CN108998425A - One plant of pyridoxol monoclonal antibody hybridoma cell strain and its application - Google Patents

Abstract

A pyridoxine monoclonal antibody hybridoma cell line and application thereof belong to the field of food safety immunoassay. The present invention prepares the complete pyridoxine antigen, mixes it with an equal amount of Freund's adjuvant and emulsifies it completely, and immunizes BALB/c mice by subcutaneous injection on the back to obtain a pyridoxine monoclonal antibody hybridoma cell line SS0708. It is preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee, with the preservation number CGMCC No.14687, and the preservation date is September 5, 2017. The monoclonal antibody secreted by this cell line has good specificity and detection sensitivity to pyridoxine (IC ₅₀ is 250 ng/mL). The achievement of the invention can be used to establish an immunological detection method for pyridoxine content in food for infants and young children, dairy products and food for special medical purposes, and has practical application value.

Description

A pyridoxine monoclonal antibody hybridoma cell line and its application

technical field

The invention relates to a pyridoxine monoclonal antibody hybridoma cell line and an application thereof, belonging to the field of food safety immunoassay.

Background technique

Pyridoxine belongs to the B vitamins and is one of the substances that have the effect of vitamin B6. It is also known as antidermatitis factor or vitamin B6. It is widely found in food and can be used as a dietary supplement. Pyridoxine is easily converted into pyridoxal-5-phosphate as a coenzyme in the body to participate in the metabolism of the body. In animals lacking pyridoxine, the transformation of protein into carbohydrates and fat will be inhibited, and rhinitis, stomatitis and dermatitis will also occur. In addition, lack of vitamin B6 can also cause arteriosclerosis in various organs. Because pyridoxine plays an important role in the central nervous system, the absence of pyridoxine can cause convulsions, which can be recovered quickly by giving pyridoxine. Pyridoxine is used to treat and prevent pyridoxine deficiency, sideroblastic anemia, pyridoxine-dependent epilepsy and certain metabolic disorders, so the control of pyridoxine in food is particularly important.

At present, there are instrumental methods such as gas chromatography (GC), high performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), and liquid mass spectrometry (LC-MS) for pyridoxine content analysis methods, but these methods require Expensive instruments, professional operators, complex sample pretreatment, high cost, and long time cannot achieve rapid detection of a large number of samples. Therefore, it is of great significance to establish a quick and easy pyridoxine detection method. Enzyme-linked immunoassay (ELISA) is an extremely efficient, sensitive, and rapid detection method. It does not require high sample purity and is easy to operate. It is suitable for on-site rapid detection of a large number of samples. It is an important prerequisite to establish an efficient immunological detection method and to screen highly specific monoclonal antibodies.

Contents of the invention

The purpose of the present invention is to provide a pyridoxine monoclonal antibody hybridoma cell line SS0708 and its application. The antibody prepared by the cell line has good specificity and detection sensitivity to pyridoxine, and can be used to establish pyridoxine Alcohol immunological detection method.

The technical solution of the present invention is a pyridoxine monoclonal antibody hybridoma cell line, which is preserved in the General Microbiology Center of the China Microbiological Culture Collection Management Committee, with a preservation number of CGMCC No. 14687 and a preservation date of September 5, 2017.

The pyridoxine monoclonal antibody is secreted and produced by the pyridoxine monoclonal antibody hybridoma cell line SS0708 with the deposit number of CGMCC No. 14687.

The application of the pyridoxine monoclonal antibody is used to establish an immunoassay method for pyridoxine content, which is applied to the detection of residual pyridoxine.

The detection field is in food for infants and young children, dairy products or food for special medical purposes.

