CN111848437A

CN111848437A - Baclofen artificial hapten, artificial antigen, preparation method and application thereof

Info

Publication number: CN111848437A
Application number: CN202010612001.7A
Authority: CN
Inventors: 王镇; 邵越水
Original assignee: Hangzhou Tongzhou Biotechnology Co ltd
Current assignee: Hangzhou Tongzhou Biotechnology Co ltd
Priority date: 2020-06-30
Filing date: 2020-06-30
Publication date: 2020-10-30
Anticipated expiration: 2040-06-30
Also published as: CN111848437B

Abstract

The invention discloses a baclofen artificial hapten, an artificial antigen, a preparation method and application thereof. The molecular structural formula of the baclofen artificial hapten is shown as a formula (I), and the molecular structural formula of the baclofen artificial antigen is shown as a formula (II). The baclofen artificial hapten of the invention furthest reserves the characteristic structure of baclofen, has an active group which can be coupled with carrier protein and can be used as an antigenic determinant; the baclofen artificial antigen further prepared can be immunized to obtain an anti-baclofen antibody with high affinity, high sensitivity and strong specificity, the titer of immune serum obtained by immunizing a New Zealand white rabbit is as high as 1: 80000, and the baclofen artificial antigen can be used for carrying out rapid and accurate immunoassay and immunoassay on baclofen.

Description

Baclofen artificial hapten, artificial antigen, preparation method and application thereof

Technical Field

The invention belongs to the technical field of biochemical engineering, and particularly relates to a baclofen artificial hapten, an artificial antigen, and preparation methods and applications thereof.

Background

Baclofen is a skeletal muscle relaxant and is used for improving spasm symptoms of muscle tension increase caused by pyramidal tract injury, spastic hemiplegia and paraplegia caused by different reasons, such as multiple sclerosis, cerebrovascular disease, spinal cord injury and myelitis sequelae, cerebral palsy of children, tetanus, intractable hiccup and the like.

The medicine is absorbed quickly and completely by the gastrointestinal tract. After the oral administration for 1-3 h, the plasma concentration reaches the peak, but the individual difference is large. 70-80% of the drug is excreted as it is from the stool and about 15% is metabolized by the liver.

Sudden withdrawal from prolonged use can cause: withdrawal syndrome, dizziness, headache, debilitation, hallucination, epilepsy, diarrhea, constipation, skin rash, anorexia, hypotension, nausea, emesis, euphoria, melancholy, insomnia, tinnitus, numbness, conversation ambiguity, liver function damage, tremor, etc.

At present, the detection of baclofen mainly depends on High Performance Liquid Chromatography (HPLC), Gas Chromatography (GC), Thin Layer Chromatography (TLC), Mass Spectrometry (MS) and the like, but the existing detection methods are expensive in instruments, time-consuming in detection, and operation is required by professional technicians, and the requirements of modern detection on rapidness and accuracy cannot be met. Therefore, it is necessary to establish a rapid, sensitive and accurate detection technique.

The immunoassay method, which is an assay method for detecting various substances (drugs, hormones, proteins, microorganisms, etc.) using an antigen-antibody specific binding reaction, can make up all of the above disadvantages, and the key to establishing an immunoassay method for small-molecule compounds is the ability to produce antibodies having high affinity and high specificity for small-molecule compounds. However, since most small molecule compounds (molecular weight less than 1000), including baclofen, are not immunogenic, i.e., lack T cell epitopes and cannot directly induce the production of specific antibodies in animal bodies, small molecule substances are called haptens. Through appropriate chemical modification, a connecting arm is connected to a certain position of a hapten molecular structure, and then the connecting arm is combined with a macromolecular carrier to synthesize a hapten-carrier conjugate (i.e. an artificial antigen), wherein the artificial antigen can indirectly induce the proliferation and differentiation of B cells by means of T cell epitopes so as to generate specific antibodies.

Related reports of baclofen artificial hapten and artificial antigen do not exist in the prior art.

Disclosure of Invention

The invention aims to overcome the defects and shortcomings in the prior art and provide a preparation method of a baclofen-bovine serum albumin artificial antigen.

