Hybridoma cell strain VAND8, vanillin-resistant monoclonal antibody generated by hybridoma cell strain VAND8 and application

Abstract

The invention relates to a hybridoma cell strain VAND8, an anti-vanillin monoclonal antibody generated by the hybridoma cell strain VAND8 and application, and belongs to the field of immunochemistry. The hybridoma cell strain VAND8 provided by the invention can be used for preparing a high-titer vanillin-resistant monoclonal antibody, and the titer measured by an enzyme-linked immunosorbent assay (ELISA) method can reach 3.2 * 10 < 5 >; the vanillin-resistant monoclonal antibody provided by the invention is high in sensitivity and good in specificity, the 50% inhibition concentration IC50 on vanillin is 22.1 mu g / mL, and the cross reaction rate with vanillin structural analogues is low or even no cross reaction exists; the method can be used for measuring the content of vanillin.

Description

Technical Field

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

[0002] Vanillin is a common food additive as a flavor. Although it is not toxic, excessive consumption can harm human health. According to a report by the European Union Expert Committee on February 24, 2000, large amounts of vanillin and other food additives can cause headaches, nausea, vomiting, breathing difficulties, and even liver and kidney damage. The maximum daily intake of vanillin is limited to 10 mg / kg by the Joint Expert Committee of the Food and Agriculture Organization (FAO) and the World Health Organization (WHO). The "National Food Safety Standard for the Use of Food Additives" (GB2760-2014) also stipulates the use limit of vanillin in food. [2] In China, food flavors and spices are not allowed to be added to pasteurized milk, sterilized milk, fermented milk, and cream. No edible spices are allowed to be added to infant formula for 0 to 6 months. The maximum amount of vanillin used in formula for older infants and toddlers is 5 mg / 100 mL, and the maximum amount of vanillin used in cereal-based complementary foods for infants and toddlers is 7 mg / 100 g. Therefore, the use of vanillin in food needs to be strictly regulated, and dairy products, baked goods, beverages, and edible oils are the focus of vanillin content testing.

[0003] Existing literature reports that the detection methods of vanillin in food include high performance liquid chromatography, high performance liquid-tandem mass spectrometry, gas chromatography, ultraviolet spectrophotometry, etc. These methods are highly sensitive and have accurate test results, but the required instruments and equipment are expensive and the sample pretreatment process is cumbersome. Immunoassay is a qualitative and quantitative detection of ultra-trace residues using the specific binding reaction of antigens and antibodies and the biological, physical or chemical amplification of markers on antibodies and antigens. Relatively speaking, immunoassay technology is low in cost and can be used for rapid detection. However, to study and establish any immunological detection technology for vanillin, high-quality anti-vanillin antibodies must be obtained first. 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 VAND8 and an application thereof 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 has been 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 list; and the light chain variable region has the amino acid sequence shown in SEQ ID NO:4 in the sequence list. It is secreted and produced by the hybridoma cell line VAND8 with a deposit number of CCTCC NO.C2023186. The anti-vanillin monoclonal antibody can recognize vanillin and has a 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%.

