CN112979457B - Bisphenol diglycidyl ether compound hapten and synthesis method thereof - Google Patents

Bisphenol diglycidyl ether compound hapten and synthesis method thereof Download PDF

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CN112979457B
CN112979457B CN202110134694.8A CN202110134694A CN112979457B CN 112979457 B CN112979457 B CN 112979457B CN 202110134694 A CN202110134694 A CN 202110134694A CN 112979457 B CN112979457 B CN 112979457B
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张灿
胡金诺
武潇潇
张祯
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Abstract

The invention belongs to the technical field of biochemical engineering, and relates to a bisphenol diglycidyl ether compound hapten and a synthetic method thereof; the hapten compound which has-COOH and contains a bisphenol diglycidyl ether compound structure to the greatest extent is prepared; furthermore, the conjugate of hapten and carrier protein is utilized, an antibody which can simultaneously recognize bisphenol A diglycidyl ether and derivatives thereof can be obtained by immunizing white rabbits in New Zealand, the preparation process is simple and economic, the titer of the antibody can reach as high as 51200, the antibody has higher affinity to the bisphenol A diglycidyl ether and the derivatives thereof, and a good basis is identified for establishing an immunoassay method based on the broad-spectrum polyclonal antibody; meanwhile, a new means is provided for the detection of the BADGE and the derivatives thereof.

Description

Bisphenol diglycidyl ether compound hapten and synthesis method thereof
Technical Field
The invention belongs to the technical field of biochemical engineering, and relates to a bisphenol diglycidyl ether compound hapten and a synthesis method thereof.
Background
Bisphenol a diglycidyl ether (BADGE) is an industrial synthesis based on epichlorohydrin and bisphenol a (bisphenol a, BPA). Are widely used as additives for epoxy resins and polycarbonate plastics. The most used in beverage cans and inner packaging materials for food products is to prevent the contents of the can from coming into direct contact with metal, thereby avoiding the occurrence of electrochemical corrosion leading to the deterioration of the food product. When the residual BADGE in the undercoating layer is mixed with a mixture containingWhen the grease or moisture contacts with the substance, the monomer is dissolved out, or hydrolysis/chlorination reaction occurs to generate various derivatives, such as: BADGE. H 2 O、BADGE·HCL、BADGE·H 2 O.HCL, and the like. BADGE and its derivatives, as an endocrine disruptor, even at low doses, can cause harm to the human environment. Easily cause the abnormality of the endocrine system, the immune system and the nervous system of the human body or the animal, and also seriously interfere the reproductive genetic function of the human body and the animal. Accordingly, the European Union EC1895/2005 specifies BADGE and its hydrolyzed derivatives (BADGE. H) in food packaging containers 2 O、BADGE·2H 2 O) should have a total migration of less than 9mg/kg or 9mg/6dm 2 Hydrochlorinated derivatives (BADGE & HCL, BADGE & 2HCL, BADGE & H 2 O.HCL) is less than 1mg/kg or 1mg/6dm 2 。
At present, methods for detecting multiple residues of BADGE and derivatives mainly comprise liquid chromatography, gas chromatography, combination of liquid chromatography and mass spectrometry, high performance liquid chromatography-fluorescence detection and the like, but due to complex pretreatment of an instrumental analysis method, long time consumption and expensive equipment, the requirement of rapid detection of multiple residues is difficult to meet. Compared with an instrument method, an enzyme-linked immunosorbent assay (ELISA) method is sensitive, is simple and convenient to operate, can be applied to large-scale detection of samples, and has developed into one of important methods for food pollution residues. The basic principle of immunological detection is established on the specific reaction of antigen and antibody, and mainly comprises the preparation of artificial antigen, the screening and purification of antibody, and the high-efficiency artificial antigen is the precondition of immunological analysis. The bisphenol glycidyl ether compound belongs to a small molecular compound, can not effectively activate the immune system of an organism to generate an antibody, and can generate the antibody aiming at a small molecular hapten only by being coupled with a carrier protein to form a complete antigen. However, the bisphenol glycidyl ether compound has no directly available active groups such as-COOH and-NH in the structure 2 or-OH, etc., therefore, to realize the coupling of the bisphenol glycidyl ether compound and the carrier protein, the design and synthesis of the hapten of the bisphenol glycidyl ether compound are firstly carried out. Based on the design of bisphenol glycidyl ether compound hapten and the establishment of immunoassay, the Kudzuvine Bright group adopts a BADGE structureSimilar diphenolic acid is taken as a starting point, BADGE hapten is synthesized, and a monoclonal antibody is prepared, but the antibody only has a good identification effect on BADGE, and cannot realize simultaneous identification of a plurality of bisphenol diglycidyl ether compounds. However, as the BADGE migrates into the food matrix and not only exists in the form of a monomer, but also generates various derivatives, an immune hapten molecule needs to be designed, an antibody capable of simultaneously recognizing the bisphenol diglycidyl ether compound is prepared, and favorable conditions are provided for establishing enzyme-linked immunoassay of the bisphenol diglycidyl ether compound.