The invention firstly provides a baclofen artificial hapten, which furthest reserves the characteristic structure of baclofen, has an active group which can be coupled with carrier protein and can be used as an antigenic determinant.

A baclofen artificial hapten, the molecular structural formula of which is shown as (I):

the invention discloses a preparation method of baclofen artificial hapten, which comprises the following steps:

(1) mixing the baclofen and the trifluoroacetic anhydride according to the mol ratio of 1: 1.1, adding benzene dried by a 3A molecular sieve, stirring and reacting for 1 hour at room temperature, then transferring the mixture into an oil bath at 80 ℃, continuously stirring and refluxing for 3 hours, cooling to room temperature after the reaction is finished, and evaporating the solvent to dryness under reduced pressure to obtain a light yellow oily substance A;

(2) Mixing the light yellow oily matter A, N-hydroxysuccinimide and 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride according to the mol ratio of 1: 1.35-1.5: 2.45-3.0, adding dichloromethane for dissolving, and stirring and reacting for 18 hours at room temperature; after the reaction is finished, transferring the reaction solution into a separating funnel, washing an organic phase by respectively using 0.1N hydrochloric acid, purified water, a saturated sodium bicarbonate solution and a saturated saline solution, drying, filtering and evaporating the organic phase under reduced pressure to obtain an off-white solid B;

(3) dissolving p-aminomethyl benzoic acid in a mixed solvent of tetrahydrofuran and purified water in a volume ratio of 2: 1, uniformly stirring at room temperature, and then adding an off-white solid B tetrahydrofuran solution, wherein the reaction solution is turbid; then adding 1N sodium hydroxide aqueous solution until the reaction solution is just clear, and stirring and reacting for 3 hours at room temperature; and (3) ending the reaction, evaporating the solvent to dryness under reduced pressure, adding double distilled water, adjusting the pH value to 6 by using 6N hydrochloric acid, extracting the mixture for three times by using ethyl acetate, collecting an organic phase, drying, filtering, evaporating to dryness under reduced pressure, and separating by TLC (thin layer chromatography) to obtain a yellow oily substance I, namely the baclofen artificial hapten as claimed in claim 1.

By the method, the connecting arm is introduced to the carboxyl of baclofen, and the characteristic structure of baclofen can be retained to a greater extent by introducing the connecting arm to the modification site, and the active site capable of being coupled with carrier protein is provided.

Compared with the method adopting general saturated chain hydrocarbon as the connecting arm, the connecting arm adopted by the invention contains a benzene ring structure, so that the specificity of the artificial hapten is enhanced, the conformation is kept stable in a solution, and the immunological characteristic of the formed artificial antigen (II) is improved.

The invention also provides a baclofen artificial antigen, the molecular structure of which is shown as (II):

in the formula (II), BSA is bovine serum albumin.

The invention also provides a preparation method of the baclofen artificial antigen, wherein the baclofen artificial antigen is obtained by combining the baclofen artificial hapten with bovine serum albumin through an active ester method.

Specifically, the method for preparing the baclofen artificial antigen by adopting the active ester method comprises the following steps:

(a) dissolving baclofen artificial hapten I, N-hydroxysuccinimide and dicyclohexylcarbodiimide in N, N-dimethylformamide according to the molar ratio of 1: 1.35-1.5, stirring at room temperature for reacting for 18 hours, and centrifuging to obtain a supernatant after the reaction is finished;

(b) and (3) dropwise adding the supernatant into a bovine serum albumin solution, standing the mixed solution at 4 ℃ overnight, dialyzing by a 0.5% sodium carbonate aqueous solution with the pH value of 12.00 and a Phosphate Buffer Solution (PBS), and centrifuging to obtain the supernatant, thereby obtaining the baclofen artificial antigen.

The bovine serum albumin solution of the present invention is prepared by dissolving bovine serum albumin in a PBS (pH 7.2-7.4) buffer solution having a phosphate ion concentration of 0.01 mol/L.

In the step (b), the concentration of the bovine serum albumin solution is 5mg/ml, and the volume ratio of the supernatant to the bovine serum albumin solution is 1: 10.