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

[0009] The specific preparation steps of the above 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, with a molecular formula of C 12 H 13NO5, with a relative molecular mass of 251.2; the active ester method was used to synthesize the complete vanillin antigen VAN-BSA: the above hapten was subjected to 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, the steps of which are as follows: injecting the obtained hybridoma cell line VAND8 into BALB / c mice that have been 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 the 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 the 50% inhibition concentration IC of vanillin is 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] The complete antigen synthesis was performed using vanillin ammonium salt, and the specific synthesis steps were as follows: 1 g (about 0.005 mol) of vanillin amine hydrochloride was weighed and dissolved in 10 mL of tetrahydrofuran, and 0.6 g (about 0.006 mol) of triethylamine was added dropwise with stirring, and stirred at room temperature for 45 min. 0.15 g (0.0015 mol) of maleic anhydride was weighed and added to the above reaction solution, and the reaction was stirred at room temperature for 7 to 8 hours, and the raw material point disappeared when monitored by TLC (developing solvent is petroleum ether: ethyl acetate = 2:1). After the reaction solution was dried by rotary evaporation at 40°C, 30 mL of water and 50 mL of ethyl acetate were added to the reaction bottle, and the mixture was magnetically stirred at room temperature for about 10 min. The mixture was allowed to stand for 15 min until the solution was separated into two layers. The ethyl acetate layer was rotary evaporated and dried to obtain a crude white powder of vanillin hapten. The crude product was recrystallized from 10 mL of anhydrous ethanol, i.e., the artificial vanillin 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 (about 0.8 mmol) into a reaction bottle, add 5 mL of DMF to dissolve, weigh 35 mg (about 0.5 mmol) of hydroxylamine hydrochloride into the reaction bottle. Reflux in an oil bath for 20 min, then cool for use. Weigh 120 mg (about 1 mmol) of NHS into the reaction bottle, stir at room temperature for 1 h, weigh 200 mg (about 1 mmol) of DCC in 2 mL of DMF, add the DCC / DMF solution dropwise to the above reaction bottle, stir at room temperature in the dark for 6 h, and stand at 4 ° C in the dark overnight. Centrifuge at 8000 r / min for 5 min, take the supernatant active ester solution and add it dropwise to 30 mL of 7 mg / mL BSA solution for reaction, and the reaction buffer is 0.05 mol / L pH 9.6 carbonate buffer. The reaction is stirred at room temperature in the dark for 6 h 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, and the dialysis solution was replaced 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 complete antigen VAN-BSA. For the first immunization, the complete antigen VAN-BSA was mixed with an equal amount of Freund's complete adjuvant and emulsified, and 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, using Freund's incomplete adjuvant and an equal volume of complete antigen VAN-BSA emulsified and injected subcutaneously at multiple points on the back of the mouse's neck. The third immunization was 2 weeks apart from the second immunization, and the immunization method and dose were the same as the second. The fourth immunization was performed 2 weeks after the third immunization, and the immunization method and immunization dose were the same as the second immunization, and each immunization dose was 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 by indirect ELISA. One week after the fourth immunization, blood was collected from the tail vein and serum was separated. The indirect ELISA method was used to monitor the mouse serum antibody titer, and the indirect competitive ELISA method was used to determine the mouse serum sensitivity. 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. The 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, take out the SP2 / 0 myeloma cells from liquid nitrogen for resuscitation. Quickly place the cryopreserved tube containing SP2 / 0 myeloma cells in a 37°C water bath and shake it continuously to thaw it quickly. Then take it to the clean bench and pour it into a 50mL centrifuge tube containing 15mL PRMI-1640 basal culture medium. Gently mix it and centrifuge it at 1000r / min for 5 minutes, and discard the supernatant. Then resuspend the myeloma cells with 30mL RPMI-1640 complete culture medium and transfer them to a cell culture bottle for culture. Culture in a 37°C 5% CO2 constant temperature incubator for 3 to 5 days. When the myeloma cells almost fill the entire culture bottle and are in good condition, the myeloma cells can be collected into a centrifuge tube, centrifuged at 1000r / min for 5 minutes, and then resuspend the cells with 10mL RPMI-1640 basal culture medium and count them. The number of SP2 / 0 myeloma cells is approximately 2 to 5×10 7 indivual.

[0025] (2) Preparation of immune spleen cells

[0026] After the boosted immunization mice were killed by dislocation, they were soaked in 75% alcohol for 5 minutes. The mice were moved to the clean bench, with the abdomen facing up, and the limbs were fixed on the plastic foam board covered with sterilized newspapers with syringe needles. The mouse peritoneum was cut open with medical scissors, and the mouse spleen was peeled off and removed with tweezers. The separated spleen was placed in a plate with RPMI-1640 base culture medium added in advance, a 2mL syringe was taken, and the spleen was evenly pierced with a syringe needle, and then gently ground with the inner core of the syringe to release the spleen cells from the spleen outer membrane. The fat and connective tissue were filtered through a 300-mesh screen, and the RPMI-1640 base culture medium with dispersed spleen cells was filtered into a centrifuge tube, centrifuged at 1000r / min for 5min, and the supernatant was discarded. Resuspended in 40mL RPMI-1640 base culture medium and centrifuged again at 1000r / min for 5min, repeated three times, and the red blood cells and connective tissue in the cells were washed away. 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 myeloma cells and spleen cells at a ratio of 1:5-10, centrifuge at 1000r / min for 5 minutes, pour out the supernatant gently, turn it upside down on a square absorbent paper to remove the liquid around the tube mouth, and then gently insert the strip of absorbent paper into the tube to absorb the remaining liquid on the tube wall and the bottom of the tube. Immerse the bottom of the centrifuge tube in a 37℃ water bath, incubate for 1 minute, and slowly add 1mL 1500PEG to the mixed cells, stirring gently while adding to disperse the cell clusters. The centrifuge tube is placed in a 37℃ water bath environment during the whole process. After adding PEG, continue to gently shake the centrifuge tube for 1 minute. Then slowly add 1mL of RPMI-1640 basal medium incubated at 37℃ within 1min, stirring while adding. Then slowly add 3mL of RPMI-1640 basal medium incubated at 37℃ within 3min, stirring while adding. Continue to slowly add 15mL of RPMI-1640 basal medium incubated at 37℃, stirring gently while adding. After adding, cover the lid, gently invert and mix several times, and finally put it in a 37℃ incubator and let it stand for 5min. Centrifuge at 1000r / min for 5min, pour out the supernatant, resuspend with 15mL RPMI-1640 basal culture solution, centrifuge again at 1000r / min for 5min, pour out the supernatant and wash off the PEG. Finally, gently resuspend the fused cells in 150mL of HAT medium. Add the HAT medium containing fused cells to a 96-well cell culture plate, 150μL per well, and place it in a 37℃ 5% CO2 constant temperature incubator for culture.