The preparation method of described pyridoxine immunogen Pyr-KLH mainly comprises the following steps:

(1) Preparation of the hapten Pyr: Dissolve pyridoxine (20 g, 118 mmol) in 200 mL of acetone, add 200 mL of 2,2-dimethoxypropane and p-toluenesulfonic acid (81.4 g, 473 mmol), and stir at 130 °C overnight. The reaction mixture was poured into ice water to cool and the aqueous phase was extracted with DCM. The organic phases were combined and washed with brine, dried _{over Na2SO4} and concentrated. The resulting product was recrystallized from diethyl ether. The mixture was filtered to obtain compound 2 as a white solid;

A solution of NaH (6.88 g, 287 mmol, 60% organic phase) was added to 70 mL THF at 0 °C under nitrogen. Compound 2 (15 g, 71.7 mmol) dissolved in 200 mL THF was added. Refluxed for 30min, cooled to room temperature, added benzyl bromide (24.5g, 143mmol), refluxed again for 4h, and then extracted with dichloromethane. The organic extracts were collected, washed with brine, dried and concentrated to give a dark brown oil. The residue was purified on a silica gel column to give 3 as a brown oil;

Add 24 mL of 98% formic acid to the aqueous solution of compound 3 (12 g, 40.1 mmol). Stirred at 50°C for 24 h, then adjusted to pH 7 with saturated NaHCO ₃ solution. After neutralization, the reaction mixture was extracted several times with dichloromethane. The organic extract was dried and concentrated to obtain a dark brown solid, and the crude product was purified by flash chromatography to obtain compound 4;

Under nitrogen at 0°C, benzyldimethylphenylammonium chloride (7.60 g, 26.6 mmol) was dissolved in 30 mL of anhydrous methanol, and this solution was added to a solution of sodium methoxide (1.80 g, 34 mmol). Then 60 mL of compound 4 (4.50 g, 17.4 mmol) in methanol was added to the mixture. After stirring at room temperature for 20 min, the mixture was concentrated by evaporation and added to a round bottom flask containing 500 ml of hot toluene. After 30 min of reaction, the mixture was concentrated. After the reaction, the oily residue was dissolved in saturated ammonium chloride solution, followed by extraction with dichloromethane. The organic extracts were dried and concentrated. The organic extract was purified by a silica gel column to obtain compound 5;

CS ₂ CO ₃ (3.70 g·114 mmol) and ethyl acrylate (2.80 g, 22.6 mmol) were added to a solution of compound 5 (2.60 g, 169 mmol) in 60 mL of toluene, and stirred overnight at 85°C. The reaction mixture was cooled and poured into water, then the solution was adjusted to pH 7 with HCl and the aqueous phase was extracted with EA. The combined organic phases were washed with brine, dried _{over Na2SO4} and concentrated. Purify the residue with a silica gel column to obtain compound 6;

LiOH H ₂ O (34.7 mg, 890 mmol) was added to a THF/H ₂ O (16.0/4.00 mL) solution containing compound 6 (1.60 g, 3.50 mmol), and stirred overnight at room temperature. The solution was then adjusted to pH 7 with HCl and the aqueous layer was extracted with EA. The mixed organic layer was washed with brine, dried over Na ₂ SO ₄ and concentrated to obtain compound 7;

Compound 7 (1 g, 2.30 mmol) in 20 mL of MeOH was added with 100 mg of Pd/C and stirred overnight at room temperature. The mixture was filtered and concentrated to afford compound 8, Pyr, as a white solid.

(2) Preparation of complete antigen:

Preparation of the immunogen Pyr-KLH: Weigh the hapten Pyr, 1-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide, and dissolve it in anhydrous N,N-dimethylformamide to obtain A solution; take keyhole limpet hemocyanin KLH, and dissolve it with boric acid buffer solution to obtain solution B; at room temperature, add solution A to solution B drop by drop, and react overnight at room temperature to obtain the conjugated Pyr-KLH mixture. The immunogen Pyr-KLH was obtained by separating the complete antigen and unconjugated small molecule hapten by dialysis.