Disclosure of Invention
The invention aims to provide a bisphenol diglycidyl ether compound hapten and a synthesis method thereof; the synthesized hapten can prepare a polyclonal antibody which can specifically recognize both BADGE and a derivative, and is a good basis for establishing an immunoassay method based on the broad-spectrum polyclonal antibody.
In order to achieve the object of the present invention, the present invention firstly provides a general hapten structure for detecting bisphenol diglycidyl ether compounds, which has a structure shown in formula 1:
Figure BDA0002926429430000021
two haptens are respectively prepared by introducing chlorine atoms and a group-OH, and the structures of the haptens are shown as formulas 2 and 3:
Figure BDA0002926429430000022
the invention also provides a preparation method of the bisphenol diglycidyl ether compound hapten, and when the hapten is a compound shown in a formula 2, a synthetic route is as follows:
Figure BDA0002926429430000031
when the bisphenol diglycidyl ether compound hapten is a compound shown as a formula 2, the preparation method specifically comprises the following steps:
(1) Synthesis of compound F01: dissolving bisphenol A in DMF (N, N-dimethylformamide), cooling to room temperature in a water bath under the protection of nitrogen, adding NaH (sodium hydride) after cooling, and stirring for the first time at room temperature; stirring, adding epoxy chloropropane, stirring for the second time at room temperature, stirring, adding water, stirring for the third time to obtain a mixed solution, adjusting the pH, extracting with ethyl acetate, combining organic phases, removing a solvent, collecting solids, and purifying by silica gel column chromatography to obtain a light brown oily substance, which is marked as a compound F01;
(2) Synthesis of compound F02: dissolving the compound F01 prepared in the step (1) in DMF, cooling in a water bath under the protection of nitrogen, adding NaH into the solution after cooling, and stirring for the first time at room temperature; stirring, adding tert-butyl 4-bromobutyrate, stirring for the second time at room temperature, stirring, adding water, stirring for the third time to obtain a mixed solution, extracting with ethyl acetate, mixing organic phases, removing the solvent, collecting solids, and purifying by silica gel column chromatography to obtain a brown oily substance, which is marked as a compound F02;
(3) Synthesis of compound F: dissolving the compound F02 prepared in the step (2) in tetrahydrofuran, then dropwise adding HCl, and stirring for the first time at room temperature; adding NaOH solution after stirring to obtain mixed solution; and stirring the mixed solution for the second time under a certain temperature condition, adjusting the pH value after stirring, standing for layering, extracting by using ethyl acetate, combining organic phases, removing the solvent, collecting the solid, and purifying by silica gel column chromatography to obtain a white solid, namely the bisphenol diglycidyl ether compound hapten which is marked as a compound F.
Preferably, the using ratio of the bisphenol A, the DMF, the NaH, the epichlorohydrin and the water in the step (1) is 7g:60ml:1.53g:3.4g:80ml.
Preferably, the time for the first stirring in the step (1) is 30-40min; the time for the second stirring is 15-16h; the third stirring time is 15-20min; the pH is specifically adjusted to 5-6 by 1N HCl.
Preferably, the compound F01, DMF, naH, tert-butyl 4-bromobutyrate and water are used in the step (2) in a ratio of 6.4g:60ml:1.13g:6.27g:80ml.
Preferably, the time for the first stirring in the step (2) is 30-40min; the time for the second stirring is 15-16h; the time for stirring for the third time is 15-20min.
Preferably, the amount ratio of the compound F02, tetrahydrofuran, hydrochloric acid and NaOH in the step (3) is 2.3g:30ml:0.50ml:10ml; the concentration of the hydrochloric acid is 12N; the concentration of NaOH was 1.6M.
