CN112574957B

CN112574957B - Hybridoma cell strain secreting clomazone monoclonal antibody and application thereof

Info

Publication number: CN112574957B
Application number: CN202011590789.2A
Authority: CN
Inventors: 胥传来; 王鹏; 匡华; 徐丽广; 孙茂忠; 刘丽强; 吴晓玲; 宋珊珊; 胡拥明; 郝昌龙; 吴爱红
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2020-12-29
Filing date: 2020-12-29
Publication date: 2022-09-27
Anticipated expiration: 2040-12-29
Also published as: CN112574957A

Abstract

A hybridoma cell strain secreting clomazone monoclonal antibody and application thereof belong to the field of food safety immunodetection. The hybridoma cell strain ABC06 secreting clomazone monoclonal antibody has been preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, CGMCC for short, and the preservation date is as follows: 11 and 28 months in 2019, and the preservation number is CGMCC No. 19181. The clomazone monoclonal antibody secreted by the cell strain is used for analyzing and detecting clomazone residue in food safety detection. The clomazone monoclonal antibody obtained by the invention can be used for immunoassay detection, and has better detection sensitivity and specificity (IC) to clomazone ₅₀ The value was 5.5 ng/mL). The achievement of the invention can be used for preparing an immunoassay kit and a colloidal gold test strip for the clomazone, and provides a powerful detection method and means for detecting the residual amount of the clomazone in grains, oil plants, vegetables, sugar materials and tobacco.

Description

Hybridoma cell strain capable of secreting clomazone monoclonal antibody and application of hybridoma cell strain

Technical Field

The invention relates to a hybridoma cell strain secreting clomazone monoclonal antibody and application thereof, belonging to the field of food safety immunodetection.

Background

Clomazone (CLO), also known as clomazone, is a foreign trade name clomazone, which is a selective preemergence herbicide. After the plant phytochrome is applied, the phytochrome can be absorbed by roots and buds of plants and is upwards conducted to each part of the plants along with the transpiration, the biosynthesis of the phytochrome of sensitive plants can be inhibited, although the phytochrome can sprout and come out of the soil, but the phytochrome is not colored and whitened and dies in a short period of time, and after the phytochrome of soybeans and other drug-resistant plants is absorbed, the phytochrome can be converted into a degradation product without the weed killing capability through the metabolic action, so that the phytochrome can be prevented from being damaged. Therefore, it is widely used in soybean fields, paddy fields, sugarcane fields, tobacco fields, and the like to control broadleaf weeds and grassy weeds. However, the herbicide belongs to a long-residue herbicide, the half-life period is about 24 d, the biological activity in soil can last for more than 6 months, phytotoxicity residue is easily caused to crops such as wheat, sunflower and the like and various vegetables in the next crop, and researches show that the herbicide has potential mutagenicity to genes of mammals and can increase cancer risks, so that the national standard makes a clear regulation on the lowest residue and requires the lowest detection line to be 10 ng/mL.

The method for analyzing the content of clomazone comprises the following steps: high Performance Liquid Chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS) and other instrument methods, and the detection methods have the defects of time consumption, complicated steps, incapability of on-site rapid detection, high cost and the like. Therefore, the establishment of a rapid and simple clomazone detection method has important significance. An enzyme-linked immunosorbent assay (ELISA) is an extremely efficient, sensitive and rapid detection method, is suitable for the field rapid detection of a large number of samples, and provides a new detection approach for the detection of clomazone.

Disclosure of Invention

The invention aims to provide a hybridoma cell strain secreting an anti-clomazone monoclonal antibody and application thereof.

According to the technical scheme, a hybridoma cell strain ABC06 secreting clomazone monoclonal antibody has been preserved in China general microbiological culture Collection center (CGMCC) for short, and is named as monoclonal cell strain by classification, wherein the CGMCC is the China academy of sciences microorganism research institute No. 3, national institute No.1, west way, north Cheng, the south China area, Beijing, and the preservation date is as follows: 11 and 28 months in 2019, and the preservation number is CGMCC No. 19181.

