CN114807051B - Hybridoma cell strain of anti-dechloridone monoclonal antibody and application thereof - Google Patents

Hybridoma cell strain of anti-dechloridone monoclonal antibody and application thereof Download PDF

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CN114807051B
CN114807051B CN202210509601.XA CN202210509601A CN114807051B CN 114807051 B CN114807051 B CN 114807051B CN 202210509601 A CN202210509601 A CN 202210509601A CN 114807051 B CN114807051 B CN 114807051B
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monoclonal antibody
dechloridone
hybridoma cell
cell strain
dechlorine
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胥传来
姚静静
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
吴爱红
郭玲玲
胥欣欣
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Jiangnan University
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Abstract

The invention relates to a hybridoma cell strain of an anti-dechloridone monoclonal antibody and application thereof, and relates to the technical field of food safety immunodetection. The hybridoma cell strain of the anti-dechloridone monoclonal antibody provided by the invention is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms for 2022, and has a preservation address of Beijing city, chaoyang region, north Chen Xi Lu No. 1,3 and a preservation number of CGMCC No.45115. The monoclonal antibody secreted by the cell strain provided by the invention has better specificity and detection sensitivity (IC) to the dechloridone 50 A value of 1.1. Mu. g L ‑1 ) Can realize the detection of the residual quantity of the dechlorine ketone, provides raw materials for the immunodetection of the residual dechlorine ketone in food, and has practical application value.

