CN113774030B

CN113774030B - Hybridoma cell strain secreting anti-picloram monoclonal antibody and application thereof

Info

Publication number: CN113774030B
Application number: CN202111117099.XA
Authority: CN
Inventors: 胥传来; 吴慧慧; 匡华; 徐丽广; 孙茂忠; 刘丽强; 吴晓玲; 宋珊珊; 胡拥明; 郝昌龙; 高巍; 马伟; 吴爱红
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2021-09-23
Filing date: 2021-09-23
Publication date: 2022-05-24
Anticipated expiration: 2041-09-23
Also published as: CN113774030A

Abstract

The invention discloses a hybridoma cell strain secreting an anti-picloram monoclonal antibody and application thereof, and belongs to the field of food safety immunodetection. The hybridoma cell strain secreting the anti-picloram monoclonal antibody has been preserved in the China general microbiological culture Collection center of the Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 20794. The monoclonal antibody cell strain of the invention can be used for immunoassay detection, and has good detection sensitivity and specificity (IC) for picloram₅₀The value was 50 ng/mL). The achievement of the invention can use the immunodetection kit for preparing the picloram and the colloidal gold test strip to provide a powerful detection method and means for detecting the residual quantity of the picloram in grains, oil, vegetables, sugar and tobacco.

Description

Hybridoma cell strain secreting anti-picloram monoclonal antibody and application thereof

Technical Field

The invention belongs to the technical field of food safety immunodetection, and particularly relates to a hybridoma cell strain secreting an anti-picloram monoclonal antibody and application thereof.

Background

Picloram is a novel pyridine carboxylic acid herbicide widely used for weed control in mountainous regions, grasslands, planting fields and non-cultivated lands, and is now being researched and developed for weed control in rape and cereal crop fields. The preparation has low toxicity, no teratogenicity, mutagenicity, carcinogenesis, no adverse side effect on endocrine and reproduction, and low toxicity to human. Picloram belongs to a synthetic hormone type herbicide. It can be quickly absorbed by stem, leaf and root of plant, and can induce the plant to produce epinasty reaction in the sensitive plant body so as to make the plant grow and die quickly.

At present, the method for detecting picloram residues is mainly an instrumental analysis method, and comprises a gas chromatography method, a gas chromatography-mass spectrometry tandem method and the like. The methods have reliable results and high sensitivity, and related technical standards are available for reference. However, expensive instruments and special operators are required, and the sample pretreatment is complicated, high in cost and long in time, so that the rapid and simple field detection requirement cannot be better met.

Therefore, the establishment of a rapid and simple picloram detection method is of great significance. An enzyme-linked immunosorbent assay (ELISA) is an extremely high-efficiency, 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 picloram.

Disclosure of Invention

In order to solve the technical problems, the invention provides a hybridoma cell strain secreting an anti-picloram monoclonal antibody, and a construction method and application thereof. The antibody secreted by the cell strain has higher detection sensitivity for picloram, and can be used for establishing an immunological detection method for picloram. The achievement of the invention can use the immunodetection kit for preparing the picloram and the colloidal gold test strip to provide a powerful detection method and means for detecting the residual quantity of the picloram in grains, oil, vegetables, sugar and tobacco.

A hybridoma cell strain secreting an anti-picloram monoclonal antibody is preserved in China general microbiological culture Collection center at 27.09.2020/27.A preservation address of No. 3 Hospital No. 1 of Beijing, Chaoyang, and a preservation number of CGMCC No. 20794.