A screening method for preparing the pyridoxine monoclonal antibody hybridoma cell line is provided, which mainly includes the following steps:

(1) Immunization of mice: mix and emulsify the immunogen Pyr-KLH with an equal amount of Freund's adjuvant, and then subcutaneously inject BALB/c mice into the back; use complete Freund's adjuvant for the first immunization, and use for multiple booster immunizations Incomplete Freund's adjuvant, the interval between the first immunization and the second booster immunization was 28 days, and the interval between multiple booster immunizations was 21 days, and the last rush immunization with Pyr-KLH complete antigen (without adjuvant); through indirect Competitive enzyme-linked immunoassay (ic-ELISA) to detect serum titer and inhibition;

(2) Cell fusion and cell line establishment: mouse splenocytes and mouse myeloma cells were fused by the polyethylene glycol (PEG 4000) method, cultured in HAT medium, and indirect competitive enzyme-linked immunoassay (ic-ELISA) ) to detect positive cell wells, and further use ic-ELISA to measure the inhibitory effect of positive cell wells, and perform three subclones on positive cell wells with the best inhibitory effect by limiting dilution method, and finally screen to obtain pyridoxine monoclonal antibody hybridomas cell line;

(3) Identification of hybridoma cell line properties: Sensitivity and specificity were determined by ic-ELISA.

Beneficial effects of the present invention: the monoclonal antibody secreted by the pyridoxine monoclonal antibody hybridoma cell line provided by the present invention has good specificity and detection sensitivity to pyridoxine (IC ₅₀ value is 250 ng/mL), An immunological method is provided for the detection of pyridoxine in food for infants and young children, dairy products and food for special medical purposes. The pyridoxine monoclonal antibody hybridoma cell line and the monoclonal antibody secreted by the present invention can be made into a kit for detecting pyridoxine, which has practical application value.

Preservation of biological material samples: a pyridoxine monoclonal antibody hybridoma cell line SS0708, the hybridoma cell line is preserved in the General Microbiology Center of the China Microbiological Culture Collection Management Committee, and the preservation address is: No. 1 Beichen West Road, Chaoyang District, Beijing No. 3, Institute of Microbiology, Chinese Academy of Sciences, the preservation number is CGMCC No.14687, the classification and naming of monoclonal cell lines, and the preservation date is September 5, 2017.

Description of drawings

Fig. 1 is the inhibition standard curve of pyridoxine monoclonal antibody to pyridoxine.

Detailed ways

The following examples of the present invention are only used as a further description of the content of the present invention, and cannot be regarded as the content or scope of the present invention. Below by embodiment the present invention will be further described.

The present invention immunizes mice with pyridoxine complete antigen, through cell fusion, cultured in HAT selective medium, and screening cell supernatant by ic-ELISA, and finally obtains a monoclonal antibody with good specificity and sensitivity to pyridoxine Antibody hybridoma cell lines.

Example 1 Preparation of hybridoma cell line SS0708

(1) Preparation of complete antigen:

a. The hapten synthesis route is as follows:

Dissolve pyridoxine (20 g, 118 mmol) in 200 mL of acetone, add 200 mL of 2,2-dimethoxypropane and p-toluenesulfonic acid (81.4 g, 473 mmol), and stir overnight at 130°C. The reaction mixture was poured into ice water to cool and the aqueous phase was extracted with DCM. The organic phases were combined and washed with brine, dried _{over Na2SO4} and concentrated. The desired product was recrystallized from ether. The mixture was filtered to obtain compound 2 as a white solid;

A solution of NaH (6.88 g, 287 mmol, 60% oil) was added to 70 mL THF at 0 °C under nitrogen. Compound 2 (15 g, 71.7 mmol) dissolved in 200 mL THF was added. Refluxed for 30 min, cooled to room temperature, added benzyl bromide (24.5 g, 143 mmol), refluxed again for 4 h, and then extracted with dichloromethane. The organic extracts were collected, washed with brine, dried and concentrated to give a dark brown oil. The residue was purified on a silica gel column to give 3 as a brown oil;

24 mL of 98% formic acid was added to the aqueous solution of compound 3 (12 g, 40.1 mmol). After stirring at 50 °C for 24 h, the solution was adjusted to pH 7 with saturated NaHCO ₃ solution. After neutralization, the reaction mixture was extracted several times with dichloromethane. The organic extract was dried and concentrated to obtain a dark brown solid, and the crude product was purified by flash chromatography to obtain compound 4;

