CN104762267A - Hybridoma cell AFB1-2A4 and aflatoxin B1 monoclonal antibody produced by hybridoma cell AFB1-2A4 - Google Patents

Abstract

The invention provides a hybridoma cell strain 2A4, an anti-aflatoxin B1 monoclonal antibody secreted by the hybridoma cell strain 2A4 and a use of the anti-aflatoxin B1 monoclonal antibody. The hybridoma cell strain 2A4 can be used for preparation of a high-titer aflatoxin antibody. The anti-aflatoxin B1 monoclonal antibody has titer of 3.12*10<6> detected by a mouse hydroperitoneum antibody enzyme-linked immunoadsorption assay (ELISA) method, has high sensitivity and aflatoxin B1 half-inhibitory concentration (IC50) of 33.2pg/mL, can be used for detecting aflatoxin B1 content and has a large practical application value.

Description

Hybridoma AFB1-2A4 and the Aflatoxin B1 Monoclonal Antibody It Produced

technical field

The invention relates to a hybridoma cell line AFB1-2A4 and the aflatoxin B1 monoclonal antibody produced therefrom.

Background technique

Aflatoxin (AF) is a highly toxic substance that has been classified as a Class 1 carcinogen by the Cancer Research Institute of the World Health Organization. Aflatoxins are metabolites of toxin-producing strains of Aspergillus flavus and Aspergillus parasiticus, and are commonly found in moldy grains, feeds and their products. Aflatoxin B1, B2, G1 and G2 are the main forms of aflatoxin in grain and food, among which aflatoxin B1 is the most toxic and carcinogenic. After aflatoxin B1 enters the human body, its main target organ is the liver, and the symptoms of acute poisoning are nausea, vomiting, gastrointestinal hemorrhage and death. Chronic poisoning mainly causes liver cancer, and can also induce bone cancer, kidney cancer, and rectal cancer.

At present, the commonly used instrument methods for detecting aflatoxin include thin-layer chromatography, high performance liquid chromatography, and micro-column chromatography. These methods have high measurement accuracy and good repeatability, but they often need to be equipped with large and expensive equipment, which is expensive and requires high requirements for operators. They are generally suitable for large-scale laboratories in national or regional research institutes or scientific research units. application. ELISA combines the high specificity of antigen-antibody reaction and the high sensitivity of enzymatic reaction. This method has high sensitivity, which is nearly 200 times higher than that of the thin-layer method; strong specificity, fluorescent substances, pigments, and structural analogues do not interfere with the results; and the recovery rate is high, the extraction method is simple, and qualitative and quantitative determination can be performed. To study and establish any immunological detection technique against aflatoxin B1 (AFB1), it is necessary to obtain anti-aflatoxin B1 (AFB1) antibody.

Contents of the invention

The problem to be solved by the present invention is to provide the monoclonal antibody of hybridoma cell line AFB1-2A4 and the aflatoxin B1 (AFB1) produced therewith

The present invention provides a hybridoma cell line AFB1-2A4, which has been preserved in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Management Committee (CGMCC) on December 12, 2014. The preservation address is Beichen, Chaoyang District, Beijing, China No. 3, No. 1 Yard, West Road, Institute of Microbiology, Chinese Academy of Sciences, the preservation number is CGMCC NO.10200, and the classification is named as S/P20 hybridoma cells.

The anti-aflatoxin B1 monoclonal antibody is secreted and produced by the hybridoma cell line AFB1-2A4 with the deposit number CGMCC NO. 10200. The AFB1 monoclonal antibody can recognize aflatoxin B1, and the 50% inhibitory concentration IC50 for aflatoxin B1 is 33.2 pg/mL.

Application of anti-aflatoxin B1 monoclonal antibody in the determination of aflatoxin B1.