Preferably, the time for the first stirring in the step (3) is 15-20min; the certain temperature condition is 40 ℃, and the time of the second stirring is 2 hours; the pH adjustment is specifically carried out with 2N HCl to pH =3 to 4.
When the hapten is a compound shown in formula 3, the synthetic route is as follows:
Figure BDA0002926429430000041
when the bisphenol diglycidyl ether compound hapten is a compound shown in a formula 3, the preparation method specifically comprises the following steps:
(1) Synthesis of compound C01: dissolving bisphenol A in DMF, cooling to room temperature in a water bath under the protection of nitrogen, adding NaH into the solution after cooling, and stirring for the first time at room temperature; adding ethyl 4-bromobutyrate after stirring, heating to a certain temperature for second stirring, cooling to room temperature after stirring, adding water, stirring for the third time to obtain a mixed solution, extracting with ethyl acetate, combining organic phases, removing the solvent, collecting solids, and purifying by silica gel column chromatography to obtain a light brown oily substance, which is marked as a compound C01;
(2) Synthesis of compound C02: dissolving the compound C01 prepared in the step (1) in DMF, performing nitrogen protection, cooling in a water bath, adding NaH into the solution after cooling, and performing first stirring at room temperature; adding epoxy chloropropane after stirring, heating to a certain temperature for second stirring, cooling to room temperature after stirring, adding water, stirring for the third time to obtain a mixed solution, extracting by using ethyl acetate, combining organic phases, removing a solvent, collecting solids, and purifying by silica gel column chromatography to obtain a colorless oily substance which is marked as a compound C02;
(3) Synthesis of compound C: dissolving the compound C02 prepared in the step (2) in ethanol, adding an aqueous solution of NaOH to obtain a mixed solution, refluxing and stirring, adjusting the pH after stirring, removing the ethanol in a rotating manner to obtain a water phase, extracting with ethyl acetate, combining organic phases, removing the solvent, collecting solids, and purifying by silica gel column chromatography to obtain a colorless oily substance, namely the bisphenol diglycidyl ether compound hapten which is marked as a compound C;
preferably, the ratio of the bisphenol A, DMF, naH, ethyl 4-bromobutyrate and water used in step (1) is 7.6g:60ml:2.28g:7.79g:80ml.
Preferably, the time for the first stirring in the step (1) is 30-40min; the heating is carried out to a certain temperature of 100 ℃, and the time for the second stirring is 6 hours; the time for stirring for the third time is 15-20min.
Preferably, the amount ratio of the compound C01, DMF, naH, epichlorohydrin and water in the step (2) is 3.9g:30ml:0.55g:3.16g:40ml.
Preferably, the time for the first stirring in the step (2) is 30-40min; heating to a certain temperature of 60 ℃, and stirring for the second time for 6 hours; the third stirring time is 15-20min.
Preferably, the amount ratio of the compound C02, ethanol and NaOH in the step (3) is 2.2g:30ml:15ml; the concentration of NaOH was 1.46M.
Preferably, the time for the first stirring in the step (3) is 15-20min; the reflux stirring time is 8 hours; the pH adjustment is specifically carried out with 2N HCl to pH =3 to 4.
Finally, the application of the hapten based on the bisphenol diglycidyl ether compound:
the invention provides a bisphenol diglycidyl ether compound artificial antigen which is obtained by coupling the bisphenol diglycidyl ether compound hapten and carrier protein; wherein, the carrier protein is preferably bovine serum albumin BSA, chicken ovalbumin OVA. Coupling a carrier protein to the carboxyl carbon of the hapten of claim 1 using an activated ester process.
The invention provides a specific antibody prepared from the BADGE compound artificial antigen, which is a polyclonal antibody or a monoclonal antibody, preferably a polyclonal antibody. The polyclonal antibody can be obtained by immunizing experimental animal (such as New Zealand white rabbit) with BADGE compound artificial antigen, collecting serum, and purifying.
The invention provides any one of the following applications of the BADGE compound hapten or the BADGE compound artificial antigen: (1) the application in preparing the anti-BADGE compound specific broad-spectrum antibody; (2) the application of the BADGE compound in food detection.
