CN114214288B - Zearalenone monoclonal antibody hybridoma cell strain and application thereof - Google Patents

Zearalenone monoclonal antibody hybridoma cell strain and application thereof Download PDF

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CN114214288B
CN114214288B CN202111605097.5A CN202111605097A CN114214288B CN 114214288 B CN114214288 B CN 114214288B CN 202111605097 A CN202111605097 A CN 202111605097A CN 114214288 B CN114214288 B CN 114214288B
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zearalenone
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胥传来
李小芳
匡华
徐丽广
孙茂忠
吴晓玲
刘丽强
马伟
朱建平
郝昌龙
宋珊珊
胡拥明
吴爱红
郭玲玲
胥欣欣
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Jiangnan University
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Abstract

The invention belongs to the technical field of immunochemistry, and hasThe body relates to a zearalenone monoclonal antibody hybridoma cell strain and application thereof. The hybridoma cell strain is preserved in China general microbiological culture Collection center (CGMCC) of China Committee for culture Collection of microorganisms, and has a preservation date of 2021, 05 months and 13 days, and a preservation number of CGMCC No.22338. The monoclonal antibody secreted by the cell strain provided by the invention has better specificity and detection sensitivity (IC) to zearalenone 50 The value is 0.057 ng/mL) and a wider linear range (0.01-0.22 ng/mL), can realize the detection of the zearalenone, and particularly can detect the residual quantity of the zearalenone in corn, wheat, barley, oat, paddy and feed, provides a raw material for the immunodetection of the zearalenone residual in food, and has practical application value.

Description

Zearalenone monoclonal antibody hybridoma cell strain and application thereof
Technical Field
The invention belongs to the technical field of immunochemistry, and particularly relates to a zearalenone monoclonal antibody hybridoma cell strain and application thereof.
Background
Zearalenone (ZEN), also known as F-2 toxin, is a secondary metabolite produced by fungi, is a mycotoxin with strong estrogenic action, has strong pollution to corn, wheat, barley, oat, rice and feed, has stable chemical properties, is difficult to decompose at high temperature, can cause acute and chronic poisoning of animals, and causes abnormal reproductive function and even death of animals. Recent studies have shown that ZEN has an effect on reproductive function, also has immunotoxicity, hepatotoxicity and cytotoxicity, and also has a certain effect on the occurrence of tumors.
The zearalenone has stable property and is difficult to decompose at high temperature, is not easy to damage in the food or feed processing process, and seriously threatens the safety of meat production and human health. For this reason, strict control of zearalenone content in food and animal feeds is required, generally requiring a detection limit in the feed of less than 500 μg/kg and a detection limit in the food of less than 60 μg/kg.
The current common methods for detecting zearalenone mainly comprise an instrument analysis method and an immunochemistry detection method. Wherein the instrumental analysis method comprises: gas Chromatography (GC), thin Layer Chromatography (TLC), high Performance Liquid Chromatography (HPLC), high performance liquid chromatography-mass spectrometry (HPLC-MS), and the like. The instrument analysis method has the advantages of good sensitivity, high accuracy, low detection limit, low false positive rate and strong specificity, but the pretreatment of the sample is complex, professional personnel are needed, and the instrument equipment is expensive and is difficult to meet the requirement of on-site rapid detection. Compared with instrument analysis, the immunological detection method has the advantages of simple pretreatment, low cost, convenience, rapidness and simple operation, thereby having unique advantages and great development potential in the aspects of large-scale sample primary screening detection and on-site rapid detection.
The immunological detection method is a method for detecting various substances by utilizing antigen-antibody specific binding reaction, and the existing monoclonal antibody aiming at zearalenone has the advantages of high affinity and high specificity, but has a narrow linear range, and can not accurately detect the content of samples with high residual quantity, and can possibly generate false negative results during detection.
Disclosure of Invention
The invention aims to solve the problems, and provides a zearalenone monoclonal antibody hybridoma cell strain and application thereof, which have better specificity and detection sensitivity to zearalenone, have a wider linear range, and can more accurately and quantitatively detect positive samples with higher residual quantity.
According to the technical scheme of the invention, the zearalenone monoclonal antibody hybridoma cell strain is preserved in China General Microbiological Culture Center (CGMCC) of China Committee for culture Collection of microorganisms, and has a preservation date of 2021, 05 and 13 days of China academy of sciences of North China, no. 1, and a preservation number of CGMCC No.22338.