The clomazone monoclonal antibody is secreted and generated by the hybridoma cell strain ABC06 with the preservation number of CGMCC No. 19181.

The application of the clomazone monoclonal antibody is used for analyzing and detecting the clomazone residual quantity in food safety detection.

The preparation of the hybridoma cell strain ABC06 secreting the clomazone monoclonal antibody provided by the invention comprises the following basic steps:

(1) preparation of clomazone hapten: dissolving 26 mu L of concentrated nitric acid into 100 mu L of concentrated sulfuric acid, and uniformly mixing to prepare a mixed acid solution for later use; dissolving 100 mg of clomazone in 325 mu L of concentrated sulfuric acid, stirring and dissolving at 4 ℃, dropwise adding a mixed acid solution into the clomazone solution, and reacting at-10 ℃ for 7 hours; recovering to 0 ℃, adding 2 mL of ice water, dropwise adding concentrated ammonia water to adjust the pH to 4.8-5.2, precipitating light yellow solid, carrying out vacuum filtration and drying to obtain the product CLO-NO ₂ (ii) a Taking 50 mg of CLO-NO ₂ Dissolving in mixed solution of ethanol/acetic acid/water (volume ratio of 2:2: 1), placing in round bottom flask, adding 20 mg zinc powder and 3 μ L concentrated hydrochloric acid, heating and refluxing at 60 deg.C for 15 min, stirring at room temperature for 40 min to clarify; filtering to remove zinc powder, extracting with ethyl acetate, collecting organic phase, washing, drying, and evaporating to dryness under reduced pressure to obtain hapten CLO-NH ₂ The hapten synthetic route is prepared by a hybridoma cell strain ABC 06;

(2) preparation of clomazone complete antigen: hapten CLO-NH using carbodiimide method ₂ Respectively coupling with carrier proteins BSA and OVA to prepare immunogen clomazone-BSA and coating antigen clomazone-OVA, and dialyzing to separate complete antigen and unconjugated hapten CLO-NH ₂ ；

(3) Immunization of mice: mixing and emulsifying an immunogen clomazone-BSA and an equivalent amount of Freund adjuvant, and injecting the mixture to immunize a BALB/c mouse through back subcutaneous injection; complete Freund adjuvant is used for the first immunization, incomplete Freund adjuvant is used for the multiple times of boosting immunization, the interval between the first immunization and the second boosting immunization is 28 days, the interval between the multiple times of boosting immunization is 21 days, and the final boosting immunization is carried out by using clomazone-BSA complete antigen (without adjuvant); detecting serum titer and inhibition by an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA);

(4) cell fusion and cell line establishment: fusing mouse spleen cells and mouse myeloma cells by a polyethylene glycol (PEG 4000) method, culturing by an HAT culture medium, detecting positive cell holes by an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), further determining the inhibition effect of the positive cell holes by the ic-ELISA, carrying out three times of subcloning on the positive cell holes with the best inhibition effect by a limiting dilution method, and finally screening to obtain a hybridoma cell strain ABC 06;

(5) and (3) identification of the properties of hybridoma cell strains: sensitivity and specificity were determined by ic-ELISA.

The invention has the beneficial effects that: the monoclonal antibody secreted by the hybridoma cell strain ABC06 has better specificity and detection sensitivity (IC) on clomazone ₅₀ The value is 5.5 ng/mL), the detection on the residual quantity of clomazone in grains, oil plants, vegetables, sugar materials and tobacco can be realized, raw materials are provided for the immunological detection on the residual clomazone in food, and the method has practical application value.

And (2) biological material sample preservation, namely a hybridoma cell strain ABC06 secreting clomazone monoclonal antibody, which is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, CGMCC for short, and the preservation address is No. 3 of Xilu No.1 of Beijing Korean district, the institute of microbiology of China academy of sciences, and is classified and named as a monoclonal cell strain, wherein the preservation date is 2019, 11 months and 28 days, and the preservation number is CGMCC No. 19181.