The hybridoma cell line AFB1-2A4 monoclonal antibody provided by the present invention is obtained by screening by a three-step method. The specific steps are: AFB1 is reacted with carboxymethylhydroxylamine hemihydrochloride (CMO) to generate AFB1 oxime, and the EDC and In the presence of NHS, it couples with BSA to generate AFB1-BSA. BLAB/c mice were immunized with it for 4 times, the last booster immunization was with 2 times the dose of AFB1-BSA of the previous immunization, and cell fusion was carried out 3 days after immunization. ELISA was used to screen fusion cells in two steps: the first step was to screen positive clones that were only resistant to aflatoxin B1 but not carrier protein BSA by indirect ELISA; the second step was to screen the first step by indirect competitive ELISA The culture medium of the positive clones was detected, and AFB1 was used as the competitive source, and the positive clones with low absorbance and high sensitivity were selected for limited dilution to continue cloning. After 10 days of cloning, the same two-step screening method was used for antibody detection, and so on for 2 more days. Second, the hybridoma cell AFB1-2A4 obtained by final screening.

The preparation method of the aflatoxin B1 monoclonal antibody provided by the present invention comprises the following steps: inject the obtained hybridoma cell line AFB1-2A4 into BALB/c mice treated with Freund's incomplete adjuvant in advance, and collect the mice The ascites fluid was purified to obtain monoclonal antibody against aflatoxin B1.

The specific operation of the above purification method is as follows: centrifuge the ascites at 4°C, 3000r/min for more than 5min, absorb the supernatant, mix the supernatant with 2 times the volume of acetate buffer, and slowly add n-octanoic acid while stirring. The required volume of n-octanoic acid is 33 μL, mix at room temperature for 30 minutes, and stand at 4°C for more than 2 hours to make it fully precipitate. Then centrifuge at 4°C and 12000r/min for more than 30min, discard the precipitate, filter the obtained supernatant with a sand core funnel, add 1/10 of the filtrate volume of 0.1mol/L pH7.4 phosphate buffer, and use 2mol /L sodium hydroxide solution to adjust the pH to 7.4, pre-cool at 4°C for 1 hour, slowly add 0.277g/mL ammonium sulfate to a final concentration of ammonium sulfate of 45%, let stand at 4°C for more than 2h, and then centrifuge at 12000r/min at 4°C After 30 minutes, discard the supernatant, resuspend the obtained precipitate with 0.01mol/L phosphate buffer saline that is 1/10 of the volume of the original ascitic fluid, put it into a dialysis bag, and dialyze it with 0.01mol/L phosphate buffer saline, and fully dialyze the protein Add an equal volume of glycerin to the solution, and store in a -20°C refrigerator for later use;

Acetate buffer: add 0.29g sodium acetate to 0.141mL acetic acid, dilute to 100mL with pure water;

0.1mol/L phosphate buffer: 0.8g sodium chloride, 0.29g disodium hydrogen phosphate dodecahydrate, 0.02g potassium chloride, 0.02g potassium dihydrogen phosphate, add water to 100mL.

0.01mol/L Phosphate Buffer: Dilute 0.1mol/L Phosphate Buffer 10 times with pure water.

The beneficial effects of the present invention are:

1 The hybridoma cell AFB1-2A4 provided by the present invention can be used to prepare high-titer anti-aflatoxin B1 monoclonal antibody, and the titer of aflatoxin B1 mouse ascites antibody ELISA can reach 3.12×10 ⁶ , that is, when the ascites was diluted to 3.12×10 ⁶ , the result of the solution was still positive.

2 The aflatoxin B1 monoclonal antibody provided by the present invention has high sensitivity and good specificity, the half-inhibitory concentration IC50 for aflatoxin B1 is 33.2 pg/mL, the cross-reactivity rate with aflatoxin B2 is 2.3%, The cross-reactivity rate of toxin G1 was 9.9%, that of aflatoxin G2 was 0.5%, and that of aflatoxin M1 was 1.6%.

3 The monoclonal antibody against aflatoxin B1 provided by the present invention can be used to determine the content of aflatoxin B1.

Description of drawings

Figure 1 AFB1-2A4 monoclonal antibody detection standard curve for AFB1.

detailed description

Embodiment 1: Screening of hybridoma cell line AFB1-2A4

1. Antigen synthesis and animal immunization

AFB1 is oximated to obtain AFB1 oxime, and then undergoes a coupling reaction with a carrier protein in the presence of EDC and NHS to obtain the corresponding artificial antigen.