The second aspect of the invention provides a preparation method of the zearalenone monoclonal antibody hybridoma cell strain, which comprises the following steps of,
s1: the zearalenone is used for derivatizing phenolic hydroxyl groups on benzene rings into carboxyl groups to obtain hapten;
s2: coupling the hapten with carrier protein to obtain a complete antigen;
s3: mixing and emulsifying the complete antigen and Freund's adjuvant, performing subcutaneous immunization on mice, and screening mice with good immunization effect;
s4: and (3) fusing the spleen cells and myeloma cells of the mice screened in the step (S3) to obtain the zearalenone monoclonal antibody hybridoma cell strain.
Further, the specific operation of the step S1 is as follows:
s1.1: dissolving zearalenone in an organic solvent, adding a catalyst, and 4-bromoethyl butyrate or 6-bromoethyl caproate, and heating to react, wherein the catalyst is an alkaline salt;
s1.2: performing alkaline hydrolysis on the product obtained in the step S1.1, and regulating the pH value to be 2-5 to obtain the hapten
Further, in the step S1.1, the temperature of the heating reaction is 55-65 ℃ and the time is 20-40h; preferably, the reflux is carried out for 24 hours at 60 ℃.
Further, the catalyst is selected from one or more of potassium carbonate, sodium carbonate and sodium bicarbonate, preferably potassium carbonate, and is added in excess.
Further, 4-6. Mu.L of ethyl 4-bromobutyrate or ethyl 6-bromohexanoate was added per 10mg of zearalenone.
Further, in the step S1.2, alkaline hydrolysis is performed by adding a methanol solution of NaOH or KOH, wherein the alkaline hydrolysis temperature is 70-90 ℃ and the alkaline hydrolysis time is 0.8-1.5h; preferably, the alkaline hydrolysis is carried out at 80℃for 1 hour.
Further, in the step S1.2, the pH is adjusted to 2-5 by adding hydrochloric acid.
Specifically, the hapten can be prepared by: 10mg of zearalenone was weighed into 300. Mu.L of acetone and added with excess K 2 CO 3 (43 mg) and 5. Mu.L of ethyl 4-bromobutyrate, refluxed at 60℃for 24 hours, allowed to stand overnight, and taken with 2mLAlkaline hydrolysis for 1h at 80 ℃, nitrogen blow-drying, dissolving with 2ml of double distilled water, adjusting the PH to 3.5 with hydrochloric acid, extracting 3 times with 3ml of ethyl acetate, discarding the water phase, adding anhydrous magnesium sulfate for drying, and blow-drying with nitrogen to obtain a product hapten S1 (haptenS 1), wherein the structural formula is as follows:
the reaction route is as follows:
further, the carrier protein is Bovine Serum Albumin (BSA) or Keyhole Limpet Hemocyanin (KLH).
Further, in the step S2, the hapten S1 is coupled to the carrier protein by using a mixed anhydride method.
Specifically, taking BSA as an example, the preparation of the complete antigen may be: 2mg of haptenS1 (the molar ratio of haptenS1 to Bovine Serum Albumin (BSA) is 60:1) is weighed and dissolved in 300 mu LN, N-Dimethylformamide (DMF), 1.2 mu L of tri-N-butylamine is added first, after 10min of reaction at 4 ℃, 0.5 mu L of isobutyl chloroformate is added, and the reaction is continued for 30min at 4 ℃ (referred to as solution A). 6mg of BSA was taken, dissolved in 2mL of 0.01M carbonate buffer (CB, pH=9.0) (referred to as solution B), and solution A was slowly added dropwise to solution B and coupled overnight; then, the solution is dialyzed by 0.01M PBS to remove unreacted small molecule hapten, thus obtaining complete antigen haptenS S1-BSA.
In step S3, the first immunization is performed by mixing and emulsifying the immunogen and the complete freund 'S adjuvant, the multiple boosting is performed by mixing and emulsifying the immunogen and the incomplete freund' S adjuvant, and finally, the immunization is diluted by normal saline and then the immunization is performed by intraperitoneal injection.
Specifically, the first immunization dose is 100 mug/dose; multiple booster doses were 50 μg/dose; one month is separated from the first immunization and the second immunization, and 21 days is separated from the multiple immunization; the interval between the sprint immunity and the last boost immunity is 18-21 days.
In the step S4, the mouse spleen cells and the mouse myeloma cells are fused by a polyethylene glycol (PEG 4000) method, the hybridoma cells are selected by using a selective medium (HAT medium), and the cells are cultured by using an HT medium. Detecting positive cell holes by using an indirect competitive enzyme-linked immunosorbent assay (ic-Elisa) after one week of fusion, further measuring the inhibition effect of the positive cell holes by using the ic-Elisa, subcloning the positive cell holes with better inhibition by using a limiting dilution method, and detecting, picking and subcloning again after one week. And (3) performing subcloning for three times according to the method to obtain the monoclonal hybridoma cell strain of the zearalenone with high secretion of specific antibodies.