Under nitrogen at 0°C, benzyldimethylphenylammonium chloride (7.60 g, 26.6 mmol) was dissolved in 30 mL of anhydrous methanol, and this solution was added to a solution of sodium methoxide (1.80 g, 34 mmol). Then 60 mL of compound 4 (4.50 g, 17.4 mmol) in methanol was added to the mixture. After stirring at room temperature for 20 min, the mixture was concentrated by evaporation and added to a round bottom flask containing 500 ml of hot toluene. After reacting for 30 min, the mixture was concentrated. After the reaction, the oily residue was dissolved in saturated ammonium chloride solution, followed by extraction with dichloromethane. The organic extracts were dried and concentrated. The organic extract was purified by a silica gel column to obtain compound 5;

CS ₂ CO ₃ (3.70 g·114 mmol) and ethyl acrylate (2.80 g, 22.6 mmol) were added to a solution of compound 5 (2.60 g, 169 mmol) in 60 mL of toluene, and stirred overnight at 85°C. The reaction mixture was cooled and poured into water, then the solution was adjusted to pH 7 with HCl and the aqueous phase was extracted with EA. The combined organic phases were washed with brine, dried _{over Na2SO4} and concentrated. Purify the residue with a silica gel column to obtain compound 6;

LiOH H ₂ O (34.7 mg, 890 mmol) was added to a THF/H ₂ O (16.0/4.00 mL) solution containing compound 6 (1.60 g, 3.50 mmol), and stirred overnight at room temperature. The solution was then adjusted to pH 7 with HCl and the aqueous layer was extracted with EA. The mixed organic layer was washed with brine, dried over Na ₂ SO ₄ and concentrated to obtain compound 7;

To a solution of compound 7 (1 g, 2.30 mmol) in 20 mL of MeOH was added 100 mg of Pd/C and stirred overnight at room temperature. The mixture was filtered and concentrated to afford compound 8, the hapten Pyr, as a white solid.

b. Weigh 2.7 mg of Pyr, 7.6 mg of 1-ethylcarbodiimide hydrochloride, and 4.6 mg of N-hydroxysuccinimide, and dissolve them in 400 μL of anhydrous N,N-dimethylformamide to obtain A1 solution , stirred at room temperature for 6-8h. Take 10 mg of keyhole limpet hemocyanin KLH (the molar ratios of Pyr and KLH are 2000:1, 4000:1 and 6000:1, respectively), dissolve in 6 mL of boric acid buffer solution to obtain B1 solution, and add A1 solution dropwise at room temperature into solution B1, and react overnight at room temperature to obtain conjugate Pyr-KLH (2000:1, 4000:1 and 6000:1) mixtures, and separate the complete antigen and unconjugated small molecule hapten by dialysis to obtain the conjugated Conjugates Pyr-KLH (2000:1), Pyr-KLH (4000:1) and Pyr-KLH (6000:1).

(2) Preparation of coated original Pyr-OVA:

Weigh 1.6 mg of Pyr, 3.8 mg of 1-ethylcarbodiimide hydrochloride, and 2.3 mg of N-hydroxysuccinimide, dissolve in 300 μL of anhydrous N,N-dimethylformamide to obtain A2 solution, and keep at room temperature Stir for 6-8h. Weigh 5 mg of chicken ovalbumin OVA (the molar ratio of Pyr to OVA is 60:1), dissolve it in 2 mL of boric acid buffer solution to obtain the B2 solution, add the A2 solution to the B2 solution drop by drop at room temperature, and react at room temperature Overnight, the conjugated Pyr-OVA mixture was obtained, and the coated original and unconjugated small molecule hapten were separated by dialysis. The original coating is used for the detection of mouse serum titer and inhibition during the preparation of monoclonal antibodies, and is not directly used in mice, and is necessary for the preparation of monoclonal antibodies.

