CN114214288A

CN114214288A - Zearalenone monoclonal antibody hybridoma cell strain and application thereof

Info

Publication number: CN114214288A
Application number: CN202111605097.5A
Authority: CN
Inventors: 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
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2021-12-24
Filing date: 2021-12-24
Publication date: 2022-03-22
Anticipated expiration: 2041-12-24
Also published as: CN114214288B

Abstract

The invention belongs to the technical field of immunochemistry, and particularly 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), China institute for microbiology, national institute for sciences, No. 3, Xilu 1, North Cheng, the south China area, Beijing, with the preservation date of 2021 year, 05 month and 13 days, and the preservation number is CGMCC No. 22338. The monoclonal antibody secreted by the cell strain provided by the invention has better specificity and detection sensitivity (IC) on zearalenone₅₀The value is 0.057ng/mL) and a wider linear range (0.01-0.22ng/mL), can realize the detection of the zearalenone, especially the detection of the residual amount of the zearalenone in corn, wheat, barley, oat, rice and feed, provides a raw material for the immunodetection of the residual amount of the zearalenone 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 estrogen 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 and even death of animal reproduction function. Recent studies have shown that ZEN has immune toxicity, liver toxicity and cytotoxicity in addition to its effect on reproductive function, and also has some effect on tumor development.

Zearalenone has stable chemical properties, is difficult to decompose at high temperature, is not easy to damage in the process of processing food or feed, and seriously threatens the safety of meat production and human health. Therefore, the content of the zearalenone in food and animal feed needs to be strictly controlled, and the detection limit in the feed is generally required to be less than 500 mu g/kg, and the detection limit in the food is required to be less than 60 mu g/kg.

The conventional methods for detecting zearalenone mainly comprise an instrumental analysis method and an immunochemical detection method. Wherein the instrumental analysis method comprises: gas Chromatography (GC), Thin Layer Chromatography (TLC), High Performance Liquid Chromatography (HPLC), and high performance liquid chromatography-mass spectrometry (HPLC-MS). 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 price, convenience, rapidness and simple operation, thereby having unique advantages and great development potential on the primary screening detection and the on-site rapid detection of a large number of samples.

The immunological detection method is a method for detecting various substances by utilizing antigen-antibody specific binding reaction, and although the existing monoclonal antibody aiming at zearalenone has the advantages of high affinity and high specificity, the linear range is narrow, the content of some samples with higher residual quantity cannot be accurately detected, and some false negative results may appear 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 for zearalenone, have a wider linear range and can be used for more accurately and quantitatively detecting a positive sample 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 collection center (CGMCC) of China Committee for culture Collection of microorganisms, China academy of sciences microorganism research institute No. 3, Xilu No. 1 Hospital, Beijing, Tokyo, with the preservation date of 2021 year, 05 month and 13 days and the 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,

s1: derivatizing phenolic hydroxyl on a benzene ring into carboxyl by zearalenone 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 the mice with good immune effect;

s4: fusing the spleen cells and myeloma cells of the mouse screened in the step S3 to obtain the zearalenone monoclonal antibody hybridoma cell strain.

Further, the specific operation of step S1 is as follows:

s1.1: dissolving zearalenone in an organic solvent, adding a catalyst and 4-ethyl bromobutyrate or 6-ethyl bromohexanoate, and heating for reaction, wherein the catalyst is an alkaline salt;

s1.2: performing alkaline hydrolysis on the product obtained in the step S1.1, and adjusting the pH to 2-5 to obtain the hapten

Further, in the step S1.1, the heating reaction temperature is 55-65 ℃ and the time is 20-40 h; preferably, the reflux is carried out for 24h 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 carried out by adding a methanol solution of NaOH or KOH, wherein the temperature of the alkaline hydrolysis is 70-90 ℃, and the time is 0.8-1.5 h; preferably, the temperature of the alkaline hydrolysis is 80 ℃ and the time is 1 h.

Further, in step S1.2, the pH is adjusted to 2-5 by adding hydrochloric acid.