The third aspect of the invention provides a zearalenone monoclonal antibody secreted by the hybridoma cell strain with the preservation number of CGMCC No.22338.
In a fourth aspect, the invention provides a composition comprising a zearalenone monoclonal antibody as described above.
In a fifth aspect, the invention provides a kit comprising the zearalenone monoclonal antibody described above.
In a sixth aspect, the invention provides the use of a zearalenone monoclonal antibody as described above, a composition as described above or a kit as described above for the detection of zearalenone.
Compared with the prior art, the technical scheme of the invention has the following advantages: the monoclonal antibody secreted by the cell strain provided by the invention has better specificity and detection sensitivity (IC) to zearalenone 50 The value is 0.057 ng/mL) and a wider linear range (0.01-0.22 ng/mL), can realize the detection of the zearalenone, and particularly can detect the residual quantity of the zearalenone in corn, wheat, barley, oat, paddy and feed, provides a raw material for the immunodetection of the zearalenone residual in food, and has practical application value.
Preservation of biological material samples: a cell strain of a zearalenone monoclonal antibody hybridoma is preserved in China general microbiological culture collection center (CGMCC) of China Committee for culture Collection of microorganisms, china academy of sciences of China, including national institute of microbiology, no. 3, no. 1, beijing, chaoyang, and with a preservation date of 2021, no. 05, and 13, and a preservation number of CGMCC No.22338.
Drawings
FIG. 1 shows inhibition standard curves of zearalenone monoclonal antibodies.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.
According to the invention, the zearalenone complete antigen is immunized on a mouse, the cell fusion is carried out, the HAT selective culture medium is used for culturing, and the cell supernatant is screened by means of ic-ELISA, so that the hybridoma cell strain with high secretion specific antibody against the zearalenone is finally obtained.
In the following examples, the solution configuration is as follows:
carbonate Buffer (CBS): weighing Na 2 CO 3 1.59 g，NaHCO 3 2.93 g, respectively dissolving in a small amount of double distilled water, mixing, adding the double distilled water to about 800mL, mixing uniformly, adjusting the pH value to 9.6, adding the double distilled water to 1000mL, and storing at 4 ℃ for later use.
Phosphate Buffer (PBS): 8.00gNaCl,0.2g KCl,0.2g KH 2 PO 4 ，2.9g Na 2 HPO 4 ·12H 2 O is dissolved in 800mL of pure water, pH is regulated to 7.2-7.4 by NaOH or HCl, and volume is regulated to 1000mL;
PBST: PBS containing 0.05% Tween 20;
TMB color development liquid: and (3) solution A: na (Na) 2 HPO 4 ·12H 2 18.43g of O, 9.33g of citric acid and pure water to 1000mL; and (2) liquid B: 60mg of TMB was dissolved in 100mL of ethylene glycol. A. The solution B is prepared from the following components in percentage by weight: 1 to obtain TMB color development liquid, and mixing immediately.
Example 1 preparation of hybridoma cell lines
(1) Derivatization of hapten: the phenolic hydroxyl group on the benzene ring is derived into carboxyl serving as hapten S1 by using zearalenone, and the steps are as follows:
10mg of zearalenone was weighed into 300. Mu.L of acetone and added with excess K 2 CO 3 (43 mg) and 5. Mu.L of ethyl 4-bromobutyrate, refluxed at 60℃for 24 hours, allowed to stand overnight, and taken with 2mLAlkaline hydrolysis of NaOH-methanol solution at 80 ℃ for 1h, blow-drying with nitrogen, and dissolving with 2ml double distilled waterAnd (3) performing solution, namely adjusting the pH to 3.5 by using hydrochloric acid, extracting for 3 times by using 3ml of ethyl acetate, discarding the water phase, adding anhydrous magnesium sulfate for drying, and drying by using nitrogen to obtain the hapten S1 (haptenS 1).