Synthesis of AFB1 oxime

Dissolve 1.5 mg of AFB1 and 3 mg of CMO in 2 mL of methanol-pyridine (1:1) solution, and react at 70°C for 5 hours. After the reaction, the reaction solution was evaporated to dryness, and the residue was dissolved with double distilled water. Adjust the pH of the solution to 3.0 and extract AFB1O 3 times with 3 mL of ethyl acetate. The ethyl acetate layers were combined, evaporated to dryness, and redissolved with 0.5 mL DMF. AFB1 oxime solution was obtained.

Synthesis of Artificial Antigens

Take 0.45mL AFB1O, add 30mg EDC and 8.64mg NHS, then add 1.5mL DMF, and react overnight at 30°C. The reaction solution was reacted with 12.5mg BSA dissolved in 3mL 0.1M PBS at room temperature for 2 hours. After the reaction was completed, centrifuge at 3000rpm for 10 minutes, and the supernatant was dialyzed 3 times with 2L PBS, each time for one day. Finally, the liquid in the dialysis bag was collected and stored at -20°C.

Identification of artificial antigens

The identification by ultraviolet spectrum scanning and mass spectrometry indicated that the complete antigen AFB1-BSA of aflatoxin B1 was prepared.

Six 6-week-old female BALB/c mice were purchased and immunized with the complete antigen AFB1-BSA of aflatoxin B1 synthesized by themselves. For the first immunization, the complete antigen AFB1-BSA was mixed and emulsified with an equal amount of Freund's complete adjuvant, and then injected subcutaneously at multiple points on the back of the mouse. The second immunization was carried out 3 weeks after the first immunization, using Freund's incomplete adjuvant to emulsify with an equal volume of complete antigen AFB1-BSA, and the immunization dose and method were the same as the first immunization. The interval between the third immunization and the second immunization was 3 weeks, and the dose and method of immunization were the same as the second time. The fourth immunization was carried out 3 weeks after the third immunization, and the immunization method and dose were the same as the third immunization, and the dose of each immunization was 30 μg per mouse. One week after each immunization for the first three times, blood was collected around the eyes, serum was separated, and the antibody titer of mouse serum was monitored by indirect ELISA method. Three days after the fourth immunization, blood was collected from the tail vein, serum was separated, and the antibody titer of mouse serum was monitored by indirect ELISA method, and the sensitivity of mouse serum was measured by indirect competitive ELISA method, and the corresponding serum with relatively high titer and sensitivity was selected. The last booster immunization was carried out in the mice, and the immunization dose was 2 times that of the previous one.

2. Cell Fusion

Three days after the last booster immunization, 50% (weight percent) polyethylene glycol (PEG, molecular weight 1460) was used as a fusion agent, and cell fusion was carried out according to conventional methods. Specific steps are as follows:

The BALB/c mice were killed by decapitation and soaked in 75% ethanol. After about 3 min, put it into the ultra-clean bench. Use tweezers to lift the abdominal skin of the mouse, cut a small opening with surgical scissors (be careful not to cut the peritoneum), and then use tweezers to tear the skin from the head and tail to fully expose the peritoneum. Use clean scissors to cut open the peritoneum, take out the spleen, wash it once in a plate filled with 10mL incomplete culture medium, and peel off the connective tissue. Transfer the spleen to another plate containing 5 mL of incomplete culture medium, take a copper mesh, place the spleen on the copper mesh, grind the spleen with the inner core of the syringe, and wash the copper mesh with the incomplete culture medium in the plate to make The splenocytes all pass through the mesh into solution. Transfer the splenocyte solution into a 50 ml centrifuge tube, add incomplete culture medium to 30 mL, and mix well. Centrifuge at 1000 rpm for 5 min and discard the supernatant. The pelleted cells were centrifuged again with incomplete culture medium, then the cells were suspended in 10 ml of complete culture medium and mixed well. Cell count, spare.

Mix myeloma cells and splenocytes at a ratio of 1:10, wash once with DMEM medium in a 50 mL centrifuge tube, and centrifuge at 1200 rpm for 8 min; discard the supernatant, and suck up the residual liquid with a pipette. Fusion with 50% PEG, resuspended in DMEM medium containing 20% bovine serum and 1% HAT. Add the above-mentioned cells to a 96-well plate with a feeder cell layer, and add 100 μL to each well. Place the culture plate in a 37°C, 5% CO2 incubator. DMEM medium is DMEM basal medium containing 2% (weight percent) growth factors. DMEM basal medium was purchased from Hyclone Company, and 1% HAT (hypoxanthine-aminopterin-thymidine) was purchased from Gibco Company.