Specifically, the hapten can be prepared by: weighing zearalenone 10mg, dissolving in 300 μ L acetone, adding excessive K₂CO₃(43mg) and 5. mu.L of 4-bromobutyric acid ethyl ester at 60 ℃ under reflux for 24h, left overnight in 2mL

Carrying out alkaline hydrolysis on the NaOH-methanol solution at the temperature of 80 ℃ for 1h, drying by using nitrogen, dissolving by using 2ml of double distilled water, adjusting the pH to 3.5 by using hydrochloric acid, extracting for 3 times by using 3ml of ethyl acetate, discarding a water phase, adding anhydrous magnesium sulfate for drying, and drying by using nitrogen for drying to obtain a product hapten S1(Hapten S1), 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 step S2, hapten S1 is coupled to the carrier protein by a mixed anhydride method.

Specifically, taking BSA as an example, the complete antigen can be prepared by: 2mg of halopten S1 (the molar ratio of halopten S1 to Bovine Serum Albumin (BSA) is 60: 1) was weighed and dissolved in 300. mu.L of N, N-Dimethylformamide (DMF), 1.2. mu.L of tri-N-butylamine was added, and after 10min reaction at 4 ℃ 0.5. mu.L of isobutyl chloroformate was added, and the reaction was continued at 4 ℃ for 30min (called solution A). 6mg of BSA was 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 dialyzed with 0.01M PBS solution to remove unreacted small molecule hapten to obtain complete antigen haptenS 1-BSA.

Further, in step S3, the first immunization is performed by mixing and emulsifying the immunogen and the complete freund adjuvant, the multiple booster immunization is performed by mixing and emulsifying the immunogen and the incomplete freund adjuvant, and finally the booster immunization is performed by injecting the immunogen into the abdominal cavity after diluting the immunogen with normal saline.

Specifically, the first immunization dose is 100 mug/mouse; the multiple booster dose is 50 mug/mouse; the interval between the first immunization and the second boosting immunization is one month, and the interval between the multiple boosting immunizations is 21 days; the interval between the sprint immunization and the last booster immunization is 18-21 days.

In step S4, the mouse spleen cells and the mouse myeloma cells are fused by a polyethylene glycol (PEG 4000) method, hybridoma cells are selected by using a selective medium (HAT medium), and cell culture is performed 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 determining the inhibition effect of the positive cell holes by using the ic-Elisa method, subcloning the positive cell holes with better inhibition by using a limiting dilution method, and detecting, selecting and subcloning again after one week. And carrying out subcloning for three times according to the method to obtain the monoclonal hybridoma cell strain of the zearalenone high-secretion specific antibody.

The third aspect of the invention provides a zearalenone monoclonal antibody which is secreted and produced by the hybridoma cell strain with the preservation number of CGMCC No. 22338.

In a fourth aspect, the present invention provides a composition comprising the zearalenone monoclonal antibody described above.

The fifth aspect of the invention provides a kit comprising the zearalenone monoclonal antibody.

The sixth aspect of the invention provides an application of the zearalenone monoclonal antibody, the composition or the kit in the detection of zearalenone.

Compared with the prior art, the technical scheme of the invention has the following advantages: the cell strain provided by the invention secretes the single peptideThe cloned antibody has better specificity and detection sensitivity (IC) on the zearalenone₅₀The value is 0.057ng/mL) and a wider linear range (0.01-0.22ng/mL), can realize the detection of the zearalenone, especially the detection of the residual amount of the zearalenone in corn, wheat, barley, oat, rice and feed, provides a raw material for the immunodetection of the residual amount of the zearalenone in food, and has practical application value.

Biological material sample preservation: a zearalenone monoclonal antibody hybridoma cell strain is deposited in China general microbiological culture collection center (CGMCC), China institute of microbiology, institute of academy of sciences, No. 3, West Lu 1, North Cheng, south China, Beijing, with the preservation date of 2021 year, 05 month and 13 days, and the preservation number of the hybridoma cell strain is CGMCC No. 22338.

