Hybridoma cell line VAND8 and anti-vanillin monoclonal antibody produced therefrom and applications thereof

Abstract

The present invention relates to a hybridoma cell line VAND8 and an anti-vanillin monoclonal antibody produced therefrom and its application, belonging to the field of immunochemistry. The hybridoma cell line VAND8 provided by the present invention can be used to prepare a high-titer anti-vanillin monoclonal antibody, with a titer of up to 3.2×10 5 The anti-vanillin monoclonal antibody provided by the present invention has high sensitivity and good specificity, and its 50% inhibitory concentration IC 50 The content of vanillin was 22.1 μg / mL, and the cross-reaction rate with vanillin structural analogues was low or even no cross-reaction; it can be used to determine the content of vanillin.

Description

Technical Field

[0001] The present invention relates to a hybridoma cell line VAND8 and an anti-vanillin monoclonal antibody produced therefrom and applications thereof, belonging to the field of immunochemistry. Background Art

[0002] Vanillin is a common food additive as a flavoring. Although it is non-toxic, excessive consumption can be harmful to human health. According to a report by the European Union Expert Committee on February 24, 2000, large amounts of vanillin and other food additives consumed by the human body can cause headaches, nausea, vomiting, breathing difficulties and even liver and kidney damage. The maximum daily intake of vanillin is limited by the Joint Expert Committee of the Food and Agriculture Organization (FAO) and the World Health Organization (WHO) to 10 mg / kg. The National Food Safety Standard for the Use of Food Additives (GB2760-2014) also stipulates the use limit of vanillin in food. [2] Therefore, the use of vanillin in food needs to be strictly regulated, and dairy products, baked goods, beverages and cooking oils are the focus of vanillin content testing.

[0003] Existing literature reports that methods for detecting vanillin in food include high-performance liquid chromatography, high-performance liquid chromatography-tandem mass spectrometry, gas chromatography, and ultraviolet spectrophotometry. These methods are highly sensitive and provide accurate results, but the required equipment is expensive and the sample pretreatment process is cumbersome. Immunoassays utilize the specific binding reaction between antigens and antibodies and the biological, physical, or chemical amplification effects of markers on the antibodies and antigens to qualitatively and quantitatively detect ultra-trace residues. Immunoassay technology is relatively low-cost and can be used for rapid detection. However, the development of any immunological detection technology for vanillin requires the acquisition of high-quality anti-vanillin antibodies. Summary of the Invention

[0004] The problem to be solved by the present invention is to provide a hybridoma cell line VAND8 and an anti-vanillin monoclonal antibody produced by the hybridoma cell line and its application in view of the deficiencies in the prior art.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention provides a hybridoma cell line VAND8, which was deposited in the China Center for Type Culture Collection (CCTCC) on September 20, 2023, with the deposit address being Wuhan University, Wuhan, China, with the deposit number being CCTCCNO.C 2023186, and the classification name being mouse hybridoma cell VAND8, Hybridoma cell line VAND8.

[0007] The second aspect of the present invention provides an anti-vanillin monoclonal antibody, wherein the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing; and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing. The antibody is secreted and produced by the hybridoma cell line VAND8, deposited with CCTCC NO. C2023186. The anti-vanillin monoclonal antibody can recognize vanillin and has a 50% inhibitory concentration (IC) of vanillin. 50 The cross-reaction rate with vanillin structural analogues was low or even no cross-reaction. The cross-reaction rate with ethyl vanillin was 21.65%, and the cross-reaction rate with methyl vanillin was less than 1%.

[0008] The hybridoma cell line VAND8 provided by the present invention was obtained using a two-step screening method. The specific steps are: BALB / c mice were immunized 4-5 times with complete VAN antigen, followed by a final booster immunization with VAN-BSA at twice the dose of the previous immunization, followed by cell fusion three days later. The fused cells were screened using an ELISA method in two steps: the first step was to screen for positive clones resistant only to vanillin but not to the carrier protein BSA using an indirect ELISA method; the second step was to test the culture fluid of the positive clones screened in the first step using an indirect competitive ELISA method, using vanillin as a competitor. Positive clones with low absorbance and high sensitivity were selected for further cloning using the limiting dilution method. Approximately 10 days after cloning, the same two-step screening method was used for antibody detection. After repeating this cloning process 2-3 times, the hybridoma cell line VAND8 was finally obtained.