(3) Animal immunization: healthy 6-8 week-old BALB/c mice were selected for immunization. Three different molar ratios of pyridoxine complete antigen were mixed and emulsified with the same amount of Freund's adjuvant, and BALB/c mice were immunized by subcutaneous injection in the back. Complete Freund's adjuvant was used for the first immunization, followed by incomplete Freund's adjuvant. The interval between the first immunization and the second booster immunization was 28 days, and the interval between multiple booster immunizations was 21 days. Blood was collected 7 days after the third immunization (5 uL of mouse tail blood collection + 995 uL antibody diluent = antiserum), and ic-ELISA was used to measure the titer and inhibition of mouse serum, and mice with high titer and inhibition were selected. 21 days after the fifth immunization, the immunization was given by intraperitoneal injection, and the immunization dose was required to be halved without any adjuvant.

(4) Cell fusion: Three days after the sprint immunization, perform cell fusion according to the conventional PEG (polyethylene glycol, molecular weight 4000) method, and the specific steps are as follows:

a. Pick up the eyeballs to take blood, kill the mice by cervical dislocation, put them into 75% alcohol immediately for disinfection, soak for about 5 minutes, take out the spleen of the mice by aseptic operation, grind them moderately with the rubber tip of the syringe and pass through 200 mesh cells Sieve to obtain splenocyte suspension, collect, centrifuge (1200 rpm, 8 min), wash splenocytes with RPMI-1640 medium three times, after the last centrifugation, dilute splenocytes to a certain volume, count, and set aside;

b. Collect SP2/0 cells: 7-10 days before fusion, put SP2/0 tumor cells in 5% CO ₂ incubator with RPMI-1640 medium containing 10% FBS (fetal bovine serum). The number of SP2/0 tumor cells is required to reach 1~4×10 ⁷ before fusion, and the SP2/0 tumor cells are in logarithmic growth phase before fusion. When confluent, tumor cells were collected, suspended in RPMI-1640 basal culture medium, and cell counted;

c. The fusion process takes 7 minutes. At 1 min, 1 mL of PEG 1500 was added dropwise to the cells from slow to fast; at 2 min, stand still. At 3 min and 4 min, add 1 mL RPMI-1640 medium dropwise within 1 min; at 5 min and 6 min, add 2 mL RPMI-1640 medium dropwise within 1 min; at 7 min, add 1 mL of RPMI-1640 medium every 10 s Add 1 mL of RPMI-1640 medium dropwise. Then incubate at 37°C for 5 min. Centrifuge (800 rpm, 8 min), discard the supernatant, resuspend in RPMI-1640 screening medium containing 20% fetal bovine serum, 2% 50×HAT, add to 96-well cell plate at 200 μL/well, set Cultured in a 37°C, 5% CO ₂ incubator.

(5) Cell screening and cell line establishment: On the 3rd day of cell fusion, half-change the RPMI-1640 screening culture medium for the fused cells, and on the 5th day, use 20% fetal bovine serum, 1% 100×HT The RPMI-1640 transition culture medium was completely changed, and the cell supernatant was collected on the 7th day for screening. The screening is divided into two steps: in the first step, ic-ELISA is used to screen positive cell wells, and in the second step, pyridoxine is selected as a standard, and the inhibitory effect of positive cells is determined by ic-ELISA. Select cell wells that have good inhibition to pyridoxine standard substances, use the limiting dilution method for subcloning, and use the same method for detection. Repeat three times to obtain cell line 2F9.

(6) Preparation and identification of monoclonal antibody: 8-10 weeks old BALB/c mice were taken, and each mouse was intraperitoneally injected with sterile paraffin oil 1 mL; 7 days later, each mouse was intraperitoneally injected with 1×10 ⁶ hybridoma Cells, ascites fluid was collected from the seventh day, and the ascites fluid was purified by octanoic acid-ammonium sulfate method. Under acidic conditions, n-octanoic acid can precipitate other miscellaneous proteins in ascites except IgG immunoglobulin, then centrifuge, and discard the precipitate; then use an equal amount of saturated ammonium sulfate solution to precipitate IgG-type monoclonal antibodies, centrifuge, and discard The supernatant was dissolved with 0.01M PBS solution (pH 7.4), dialyzed and desalted, and finally the purified monoclonal antibody was stored at -20°C.