The Bovine Serum Albumin (BSA) selected by the invention is taken as a macromolecular carrier, and compared with other carrier proteins, the bovine serum albumin has the following advantages: the BSA has 583 amino acid residues, is easy to couple with baclofen hapten, can prepare baclofen artificial antigens with different coupling ratios, and has higher immunogenicity; secondly, the BSA is economical and practical and has low cost; and the BSA has stable chemical properties, good solubility and stability in acidic and weakly alkaline environments, and is suitable for long-term storage.

The invention also provides application of the baclofen artificial antigen in preparing an anti-baclofen antibody.

The invention also provides an anti-baclofen antibody, which is globulin obtained by animal immunization of baclofen artificial antigen and capable of generating specific immunoreaction with baclofen.

The invention also provides application of the anti-baclofen antibody in baclofen detection.

Experiments show that the titer of immune serum obtained by immunizing new zealand white rabbits with the baclofen artificial antigen is 1: 80000. The baclofen artificial antigen can be used for immunizing to obtain the baclofen-resistant antibody with high affinity, high sensitivity and strong specificity, and the baclofen-resistant antibody can be used for immune detection and analysis of baclofen.

Compared with the prior art, the invention has the beneficial effects that:

the baclofen artificial hapten of the invention furthest reserves the characteristic structure of baclofen, has an active group which can be coupled with carrier protein and can be used as an antigenic determinant; the baclofen artificial antigen further prepared can be immunized to obtain an anti-baclofen antibody with high affinity, high sensitivity and strong specificity, the titer of immune serum obtained by immunizing a New Zealand white rabbit is as high as 1: 80000, and the baclofen artificial antigen can be used for carrying out rapid and accurate immunoassay and immunoassay on baclofen.

Drawings

FIG. 1 is a flow chart of the preparation of baclofen artificial antigen of the present invention;

wherein THF represents tetrahydrofuran, EDCI represents 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, MeOH represents methanol, DCM represents dichloromethane, DCC represents dicyclohexylcarbodiimide, DMF represents N, N-dimethylformamide, Et ₃N represents triethylamine, BSA represents bovine serum albumin, BGG represents bovine gamma globulin, the same as the following;

FIG. 2 is a liquid chromatogram of the baclofen artificial hapten of the invention;

wherein mAU represents milliabsorbance units, min represents minutes;

FIG. 3 is a mass spectrum of baclofen hapten of the present invention;

wherein Relative Absundance represents Relative Abundance; m/z represents a charge-to-mass ratio;

FIG. 4 is an ultraviolet scan of bovine serum albumin, baclofen artificial hapten and baclofen artificial antigen;

wherein A represents ultraviolet-visible absorbance, and λ (nm) represents wavelength (nm);

FIG. 5 is a flow chart of the preparation of a baclofen artificial antigen of comparative example 1;

FIG. 6 is a flow chart of the preparation of a comparative example 2 baclofen artificial antigen;

FIG. 7 is a flow chart of the preparation of a comparative example 3 baclofen artificial antigen;

FIG. 8 is a flow chart of the preparation of a comparative example 4 baclofen artificial antigen;

FIG. 9 is a flow chart of the preparation of a comparative example 5 baclofen artificial antigen;

FIG. 10 is a flow chart of the preparation of a comparative example 6 baclofen artificial antigen;

FIG. 11 is a flow chart of the preparation of the artificial antigen of baclofen in comparative example 7.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.

Example 1

The preparation method of the baclofen artificial antigen (the reaction process is shown in figure 1) comprises the following steps:

(1) Preparing artificial hapten:

weighing 200mg (0.94mmol) of baclofen into a 50ml single-neck round-bottom single-neck flask, adding 10ml of benzene dried by a 3A molecular sieve, installing a condenser tube and a drying tube, adding a stirrer, stirring for 5 minutes in an ice-water bath at 0 ℃, then adding 143 mu L (1.03mmol) of trifluoroacetic anhydride, removing the ice-water bath, stirring for reaction for 1 hour at room temperature, and then transferring into an oil bath at 80 ℃ to continue stirring and refluxing for 3 hours; after the reaction is finished, cooling to room temperature, taking out the rotor, and evaporating the solvent to dryness under reduced pressure to obtain a light yellow oily substance A262mg (0.85 mmol);

the light yellow oil A was subjected to TLC detection, and the chromatographic solution was ethyl acetate: petroleum ether is 10: 3(v/v), product Rf is 0.9;

