liquid-mass spectrometer (i.e., LC-MS), water (100mL) was added to the reaction solution, the aqueous phase was extracted with EA (100mL × 3), and the organic layer was washed with saturated brine (100mL), dried over anhydrous sodium sulfate, and then concentrated by filtration. Remainder ofThe product was separated and purified by reverse phase column chromatography (ammonia water: acetonitrile 100:0-30:70) to obtain intermediate 30(2g, yield: 53.8%, 1.98mmol) as a pale yellow solid. MS (ESI) M/z (M + H)+1009.5。
S14. preparation of intermediate 31
Intermediate 30(2g, 1.98mmol) was dissolved in EtOH (20mL), diethylamine (5mL) was added and the reaction was stirred at room temperature for an additional 12 hours, and after monitoring the completion of the reaction by a mass spectrometer (i.e. LC-MS), the reaction was concentrated and taken up twice with EtOH. The residue was dissolved in water (20mL), washed with EA to remove impurities and lyophilized to give intermediate 31(1.5g, yield: 96.2%, 1.90mmol) as a pale yellow solid. MS (ESI) M/z (M + H) + 787.4.
S15. preparation of intermediate 33
Intermediate 31(1.5g,1.9mmol), methyl 4-oxobutyrate, compound 20(2g,17.2mmol) and STAB (4g,9.5mmol) were stirred in DCM (100mL) and methanol (10mL) overnight at room temperature, and the reaction was spun dry after completion of the reaction was monitored by a liquid-mass spectrometer (i.e., LC-MS). The residue was separated and purified by a reverse phase column (ammonia water, water: acetonitrile 100:0-70:30) to obtain intermediate 33(1g, yield: 58.9%, 1.12mmol) 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).
S16. preparation of AMC-6C
At room temperature, intermediate 33(1g, 1.12mmol) and LiOH (90mg,2.8mmol) were dissolved in water (10mL) and THF (20mL) and stirred for reaction for 6h, and after completion of the reaction was monitored by a liquid-mass spectrometer (i.e., LC-MS), the reaction solution was subjected to preparative separation and purification by means of a high performance reverse phase column to give a pale yellow solid, i.e., AMC-6C (400mg, yield: 40.9%, 0.458 mmol). MS (ESI) M/z (M + H) + 873.7. 1H NMR (400MHz, DMSO) δ 10.62(s,1H),8.81(t, J ═ 5.8Hz,1H),8.59(d, J ═ 2.0 Hz,1H),8.11(d, J ═ 8.8Hz,2H),8.04(s,1H),7.93(dt, J ═ 9.1,5.6Hz,2H),7.60(d, J ═ 8.7Hz,2H),7.34(s,1H),5.76(s,2H),5.03(d, J ═ 2.2Hz,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), 3.57 (s,2H), 7.9.3, 3.3H), 7.9 (dd, 1H), 9.3.3H, 9, 1H),7.9, 1H, 9 (dd, 1H), 7.3H, 9H, 1H).
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 immunogen and coatingen. The preparation of the immunogen is different from that of the coating antigen in the types of carriers, wherein the carriers adopted by the immunogen are bovine Lactoferrin (LF) and Keyhole Limpet Hemocyanin (KLH); the coating antigen adopts carrier protein as Ovalbumin (OVA). 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 recorded as solution A. 10mg of carrier protein was weighed and dissolved in 4mL of PBS buffer solution (0.01M, pH 7.4), and the solution was dissolved with stirring to prepare solution B. And (3) under magnetic stirring, sucking the solution A, dropwise adding the solution A into the solution B, and reacting for 12 hours under magnetic stirring at 4 ℃. The reaction mixture was dialyzed against PBS at 4 ℃ for 3 days, and the dialyzate was changed 2 times a day. The immunogen can be obtained and frozen in a refrigerator at the temperature of-20 ℃ for later use. The hapten prepared in example 1 and the artificial antigen prepared in example 2 are respectively subjected to ultraviolet wavelength scanning (150-400 nm) for identification, and the results are shown in figure 1, compared with the carrier protein and the hapten, the characteristic absorption peak and the peak shape are changed after coupling, and the results indicate that the antigen coupling is successful.
EXAMPLE 3 preparation of Multi-Cluster tandem Linear antigen-specific polyclonal antibody
Animal immunization: healthy 6-week-old Balb/c female mice were used as experimental animals, and the two immunogens prepared in example 2 were mixed and emulsified with equal amounts of adjuvant (complete Freund's adjuvant for the first time, followed by incomplete Freund's adjuvant) and injected subcutaneously into the neck, back and abdominal cavity of mice at an immunization dose of 0.5mL (containing 0.5mg of immunogen) each time. Emulsifying 0.5mL of complete Freund's adjuvant and antigen for immunization for the first immunization, emulsifying 0.5mL of incomplete Freund's adjuvant and antigen for 4 weeks for boosting immunization, then immunizing once every 2 weeks, taking a small amount of blood from tail vein during the immunization for antibody quality identification, after the antibody is stable, collecting serum of a mouse with the best performance, carrying out warm bath at 37 ℃ for 30min, centrifuging at room temperature for 20min, taking supernatant, separately filling into a centrifuge tube, and storing at-20 ℃ for use.