Example 2 Determination of IC ₅₀ of Pyridoxine Monoclonal Antibody

Carbonate buffer solution (CBS): Weigh 1.59 g of Na ₂ CO ₃ and 2.93 g of NaHCO ₃ , dissolve them in a small amount of double-distilled water and mix them. Add double-distilled water to about 800 mL and mix well. Adjust the pH value to 9.6. Add double distilled water to make up to 1000 mL, and store at 4°C for later use.

Phosphate buffered saline (PBS): 8.0 g NaCl, 0.2 g KCl, 0.2 g KH ₂ PO ₄ , 2.9 g Na ₂ HPO ₄ • 12H ₂ O, dissolved in 800 mL pure water, adjusted to pH with NaOH or HCl 7.2～7.4, set the volume to 1000 mL;

Washing solution (PBST): Add 0.5 mL of Tween-20 to 1000 mL of 0.01 mol/L PBS solution with pH7.4;

PBST: PBS containing 0.05% Tween-20;

Antibody diluent: wash buffer containing 0.1% gelatin;

TMB chromogenic solution: A solution: Na ₂ HPO ₄ .12H ₂ O 18.43 g, citric acid 9.33 g, dilute to 1000 mL with pure water; B solution: 60 mg TMB dissolved in 100 mL ethylene glycol. A and B liquids are mixed at a volume ratio of 1:5 to form TMB

Chromogenic solution, ready to use and mix.

(1) Coating: The coated original Pyr-OVA was diluted with 0.05M pH9.6 carbonate buffer starting from 1 μg/mL, 100 μL/well, and reacted at 37°C for 2 h.

(2) Washing: Pour off the solution in the plate, and wash 3 times with washing solution, 3 min each time.

(3) Blocking: After patting dry, add 200 μL/well blocking solution and react at 37°C for 2 h. Wash and tumble dry for later use.

(4) Adding samples: Dilute the antiserum (after blood collection from the tail of the mouse, dilute the corresponding times with the antibody diluent) starting from 1:1000, and add it to the coated wells of each dilution. 100 μL/well, react at 37°C for 30 min; after washing thoroughly, add 1:3000 diluted HRP-goat anti-mouse IgG, 100 μL/well, react at 37°C for 30 min.

(5) Color development: Take out the ELISA plate, after washing thoroughly, add 100 μL of TMB color development solution to each well, and react in the dark at 37 °C for 15 min.

(6) Termination and measurement: 50 μL of stop solution was added to each well to terminate the reaction, and then the OD450 value of each well was measured with a microplate reader.

The IC ₅₀ of the monoclonal antibody pyridoxine determined by ic-ELISA was 250 ng/mL, indicating that it has good sensitivity to pyridoxine and can be used for pyridoxine immunoassay detection.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore The scope of protection of the present invention should be defined by the claims.

Claims (5)

1. one plant of pyridoxol monoclonal antibody hybridoma cell strain, it is common to be preserved in China Committee for Culture Collection of Microorganisms Microorganism center, deposit number are CGMCC No.14687, and the deposit date is on September 5th, 2017.

2. pyridoxol monoclonal antibody, which is characterized in that the deposit number as described in claim 1 is CGMCC No.14687, pyrrole The alcohol monoclonal antibody hybridoma cell strain SS0708 that trembles secretion generates.

3. the application of pyridoxol monoclonal antibody described in claim 2, it is characterized in that: the immune inspection for establishing pyridoxol content Survey method, applied in the detection for remaining pyridoxol.

4. the application of pyridoxol monoclonal antibody according to claim 3, it is characterized in that: the detection field is infant's food In product, dairy products or special medicine purposes food.