[0009] The specific preparation steps of the above-mentioned VAN complete antigen are:

[0010] Vanillinamine hydrochloride was weighed and reacted with maleic anhydride at room temperature under the catalysis of triethylamine to obtain the hapten: (2Z)-4-[(4-hydroxy-3-methoxybenzyl)amino]-4-oxobut-2-enoic acid ((2Z)-4-[(4-hydroxy-3-methoxybenzyl)amino]-4-oxobut-2-enoic acid), with the molecular formula C 12 H 13 NO5, with a relative molecular mass of 251.2; the vanillin complete antigen VAN-BSA was synthesized by the active ester method: the above-mentioned hapten was subjected to an oximation reaction with NH2OH, and then reacted with N-hydroxysuccinimide (NHS) in the dark under the action of dicyclohexylcarbodiimide (DCC), and the supernatant active ester solution was coupled with BSA to obtain the complete antigen VAN-BSA.

[0011]

[0012] The present invention further provides a method for preparing an anti-vanillin monoclonal antibody, comprising the following steps: injecting the obtained hybridoma cell line VAND8 into BALB / c mice pre-treated with Freund's incomplete adjuvant, collecting the ascites of the mice, and obtaining the anti-vanillin monoclonal antibody after purification.

[0013] The beneficial effects of the present invention are:

[0014] (1) The hybridoma cell line VAND8 provided by the present invention can be used to prepare high-titer anti-vanillin monoclonal antibodies. The titer of anti-vanillin mouse ascites antibody enzyme-linked immunosorbent assay (ELISA) can reach 3.2×10 5 It provides core raw materials for the future development of vanillin immunological detection technology with simple operation, wide application range and high sensitivity.

[0015] (2) The anti-vanillin monoclonal antibody provided by the present invention has high sensitivity and good specificity, and its 50% inhibitory concentration IC 50 The cross-reaction rate with vanillin structural analogues was low or even no cross-reaction. The cross-reaction rate with ethyl vanillin was 21.65%, and the cross-reaction rate with methyl vanillin was less than 1%.

[0016] (3) The anti-vanillin monoclonal antibody provided by the present invention can be used to determine the content of vanillin. DETAILED DESCRIPTION

[0017] Example 1: Screening of hybridoma cell line VAND8

[0018] 1. Antigen synthesis and animal immunization

[0019] Complete antigen synthesis was performed using vanillin ammonium salt. The specific synthesis steps are as follows: Weigh 1g (approximately 0.005mol) of vanillin amine hydrochloride and dissolve it in 10mL of tetrahydrofuran. Stir and dropwise add 0.6g (approximately 0.006mol) of triethylamine. Stir at room temperature for 45 minutes. Weigh 0.15g (0.0015mol) of maleic anhydride and add it to the reaction mixture. Stir at room temperature for 7-8 hours. TLC (developing solvent: petroleum ether:ethyl acetate = 2:1) indicates the disappearance of the starting material spot. The reaction solution was rotary evaporated to dryness at 40°C, and then 30 mL of water and 50 mL of ethyl acetate were added to the reaction flask. The mixture was magnetically stirred at room temperature for about 10 minutes, and allowed to stand for 15 minutes until the solution separated into two layers. The ethyl acetate layer was rotary evaporated to dryness to obtain a crude white powder of vanillin hapten. The crude product was recrystallized from 10 mL of anhydrous ethanol, namely, the vanillin artificial hapten (2Z)-4-[(4-hydroxy-3-methoxybenzyl)amino]-4-oxobut-2-enoic acid, with a molecular formula of C 12 H 13 NO5, relative molecular mass is 251.2.

[0020] Weigh 200 mg of VAN hapten powder (approximately 0.8 mmol) into a reaction flask and dissolve it in 5 mL of DMF. Weigh 35 mg (approximately 0.5 mmol) of hydroxylamine hydrochloride into the flask. Reflux the mixture in an oil bath for 20 minutes, then cool it for later use. Separately weigh 120 mg (approximately 1 mmol) of NHS and add it to the flask. Stir and react at room temperature for 1 hour. Weigh 200 mg (approximately 1 mmol) of DCC in 2 mL of DMF and add the DCC / DMF solution dropwise to the reaction flask. Stir at room temperature in the dark for 6 hours and then stand at 4°C in the dark overnight. Centrifuge at 8000 rpm for 5 minutes. The supernatant active ester solution is added dropwise to 30 mL of 7 mg / mL BSA solution in 0.05 mol / L carbonate buffer (pH 9.6). The reaction is allowed to proceed at room temperature in the dark for 6 hours under magnetic stirring. The reaction solution was placed in a dialysis bag and dialyzed in 0.01 mol / L pH 7.4 PBS at 4°C with stirring. The dialysis solution was changed every 4 hours for a total of 48 hours. After the dialysis, the vanillin artificial complete antigen immune antigen VAN-BSA was obtained.