Drawings

FIG. 1 is a standard curve for inhibition of zearalenone monoclonal antibodies.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

The hybridoma cell strain with high secretion specificity antibody for zearalenone is finally obtained by immunizing a mouse with complete antigen of zearalenone, performing cell fusion, culturing in HAT selective medium and screening cell supernatant through ic-ELISA.

In the following examples, the solution was prepared as follows:

carbonate Buffer (CBS): weighing Na₂CO₃1.59 g，NaHCO₃2.93 g, respectively dissolving in a small amount of double distilled water, mixing, adding the double distilled water to about 800mL, uniformly mixing, adjusting the pH value to 9.6, adding the double distilled water to a constant volume of 1000mL, and storing at 4 ℃ for later use.

Phosphate Buffered Saline (PBS): 8.00g NaCl, 0.2g KCl, 0.2g KH₂PO₄，2.9g Na₂HPO₄·12H₂Dissolving O in 800mL of pure water, adjusting pH to 7.2-7.4 with NaOH or HCl, and diluting to 1000mL；

PBST: PBS containing 0.05% tween 20;

TMB color development liquid: solution A: na (Na)₂HPO₄·12H₂18.43g of O, 9.33g of citric acid and pure water to reach the constant volume of 1000 mL; and B, liquid B: 60mg of TMB was dissolved in 100mL of ethylene glycol. A. The liquid B is prepared according to the following steps: 1 to obtain the TMB color developing solution which is mixed at present.

EXAMPLE 1 preparation of hybridoma cell lines

(1) Derivation of hapten: the method comprises the following steps of (1) deriving a carboxyl group from a phenolic hydroxyl group on a benzene ring by using zearalenone to obtain a hapten S1:

weighing zearalenone 10mg, dissolving in 300 μ L acetone, adding excessive K₂CO₃(43mg) and 5. mu.L of 4-bromobutyric acid ethyl ester at 60 ℃ under reflux for 24h, left overnight in 2mL

And (3) carrying out alkaline hydrolysis on the NaOH-methanol solution at the temperature of 80 ℃ for 1h, drying by using nitrogen, dissolving by using 2ml of double distilled water, adjusting the pH to 3.5 by using hydrochloric acid, extracting for 3 times by using 3ml of ethyl acetate, removing a water phase, adding anhydrous magnesium sulfate for drying, and drying by using nitrogen for drying to obtain a product hapten S1 (halopten S1).

(2) Preparation of complete antigen Haptens 1-BSA: coupling hapten S1 with carrier protein by a mixed anhydride method to obtain the zearalenone artificial antigen, which comprises the following steps:

2mg of halopten S1 (the molar ratio of halopten S1 to Bovine Serum Albumin (BSA) is 60: 1) was weighed and dissolved in 300. mu.L of N, N-Dimethylformamide (DMF), 1.2. mu.L of tri-N-butylamine was added, and after 10min reaction at 4 ℃ 0.5. mu.L of isobutyl chloroformate was added, and the reaction was continued at 4 ℃ for 30min (called solution A). 6mg of BSA was 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 dialyzing with 0.01M PBS solution to remove unreacted small molecule hapten to obtain complete antigen haptenS1-BSA, and identifying by ultraviolet absorption scanning method.

(3) Animal immunization: after mixing and emulsifying the complete antigen of haptenS1-BSA with equal amount of Freund's adjuvant, BALB/c mice were immunized by subcutaneous multipoint injection at the back of the neck (except for the puncture immunization).

Complete Freund adjuvant is used for the first immunization, and the dosage is 100 ug/mouse; incomplete Freund's adjuvant is used for multiple times of boosting immunization, and the dosage is reduced by half to be 50 ug/mouse; the thorny immunity does not use any adjuvant, and the thorny immunity is directly diluted by normal saline and injected into the abdominal cavity, and the dosage is reduced by half to obtain 25 ug/mouse. The interval between the first immunization and the second boosting immunization is one month, the interval between the multiple boosting immunizations is 21 days, and the interval between the sprint immunization and the last boosting immunization is 18-21 days. The titer and inhibition of the mouse serum are detected by observing the immune effect of the mouse by an indirect competitive enzyme-linked immunosorbent assay (ic-Elisa).