The invention has the beneficial effects that: the method discloses two novel BADGE compound haptens, artificial antigens and preparation methods thereof for the first time, and the BADGE compound artificial antigens are used for immunizing animals to obtain specific antibodies which can simultaneously recognize BADGE and derivatives thereof and have high sensitivity. The preparation process is simple and economic, the antibody titer can reach 51200 at most, the antibody has higher affinity to bisphenol A diglycidyl ether and derivatives thereof, and a new means is provided for establishing a rapid, simple, convenient, cheap, sensitive and specific BADGE and derivative detection method thereof.
Drawings
FIG. 1 is a NMR chart of hapten F of formula 2 prepared in example 1;
FIG. 2 is a NMR chart of hapten C prepared in example 1 and represented by formula 3;
FIG. 3 is a standard curve of DCC-C-BSA-2# antiserum against BADGE and its derivatives;
FIG. 4 is a standard curve of DCC-F-BSA-3# antiserum against BADGE and its derivatives.
Detailed Description
The invention is further illustrated by the following examples.
Example 1: preparation and characterization of haptens of BADGE compounds
1. Preparation of BADGE compound hapten
When the bisphenol diglycidyl ether compound hapten is a compound shown as a formula 2, the preparation method specifically comprises the following steps:
(1) Synthesis F01: 7.00g of bisphenol A (30.66mmol, 1.0 eq.) was dissolved in 60ml of DMF, protected with nitrogen, cooled in a water bath, to which was added 1.53g of NaH (60%) (38.33mmol, 1.25eq.) after which stirring was carried out at room temperature for 40min. 3.40g of epichlorohydrin (36.79mmol, 1.2eq.) was added to the system, and the system was stirred at room temperature for 16 hours after the addition. Adding 80ml of water into the system, stirring for 15min, adjusting the pH of the system to be 5-6 by using 1N HCl (aq.), extracting the mixture for 3 times by using ethyl acetate, wherein the volume of the mixture is 100ml, combining organic phases, removing the solvent, collecting solid residues, and purifying the solid residues by using a silica gel column chromatography (the particle size of the silica gel is 200-300 meshes, and the used eluent and the volume ratio of the eluent are petroleum ether/ethyl acetate = 5/1-1/1) to obtain light brown oily substances, 7.59g and the yield is 87.0%.
(2) Synthesis F02: 6.40g of F01 (22.51mmol, 1.0eq.) was dissolved in 60ml of DMF, and the solution was cooled in a water bath under nitrogen, and 1.13g of NaH (60%) (28.14mmol, 1.25eq.) was added thereto, followed by stirring at room temperature for 40min. 6.27g of tert-butyl 4-bromobutyrate (27.01mmol, 1.2 eq.) was added to the system, and the system was stirred at room temperature for 16h after the addition. Adding 80ml water into the system, stirring for 15min, extracting with ethyl acetate for 3 times, each time 100ml, combining organic phases, removing solvent, collecting solid residue, and purifying with silica gel column chromatography (silica gel particle size is 200-300 mesh, eluent and volume ratio are petroleum ether/ethyl acetate = 5/1-2/1) to obtain light brown oil, 2.36g, and yield 24.6%.
(3) Synthesis F: 2.30g of F02 (5.39mmol, 1.0eq.) was dissolved in 30ml of tetrahydrofuran, and 0.50ml of 12N HCl (aq), (6.00mmol, 1.1eq.) was slowly added dropwise thereto, and after addition, stirring was carried out at room temperature for 15min, and TLC showed disappearance of F02. Then, 1.6M NaOH (aq.) was added to the system in an amount of 10ml, and after completion of the addition, the system was stirred at 40 ℃ for 2h, and TLC showed disappearance of the intermediate, and the reaction was stopped. The system is adjusted to pH = 3-4 by 2NHCl (aq), the mixture is stood for layering, the water phase is extracted by ethyl acetate for 2 times, 50ml each time, the organic phase is combined, the solvent is removed, the solid residue is collected and purified by silica gel column chromatography (the particle size of the silica gel is 200-300 meshes, and the used eluent and the volume ratio thereof are dichloromethane/methanol = 15/1-10/1), 0.75g of white solid is obtained, and the yield is 34.2%.