3. Screening and cloning of cell lines

About 10 days after cell fusion, the cell colony grows to occupy 1/2 of the area of the bottom of the well, and then the antibody detection can be performed. Use the ELISA method to screen the culture wells with hybridoma cell growth. 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 aflatoxin B1 but not the carrier protein BSA; Competitive ELISA method was used to detect the positive wells screened in the first step, using aflatoxin B1 as the competitive source, and selecting wells with higher absorbance and sensitivity (a higher absorbance value refers to the wells with a competitive source of 0, i.e. negative control wells The final measured value is higher, and the higher sensitivity refers to the concentration of the competitive source when the inhibition rate is 50%, that is, the IC 50 value is smaller), the hybridoma cells were cloned by the limiting dilution method, and the same two samples were used about 10 days after cloning. The detection was carried out by step method, and the hybridoma cell line AFB1-2A4 was obtained after repeating the cloning twice.

Example 2: Preparation, purification and identification of antibodies

The obtained hybridoma cell strain AFB1-2A4 obtained in Example 1 was injected into BALB/c mice treated with Freund's incomplete adjuvant in advance, the ascites of the mice was collected, purified and treated to obtain anti-aflatoxin B1 of monoclonal antibodies.

The specific operation is as follows: centrifuge the ascites at 3000r/min at 4°C for more than 5 minutes, draw the supernatant, mix the supernatant with 2 times the volume of acetate buffer, and slowly add n-octanoic acid while stirring, the n-octanoic acid required per milliliter of ascites The volume is 33 μL, mix at room temperature for 30 min, and stand at 4 °C for 2 h to fully precipitate. Then centrifuge at 12000r/min at 4°C for 30min, discard the precipitate, filter the obtained supernatant with a sand core funnel, add 0.1mol/L pH7.4 phosphate buffer of 1/10 volume of the filtrate, and use 2mol/L Adjust the pH to 7.4 with sodium hydroxide solution, pre-cool at 4°C for 1 hour, slowly add 0.277g/mL ammonium sulfate until the final concentration of ammonium sulfate is 45%, let stand at 4°C for 2h, centrifuge at 12000r/min for 30min at 4°C, discard clear, resuspend the obtained precipitate with 0.01mol/L phosphate buffer saline that was 1/10 of the volume of the original ascitic fluid, put it into a dialysis bag, dialyze with 0.01mol/L phosphate buffer saline, add the fully dialyzed protein solution into an equal volume Glycerin, stored in -20°C refrigerator for later use;

Acetate buffer: add 0.29g sodium acetate to 0.141mL acetic acid, dilute to 100mL with pure water;

0.1mol/L phosphate buffer: 0.8g sodium chloride, 0.29g disodium hydrogen phosphate dodecahydrate, 0.02g potassium chloride, 0.02g potassium dihydrogen phosphate, add water to 100mL.

0.01mol/L Phosphate Buffer: Dilute 0.1mol/L Phosphate Buffer 10 times with pure water.

The titer of 2A4 mouse ascites antibody measured by conventional non-competitive enzyme-linked immunosorbent assay (ELISA) method can reach 3.12×10 ⁶ , that is, the assay result is positive when the ascites is diluted to 3.12×10 ⁶ . The half-inhibitory concentration IC50 of aflatoxin B1 identified by conventional indirect competition ELISA was 33.2 pg/mL, the cross-reaction rate with aflatoxin B2 was 2.3%, and the cross-reaction rate with aflatoxin G1 was 9.9%. The cross-reactivity rate of aspergillus toxin G2 was 0.5%, and that of aflatoxin M1 was 1.6%.