② the light yellow oily matter A262mg (0.85mmol), N-hydroxysuccinimide 132mg (1.15mmol) and 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride 399mg (2.08mmol) are put into a 50ml single-neck round-bottom flask, added with 20ml dichloromethane for dissolution, and then added with a stirrer for stirring reaction at room temperature for 18 hours; after the reaction, the reaction mixture was transferred to a 125ml separatory funnel, the organic phase was washed with 20ml of 0.1N hydrochloric acid, 20ml of purified water, 20ml of saturated sodium bicarbonate solution and 20ml of saturated saline solution, and the organic phase was dried over anhydrous magnesium sulfate, filtered, and evaporated to dryness under reduced pressure to obtain an off-white solid B312mg (0.77 mmol);

TLC detection was performed on the off-white solid B, and the chromatography liquid was ethyl acetate: petroleum ether is 10: 3(v/v), product Rf ═ 0.3;

③ 174mg (1.15mmol) of p-aminomethyl benzoic acid is weighed, suspended in 16ml of tetrahydrofuran and 8ml of purified water, and a stirrer is added to stir uniformly at room temperature, and the solution is marked as solution A. The off-white solid from above, B312mg (0.77mmol), was dissolved in 16ml of tetrahydrofuran and designated as liquid B. Slowly adding the solution B into the solution A, adding 1.92ml of 1N sodium hydroxide aqueous solution when the reaction solution is turbid, clarifying the reaction solution, and stirring at room temperature for reaction for 3 hours; after the reaction, the solvent was evaporated to dryness under reduced pressure, 16ml of double distilled water was added, pH was adjusted to 6 with 6N hydrochloric acid, a large amount of white precipitate was generated, the reaction solution was transferred to a 125ml separatory funnel, extracted three times with 20ml of ethyl acetate, the organic phase was collected, dried over anhydrous magnesium sulfate, filtered, evaporated to dryness under reduced pressure to obtain a crude product, and the crude product was subjected to TLC thin layer chromatography to obtain 168mg (0.38mmol) of a yellow oily substance i (anhydrous ethanol was used as the solvent and the elution machine, and the developing agent was dichloromethane: 95% ethanol: 1, 4-dioxane: concentrated ammonia water: 10: 8: 1: 1, and Rf: 0.4 was collected), which was the baclofen artificial hapten according to claim 1.

The yellow oil I was subjected to TLC detection, and the chromatography liquid was dichloromethane: 95% ethanol: 1, 4-dioxane: ammonia water 10: 8: 1: 1(v/v), product Rf ═ 0.4;

the liquid chromatogram of baclofen artificial hapten is shown in figure 2 (ultraviolet detector, wavelength 215 nm).

From fig. 2, it can be seen that the purity of the baclofen artificial hapten obtained by purification reaches more than 99.9%.

The mass spectrum of baclofen hapten is shown in figure 3. It can be seen from fig. 3 that the mass-to-charge ratio (m/z) of the molecular ion peak of the baclofen artificial hapten obtained in this example is 442, which is consistent with the theoretical molecular weight thereof, and the mass-to-charge ratio (m/z) of the ion peak of the main fragment thereof is consistent with the theoretical value of the main fragment thereof, and it can be determined that the final compound obtained in the third step is the baclofen artificial hapten designed by the present invention.

(2) Preparation of baclofen artificial antigen:

fourthly, putting 168mg (0.38mmol) of baclofen artificial hapten I into a 50ml round-bottom flask, adding a stirrer, adding 8.4ml of N, N-Dimethylformamide (DMF) for dissolving, then adding 59mg (0.51mmol) of N-hydroxysuccinimide and 105mg (0.51mmol) of dicyclohexylcarbodiimide, stirring at 25 ℃ for reacting for 18 hours, centrifuging after the reaction is finished, and taking a supernatant for later use.