Evaluation of antiserum effects: the multi-cluster tandem linear envelope antigen prepared in example 2 was used, the collected mouse serum was used as a detection antibody, and the antiserum titer and inhibition ratio of the mouse serum were measured by an indirect competitive ELISA method, and the titer and inhibition ratio of each antiserum were comprehensively considered and evaluated. The specific operation steps are as follows:
1) wrapping a plate: the multi-cluster tandem linear coating antigen is diluted to 1000ng/mL by 0.05M carbonate buffer (pH 9.6), coated overnight at 4 ℃ according to 100 mu L/hole; discarding the coating solution, washing with PBST for 2 times, adding 120 μ L of blocking solution (5% skimmed milk) into each well, and blocking at 37 deg.C for 3 hr; removing the sealing liquid, drying at 37 ℃ for 60min, and packaging with a sealing bag at 4 ℃ for later use to obtain the wrapped ELISA plate.
2) Serum titer and inhibition detection: the titer of the ELISA plate coated in the step 1) is listed as follows: 50 μ L PBS and 50 μ L serum diluted by gradient fold were added to each well; inhibition column: each well was filled with 50. mu.L of diluted 1000ng/mL drug (multiple clusters of linear haptens in tandem) and 50. mu.L of serum diluted in multiples of a gradient, and 2 replicates were made. Incubating for 40min at 37 ℃, washing for five times by PBST, patting off the liquid in the hole, adding enzyme-labeled II diluted by 1:5000Anti (goat anti-mouse IgG-HRP), incubation for 30min at 37 ℃, washing with PBST five times, patting off liquid in the wells, adding 100 μ L of TMB substrate solution, and developing for 10min at 37 ℃ in the dark; add 50. mu.L of stop solution (10% H)2SO4) Terminating the reaction; the absorbance at 450nm was read with a microplate reader.
3) Results of the experiment
The antiserum titer and inhibition rate of the mouse serum are determined based on an indirect competition ELISA method, and the result is shown in Table 1, wherein the titer is between 8 and 32K, and the inhibition rate of the antiserum titer to the multi-cluster tandem linear hapten is 63 to 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 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, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A hapten for simultaneously detecting amantadine, olaquindox and chloramphenicol is characterized in that the structural general formula is shown as formula I:
wherein R is1And R2Are respectively selected from alkyl with 2-5 carbon atoms.
2. The hapten of claim 1, wherein R is1Selected from propyl or pentyl, R2Selected from ethyl or butyl.
3. The method for producing the hapten according to claim 1, comprising the steps of:
s1, preparing olaquindox metabolite, amantadine and chloramphenicol intermediates respectively: taking methyl acetoacetate as a raw material, and performing NBS bromination, 4-nitro o-phenylenediamine cyclization, nitro reduction, amino protecting group addition and ester bond hydrolysis to obtain a olaquindox metabolite intermediate shown in a formula II; taking adamantane as a raw material, carrying out nitration, nitro reduction, amino acylation and hydrolysis, connecting an amino acid arm protected by tert-butyloxycarbonyl on 1 amino group, then connecting fluorenylmethoxycarbonyl acyl on the other 1 amino group, and finally removing a Boc protecting group to obtain an adamantanamine intermediate shown in a formula III; hydrolyzing and removing dichloroacetyl of chloramphenicol to obtain chloramphenicol intermediate chloramphenicol amine shown in formula IV;
wherein R is1And R2Respectively selecting alkyl with 2-5 carbon atoms;
s2, synthesis of hapten: 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 on molecules and carboxyl groups, removing DMB protecting groups of aromatic ring amino groups of the olaquindox metabolite intermediate, coupling through acylation condensation reaction to obtain an intermediate V, subjecting the intermediate V to acylation condensation through 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, reducing and aminating with 4-oxobutyric acid methyl ester respectively to obtain an intermediate VII, and finally hydrolyzing ester groups to obtain the hapten shown in the formula I.
4. Use of a hapten according to claim 1 or 2 for the preparation of an artificial antigen.
5. An artificial antigen obtained by coupling the hapten of claim 1 with a carrier protein, and having the structure shown in formula VIII:
wherein R is1And R2Are respectively selected from alkyl 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 producing the artificial antigen according to claim 5 or 6, which is characterized by coupling a carrier protein to the carboxyl group of the hapten according to claim 1.
8. The method of claim 7, wherein the coupling is by an active ester method.
9. Use of the artificial antigen according to claim 5 or 6 for the preparation of mono/polyclonal antibodies, single chain antibodies or nanobodies.
10. A polyclonal antibody prepared by immunizing a mouse with the artificial antigen of claim 5 or 6.
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