A preparation method of a hybridoma cell strain secreting an anti-picloram monoclonal antibody comprises the following steps:

s1: adding Picloram solution to SOCl₂Heating and refluxing to form an acidizing solution of picloram, performing solid-liquid separation after the reaction is finished to obtain a solid phase, dissolving the solid phase in an organic solvent to obtain a picloram chlorine solution, uniformly stirring the picloram chlorine solution and a bovine serum albumin BSA (bovine serum albumin) alkaline solution, performing solid-liquid separation after the reaction is finished to obtain a filtrate, and dialyzing the obtained filtrate to obtain a picloram hapten;

s2: mixing and dissolving the picloram hapten, the N-hydroxysuccinimide and the 1-ethyl carbodiimide hydrochloride obtained in the step S1 in an organic solvent, reacting for 6-8 hours to obtain a mixture, adding the picloram hapten into a protein solution, and reacting to obtain a conjugate of the picloram hapten and bovine serum albumin BSA;

s3: mixing and emulsifying the conjugate obtained in S2 and complete Freund 'S adjuvant to obtain immunogen 1, and mixing and emulsifying the conjugate obtained in S2 and incomplete Freund' S adjuvant to obtain immunogen 2;

s4: the immunogen 1 obtained in the S3 is used for subcutaneous immunization of mice, the immunized mice are further boosted by using the immunogen 2 in the S3, and the conjugate of the picloram hapten and the protein in the S2 is used for sprint immunization;

s5: and (3) taking spleen cells and myeloma cells of the immunized mouse punched in S4, and carrying out cell fusion to obtain the hybridoma cell strain.

In one embodiment of the present invention, the picloram in S1: the mol ratio of the thionyl chloride is 1:1.5-1: 2.

in one embodiment of the present invention, the molar ratio of the picloram to the RSA in the picloram solution in S1 is 1:1.5-1: 2.

In one embodiment of the present invention, the alkali in the alkaline solution in S1 is sodium hydroxide or potassium hydroxide.

In one embodiment of the present invention, the protein in the protein solution in S2 is chicken ovalbumin or bovine serum albumin BSA.

In one embodiment of the present invention, the molar ratio of picloram hapten to N-hydroxysuccinimide in S2 is 3:1-4: 1.

In one embodiment of the present invention, the molar ratio of the picloram hapten to the 1-ethylcarbodiimide hydrochloride in S2 is 3:1 to 4: 1.

The hybridoma cell strain is applied to the preparation of the amlodipine acid monoclonal antibody.

The monoclonal antibody against picloram is obtained by secreting the hybridoma cell strain.

The monoclonal antibody against picloram is applied to the detection of picloram drugs.

A method for detecting picloram drugs utilizes the anti-picloram monoclonal antibody to carry out detection.

A kit containing the monoclonal antibody against aminopyralid.

In one embodiment of the invention, the kit is used for detecting picloram.

A test strip comprises the monoclonal antibody against the aminopyralid.

In one embodiment of the invention, the test strip is used for detecting picloram.

Compared with the prior art, the technical scheme of the invention has the following advantages:

the monoclonal antibody secreted by the hybridoma cell strain 1A8 has good specificity and detection sensitivity (IC) on picloram₅₀The value is 50ng/mL), the detection of the residual quantity of the picloram in the grains, the oil, the vegetables and the sugar can be realized, the raw material is provided for the immunological detection of the residual picloram in the food, and the practical application value is realized. The monoclonal antibody has strong specificity, can greatly improve the specificity of antigen-antibody reaction, reduces possible cross reaction, ensures that the reliability of a test result in a detection process is higher, and simultaneously ensures that the uniformity and the biological activity unicity of the monoclonal antibody ensure that the quality of the antigen-antibody reaction result is convenient to control, thereby being beneficial to standardization and normalization.

Drawings

In order that the present disclosure may be more readily and clearly understood, reference is now made to the following detailed description of the embodiments of the present disclosure taken in conjunction with the accompanying drawings, in which

FIG. 1 is a standard curve of the inhibition of picloram by the monoclonal antibody 1A8 of the present invention.

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 experimental raw materials adopted by the invention can be purchased from the market or prepared according to the existing method.

Solution preparation:

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): 8.0g NaCl, 0.2g KCl, 0.2g KH₂PO₄，2.9gNa₂HPO₄·12H₂Dissolving O in 800mL of pure water, adjusting the pH value to 7.2-7.4 by using NaOH or HCl, and fixing the volume to 1000 mL;

washing solution (PBST): adding 0.5mL of Tween-20 into 1000mL 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)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. Mixing the solution B according to the volume ratio of 1:5 to obtain TMB. The color developing liquid is mixed at present.