[0021] Three 6-week-old female BALB / c mice were purchased and immunized with the complete antigen VAN-BSA. For the first immunization, the complete antigen VAN-BSA was mixed with an equal volume of Freund's complete adjuvant and emulsified, then injected subcutaneously at multiple points on the back of the mouse's neck. The second immunization was performed 3 weeks after the first immunization. The complete antigen VAN-BSA was emulsified with an equal volume of Freund's incomplete adjuvant and injected subcutaneously at multiple points on the back of the mouse's neck. The third immunization was performed 2 weeks after the second immunization, using the same immunization method and dose as the second immunization. The fourth immunization was performed 2 weeks after the third immunization, using the same immunization method and dose as the second immunization, with each immunization dose of 100 μg per mouse. One week after the third immunization, blood was collected from the tail vein, serum was separated, and the mouse serum antibody titer was monitored using indirect ELISA. One week after the fourth immunization, blood was collected from the tail vein and serum was separated. The mouse serum antibody titer was monitored by indirect ELISA, and the mouse serum sensitivity was determined by indirect competitive ELISA. Mice corresponding to sera with relatively high titer and sensitivity were selected for the final booster immunization. The immunization dose was twice that of the previous one. VAN-BSA antigen was diluted to 500 μg / mL with 0.01 mol / L PBS, and 0.2 mL was injected intraperitoneally into each mouse.

[0022] 2. Cell Fusion

[0023] (1) Preparation of myeloma cells (SP2 / 0)

[0024] First, remove the SP2 / 0 myeloma cells from liquid nitrogen for resuscitation. Quickly place the cryovial containing SP2 / 0 myeloma cells in a 37°C water bath and shake continuously to thaw quickly. Then take it to the clean bench and pour it into a 50mL centrifuge tube containing 15mL RPMI-1640 basal culture medium. Gently mix and centrifuge at 1000r / min for 5 minutes. Discard the supernatant. Then resuspend the myeloma cells in 30mL RPMI-1640 complete culture medium and transfer them to a cell culture flask for culture. Culture in a 37°C 5% CO2 constant temperature incubator for 3-5 days. When the myeloma cells have almost filled the entire culture flask and are in good condition, collect the myeloma cells into a centrifuge tube, centrifuge at 1000r / min for 5 minutes, resuspend the cells in 10mL RPMI-1640 basal culture medium and count them. The number of SP2 / 0 myeloma cells is approximately 2-5×10 7 indivual.

[0025] (2) Preparation of immune spleen cells

[0026] After boosting, mice were sacrificed by dislocation and soaked in 75% alcohol for 5 minutes. The mice were placed in a laminar flow hood, facing upwards, with their limbs secured to a plastic foam board covered in sterile newspaper using syringe needles. The mouse peritoneum was cut open with medical scissors, and the spleen was removed using forceps. The isolated spleen was placed in a dish pre-filled with RPMI-1640-based culture medium. A 2 mL syringe was used to evenly puncture the spleen with the needle. The spleen was then gently triturated with the syringe plunger to release splenocytes from the outer membrane. Fat and connective tissue were removed by filtering through a 300-mesh sieve. The RPMI-1640-based culture medium containing the dispersed splenocytes was filtered into a centrifuge tube and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded. The cells were resuspended in 40 mL of RPMI-1640-based culture medium and centrifuged again at 1000 rpm for 5 minutes. This process was repeated three times to wash away red blood cells and connective tissue. Finally, the spleen cells were resuspended in 10 mL RPMI-1640 medium and counted. The number of spleen cells was about 2 to 5 × 10 8 indivual.