Embodiment 3: the application of antibody

The anti-aflatoxin B1 monoclonal antibody secreted by the hybridoma cell line AFB1-2A4 was used in the aflatoxin B1 monoclonal ELISA addition and recovery test, and the specific steps were as follows:

(1) Coating: Use a 96-well polystyrene microplate, dilute AFB1-BSA to 1 μg/mL with 0.05mol/L pH 9.6 carbonic acid buffer, and use a multi-channel adjustable pipette (referred to as the discharge gun) to Add 100 μL to each well, cover the microtiter plate with plastic wrap, and place it at 4°C overnight.

(2) Washing the plate: The next day, take the ELISA plate out from 4°C, wash with PBST, 250 μl per well, wash 4 times, and pat dry on a towel after washing.

(3) Sealing: add 200 μL of 1% BSA PBS to each well with a row gun, cover the microplate plate with a lid after adding, and place it at 37°C for 2 hours. Wash with PBST, 250 μl per well, wash 4 times, and pat dry on a towel after washing.

(4) Prepare 0, 0.005, 0.015, 0.045, 0.14, 0.41 μg/L standard solutions of aflatoxin B1 with 10% methanol. Add the standard solution and the extract of the sample to be tested into the sealed microtiter plate, 50mL/well, 3 replicate wells for each sample, and then add 50mL/well of aflatoxin B1 monoclonal antibody diluted to 1:160000 , add 50 mL/well of the enzyme-labeled secondary antibody diluted to 1:4000, gently tap the enzyme-labeled plate and react in a 37°C incubator for 40 minutes.

(5) Plate washing: the operation is the same as above.

(6) Color development: Mix color development solution A and color development solution B at a volume ratio of 1:1, add 100 mL/well of the mixed color development solution with a row gun, and place in a 37°C thermostat to avoid light for 10 minutes.

(7) Determination: Add 2mol/L H ₂ SO ₄ 50mL/well with a discharge gun, gently shake the microplate plate, and use a microplate reader to detect the OD value at the wavelength of 450/630nm.

(8) Addition recovery and sample pretreatment: Weigh 5g sample into a clean 50mL centrifuge tube, add 1, 4, 16ng AFB1 respectively, add 25mL sample extract, cover and shake for 5min, centrifuge at 4000rpm for 5min , take 50mL supernatant, add 950mL sample diluent, mix well and use it as ELISA sample extract to be tested. The direct competition ELISA was used for the addition recovery experiment, and the recoveries were 82.4, 102.3, and 95.7%, respectively.

(9) Solution configuration: Carbonic acid buffer: Weigh 1.59g Na ₂ CO ₃ and 2.93g NaHCO ₃ , add pure water to 990mL, adjust the pH to 9.6, then dilute to 1000mL with pure water, store at 4°C for later use. Phosphate buffer solution (PBS): Dissolve 8.5g NaCl, 2.2g Na ₂ HPO ₄ 12H ₂ O, 0.2g NaH ₂ PO ₄ 2H ₂ O in 900mL pure water, adjust the pH to 7.4, and dilute to 1000mL. PBST: Take 500mL PBS, add 0.25mL Tween 20, mix well and set aside.

Chromogenic Solution A: Dissolve 20mg TMB in 10mL DMSO, store at 4°C, take it out before use and return to room temperature, and dilute to 100mL with pure water.

Chromogenic solution B: 21g citric acid monohydrate, 71.1g Na ₂ HPO ₄ 12H ₂ O, 2g urea hydrogen peroxide, add pure water to 1000mL.

Sample extract: Take 4g NaCl, dissolve it in 30mL water, add 70mL methanol, mix well and set aside.

Enzyme-labeled secondary antibodies were purchased from SoutherBiotech.

Claims (3)

1. hybridoma cell strain AFB1-2A4, is characterized in that: be preserved in China General Microbiological culture presevation administrative center, and deposit number is CGMCC NO. 10200.

2. the aspergillus flavus resisting toxin B1 monoclonal antibody that the hybridoma cell strain AFB1-2A4 described in claim 1 secretes, is characterized in that: it produces secreted by described hybridoma cell strain AFB1-2A4.

3. the application of the aspergillus flavus resisting toxin B1 monoclonal antibody described in claim 2, it is characterized in that the application in aflatoxin B1 analyzing and testing, to aflatoxin B1, there is good avidity and detection sensitivity, for aflatoxin B1 immunodetection in food safety.