(4) Cell fusion: after three days of the spurting immunization, cell fusion is carried out according to a conventional PEG (polyethylene glycol, molecular weight is 4000) method, and the specific steps are as follows:

a. the method comprises the following steps of (1) taking eyeballs of a mouse, taking blood, killing the mouse by a cervical vertebra dislocation method, immediately placing the mouse into 75% alcohol for disinfection, soaking for about 5min, taking out the spleen of the mouse by aseptic operation, properly grinding the spleen by using a syringe rubber head, passing through a 200-mesh cell screen to obtain a spleen cell suspension, collecting, centrifuging (1200rpm, 8min), washing the spleen cells for three times by using an RPMI-1640 culture medium, finally centrifuging for the last time, diluting the spleen cells to a certain volume, and counting for later use;

b. collection of SP_2/0Cell: 7-10 days before fusion, SP is added_2/0Tumor cells were cultured in RPMI-1640 medium containing 10% FBS (fetal bovine serum) at 5% CO₂Culturing in an incubator. Pre-fusion requirements SP_2/0The number of tumor cells reaches 1-4 x 10⁷Ensuring SP before fusion_2/0The tumor cells are in logarithmic growth phase. During fusion, tumor cells are collected and suspended in RPMI-1640 basic culture solution for cell counting;

c. the fusion process is 7 min: 1min, 1mL of PEG 4000 was added to the cells dropwise from slow to fast; standing for 2 min. Dropping 1mL of RPMI-1640 culture medium within 1min at 3min and 4 min; dropping 2mL of RPMI-1640 culture medium within 1min at 5min and 6 min; at 7min, 1mL of RPMI-1640 medium was added dropwise every 10 s. Then, the mixture is incubated at 37 ℃ for 5min. Centrifuging (800rpm, 10min), discarding the supernatant, gently tapping the cells, and adding 20% fetal bovine serum, 2% 50 × HAT in RPMI-1640 selection Medium (HAT Medium), 200. mu.L/well to 96-well cell plate, 5% CO at 37 ℃₂Culturing in an incubator.

(5) Cell screening and cell strain establishment: half-changing the fused cells with HAT medium on day 3 after cell fusion; on day 5, the whole culture medium was changed with RPMI-1640 medium (HT medium) containing 20% fetal bovine serum and 1% 100 × HT; cell supernatants were taken on day 7 for screening. The screening is divided into two steps: firstly, screening out positive cell holes by using an ic-ELISA method, secondly, selecting zearalenone as a standard substance, and determining the inhibition effect and the linear range of the positive cells by using the ic-ELISA method. Selecting cell pores with better inhibition and linear range on the zearalenone standard, performing subcloning by adopting a limiting dilution method, and detecting by using the same method after seven days. And carrying out subcloning for three times according to the method to finally obtain the zearalenone monoclonal antibody cell strain.

EXAMPLE 2 preparation and characterization of monoclonal antibodies

Taking BALB/c mice 8-10 weeks old, and injecting 1mL of sterile paraffin oil into the abdominal cavity of each mouse; 7 days later, each mouse was injected intraperitoneally with 1X 10⁶Zearalenone hybridoma cells, ascites fluid was collected from the seventh day, and antibody purification was performed on the ascites fluid by the octanoic acid-saturated ammonium sulfate method. Under the condition of partial acid, the caprylic acid can precipitate other hybrid proteins except IgG immunoglobulin in the ascites, then the centrifugation is carried out, and the precipitate is discarded; then, the IgG type monoclonal antibody was precipitated with an ammonium sulfate solution of the same saturation, centrifuged, the supernatant was discarded, and the supernatant was dissolved in a 0.01M PBS solution (pH7.4), dialyzed and desalted to finally obtain a purified monoclonal antibody, which was stored at-20 ℃.

5.1 coating: the original coating haptenS1-OVA was diluted 3-fold with 0.05M pH 9.6 carbonate buffer starting from 1. mu.g/mL, 100. mu.L/well, reacted at 37 ℃ for 2 h.