[0027] (3) Cell fusion

[0028] Mix the above-mentioned myeloma cells and spleen cells at a ratio of 1:5-10 and centrifuge at 1000 rpm for 5 minutes. Gently discard the supernatant and place the tube upside down on a square of absorbent paper to remove any liquid around the tube opening. Gently insert a strip of absorbent paper into the tube to remove any remaining liquid from the tube walls and bottom. Immerse the bottom of the centrifuge tube in a 37°C water bath and incubate for 1 minute. Slowly add 1 mL of 1500 PEG to the mixed cells, gently stirring to disperse cell clumps. Keep the centrifuge tube in a 37°C water bath throughout the process. Continue to gently shake the centrifuge tube for 1 minute after adding PEG. Then, slowly add 1 mL of RPMI-1640 basal medium incubated at 37°C over 1 minute while stirring. Then, slowly add 3 mL of RPMI-1640 basal medium incubated at 37°C over 3 minutes while stirring. Continue to slowly add 15 mL of RPMI-1640 basal medium incubated at 37°C while stirring. After additions are complete, cover the plate and gently invert several times to mix thoroughly. Finally, place the plate in a 37°C incubator and let it rest for 5 minutes. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, resuspend in 15 mL of RPMI-1640 basal medium, and centrifuge again at 1000 rpm for 5 minutes. Discard the supernatant to remove the PEG. Finally, gently resuspend the fused cells in 150 mL of HAT medium. Add 150 μL of HAT medium containing the fused cells to a 96-well cell culture plate and incubate in a 37°C, 5% CO2 incubator.

[0029] 3. Screening and cloning of cell lines

[0030] Around day 12 after cell fusion, when the cell colonies have grown to occupy 1 / 2 of the area at the bottom of the well and the culture medium turns yellow, antibody detection can be performed. The culture wells where hybridoma cells grow are screened using the ELISA method. The screening is carried out in two steps. The first step is to use the indirect ELISA method to screen the positive wells that are resistant to vanillin but not to the carrier protein BSA. The second step is to use the indirect competitive ELISA method to detect the positive wells screened in the first step, using vanillin as the competitor, and selecting wells with higher absorbance and sensitivity (higher absorbance means that the final measured value of the well with zero competitor, i.e., the negative control well, is higher, and higher sensitivity means that the competitor concentration at which the inhibition rate is 50%, i.e., the IC 50 The value was small), and the hybridoma cell line VAND8 was obtained by limiting dilution method for cloning. The same two-step method was used for detection about 10 days after cloning. After repeating the cloning 2-3 times, the hybridoma cell line VAND8 was obtained.

[0031] Example 2: Determination of variable region sequence of anti-vanillin monoclonal antibody:

[0032] (1) Hybridoma culture. After cell recovery, the hybridoma cell line VAND8 was cultured and the cell number was expanded to about 1x10 7When the cells were centrifuged at 1000 r / min for 5 min, the cells were collected. (2) Cell RNA was extracted. Under a clean bench environment, 1 mL of Trizol reagent was added to the centrifuged cells, allowed to stand for 5 min, 2 mL of chloroform was added, and the cells were shaken vigorously for 15 s. The cells were allowed to stand for 3 min at room temperature, and centrifuged at 12000 r / min for 15 min. The upper aqueous layer was transferred to a new EP tube, 0.5 mL of isopropanol was added, and the cells were allowed to stand for 10 min at room temperature. The cells were centrifuged at 12000 r / min for 10 min. The supernatant was discarded, 1 mL of 75% ethanol was added, and the cells were centrifuged at 7500 r / min for 5 min. The precipitate was dried and 50 μL of double-distilled water was added. The purity was determined by agarose electrophoresis and the precipitate was quantified. The cells were stored at -70°C for later use. (3) Reverse transcription was performed to prepare cDNA. 1 μL of total cell RNA, 6 μL of RNaseFree ddH2O, 0.5 μL of oligo dT Primer, 10.5 μL of PRIME Script RTEnzyme Mix, 2 μL of 5x Prime Script Buffer, mix well, incubate at 37°C for 15 min, and then at 85°C for 5 s. (4) Amplify cDNA. Amplify the above cDNAs using the mouse IgG VH VL primer library. 10 μL of 5x Prime Star Buffer, 4 μL of dNTPs, 1 μL of cDNA, 1 μL of upstream primer, 1 μL of downstream primer, 0.5 μL of PrimeSTAR, add water to 50 μL, and perform PCR reaction according to the following reaction conditions: incubate at 94°C for 5 min, denature at 94°C for 45 s, anneal at 63°C for 45 s, extend at 72°C for 1 min, and then extend at 72°C for 10 min after 30 cycles. (5) Agarose gel electrophoresis and gel recovery. The PCR products were subjected to agarose gel electrophoresis, and the amplified products with a molecular weight of 250-350 bp were sent for sequencing by Wuhan Dai'an Technology Co., Ltd.