Weighing 14.500g (0.0405mol) of disodium hydrogen phosphate dodecahydrate, 43.875g (0.75mol) of sodium chloride and 1.495g (0.00958mol) of sodium dihydrogen phosphate dihydrate, dissolving with double distilled water and fixing the volume to 5.0L to obtain PBS buffer solution with phosphate ion concentration of 0.01mol/L and sodium ion concentration of 0.17mol/L and pH of 7.4.

Sixthly, 25g of Na is weighed₂CO₃The solid powder was dissolved in purified water to a volume of 5L, and the pH was adjusted to 12.00 ± 0.01 with a 2N NaOH aqueous solution to prepare an alkaline dialysate.

Seventhly, 0.42g of bovine serum albumin is weighed and dissolved in 84ml of PBS buffer solution in the fifth step to obtain bovine serum albumin solution with the concentration of 5 mg/ml.

And eighthly, slowly dripping the supernatant obtained in the step (iv) into the bovine serum albumin solution with rapid stirring, wherein the volume ratio of the supernatant to the bovine serum albumin solution is 1: 10, and standing and storing the obtained mixed solution at 4 ℃ overnight to obtain the artificial antigen mixed solution.

Ninthly, transferring the mixed liquid of the artificial antigens in the last step into a dialysis bag, dialyzing the mixed liquid for two days by using the alkaline dialysate obtained in the step (c), dialyzing the mixed liquid for 7 times in the PBS buffer solution obtained in the fifth step by transferring the dialysis bag every day, wherein the time interval of each time is not less than two hours, and after the dialysis is finished, centrifuging and taking the supernatant to obtain the artificial antigens: baclofen-bovine serum albumin conjugate (formula II).

The ultraviolet scan before and after preparation of baclofen artificial antigen is shown in figure 4. Wherein, the curve a is an ultraviolet scanning image of the baclofen artificial hapten, the curve b is an ultraviolet scanning image of bovine serum albumin, and the curve c is an ultraviolet scanning image of the baclofen artificial antigen. The maximum absorption wavelength of the baclofen artificial hapten is 245nm, the maximum absorption wavelength of the baclofen artificial antigen is 253nm, and compared with the baclofen hapten and bovine serum albumin, the maximum absorption wavelength and the absorbance of the baclofen artificial antigen are obviously changed, which indicates that the baclofen hapten and the bovine serum albumin are successfully coupled.

Comparative example 1

The preparation method of the baclofen artificial antigen (the reaction process is shown in figure 5) comprises the following steps:

(1) preparing artificial hapten:

weighing 200mg (0.94mmol) of baclofen into a 50ml single-neck round-bottom flask, adding 6.4ml of anhydrous methanol for dissolving, adding 300 mu L of 98% concentrated sulfuric acid, putting into a stirrer, installing a condenser tube and a drying tube, and placing into an oil bath at 68 ℃ for stirring reflux reaction for 18 hours; after the reaction was completed, the reaction was cooled to room temperature, 928mg of a solid sodium bicarbonate was added to the reaction flask until no more air bubbles were blown out, and the residue obtained by filtration and drying was extracted with 15ml of tetrahydrofuran to obtain a pale yellow oily substance C207mg (0.91 mmol);

The light yellow oil C was checked by TLC, and the chromatography liquid was ethyl acetate: petroleum ether is 10: 3(v/v), product Rf ═ 0.8;

② adding 15ml of anhydrous pyridine into the light yellow oily matter C207mg (0.91mmol) obtained in the previous step to dissolve in a 50ml single-neck round-bottom flask, adding 136mg of succinic anhydride, and stirring and refluxing in an oil bath at 100 ℃ for reaction for 20 hours; after the reaction is finished, cooling to room temperature, decompressing and evaporating the solvent, adding 20ml of purified water and 20ml of dichloromethane for extraction, extracting the water phase twice with 20ml of dichloromethane, collecting the organic phase, washing with 30ml of purified water and 30ml of saturated saline solution respectively, taking the organic phase, drying with anhydrous magnesium sulfate, filtering, transferring to dryness to obtain a crude product, separating the crude product by TLC thin-layer chromatography to obtain 212mg (0.65mmol) of yellow oily matter III, (the solvent and the washing and dehydrating machine are anhydrous ethanol, the developing agent is dichloromethane, 95% ethanol, 1, 4-dioxane, concentrated ammonia water, 10: 8: 1: 1, and the point of collecting Rf, 0.3) is baclofen artificial hapten (shown in the formula III);