5. being used to prepare the immunogene Pyr-KLH of pyridoxol monoclonal antibody hybridoma cell strain SS0708 described in claim 1 Preparation method, it is characterized in that steps are as follows:

(1) preparation of haptens Pyr:

A, pyridoxol 20g, 118mmol are dissolved in 200mL acetone, 200mL 2,2-dimethoxypropane are added and to toluene 81.4g, 473mmol, 130 DEG C of sulfonic acid are stirred overnight；Reaction mixture is poured into ice water and cools down and extracts water phase with DCM, is mixed It closes organic phase and is washed with brine, Na₂SO₄Dry concentration, with Diethyl ether recrystallization products therefrom, mixture is filtered, obtains white Solid chemical compound 2；

B, under 0 DEG C of nitrogen, by 6.88 g of NaH, 287 mmol, the solution of 60% organic phase is added in 70 mL THF, is added molten 15 g compound 2,71.7 mmol of the solution in 200 mL THF；Flow back 30min, and after being cooled to room temperature, benzyl bromide is added 24.5g, 143mmol, flow back 4h again, is then extracted with dichloromethane；Organic extract is collected, is washed with brine, drying is dense Contracting, obtains dark brown oil, with silicagel column purification residues, obtains brown oil compound 3；

C, 24 mL, 98% formic acid, 50 DEG C of 24 h of stirring, then with saturation are added in the aqueous solution of 12g, 40.1mmol compound 3 NaHCO₃Solution is adjusted to pH 7；After neutralization, reaction mixture is repeatedly extracted with methylene chloride, organic extract is dry Concentration, obtains dark brown solid, with purified by flash chromatography crude product, obtains compound 4；

D, under 0 DEG C of nitrogen, benzyl dimethyl Phenyl chloride 7.60g, 26.6mmol are dissolved in 30mL anhydrous methanol, by this Solution is added to sodium methoxide 1.80g, in 34mmol solution, is then added into mixture and contains 4.50g, 17.4mmol compound 4 60mL methanol solution in；20min is stirred at room temperature, and evaporation concentrated mixture is simultaneously added it to containing 500mL hot toluene In round-bottomed flask, after reacting 30min, mixture is concentrated；After reaction, oil residue is dissolved in saturated ammonium chloride solution, then It is extracted with dichloromethane, by the dry concentration of organic extract, by organic extract through silica gel column purification, obtains compound 5；

E, contain 2.60g, CS is added in the 60mL toluene solution of 169mmol compound 5₂CO₃3.70g114mmol and acrylic acid second 2.80g, 22.6mmol, 85 DEG C of ester are stirred overnight；Reaction mixture is cooled and poured into water, is then adjusted to solution with HCl PH7, with EA aqueous phase extracted；Mixing organic phase is washed with brine, and uses Na₂SO₄It is dried and concentrated；With silicagel column purification residues, obtain To compound 6；

D, contain 1.60g, the THF/H of 3.50mmol compound 6₂LiOH H is added in O 16.0/4.00mL meter, solution₂O 34.7mg, 890mmol is stirred overnight at room temperature；Then solution is adjusted to pH7 with HCl, extracts water layer with EA；Mix organic layer salt water Washing, through Na₂SO₄Dry concentration, obtains compound 7；

Containing 1g, 100mg Pd/C is added in the 20mL MeOH solution of 2.30mmol compound 7, is stirred overnight at room temperature；It will mixing Object is filtered and is concentrated, and obtains compound as white solid 8 i.e. Pyr；

(2) haptens Pyr, 1- ethyl-carbodiimide hydrochloride and N- hydroxysuccinimidyl the preparation of immunogene Pyr-KLH: are weighed Acid imide is dissolved to obtain A liquid with anhydrous n,N-Dimethylformamide；Keyhole limpet hemocyanin KLH is taken, is dissolved with borate buffer solution Obtain B liquid；In room temperature condition, A liquid is added in B liquid dropwise, room temperature reaction is stayed overnight to get conjugate Pyr-KLH mixed liquor, Immunogene Pyr-KLH is obtained by dialysis separation comlete antigen and the small haptens not being coupled.