Drawings

Figure 1 standard curve for inhibition of clomazone by ABC06 secreted monoclonal antibody.

Detailed Description

The following examples are included merely as further illustrations of the present invention and are not intended to serve as limitations or illustrations of the present invention. The invention is further illustrated by the following examples.

According to the invention, a mouse is immunized by a clomazone complete antigen, cell fusion and HAT selective culture medium culture are carried out, and cell supernatant is screened by ICELISA, so that a monoclonal antibody hybridoma cell strain ABC06 with high sensitivity to clomazone is finally obtained.

EXAMPLE 1 preparation of hybridoma cell line ABC06

1. Preparation of complete antigens

(1) Synthesis of hapten, the reaction equation is shown as the following formula:

the derivation procedure is briefly as follows:

preparation of clomazone hapten: dissolving 26 mu L of concentrated nitric acid in 100 mu L of concentrated sulfuric acid, and uniformly mixing to prepare a mixed acid solution for later use; dissolving 100 mg of clomazone in 325 mu L of concentrated sulfuric acid, stirring and dissolving at 4 ℃, dropwise adding a mixed acid solution into the clomazone solution, and reacting at-10 ℃ for 7 hours; recovering to 0 ℃, adding 2 mL of ice water, dropwise adding concentrated ammonia water to adjust the pH to 4.8-5.2, precipitating light yellow solid, carrying out vacuum filtration and drying to obtain the product CLO-NO ₂ (ii) a Taking 50 mg CLO-NO ₂ Dissolving in mixed solution of ethanol/acetic acid/water (volume ratio of 2:2: 1), placing in round bottom flask, adding 20 mg zinc powder and 3 μ L concentrated hydrochloric acid, heating and refluxing at 60 deg.C for 15 min, stirring at room temperature for 40 min to clarify; filtering to remove zinc powder, extracting with ethyl acetate, collecting organic phase, washing, drying, and evaporating under reduced pressure to obtain hapten CLO-NH ₂ ；

(2) Preparation of immunogen clomazone-BSA:

3.4 mg of prepared CLO-NH were weighed ₂ Dissolving in 200 μ L DMF, adding 4.6 mg N-hydroxysuccinimide and 7.7 mg 1-ethylcarbodiimide hydrochloride in sequence under stirring, reacting at room temperature for 4 h to obtain a mixture called solution A; then weighing 10 mg bovine serum albumin BSA and dissolving in 2 mL carbonate buffer solution, which is called solution B; slowly dropping the solution A into the solution B, reacting for 12 h at room temperature under stirring, dialyzing for 3 d with 0.01 mol/L phosphate buffer PBS to obtain a conjugate clomazone-BSA, and freezing and storing at-20 ℃ for later use;

2. preparation of coating antigen:

weighing 1.7 mg prepared CLO-NH ₂ Dissolving the mixture in 200 mu L of DMF, adding 2.3 mg of N-hydroxysuccinimide and 3.4 mg of 1-ethyl carbodiimide hydrochloride in sequence under stirring, and reacting for 4 hours at room temperature to obtain a mixture called solution A; then 10 mg of ovalbumin OVA was weighed out and dissolved in 2 mL of carbonIn an acid salt buffer solution, called as solution B; slowly dropping the solution A into the solution B, reacting at room temperature for 12 h under stirring, dialyzing with 0.01 mol/L phosphate buffer PBS for 3 d to obtain the conjugate clomazone-OVA, and freezing and storing at-20 ℃ for later use.