TLC detection was performed on the yellow oil III, and the chromatography was dichloromethane: 95% ethanol: 1, 4-dioxane: ammonia water 10: 8: 1: 1(v/v), product Rf ═ 0.3;

preparation of baclofen artificial antigen:

③ putting the baclofen artificial hapten III into a 50ml single-neck round-bottom flask, adding a stirrer, adding 10.6ml of N, N-Dimethylformamide (DMF) for dissolving, then adding 101mg (0.88mmol) of N-hydroxysuccinimide and 181mg (0.88mmol) of dicyclohexylcarbodiimide, stirring at 25 ℃ for reacting for 18 hours, centrifuging after the reaction is finished, and taking a supernatant for later use.

14.500g (0.0405mol) of disodium hydrogen phosphate dodecahydrate, 43.875g (0.75mol) of sodium chloride and 1.495g (0.00958mol) of sodium dihydrogen phosphate dihydrate are weighed and dissolved by double distilled water to be constant volume to 5.0L, and PBS buffer solution with 0.01mol/L phosphate radical ion concentration and 0.17mol/L sodium ion concentration is obtained, and the pH value is 7.4.

Fifthly, weighing 25gNa₂CO₃The solid powder was dissolved in purified water to a volume of 5L, and the pH was adjusted to 12.00 ± 0.01 with a 2N NaOH aqueous solution to prepare an alkaline dialysate.

Sixthly, 0.53g of bovine serum albumin is weighed and dissolved in 106ml of PBS buffer solution obtained in the step (four), and the bovine serum albumin solution with the concentration of 5mg/ml is obtained.

Seventhly, slowly dripping the supernatant obtained in the step III into the bovine serum albumin solution under the condition of rapid stirring, wherein the volume ratio of the supernatant to the bovine serum albumin solution is 1: 10, and standing and storing the obtained mixed solution at 4 ℃ overnight to obtain the artificial antigen mixed solution.

Transferring the artificial antigen mixed solution into a dialysis bag, dialyzing with the alkaline dialysate of the fifth step for two days, each time every day, then transferring the dialysis bag into the PBS buffer solution obtained in the fourth step for 7 times, wherein the time interval of every two times is not less than two hours, and after the dialysis is finished, centrifuging and taking supernate to obtain the artificial antigen IV: baclofen-bovine serum albumin conjugate.

Comparative example 2

The preparation method of the baclofen artificial antigen (the reaction process is shown in figure 6) comprises the following steps:

(1) preparation of baclofen artificial hapten:

first to second are the same as comparative example 1, respectively.

(2) Preparation of baclofen artificial antigen:

coupling the baclofen artificial hapten III by using bovine albumin as a carrier, wherein the coupling step is the same as that of the comparative example 1, and obtaining a baclofen artificial antigen V: baclofen-bovine gamma globulin conjugates.

Comparative example 3

The preparation method of the baclofen artificial antigen (the reaction process is shown in figure 7) comprises the following steps:

(1) preparation of baclofen artificial hapten:

first to second are the same as comparative example 1, respectively.

(2) Preparation of baclofen artificial antigen:

③ 212mg (0.65mmol) of baclofen artificial hapten III is weighed and placed in a 50ml round-bottom flask, 10.6ml of N, N-Dimethylformamide (DMF) is added, 89 mu L (0.65mmol) of triethylamine and 85 mu L (0.65mmol) of isobutyl chloroformate are added, the mixture is stirred and reacted for 18 hours at room temperature, after the reaction is finished, the mixture is centrifuged, and the supernatant is taken for standby.

(iv) -viii are the same as in comparative example 1, respectively; obtaining baclofen artificial antigen VI, baclofen-bovine serum albumin conjugate.

Comparative example 4

The preparation method of the baclofen artificial antigen (the reaction process is shown in figure 8) comprises the following steps:

(1) preparation of baclofen artificial hapten:

first to second are the same as comparative example 1, respectively.

(2) Preparation of baclofen artificial antigen:

③ same as comparative example 3.