(2) Preparation of complete antigen Haptens 1-BSA: coupling hapten S1 with carrier protein by using a mixed anhydride method to obtain zearalenone artificial antigen, wherein the steps are as follows:
2mg of haptenS1 (the molar ratio of haptenS1 to Bovine Serum Albumin (BSA) is 60:1) is weighed and dissolved in 300 mu LN, N-Dimethylformamide (DMF), 1.2 mu L of tri-N-butylamine is added first, after 10min of reaction at 4 ℃, 0.5 mu L of isobutyl chloroformate is added, and the reaction is continued for 30min at 4 ℃ (referred to as solution A). 6mg of BSA was taken, dissolved in 2mL of 0.01M carbonate buffer (CB, pH=9.0) (referred to as solution B), and solution A was slowly added dropwise to solution B and coupled overnight; then, the unreacted small molecule hapten is removed by 0.01M PBS solution dialysis, so that complete antigen haptenS1-BSA is obtained, and the complete antigen is identified by an ultraviolet absorption scanning method.
(3) Animal immunization: after mixing and emulsifying the complete antigen of haptenS S1-BSA and equivalent Freund's adjuvant, BALB/c mice were subjected to subcutaneous multi-point injection immunization (except sprint immunization) on the back of the neck.
The first immunization was performed with complete Freund's adjuvant at a dose of 100 ug/dose; multiple boosting with incomplete Freund's adjuvant and halving the dose to 50 ug/dose; the sprint immunity is directly diluted by normal saline without adjuvant, and then the dosage is halved to 25 ug/patient. One month is separated from the first immunization and the second immunization, 21 days is separated from the multiple boosting, and 18-21 days is separated from the final boosting. The immune effect of the mice is observed by an indirect competitive enzyme-linked immunosorbent assay (ic-Elisa), namely the titer and inhibition of the serum of the mice are detected.
(4) Cell fusion: three days after sprint immunization, cell fusion was performed according to the conventional PEG (polyethylene glycol, molecular weight 4000) method, as follows:
a. taking out eyeballs of a mouse to obtain blood, killing the mouse by a cervical dislocation method, immediately putting the mouse into 75% alcohol for disinfection, soaking for about 5 minutes, taking out the spleen of the mouse by aseptic operation, moderately grinding the spleen by a syringe rubber head, obtaining spleen cell suspension by a 200-mesh cell screen, collecting, centrifuging (1200 rpm,8 minutes), washing the spleen cells for three times by using an RPMI-1640 culture medium, and diluting the spleen cells to a certain volume and counting for later use after the last centrifugation;
b. collecting SP 2/0 And (3) cells: 7-10 days before fusion, SP was pooled 2/0 Tumor cells were cultured in 10% FBS (fetal bovine serum) RPMI-1640 medium in 5% CO 2 Culturing in an incubator. Pre-fusion requirement SP 2/0 The number of tumor cells reaches 1-4 x 10 7 Guarantee of pre-fusion SP 2/0 Tumor cells are in logarithmic growth phase. During fusion, collecting tumor cells, suspending in RPMI-1640 basic culture solution, and performing cell count;
c. fusion process 7min: 1min, 1mL of PEG 4000 was added dropwise to the cells from slow to fast; and (2) standing for 2 min. Dripping 1mL of RPMI-1640 culture medium in the period of 1min for 3min and 4 min; dripping 2mL of RPMI-1640 culture medium in the period of 1min at the 5 th and 6 th min; at 7min, 1mL of RPMI-1640 medium was added dropwise every 10 s. Then, the mixture was incubated at 37℃for 5min. Centrifuging (800 rpm,10 min), removing supernatant, gently tapping cells, and adding RPMI-1640 selective medium (HAT medium) containing 20% fetal bovine serum and 2%50 XHAT to 96-well cell plate at 200 μl/well, placing at 37deg.C, 5% CO 2 Culturing in an incubator.
(5) Cell screening and cell strain establishment: half-changing the fused cells by HAT medium on the 3 rd day after cell fusion; full exchange with RPMI-1640 medium (HT medium) containing 20% fetal bovine serum, 1% 100×ht on day 5; cell supernatants were taken on day 7 for screening. Screening is carried out in two steps: the first step is to screen out positive cell holes by using an ic-ELISA method, and the second step is to select zearalenone as a standard substance, and to measure the inhibition effect and linear range of positive cells by using the ic-ELISA method. Cell holes with better inhibition and linear range on the zearalenone standard are selected, subcloning is carried out by adopting a limiting dilution method, and detection is carried out by adopting the same method after seven days. And performing subcloning for three times according to the method to finally obtain the zearalenone monoclonal antibody cell strain.