[0033] The heavy chain sequencing results are:

[0034]

[0035]

[0036] As shown in SEQ ID No.1.

[0037] Heavy chain variable region sequence:

[0038]

[0039] As shown in SEQ ID No. 3, wherein:

[0040] CDR1:GFIFSTYY

[0041] CDR2:IHGNGNSI

[0042] CDR3:VRHDGYYVDHAMDC

[0043] The light chain sequencing results are:

[0044]

[0045] As shown in SEQ ID No. 2

[0046] Light chain variable region sequence:

[0047]

[0048] As shown in SEQ ID No. 4, wherein:

[0049] CDR1:QRIVHVNGNTY

[0050] CDR2:KVS

[0051] CDR3:FQGSHVPRT

[0052] The resulting gene sequence encoding the heavy chain variable region is 363 bp long, as shown in SEQ ID NO: 1. Based on the obtained gene sequence, it was deduced that the heavy chain variable region encoded by this gene sequence consists of 121 amino acids, as shown in SEQ ID NO: 3. The light chain variable region encoding gene sequence is 336 bp long, as shown in SEQ ID NO: 2. Based on the obtained gene sequence, it was deduced that the light chain variable region encoded by this gene sequence consists of 112 amino acids, as shown in SEQ ID NO: 4.

[0053] Example 3: Preparation, purification, subtype and characterization of anti-vanillin monoclonal antibodies

[0054] The hybridoma cell line VAND8 was injected into BALB / c mice pretreated with Freund's incomplete adjuvant. Ascites fluid was collected and purified to obtain anti-vanillin monoclonal antibodies. The ascites fluid was filtered through double-layer filter paper and centrifuged at 12,000 rpm for at least 15 minutes at 4°C. The supernatant was aspirated and mixed with three volumes of acetate buffer. The pH was adjusted to 4.5-4.8 with 2 mol / L HCl. Then, 33 μL of octanoic acid per mL of ascites was slowly added with stirring. The mixture was stirred at room temperature for 30 minutes and allowed to stand at 10°C for at least 4 hours. After standing, the mixture was centrifuged at 12,000 rpm for 30 minutes at 4°C. The precipitate was discarded and the supernatant was filtered through double-layer filter paper. The antibody filtrate was concentrated by ultrafiltration in an ice bath. The IgG content of the concentrate was determined and then freeze-dried under vacuum. The lyophilized powder was collected to obtain anti-vanillin monoclonal antibody powder.

[0055] The acetate buffer solution is formulated as follows: 0.29 g sodium acetate, 0.141 mL acetic acid, and pure water to 100 mL.

[0056] The 0.01 mol / L phosphate buffer (PBS) is composed of 0.8 g sodium chloride, 0.29 g disodium hydrogen phosphate dodecahydrate, 0.02 g potassium chloride, and 0.02 g potassium hydrogen phosphate, and water is added to make the volume to 1000 mL.

[0057] The titer of VAND8 mouse ascites antibody was measured by conventional non-competitive enzyme-linked immunosorbent assay (ELISA) method, which was up to 3.2×10 5 , i.e. mouse ascites antibody dilution 3.2×10 5 The sensitivity of the conventional indirect competitive ELISA method to vanillin was 22.1 μg / mL, the cross-reaction rate with ethyl vanillin was 21.65%, and the cross-reaction rate with methyl vanillin was less than 1%.

[0058] The subtype of the anti-vanillin monoclonal antibody secreted by the hybridoma cell line VAND8 was identified as IgG2a using a commercially available subtype identification kit.

[0059] It should be understood that those skilled in the art can make improvements or changes based on the above description, and all such improvements and changes should fall within the scope of protection of the appended claims of the present invention.

Claims

1. A hybridoma cell line VAND8, characterized in that: It is deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO.C2023186.

2. Anti-vanillin monoclonal antibody, characterized in that: The heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 3; the light chain variable region has the amino acid sequence shown in SEQ ID NO:

4.

3. Use of the anti-vanillin monoclonal antibody according to claim 2 in vanillin determination.

4. The method for preparing the anti-vanillin monoclonal antibody according to claim 2, characterized in that: The hybridoma cell line VAND8 according to claim 1 is injected into BALB / c mice that have been treated with Freund's incomplete adjuvant in advance, and the ascites of the mice is collected and purified to obtain anti-vanillin monoclonal antibodies.

Images