5.2 washing: the plate solution was decanted and washed 3 times for 3min each with washing solution.

5.3 sealing: after patting to dryness, 200. mu.L/well blocking solution was added and reacted at 37 ℃ for 2 hours. And drying after washing for later use.

5.4 sample adding: diluting antiserum from 1:1000 times, adding into coated wells of each dilution, reacting at 37 deg.C for 30min at 100 μ L/well; after washing sufficiently, HRP-goat anti-mouse IgG diluted at a ratio of 1:3000 was added thereto at 100. mu.L/well, and the reaction was carried out at 37 ℃ for 30 min.

5.5 color development: the ELISA plate was removed, washed thoroughly, and 100. mu.L of TMB developing solution was added to each well, and the reaction was carried out at 37 ℃ in the dark for 15min.

5.6 termination and determination: the reaction was stopped by adding 50. mu.L of a stop buffer to each well, and the OD 450 value of each well was measured by a microplate reader.

IC-ELISA determination of monoclonal antibody zearalenone₅₀Comprises the following steps: 0.057ng/mL, which shows that the kit has good sensitivity to zearalenone and can be used for immunoassay detection of zearalenone.

The linear range (IC) of the zearalenone monoclonal antibody can be obtained by establishing an inhibition standard curve of the antibody₂₀-IC₈₀) Is 0.01-0.22 ng/mL. Compared to antibodies with a narrow linear range (antibodies prepared in the same manner using formula I as the immunogen, IC)₅₀0.03ng/mL and the linear range of 0.01-0.06ng/mL), the antibody has a wider linear range, improves the accuracy and reliability of a detection result, and can more accurately and quantitatively detect a positive sample with higher residual quantity.

The results of the cross reaction of zearalenone monoclonal antibodies to DON (vomitoxin), OTA (ochratoxin), aflatoxin B1 and T-2 toxin are shown in Table 1:

TABLE 1

The result shows that the zearalenone monoclonal antibody has extremely low cross reaction rate to DON, OTA, aflatoxin B1 and T-2 toxins, so the antibody prepared by the invention has better specificity to zearalenone.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.

Claims

1. A zearalenone monoclonal antibody hybridoma cell strain is deposited in China general microbiological culture collection center (CGMCC), China institute of microbiology, institute of academy of sciences, No. 3, West Lu 1, North Cheng, south China, Beijing, with the preservation date of 2021 year, 05 month and 13 days, and the preservation number of the hybridoma cell strain is CGMCC No. 22338.

2. A method for preparing a zearalenone monoclonal antibody hybridoma cell line according to claim 1, comprising the steps of,

s2: coupling the hapten with carrier protein to obtain a complete antigen;

3. The method according to claim 2, wherein the step S1 is specifically performed as follows:

s1.2: and (4) carrying out alkaline hydrolysis on the product obtained in the step (S1.1), and adjusting the pH value to 2-5 to obtain the hapten.

4. The method of claim 3, wherein in step S1.1, the heating reaction is carried out at a temperature of 55-65 ℃ for 20-40 h.

5. The method of claim 3, wherein in step S1.2, the alkaline hydrolysis is carried out by adding NaOH or KOH in methanol at a temperature of 70-90 ℃ for a time of 0.8-1.5 h.

6. The method of claim 2, wherein in step S3, the first immunization is performed by mixing and emulsifying the immunogen with complete freund 'S adjuvant, the multiple booster immunization is performed by mixing and emulsifying the immunogen with incomplete freund' S adjuvant, and finally the booster immunization is performed by intraperitoneal injection after diluting the immunogen with physiological saline.

7. A zearalenone monoclonal antibody secreted by the hybridoma cell strain with the preservation number of CGMCC No.22338 as claimed in claim 1.

8. A composition comprising the zearalenone monoclonal antibody of claim 7.

9. A kit comprising the zearalenone monoclonal antibody of claim 7.

10. Use of the zearalenone monoclonal antibody of claim 7, the composition of claim 8 or the kit of claim 9 for detecting zearalenone.