EXAMPLE 2 preparation and identification of monoclonal antibodies
Taking 8-10 week old BALB/c mice, and injecting 1mL of sterile paraffin oil into the abdominal cavity of each mouse; intraperitoneal injection of 1X 10 per mouse after 7 days 6 The zearalenone hybridoma cells were collected for ascites from the seventh day, and antibody purification was performed on the ascites by octanoic acid-saturated ammonium sulfate method. Under the condition of meta-acid, the n-octanoic acid can precipitate other hetero proteins except IgG immunoglobulin in ascites, and then the mixture is centrifuged and the precipitate is discarded; then the monoclonal antibody of IgG type is precipitated by an ammonium sulfate solution with equivalent saturation, centrifugated, the supernatant is discarded, dissolved by a PBS solution (pH 7.4) of 0.01M, and then dialyzed and desalted, and finally the purified monoclonal antibody is preserved at-20 ℃.
5.1 coating: the coated original haptenS S1-OVA was diluted 3-fold from 1. Mu.g/mL with 0.05M pH 9.6 carbonate buffer, 100. Mu.L/well, and reacted at 37℃for 2 hours.
5.2 washing: the plate solution was poured off and washed 3 times with wash solution for 3min each.
5.3 closing: after drying, 200. Mu.L/well of blocking solution was added thereto and reacted at 37℃for 2 hours. And (5) drying for standby after washing.
5.4 sample addition: the antisera was diluted from 1:1000 in a double ratio and added to the coated wells at each dilution, 100. Mu.L/well, and reacted at 37℃for 30min; after extensive washing, HRP-goat anti-mouse IgG diluted 1:3000 was added, 100. Mu.L/well, and reacted at 37℃for 30 minutes.
5.5 color development: and taking out the ELISA plate, fully washing, adding 100 mu L of TMB color developing solution into each hole, and carrying out light-shielding reaction for 15min at 37 ℃.
5.6 termination and measurement: 50. Mu.L of stop solution was added to each well to terminate the reaction, and the OD 450 value of each well was measured by using a microplate reader.
IC for determining monoclonal antibody zearalenone by IC-ELISA 50 The method comprises the following steps: 0.057ng/mL, which shows that the kit has good sensitivity to zearalenone and can be used for the immunoassay detection of zearalenone.
The linear range (IC) of the zearalenone monoclonal antibody can be obtained by establishing an inhibition standard curve of the antibody 20 -IC 80 ) 0.01-0.22ng/mL. Compared to antibodies of narrow linear range (antibodies produced in the same way with formula I as immunogen,IC 50 the linear range is 0.03ng/mL and 0.01-0.06 ng/mL), the antibody has a wider linear range, the accuracy and the reliability of a detection result are improved, and positive samples with higher residual quantity can be more accurately and quantitatively detected.
Cross-reactions of zearalenone monoclonal antibodies to DON (vomitoxin), OTA (ochratoxin), aflatoxin B1, T-2 toxins, respectively, are shown in table 1:
TABLE 1
The results show that the cross reaction rate of the zearalenone monoclonal antibody to DON, OTA and aflatoxin B1 and T-2 is extremely low, so that the antibody prepared by the invention has better specificity to zearalenone.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations and modifications of the present invention will be apparent to those of ordinary skill in the art in light of the foregoing description. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.

Claims (5)

1. A cell strain of a zearalenone monoclonal antibody hybridoma is preserved in China general microbiological culture collection center (CGMCC) of China Committee for culture Collection of microorganisms, china academy of sciences of China, including national institute of microbiology, no. 3, no. 1, beijing, chaoyang, and with a preservation date of 2021, no. 05, and 13, and a preservation number of CGMCC No.22338.
2. A zearalenone monoclonal antibody, characterized in that it is secreted by the hybridoma cell line with the preservation number of CGMCC No.22338 according to claim 1.
3. A composition comprising the zearalenone monoclonal antibody of claim 2.
4. A kit comprising the zearalenone monoclonal antibody of claim 2.
5. Use of a zearalenone monoclonal antibody according to claim 2 or a composition according to claim 3 for the preparation of a formulation for detecting zearalenone.
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CN106950375A (en) * 2017-03-03 2017-07-14 重庆市畜牧科学院 A kind of zearalenone monoclonal antibody and its application
CN107915774A (en) * 2017-07-26 2018-04-17 华中农业大学 For detecting the monoclonal antibody and enzyme-linked immunoassay method and kit of zearalenone and its metabolite

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102690788A (en) * 2012-04-25 2012-09-26 暨南大学 Zearalenone anti-idiotypic antibody, preparation method thereof, and application thereof
CN103232975A (en) * 2013-04-03 2013-08-07 中国农业科学院油料作物研究所 Hybridoma cell strain 2D3, monoclonal antibody to zearalenone secreted by same and application of monoclonal antibody
CN106950375A (en) * 2017-03-03 2017-07-14 重庆市畜牧科学院 A kind of zearalenone monoclonal antibody and its application
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