[0029] 3. Screening and cloning of cell lines

[0030] Around the 12th day after cell fusion, when the cell colonies grow to occupy 1 / 2 of the area at the bottom of the well and the culture fluid 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 a competitor of 0 is higher, that is, the negative control well, and higher sensitivity means that the competitor concentration when the inhibition rate is 50%, that is, the IC 50 The value was small), and the limiting dilution method was used 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) Cultivate hybridomas. After cell recovery, the hybridoma cell line VAND8 was cultured until the cell number expanded to about 1x10 7When the cells were centrifuged at 1000r / min for 5min, they were collected. (2) Extracting cellular RNA. Under a clean bench environment, 1mL of Trizol reagent was added to the centrifuged cells, allowed to stand for 5min, 2mL of chloroform was added, and the mixture was shaken vigorously for 15s. The mixture was allowed to stand for 3min at room temperature, and centrifuged at 12000r / min for 15min. The upper aqueous layer was transferred to a new EP tube, and 0.5mL of isopropanol was added. The mixture was allowed to stand for 10min at room temperature. Centrifuged at 12000r / min for 10min. The supernatant was discarded, 1mL of 75% ethanol was added, and the mixture was centrifuged at 7500r / min for 5min. The precipitate was dried and 50μL of double distilled water was added. The purity was identified and quantified by agarose electrophoresis, and the mixture was stored at -70℃ for later use. (3) Reverse transcription was used to prepare cDNA. Total cell RNA 1μL, RNaseFree ddH2 0 6μL, oligo dT Primer 0.5μL, PRIME Script RTEnzyme Mix 10.5μL, 5xPrime Script Buffer 2μL, mix well, 37℃15min, 85℃, 5s. (4) Amplify cDNA. Use mouse IgG VH VL primer library to amplify the above cDNAs respectively. 5x Prime Star Buffer 10μL, dNTP 4pL, cDNA 1μL, upstream primer 1pL, downstream primer 1μL, PrimeSTAR 0.5μL, add water to 50μL, and perform PCR reaction according to the following reaction conditions: incubate at 94℃ for 5min, denature at 94℃ for 45s, anneal at 63℃ for 45s, extend at 72℃ for 1min, and extend at 72℃ for 10min after 30 cycles. (5) Agarose gel electrophoresis and gel recovery. The PCR products were subjected to agarose gel electrophoresis, and the electrophoresis results were observed. The amplified products with a molecular weight of 250-350 bp were sent for sequencing. The sequencing was performed 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 obtained gene sequence results showed that the heavy chain variable region encoding gene sequence was 363 bp long, and the sequence was shown in SEQ ID NO: 1. According to the obtained gene sequence, it was deduced that the heavy chain variable region encoded by the gene sequence consisted of 121 amino acids, and the sequence was shown in SEQ ID NO: 3. The light chain variable region encoding gene sequence was 336 bp long, and the sequence was shown in SEQ ID NO: 2. According to the obtained gene sequence, it was deduced that the light chain variable region encoded by the gene sequence consisted of 112 amino acids, and the sequence was shown in SEQ ID NO: 4.

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

[0054] The obtained hybridoma cell line VAND8 was injected into BALB / c mice treated with Freund's incomplete adjuvant in advance, and the ascites of the mice was collected, and then the ascites was purified to obtain anti-vanillin monoclonal antibodies. The specific operation is: the mouse ascites is filtered with double-layer filter paper, the filtered ascites is centrifuged at 4°C, 12000r / min for more than 15min, the supernatant is aspirated, the supernatant is mixed with 3 times the volume of acetate buffer, and the pH is adjusted to 4.5-4.8 with 2mol / L HCl, and then octanoic acid is slowly added while stirring. The volume of octanoic acid required for each milliliter of ascites is 33μL. After adding, stir at room temperature for 30min and stand at 10°C for more than 4h. After standing, centrifuge at 4°C, 12000r / min for 30min, discard the precipitate and take the supernatant to filter with double-layer filter paper, and the filtered antibody filtrate is concentrated by ultrafiltration membrane in an ice bath. After determining the IgG content of the concentrate, the concentrate is freeze-dried in a vacuum and the lyophilized powder is 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 measured by conventional non-competitive enzyme-linked immunosorbent assay (ELISA) was 3.2×10 5 , i.e. mouse ascites antibody dilution 3.2×10 5 The result of the solution was positive. The sensitivity of the vanillin was 22.1 μg / mL by conventional indirect competitive ELISA method, 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 these 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 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 the determination of vanillin.

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

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