When the bisphenol diglycidyl ether compound hapten is a compound shown in a formula 3, the preparation method specifically comprises the following steps:
(1) Synthesis C01: 7.6g of bisphenol A (33.29mmol, 1.0 eq.) were dissolved in 60ml of DMF, protected with nitrogen, cooled in a water bath, and 2.28g of NaH (60%) (56.93mmol, 1.71eq.) were added thereto, after which stirring was carried out at room temperature for 40min. 7.79g of ethyl 4-bromobutyrate (39.95mmol, 1.2eq.) was added to the system, and the system was stirred at 100 ℃ for 6 hours after the addition. Cooling the system to room temperature, adding 80ml of water into the system, stirring for 15min, extracting for 3 times with 150ml of ethyl acetate, combining organic phases, removing the solvent, collecting solid residue, and purifying by silica gel column chromatography (silica gel particle size is 200-300 meshes, and the used eluent and the volume ratio thereof are petroleum ether/ethyl acetate = 5/1-1/1) to obtain light brown oily substance, 3.98g, and the yield is 34.9%.
(2) Synthesis of C02: 3.90g of C01 (11.39mmol, 1.0 eq.) was dissolved in 30ml of DMF, protected with nitrogen, cooled in a water bath, and 0.55g of NaH (60%) (13.67mmol, 1.2 eq.) was added thereto, after which stirring was carried out at room temperature for 30min. 3.16g of epichlorohydrin (34.17mmol, 3.0 eq.) was added to the system, and the system was stirred at 60 ℃ for 6 hours after the addition. Cooling the system to room temperature, adding 40ml of water into the system, stirring for 15min, extracting with ethyl acetate for 3 times, 50ml each time, combining organic phases, removing the solvent, collecting solid residue, and purifying by silica gel column chromatography (silica gel particle size is 200-300 meshes, and the used eluent and volume ratio thereof are petroleum ether/ethyl acetate = 5/1-2/1) to obtain colorless oily matter, 2.20g, and the yield is 48.5%.
(3) Synthesis of C: 2.20g of C02 (5.52mmol, 1.0eq.) was dissolved in 30ml of ethanol, and an aqueous solution containing 0.88g of NaOH (22.09mmol, 4.0eq.) was added thereto, the concentration of NaOH being 1.46M; after the addition, the reaction was stopped by stirring the system under reflux for 8 hours. The system was adjusted to pH = 3-4 with 2N HCl (aq), the ethanol was removed by centrifugation, the aqueous phase was extracted 2 times with 50ml of ethyl acetate, the organic phases were combined, the solvent was stripped off, the solid residue was collected and purified by silica gel column chromatography (silica gel particle size 200-300 mesh, eluent used and its volume ratio dichloromethane/methanol = 15/1-10/1) to give 0.53g of colorless oil in 24.7% yield.
2. Characterization of haptens
And (3) identifying and characterizing the synthesized hapten by adopting a nuclear magnetic resonance hydrogen spectrometry:
nuclear magnetic identification of the prepared hapten shown in the formula 2: 1H NMR (400MHz, CDCl) 3 )δ7.26– 7.11(m,4H，ArH),6.82–6.76(m，4H，ArH),4.15–4.02(m,3H，CH,CH2), 4.00-3.95(m,2H,CH 2 ),3.82-3.69(m,2H，CH 2 ),2.59-2.55(m,2H,CH 2 ),2.11-2.08 (m,2H,CH 2 ),1.63,1.2(2s,6H，2xCH 3 ). The nuclear magnetic data shows that the compound synthesized by the method is a target product, and the magnetic resonance identification result is shown in figure 1. Nuclear magnetic identification of the prepared hapten shown in the formula 3: 1H NMR (400mhz, cdcl3) δ 7.15-7.10 (m, 4h, arh), 6.81-6.77 (m, 4h, arh), 4.10-4.08 (m, 1h, CH), 4.02-3.97 (m, 4h, ch2), 3.83 (dd, J1=11.4hz, j2=5.4hz,1h, ch2), 3.74 (dd, J1=11.4hz, j2=5.4hz,1h, ch2), 2.57 (t, J =7.2hz,2h, CH 2), 2.11-2.08 (m, 2h, ch2), 1.62 (s, 6h, 2hxc3). The nuclear magnetic data shows that the compound synthesized by the method is the target product, and the magnetic resonance identification result is shown in figure 2.