And (iv) -replacing all bovine serum albumin with bovine gamma globulin similarly to the comparative example 1 to obtain the baclofen artificial antigen VII: baclofen-bovine gamma globulin conjugates.

Comparative example 5

The preparation method of the baclofen artificial antigen (the reaction process is shown in figure 9) comprises the following steps:

(1) preparation of baclofen artificial hapten:

(iii) and (iii) are the same as in example 1, respectively.

(2) Preparation of baclofen artificial antigen:

and fourthly, coupling the baclofen artificial hapten I by using bovine albumin as a carrier, wherein the coupling step is the same as that of the example 1, and obtaining baclofen artificial antigen VIII: baclofen-bovine gamma globulin conjugates.

Comparative example 6

The preparation method of the baclofen artificial antigen (the reaction process is shown in figure 10) comprises the following steps:

(1) Preparation of baclofen artificial hapten:

(iii) and (iii) are the same as in example 1, respectively.

(2) Preparation of baclofen artificial antigen:

weighing 168mg (0.38mmol) of baclofen artificial hapten I, placing the baclofen artificial hapten I in a 50ml round-bottom flask, adding 10ml of N, N-Dimethylformamide (DMF), adding 52 mu L (0.38mmol) of triethylamine and 50 mu L (0.38mmol) of isobutyl chloroformate, stirring at room temperature for reacting for 18 hours, centrifuging after the reaction is finished, and taking a supernatant for later use.

Fifthly, the method is the same as the example 1 respectively to obtain baclofen artificial antigen IX: baclofen-bovine serum albumin conjugate.

Comparative example 7

The preparation method of the baclofen artificial antigen (the reaction process is shown in figure 11) comprises the following steps:

(1) preparation of baclofen artificial hapten:

(iii) and (iii) are the same as in example 1, respectively.

(2) Preparation of baclofen artificial antigen:

the fourth step is the same as the fourth step in comparative example 6;

ninthly, coupling the baclofen artificial hapten I by using bovine albumin as a carrier, wherein the coupling step is the same as that of the example 1 to obtain a baclofen artificial antigen X: baclofen-bovine gamma globulin conjugates.

Example 2 Performance assay of baclofen Artificial antigen

(1) Identification of baclofen artificial antigen:

Molar absorption coefficient: the maximum absorption wavelength of baclofen hapten is 245nm through an ultraviolet scanning chart, and the absorbance value is measured at 245nm by using PBS as a control sample, wherein each concentration is used as a parallel sample. The molar absorption coefficient (i.e., molar absorption coefficient) is calculated by the formula: absorbance/molar concentration.

Determination of conjugate protein concentration: 1ml each of bovine serum albumin solutions prepared with PBS buffer solution at concentrations of 0. mu.g/ml, 10. mu.g/ml, 20. mu.g/ml, 30. mu.g/ml, 40. mu.g/ml, 60. mu.g/ml, 80. mu.g/ml, 100. mu.g/ml and 120. mu.g/ml, 3ml Coomassie brilliant blue staining solution was added, immediately mixed, warmed in a water bath at 30 ℃ for 5 minutes, each concentration was sampled in parallel, absorbance was measured at 655nm, and a curve was drawn relating the protein concentration to the absorbance. Diluting the artificial antigen solution (prepared by PBS buffer solution) according to a certain proportion, measuring the light absorption value of the artificial antigen at 655nm, and reading the corresponding protein concentration value of the artificial antigen solution from the curve.

And (3) coupling ratio determination: preparing 100 mu g/ml bovine serum albumin PBS solution, diluting the conjugate (namely baclofen artificial antigen) to 100 mu g/ml by PBS, taking the PBS as a blank control, and respectively measuring the absorbance values A2 and A1 at 253nm, wherein the coupling ratio gamma is as follows: γ ═ [ (A1-A2) | ]/(100×10^-3/65000)。

Wherein the molar absorption coefficient (L/mol) is 65000, the molecular weight of bovine serum albumin is 100 × 10^-3Bovine serum albumin concentration (g/L).

The bovine gamma globulin is used as carrier, and the coupling ratio is calculated as gamma [ (A1-A2)]/(100×10^-3150000), wherein 150000 is the molecular weight of bovine gamma globulin, 100 × 10^-3Is the bovine gamma globulin concentration (g/L).