Example 1

Preparation of hybridoma cell line 1A8

1. Preparation of complete antigens

(1) Synthesis of hapten, the reaction equation is shown in the following chart:

the derivation procedure is briefly as follows:

1. preparation of picloram hapten:

picloram (50mg) was treated with 5mL of thionyl chloride (SOCI)₂) Dissolved in a small flask. The solution was refluxed at 85 ℃ for 2.5h to form an acidified solution of picloram. Excess chloride was removed on a rotary evaporator under vacuum at 60 ℃ and the residue was dissolved in 2mL of Tetrahydrofuran (THF). Mixing the picloram chloride solution with 200mg bovine serum albumin BSA in 10mL 0.02M NaOH solutionSlowly stirring. An insoluble precipitate formed after stirring for 18 hours at room temperature before the addition of the acid chloride solution was complete. The reaction mixture was diluted to 200mL with 0.02M NaOH solution, successfully dissolving most of the precipitate. The resulting suspension was centrifuged to remove any precipitate. The supernatant was dialyzed against cold flowing tap water for 24h and lyophilized.

2. Preparation of coating antigen:

weighing 2.9mg of prepared picloram hapten, dissolving in 200 microliter of DMF, slowly stirring, sequentially adding 4.5mg of N-hydroxysuccinimide and 5.4mg of 1-ethyl carbodiimide hydrochloride, and reacting at room temperature for 6-8h to obtain a mixture; then weighing 10mg of chicken ovalbumin OVA, dissolving in 2mL of carbonate buffer solution, slowly dropping the obtained mixture into the chicken ovalbumin solution, reacting for 14h at room temperature under stirring, dialyzing for 3d by 0.01mol/L phosphate buffer PBS to obtain the conjugate of picloram-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. After the complete antigen of the picloram is mixed and emulsified with the same amount of Freund's adjuvant, BALB/c mice are immunized respectively through 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. Blood was collected 7 days after the third immunization (mice were bled with tail-off 5 μ L +995 μ L antibody diluent ═ antiserum), serum titers and inhibition were determined in mice using ic-ELISA, mice with high titers and good inhibition were selected, immunized by puncture 21 days after the fifth immunization, injected intraperitoneally, requiring a halved priming dose 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 the mice are killed by a cervical vertebra dislocation method, soaking for about 5min, taking out the spleen of the mice by aseptic operation, properly grinding the spleen by using a rubber head of an injector, 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, diluting the spleen 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% CO₂An incubator. Before fusion, the number of SP2/0 tumor cells is required to reach 1-4 multiplied by 10⁷Ensuring 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. 1min, 1mL of PEG 1500 is added to the cells in a dropwise manner 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. After centrifugation (800rpm, 8min), the supernatant was discarded and resuspended in RPMI-1640 screening medium containing 20% fetal bovine serum and 2% 50 XHAT, 200. mu.L/well of the medium was applied to a 96-well cell plate and cultured in a 5% CO2 incubator at 37 ℃.

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 for screening. The screening is divided into two steps: firstly, screening out positive cell holes by using ic-ELISA, and secondly, selecting picloram as a standard substance and measuring the inhibition effect of the positive cells by using the ic-ELISA. And selecting cell pores which have better inhibition on the picloram standard, carrying out subcloning by adopting a limiting dilution method, and carrying out detection by using the same method. This was repeated three times to obtain cell line 1A 8.

6. Preparation and identification of monoclonal antibody: 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 10⁶Hybridoma 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, caprylic acid can precipitate other hybrid proteins except IgG immunoglobulin in ascites, and then is separatedRemoving the precipitate; then, the IgG type monoclonal antibody is precipitated by an ammonium sulfate solution of the same saturation, centrifuged, the supernatant is discarded, dissolved by a 0.01MPBS solution (pH 7.4), dialyzed and desalted, and finally the purified monoclonal antibody is stored at-20 ℃.