Example 2: preparation and characterization of BADGE Artificial antigen
The immunogen carrier protein is BSA, and the coupling mode is an active ester method. Dissolving 0.01mM hapten F (about 4.06 mg), 0.02mM DCC and 0.02mM NHS in anhydrous DMF, and reacting for about 4h under the condition of introducing nitrogen by magnetic stirring to obtain a solution I; approximately 15mg of BSA was dissolved in NaHCO 3 Fully dissolving to obtain a solution II; slowly dripping the solution I into the solution II until the solution is turbid, adding about 1ml of the solution I, stirring in an ice bath for about 3 hours, then filling into a dialysis bag, dialyzing in PBS (dialyzate is replaced for 6 times in the middle) for 48 hours, centrifuging after the dialysis is finished to obtain an immunogen solution, namely DCC-F-BSA, and storing at-20 ℃; coupling hapten C and BSA in the same coupling mode to obtain immunogen DCC-C-BSA;
and replacing the carrier protein with OVA to obtain the coating antigen DCC-F-OVA and DCC-C-OVA. And determining the binding ratio of BSA to hapten in the DCC-F-BSA solution and the DCC-C-BSA solution by means of time-of-flight mass spectrometry, wherein the binding ratio is = { M (conjugate) -M (protein) }/M (hapten), and M is the molecular weight.
The molecular weight of hapten of formula 2 is 406.9, the molecular weight of BSA is 66316.560, the molecular weight of conjugate DCC-F-BSA is 69709.765, and the binding ratio of BSA to hapten is 8.3 through calculation; formula 3 the hapten has a molecular weight of 388.46, BSA has a molecular weight of 66316.560, the conjugate DCC-C-BSA has a molecular weight of 68356.856, and the binding ratio of BSA to hapten would be calculated to be 5.2.
Example 3: preparation of BADGE and its derivative antiserum
Four male, healthy, new zealand white rabbits were selected as experimental animals, and were divided into two groups on average. The immune experiment is carried out by DCC-C-BSA immunogen and DCC-F-BSA immunogen respectively. Prior to immunization of rabbits, relevant immune emulsions were prepared: 1mg of immunogen was dissolved in physiological saline and an equal volume of Freund's complete adjuvant was added, and then the emulsion was immunized by multiple subcutaneous injections in the back. The rabbits were given a total of seven immunizations every two weeks. Serum titers and specificities were monitored by ear-marginal venous bleeds on day 7 after each immunization, starting with the third immunization, and whole blood was drawn from the heart into centrifuge tubes and allowed to stand overnight at 4 ℃ within 7 days after the end of the final immunization. Centrifuging at 4500r/min at 4 deg.C the next day, packaging the upper layer antiserum, and freezing at-20 deg.C.
Example 4: assay of BADGE and its derivatives antiserum
1. The indirect ELISA method for measuring the antiserum titer comprises the following specific operation steps:
1) Coating: diluting the coating stock to 3. Mu.g/ml, 100. Mu.L/well, coating overnight at 4 ℃ with carbonate buffer (0.05M, pH = 9.6), washing the plate 4 times with PBST buffer (0.05% Tween-20 in PBS, pH 7.4);
2) And (3) sealing: adding 3% skimmed milk powder, incubating at 37 deg.C for 1h, discarding blocking solution, and washing the plate with PBST buffer solution for 4 times;
3) Adding the serum to be detected: diluting antiserum in a multiple ratio from 1;
4) Color development: adding 150 mu L of TMB color development solution into each hole, and reacting for 10min at 37 ℃ in a dark place;
5) Termination and measurement: adding 50 mu L of stop solution into each hole, measuring the absorbance under the condition of 450nm/650nm (reference) of an enzyme labeling instrument, and obtaining the antiserum titer by the antiserum dilution factor with the OD value about 1.0.
The antiserum titer is shown in table 1, and the data in the table show that the dilution of all antiserum is 1. And by comparing the effect of the homologous coating and the heterologous coating on the antiserum titer, the antiserum titer measured by the homologous coating is found to be higher. Wherein the titer of the antiserum DCC-C-BSA-2# and DCC-F-BSA-3# is the highest and is 1.
2. Indirect ELISA method for determination of antiserum IC 50 The method comprises the following specific operation steps:
1) The coating and sealing process is the same as above.
2) Preparing a BADGE standard solution and a derivative thereof: dissolving BADGE and its derivative standard in methanol to obtain 1mg/mL mother solution, and diluting the standard with 10% methanol/PBS gradient (standard concentration of 0.1, 1, 10, 100, 200, 500, 1000, 10000, 20000, 50000 ng/mL)
3) Sample adding: : each well was added 100. Mu.L of each concentration standard of BADGE and its derivatives, and then 100. Mu.L of antiserum (diluted according to the titer of the antiserum in Table 1) was added and incubated at 37 ℃ for 1h.