3. Immunization of mice: healthy BALB/c mice 6-8 weeks old were selected for immunization. Mixing and emulsifying a clomazone complete antigen and an equivalent amount of Freund adjuvant, and respectively immunizing BALB/c mice by back subcutaneous injection. Complete Freund's adjuvant was used for the first immunization, followed by incomplete Freund's adjuvant. The interval between the first immunization and the second boosting immunization is 28 days, and the interval between the boosting immunization is 21 days. The primary immunization dose is 100 mu L/mouse, the booster immunization dose is 50 mu L/mouse, and the sprint immunization dose is 25 mu L/mouse. Blood was collected 7 days after the third immunization (mice tail-cut blood 5 μ L + 995 μ L antibody dilution = antiserum), mouse serum titers and inhibition were determined using ic-ELISA, mice with high titers and good inhibition were selected, immunized by puncture 21 days after the fifth immunization, i.p., the dose of the immunization required was halved and without any adjuvant.

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:

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

(2) collecting mouse myeloma SP2/0 cells: 7-10 days before fusion, SP2/0 tumor cells were cultured in RPMI-1640 medium containing 10% FBS (fetal bovine serum) at 5% CO ₂ Culturing in an incubator. Before fusion, SP2/0 tumor cell number is required to reach (1-4) × 10 ⁷ Ensuring that SP2/0 tumor cells are in logarithmic growth phase before fusion. During fusion, tumor cells are collected and suspended in RPMI-1640 basic culture solution for cell counting;

（3）the fusion process is 7 min. 1 min, 1 mL of PEG 4000 was added to the cells dropwise from slow to fast; standing for 2 min. Dropping 1 mL of RPMI-1640 culture medium within 1 min at 3 min and 4 min; dropping 2 mL of RPMI-1640 culture medium within 1 min at 5 min and 6 min; at 7 min, 1 mL of RPMI-1640 medium was added dropwise every 10 s. Then, the mixture is incubated at 37 ℃ for 5 min. Centrifuging (800 rpm, 8 min), discarding supernatant, resuspending in RPMI-1640 screening medium containing 20% fetal calf serum, 2% 50 XHAT, adding to 96-well cell plate at 200. mu.L/well, placing at 37 ℃ and 5% CO ₂ Culturing in an incubator.

5. Cell screening and cell strain establishment: on day 3 of cell fusion, the fused cells were subjected to RPMI-1640 screening medium half-replacement, on day 5, to total-replacement with RPMI-1640 medium containing 20% fetal bovine serum and 1% 100 XHT, and on day 7, cell supernatants were collected and screened. The screening is divided into two steps: firstly, screening out positive cell holes by using ic-ELISA, secondly, selecting clomazone as a standard substance, and measuring the inhibition effect of the positive cells by using ic-ELISA. And selecting cell pores which have better inhibition on the clomazone standard substance, performing subcloning by adopting a limiting dilution method, and detecting by using the same method. The cell strain ABC06 was obtained by repeating the above steps three times.

6. Preparation and identification of monoclonal antibody: taking BALB/c mice 8-10 weeks old, and injecting 1 mL of sterile paraffin oil into the abdominal cavity of each mouse; 7 days later, each mouse was injected intraperitoneally with 1X 10 ⁶ Hybridoma cells, ascites fluid was collected from the seventh day, and the ascites fluid was purified by the octanoic acid-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 (pH 7.4), dialyzed and desalted to obtain a purified monoclonal antibody, which was stored at-20 ℃.

IC determination of monoclonal antibodies to clomazone using indirect competition ELISA ₅₀ 5.5 ng/mL, and verified its IC for famoxadone and the like ₅₀ And the cross-reactivity ratio are shown in Table 1.