Description

Hybridoma cell strain of anti-dechloridone monoclonal antibody and application thereof
Technical Field
The invention relates to the technical field of food safety immunodetection, in particular to a hybridoma cell strain of an anti-dechlorine ketone monoclonal antibody and application thereof.
Background
Tectone (CLD), also known as Kaempferia galanga (Kepone), is a synthetic organochlorine pesticide. Since decachloroketone is poorly soluble in water, has strong hydrophobicity, is particularly easy to enrich in organisms, is difficult to chemically and biologically degrade, has a possibility of long-distance cross-boundary migration, and has a property of being able to permanently present Persistent Organic Pollutants (POPs) in the environment has been disabled. Tectone has been widely used for controlling banana root borers, it is also used for controlling fly larvae pesticides, bactericides, potato beetles, rust mites, potato and tobacco rootworm, and in ants and cockroach attractants, and it is a highly toxic organic substance, having a certain influence on the fetal central nervous system, urogenital system, immune system. The dechlorine ketone can affect the nervous system of human body, and can cause symptoms such as tremble, weight loss, eyeball spasm, costal membrane inflammation, arthralgia, hepatomegaly, oligospermia, etc. Experimental researches on human bodies and animals show that after being absorbed, the decachloroketone is mainly enriched in the liver and is slowly metabolized through bile and discharged out of the body through excrement, and the decachloroketone has obvious carcinogenicity on rats and mice and can cause malignant tumors of other organs such as the liver. Therefore, the establishment of a method for rapidly and effectively detecting the content of the decachloroketone has important significance and market value.
The efficient and sensitive detection method for the dechloridone is a problem to be solved urgently. Current detection methods include instrumental and immunoassay. Instrumental analyses such as high performance liquid chromatography (Wang Hui, peng Ying, mu Su, et al gas chromatography-tandem mass spectrometry for determining the presence of decachloroketone [ J ]. Chinese environmental monitoring, 2015 (5): 129-133; saint-Hilaire M, inthavong C, bertin T, lavison-Bompard G, guerin T, fournier A, feidint C, rychen G, parinet J.development and validation of an HPLC-MS/MS method with QuEChERS extraction using isotopic dilution to simultaneously analyze chlordecone and chlordecol in animal lists.food chem.2018,30; 252:147-153.), gas chromatography-tandem mass spectrometry (Martin-Laurent F, sahnoun MM, merlin C, vollmer G, L bke M.Detec and quantification of chlordecone in contaminated soils from the French West Indies by GC-MS using 13C10-chlordecone stable isotope as a tracer [ J ]. Environ Sci Pollut Res Int.2014,21 (7.4928-4933;ALAIN SOLER,MARC LEBRUN,YOAN LABROUSSE,et al.Solid-phase microextraction and gas chromatography-mass spectrometry for quantitative determination of chlordecone in water; plant and soil samples): 2014). Because of complicated pretreatment of the sample, more interferents and limitation of the working conditions of the instrument, and higher technical requirements on operators, the instrument method is not suitable for on-site detection.
Compared with an instrument detection method, the immunoassay method has the characteristics of low cost, high flux, high sensitivity, low requirements on technicians and the like, and is suitable for rapid screening of a large number of samples. Therefore, the immunoassay method has important significance for detecting the dechloridone.
The enzyme-linked immunosorbent assay (ELISA) is a low-cost, rapid and portable immunological detection method, has low requirements on the purity of samples during detection, is simple and convenient to operate, and is suitable for rapidly detecting results on site of a large number of samples. An efficient immunological detection method is established, and the screening of monoclonal antibodies with high specificity is an important precondition.
Disclosure of Invention
In order to solve the technical problems, the invention provides a hybridoma cell strain of an anti-dechloridone monoclonal antibody and application thereof. The antibody secreted by the hybridoma cell strain has higher detection sensitivity against the dechlorine ketone, and can be used for establishing an immunological detection method against the dechlorine ketone.
The first object of the present invention is to provide a hybridoma cell strain of an anti-dechloridone monoclonal antibody, wherein the hybridoma cell strain is preserved in the China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) at the year 03 and the preservation address is 1,3 and the preservation number is CGMCC No.45115 in the Korean region of Beijing city, and the obtained hybridoma cell strain belongs to the monoclonal cell strain.
The second object of the present invention is to provide a method for preparing a hybridoma cell strain of an anti-dechloridone monoclonal antibody, comprising the steps of:
s1, adding gram Fan Rong into an organic solvent, adding an alkaline solution, heating to 70-90 ℃, stirring and reacting for 0.5-2h, and regulating the pH value to 4-5 by using an acidic solution to obtain a decachloroketone hapten;
s2, preparing a complete antigen of the dechloridone by using the hapten of the dechloridone in the S1, and preparing the complete antigen of the dechloridone into a Freund complete adjuvant containing an antigen and a Freund incomplete adjuvant containing an antigen;
s3, performing primary immunization, reinforcing immunization and sprint immunization on the immunized animal, wherein the primary immunization adopts the antigen-containing Freund complete adjuvant in S2, the reinforcing immunization adopts the antigen-containing Freund incomplete adjuvant in S2, and the sprint immunization adopts the dechloridone complete antigen in S2;
s4, taking spleen cells and myeloma cells of the immunized animal subjected to sprint immunization in the S3 to perform cell fusion, so as to obtain the hybridoma cell strain of the anti-dechlorine ketone monoclonal antibody.
In one embodiment of the invention, in S1, extraction, purification and drying steps are also included.