Determination of IC of monoclonal antibody against Picloram Using Indirect competitive ELISA₅₀50ng/mL, and IC for picloram and the like was verified₅₀And the cross-reactivity ratio are shown in Table 1.

TABLE 1 IC of monoclonal antibody 1A8 for Picloram, Picloram₅₀And cross reaction rate

7. The application of the antibody comprises the following steps: the monoclonal antibody prepared from the hybridoma cell strain 1A8 through ascites in vivo is applied to an addition recovery test of picloram, and the method comprises the following specific steps:

(1) coating: the coated amlodipine acid-OVA was diluted at a rate of 1. mu.g/mL in 0.05M carbonate buffer pH 9.6, and reacted at 37 ℃ for 2 hours at 100. mu.L/well.

(2) Washing: the plate solution was decanted and washed 3 times for 3min 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. And drying after washing for later use.

(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 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) 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.

(6) Termination and measurement: the reaction was stopped by adding 50. mu.L of a stop solution to each well, and the OD of each well was measured by a microplate reader₄₅₀The value is obtained.

Determination of IC of monoclonal antibody to Picloram by IC-ELISA₅₀The concentration is 50ng/mL, which shows that the sensitivity to picloram is good, and the method can be used for the immunoassay detection of picloram. The standard curve of the inhibition of picloram by the monoclonal antibody 1A8 obtained in this example is shown in fig. 1.

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 scope of the invention.

Claims

1. A hybridoma cell strain secreting an amlodipine acid-resistant monoclonal antibody is characterized in that the hybridoma cell strain is preserved in China general microbiological culture Collection center at 09 months and 27 days in 2020, with the preservation address of No. 3 Xilu No. 1 Beijing of the Chaoyang district in Beijing, and the preservation number is CGMCC No. 20794.

2. A preparation method of a hybridoma cell strain secreting an anti-picloram monoclonal antibody is characterized by comprising the following steps:

s1: adding an picloram acid solution into thionyl chloride, heating and refluxing to form an acidizing solution of picloram, performing solid-liquid separation after the reaction is finished, taking a solid phase, dissolving the solid phase in an organic solvent to obtain a picloram chlorine solution, uniformly stirring the picloram chlorine solution and an alkaline solution of bovine serum albumin BSA (bovine serum albumin), performing solid-liquid separation after the reaction is finished, and taking a filtrate to obtain a picloram hapten;

s2: mixing and dissolving the obtained picloram hapten, N-hydroxysuccinimide and 1-ethyl carbodiimide hydrochloride in the S1 in an organic solvent, reacting to obtain a mixture, and adding the picloram hapten into a bovine serum albumin solution for reacting to obtain a conjugate of the picloram hapten and the bovine serum albumin;

s4: the immunogen 1 obtained in the S3 is used for subcutaneous immunization of a mouse, the immunized mouse is further boosted by using the immunogen 2 in the S3, and the conjugate of the picloram hapten and bovine serum albumin in the S2 is used for sprint immunization;

s5: taking splenocytes and myeloma cells of the mice which are subjected to the sprint immunization in S4, and carrying out cell fusion to obtain the hybridoma cell strain; the structural formula of the picloram hapten in S1 is as follows:

。

3. the hybridoma cell strain of claim 1, wherein the hybridoma cell strain is used for preparing an anti-picloram monoclonal antibody.

4. An anti-picloram monoclonal antibody, wherein the anti-picloram monoclonal antibody is secreted by the hybridoma cell line of claim 1.

5. The use of the monoclonal antibody against picloram according to claim 4 for the analytical detection of picloram.

6. A kit comprising the monoclonal antibody against aminopyralid according to claim 4.

7. The kit of claim 6, wherein the kit is for detecting picloram.

8. A test strip comprising the monoclonal antibody against aminopyralid of claim 4.

9. The test strip of claim 8, wherein the test strip is for detecting picloram.