TABLE 1 monoclonal antibodiesIC of ABC06 for clomazone, famoxadone and hymexazol ₅₀ And cross reaction rate

7. The application of the antibody comprises the following steps: the monoclonal antibody prepared from hybridoma cell strain ABC06 through ascites in vivo is applied to an addition recovery test of clomazone, and the method comprises the following specific steps:

(1) coating: diluting the coating source clomazone-OVA by a 0.05M carbonate buffer solution with pH of 9.6 from 1 mu g/mL by a multiple ratio, reacting at the temperature of 37 ℃ for 2 h at 100 mu L/hole;

(2) washing: the plate solution was decanted and washed 3 times for 3 min each with washing solution;

(3) and (3) sealing: after patting to dryness, 200. mu.L/well blocking solution was added and reacted at 37 ℃ for 2 hours. Drying for later use after washing;

(4) sample adding: diluting antiserum (which is obtained by diluting the antiserum by a corresponding multiple with an antibody diluent after tail-cutting blood collection of a mouse) from 1:1000 by multiple, adding the diluted antiserum into coated holes of each dilution, reacting at the temperature of 100 mu L/hole for 30 min; after fully washing, adding HRP-goat anti-mouse IgG diluted by 1:3000, 100 mu L/hole, and reacting for 30 min at 37 ℃;

(5) color development: taking out the ELISA plate, fully washing, adding 100 mu L of TMB color development liquid into each hole, and reacting for 15 min at 37 ℃ in a dark place;

(6) termination and measurement: the reaction was stopped by adding 50. mu.L of a stop buffer to each well, and the OD450 value of each well was measured by a microplate reader.

IC-ELISA for determination of IC of monoclonal antibody to clomazone ₅₀ The concentration is 5.5 ng/mL, which shows that the sensitivity to clomazone is very good, and the clomazone can be used for immunoassay detection of clomazone.

The standard curve for inhibition of clomazone by monoclonal antibody secreted from ABC06 is shown in fig. 1.

Solution preparation:

carbonate Buffer (CBS): weighing Na ₂ CO ₃ 1.59 g，NaHCO ₃ 2.93 g, respectively dissolving a small amount of double distilled water, mixing, adding the double distilled water to about 800 mL, uniformly mixing, adjusting the pH value to 9.6, adding the double distilled water to a constant volume of 1000 mL, and storing at 4 ℃ for later use;

phosphate Buffered Saline (PBS): 8.0 g NaCl, 0.2g KCl, 0.2g KH ₂ PO ₄ ，2.9 g Na ₂ HPO ₄ •12 H ₂ Dissolving O in 800 mL of pure water, adjusting the pH value to 7.2-7.4 by using NaOH or HCl, and fixing the volume to 1000 mL;

wash solution (PBST): adding 0.5 mL of Tween-20 into 1000 mL of PBS solution with the concentration of 0.01 mol/LpH7.4;

PBST: PBS containing 0.05% Tween-20;

antibody dilution: wash buffer containing 0.1% gelatin;

TMB color development liquid: solution A: na (Na) ₂ HPO ₄ •12H ₂ 18.43 g of O, 9.33 g of citric acid and pure water to reach the constant volume of 1000 mL; and B, liquid B: 60 mg of TMB was dissolved in 100 mL of ethylene glycol. A. Mixing the liquid B according to the volume ratio of 1:5 to obtain the TMB color developing solution, and mixing the liquid B at the present time.

In summary, although the present invention has been disclosed in terms of the preferred embodiments, it is not intended to limit the invention, and various changes and modifications can be made by one skilled in the art without departing from the spirit and scope of the invention.

Claims

1. A hybridoma cell strain ABC06 secreting clomazone monoclonal antibody is deposited in China general microbiological culture Collection center (CGMCC), China academy of sciences microorganism institute No. 3, Xilu No.1 Hospital, the morning area, Beijing, and is classified and named as monoclonal cell strain, and the preservation date is as follows: 28 months 11 and 2019, and the preservation number is CGMCC No. 19181.

2. Clomazone monoclonal antibody, characterized in that: it is secreted and produced by hybridoma cell strain ABC06 with the preservation number of CGMCC No.19181 as claimed in claim 1.

3. The use of the clomazone monoclonal antibody according to claim 2, which is characterized in that the clomazone monoclonal antibody is used for analyzing and detecting clomazone residue in food safety detection.