TABLE 1 coupling ratio and molar absorption coefficient of each baclofen artificial antigen

As can be seen from Table 1, the structure of the artificial hapten, the method of activating the artificial hapten and the structure of the carrier protein all affect the coupling ratio of the artificial hapten to the carrier protein when they are crosslinked.

(2) Animal immunization

The prepared baclofen artificial antigens are used for immunizing New Zealand white rabbits, the titer of the obtained immune serum is detected by an ELISA method, and the detection result is shown in Table 2.

TABLE 2 results of potency assay of each immune serum

Numbering	Baclofen artificial antigen	Immune serum titer
			Example 1	Ⅱ	1∶80000
Comparative example 1	Ⅳ	1∶50000
			Comparative example 2	Ⅴ	1∶20000
Comparative example 3	Ⅵ	1∶30000
			Comparative example 4	Ⅶ	1∶7000
Comparative example 5	Ⅷ	1∶40000
			Comparative example 6	Ⅸ	1∶8000
Comparative example 7	Ⅹ	1∶7000

As can be seen from Table 2, compared to example 1, the immune sera obtained by immunizing animals with each of the ratios of baclofen artificial antigen had lower titer and could not be used in the immunoassay. The titer of immune serum obtained by using the baclofen artificial antigen II to carry out animal immunization reaches 1: 80000, and the immune serum can be completely used in immunoassay and can provide a more convenient, rapid and accurate way for baclofen detection.

Claims

1. The baclofen artificial hapten is characterized in that the molecular structural formula is shown as (I):

2. the method of preparing the baclofen artificial hapten as claimed in claim 1, comprising the steps of:

(3) dissolving p-aminomethyl benzoic acid in a mixed solvent of tetrahydrofuran and purified water in a volume ratio of 2: 1, uniformly stirring at room temperature, and then adding an off-white solid B tetrahydrofuran solution, wherein the reaction solution is turbid; then adding 1N sodium hydroxide aqueous solution, and stirring and reacting for 3 hours at room temperature; and (3) ending the reaction, evaporating the solvent to dryness under reduced pressure, adding double distilled water, adjusting the pH value to 6 by using 6N hydrochloric acid, extracting for three times by using ethyl acetate, collecting an organic phase, drying, filtering, evaporating to dryness under reduced pressure, and separating by TLC (thin layer chromatography) to obtain a yellow oily substance I, namely baclofen artificial hapten.

3. A baclofen artificial antigen is characterized in that the molecular structural formula is shown as (II):

in the formula (II), BSA is bovine serum albumin.

4. The baclofen artificial antigen of claim 3 wherein the baclofen artificial antigen is obtained by combining a baclofen artificial hapten with bovine serum albumin by an active ester method.

5. The baclofen artificial antigen of claim 4, comprising the steps of:

(a) dissolving baclofen artificial hapten I, N-hydroxysuccinimide and dicyclohexylcarbodiimide in N, N-Dimethylformamide (DMF) according to the molar ratio of 1: 1.35-1.5, stirring at room temperature for reacting for 18 hours, and centrifuging to obtain a supernatant after the reaction is finished;

(b) and (3) dropwise adding the supernatant into a bovine serum albumin solution, quickly and uniformly mixing, standing overnight at 4 ℃, dialyzing by a 0.5% sodium carbonate aqueous solution with the pH value of 12.00 and a Phosphate Buffer Solution (PBS), and centrifuging to obtain the supernatant, thereby obtaining the baclofen artificial antigen.

6. The baclofen artificial antigen of claim 5 wherein in step (b) the concentration of the bovine serum albumin solution is 5mg/ml and the volume ratio of the supernatant to the bovine serum albumin solution is 1: 10.

7. Use of the baclofen artificial antigen of claim 3 in the preparation of an anti-baclofen antibody.

8. An anti-baclofen antibody, which is a globulin obtained by immunizing an animal with the baclofen artificial antigen of claim 3 and specifically immunoreactive with baclofen.

9. Use of the anti-baclofen antibody of claim 8 for detecting baclofen.