4) Adding enzyme labeled secondary antibody, developing, terminating and reading.
The measured data were fitted using the concentration of competitor as abscissa and the inhibition ratio as ordinate using the four-parameter equation of Origin 8.5 to obtain IC 50 The value is obtained. The results are shown in Table 2, and when the specificity of the antiserum is determined by using BADGE as a competitor, IC of 2# antiserum obtained by DCC-C-BSA immunization and 3# antiserum obtained by DCC-F-BSA immunization are compared with those of the parallel group 50 The concentrations were as low as 55ng/mL and 10ng/mL, respectively, and the recognition effects of these two antibodies on BADGE derivatives were determined to draw a standardThe quasi-curve is shown in FIG. 3, the specificity results are shown in Table 3, the antiserum DCC-C-BSA-2# and DCC-F-BSA-3# have higher affinity to the BADGE derivatives, wherein, when the competitor micromolecules are BADGE and BADGE HCL, the antiserum DCC-F-BSA-3# has better recognition effect and IC 50 10ng/ml and 51ng/ml respectively; when the competitor is BADGE. H 2 O and BADGE. H 2 In case of O.HCL, antiserum DCC-C-BSA-2# has better recognition effect on the antigen and IC 50 22ng/ml and 18ng/ml respectively. Therefore, the antiserum obtained by immunizing a white rabbit with immunogen prepared by the newly synthesized hapten has higher sensitivity and can simultaneously recognize BADGE and derivatives thereof.
TABLE 1 antiserum titers under homologous and heterologous coatings
Figure BDA0002926429430000101
TABLE 2 IC of antisera coated with homologous and heterologous proteins 50
Figure BDA0002926429430000111
TABLE 3 identification of BADGE and its derivatives by two groups of antisera
Figure BDA0002926429430000112
In conclusion, the hapten and the artificial antigen designed and synthesized by the invention can prepare the specific antibody which has high affinity and high sensitivity and can simultaneously identify the BADGE and the derivatives thereof.
Although the invention has been described in detail with respect to the general description and the specific embodiments thereof, it will be apparent to those skilled in the art that modifications and improvements can be made based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims (10)

1. A bisphenol diglycidyl ether compound hapten is characterized in that two haptens are respectively prepared by introducing chlorine atoms and groups-OH, and the structures of the haptens are shown as formulas 2 and 3:
Figure FDA0003711900650000011
2. the method for preparing the hapten according to claim 1, wherein when the hapten is a compound represented by formula 2, the method comprises the following steps:
(1) Synthesis of compound F01: dissolving bisphenol A in DMF, cooling to room temperature in a water bath under the protection of nitrogen, adding NaH into the solution after cooling, and stirring for the first time at room temperature; stirring, adding epoxy chloropropane, stirring for the second time at room temperature, stirring, adding water, stirring for the third time to obtain a mixed solution, adjusting the pH, extracting with ethyl acetate, combining organic phases, removing a solvent, collecting solids, and purifying by silica gel column chromatography to obtain a light brown oily substance, which is marked as a compound F01;
(2) Synthesis of compound F02: dissolving the compound F01 prepared in the step (1) in DMF, performing nitrogen protection, cooling in a water bath, adding NaH into the solution after cooling, and performing first stirring at room temperature; stirring, adding tert-butyl 4-bromobutyrate, stirring for the second time at room temperature, stirring, adding water, stirring for the third time to obtain a mixed solution, extracting with ethyl acetate, combining organic phases, removing a solvent, collecting a solid, and purifying by silica gel column chromatography to obtain a brown oily substance, which is marked as a compound F02;
(3) Synthesis of compound F: dissolving the compound F02 prepared in the step (2) in tetrahydrofuran, then dropwise adding HCl, and stirring for the first time at room temperature; adding NaOH solution after stirring to obtain mixed solution; and stirring the mixed solution for the second time under a certain temperature condition, adjusting the pH value after stirring, standing for layering, extracting by using ethyl acetate, combining organic phases, removing the solvent, collecting the solid, and purifying by silica gel column chromatography to obtain a white solid, namely the bisphenol diglycidyl ether compound hapten which is marked as a compound F.
3. The method for preparing the hapten according to claim 2, wherein the ratio of the amounts of the bisphenol A, the DMF, the NaH, the epichlorohydrin and the water in the step (1) is 7g:60ml:1.53g:3.4g:80ml; the time for the first stirring is 30-40min; the time for the second stirring is 15-16h; the third stirring time is 15-20min; the pH adjustment is specifically to adjust the pH of the mixed solution to 5-6 by using 1N HCl.
4. The method for preparing the hapten according to claim 2, wherein the compound F01, DMF, naH, tert-butyl 4-bromobutyrate and water are used in the step (2) in a ratio of 6.4g:60ml:1.13g:6.27g:80ml of the solution; the time for the first stirring is 30-40min; the time for the second stirring is 15-16h; the third stirring time is 15-20min.
5. The method for preparing the hapten according to claim 2, wherein the compound F02, tetrahydrofuran, hydrochloric acid and NaOH solution are used in a ratio of 2.3g:30ml:0.50ml:10ml; the concentration of the hydrochloric acid is 12N; the concentration of the NaOH solution is 1.6M; the first stirring time is 15-20min; the certain temperature condition is 40 ℃, and the time of the second stirring is 2 hours; the pH adjustment is specifically carried out with 2N HCl to pH =3 to 4.
6. The method for preparing the hapten according to claim 1, wherein when the hapten is a compound represented by formula 3, the method comprises the following steps:
(1) Synthesis of compound C01: dissolving bisphenol A in DMF, cooling to room temperature in a water bath under the protection of nitrogen, adding NaH after cooling, and stirring for the first time at room temperature; adding ethyl 4-bromobutyrate after stirring, heating to a certain temperature for second stirring, cooling to room temperature after stirring, adding water, stirring for the third time to obtain a mixed solution, extracting with ethyl acetate, combining organic phases, removing the solvent, collecting solids, and purifying by silica gel column chromatography to obtain a light brown oily substance, which is marked as a compound C01;
(2) Synthesis of compound C02: dissolving the compound C01 prepared in the step (1) in DMF, performing nitrogen protection, cooling in a water bath, adding NaH into the solution after cooling, and performing first stirring at room temperature; adding epoxy chloropropane after stirring, heating to a certain temperature for second stirring, cooling to room temperature after stirring, adding water, stirring for the third time to obtain a mixed solution, extracting by using ethyl acetate, combining organic phases, removing a solvent, collecting solids, and purifying by silica gel column chromatography to obtain a colorless oily substance which is marked as a compound C02;
(3) Synthesis of compound C: dissolving the compound C02 prepared in the step (2) in ethanol, adding an aqueous solution of NaOH to obtain a mixed solution, refluxing and stirring, adjusting the pH after stirring, removing the ethanol in a rotating manner to obtain a water phase, extracting with ethyl acetate, combining organic phases, removing the solvent, collecting solids, and purifying by silica gel column chromatography to obtain a colorless oily substance, namely the bisphenol diglycidyl ether compound hapten which is marked as a compound C;
7. the method for producing the hapten according to claim 6, wherein the ratio of the amounts of bisphenol A, DMF, naH, ethyl 4-bromobutyrate and water used in step (1) is 7.6g:60ml:2.28g:7.79g:80ml; the first stirring time is 30-40min; the heating is carried out until the certain temperature is 100 ℃, and the time for the second stirring is 6h; the third stirring time is 15-20min.
8. The process for the preparation of the hapten according to claim 6, wherein the ratio of the amounts of the compound C01, DMF, naH, epichlorohydrin and water used in step (2) is 3.9g:30ml:0.55g:3.16g:40ml; the time for the first stirring is 30-40min; the heating is carried out to a certain temperature of 60 ℃, and the time of the second stirring is 6h; the time for stirring for the third time is 15-20min.
9. The method for preparing the hapten according to claim 6, wherein the dosage ratio of the compound C02, ethanol and NaOH aqueous solution in the step (3) is 2.2g:30ml:15ml; the concentration of the NaOH is 1.46M; the first stirring time is 15-20min; the reflux stirring time is 8 hours; the pH adjustment is specifically carried out with 2N HCl to pH =3 to 4.
10. Use of the hapten according to claim 1 for the preparation of anti-BADGE-like compound specific broad-spectrum antibodies and for the detection of BADGE and derivatives thereof.
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