In one embodiment of the present invention, in S1, the organic solvent is one or more of dimethyl sulfoxide, methanol, and tetrahydrofuran.
In one embodiment of the present invention, in S1, the alkaline solvent in the alkaline solution is sodium hydroxide and/or potassium hydroxide.
In one embodiment of the invention, the molar ratio of the substances of the gram and of the basic solvent is 1:1-3.
In one embodiment of the invention, the molar ratio of the substances of the gram and of the basic solvent is 1:2.
in one embodiment of the invention, in S1, the concentration of the organic solvent in grams ranges from 0.01 to 0.05g/mL.
In one embodiment of the invention, in S1, the concentration of the organic solvent in grams is 0.02g/mL.
In one embodiment of the present invention, in S1, the acidic solution is a hydrochloric acid solution.
In one embodiment of the present invention, in S1, the decachloroketone hapten has the following structural formula:
in one embodiment of the present invention, in S1, the preparation method of the decachloroketone hapten specifically includes the steps of adding 1 equ.g. Fan Rong to dimethyl sulfoxide, adding 2 equ.sodium hydroxide solution, heating to 80 ℃ and stirring for reaction for 1h; adding pure water, adjusting pH to 4-5 with hydrochloric acid solution, extracting, purifying, and drying to obtain white solid as decachloroketone hapten.
In one embodiment of the invention, in S2, the complete decachloroketone antigen is obtained by coupling the decachloroketone hapten to a carrier protein; the structural formula of the dechloridone complete antigen is as follows:
in one embodiment of the invention, the carrier protein comprises Bovine Serum Albumin (BSA).
In one embodiment of the present invention, in S2, the method for preparing the complete decachloroketone antigen includes the steps of dissolving the complete decachloroketone antigen, N-hydroxysuccinimide (NHS) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI) in a solvent to obtain a mixed solution, and adding the mixed solution into a carrier protein solution to react to obtain the complete decachloroketone antigen.
In one embodiment of the invention, the solvent is N, N-Dimethylformamide (DMF).
In one embodiment of the invention, the mass ratio of the decachloroketone hapten, N-hydroxysuccinimide (NHS) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI) is 1:1-1.5:1-1.5.
In one embodiment of the invention, the mass ratio of the decachloroketone hapten, N-hydroxysuccinimide (NHS) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI) is 1:1.2:1.2.
in one embodiment of the invention, the concentration of the decachloroketone hapten in the solvent is 10.0-14.0g/L.
In one embodiment of the invention, the concentration of the decachloroketone hapten in the solvent is 12.0g/L.
In one embodiment of the invention, the mass ratio of the decachloroketone hapten to the carrier protein is from 30 to 80:1.
in one embodiment of the invention, the mass ratio of the decachloroketone hapten to the carrier protein is 50:1.
in one embodiment of the present invention, in S2, the preparation method of the complete decachloroketone antigen specifically includes the steps of dissolving the complete decachloroketone antigen KLV in anhydrous N, N-dimethylformamide, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), and reacting N-hydroxysuccinimide (NHS) with stirring at room temperature, which is referred to as solution a; weighing carrier protein, and adding boric acid buffer solution called solution B; slowly dropwise adding the solution A into the solution B under stirring, and reacting at room temperature to obtain a mixed solution containing KLV-BSA; dialyzing to obtain the complete antigen of the dechloridone.
In one embodiment of the invention, the volume ratio of the anhydrous N, N-dimethylformamide to the boric acid buffer solution is 1:5-15.
In one embodiment of the invention, the volume ratio of the anhydrous N, N-dimethylformamide to the boric acid buffer solution is 1:10.
in one embodiment of the invention, in S2, the antigen-containing freund 'S complete adjuvant is an emulsion of equal volumes of freund' S complete adjuvant and decachloroketone complete antigen.
In one embodiment of the invention, in S2, the antigen-containing incomplete Freund 'S adjuvant is an emulsion of equal volumes of incomplete Freund' S adjuvant and complete decachloroketone antigen.
In one embodiment of the invention, in S3, the entire immunization process includes 1 first immunization, 4-6 booster immunizations, and 1 sprint immunization.
In one embodiment of the invention, the number of booster immunizations is 5.
In one embodiment of the invention, in S3, the interval between the first immunization and the boost is 28-31 days, the interval between the boost is 20-22 days, and the interval between the boost and the sprint is 18-21 days during the whole immunization process.
In one embodiment of the invention, in S3, the dose of the first immunization is 95-105 μg/dose of the whole immunization process, the dose of the booster immunization is 45-55 μg/dose of the sprint immunization is 20-30 μg/dose of the whole immunization process.
In one embodiment of the present invention, in S3, the immunized animal is collected during the booster immunization, serum titer and inhibition rate are detected by indirect competitive enzyme-linked immunosorbent assay (iceelisa), and the immune animal epidemic with high content of the oxychloroketone antibody in serum is screened.
In one embodiment of the invention, the blood collection is performed on days 6-8 after the end of the booster immunization procedure.
In one embodiment of the invention, in S3, the immunization is injected subcutaneously into the immunized animal via the back.
In one embodiment of the present invention, in S4, the cell fusion is to culture the fused cells in HAT medium, detect positive cells by indirect ELISA, further measure the inhibition effect of positive cells by indirect competition ELISA, subclone the positive cells with the best inhibition by limiting dilution, and obtain hybridoma cell lines.
In one embodiment of the invention, the HAT medium is RPMI-1640 medium.
In one embodiment of the invention, the number of subclones is 2-4.
In one embodiment of the invention, in S4, the cell fusion is performed by the polyethylene glycol (PEG 4000) method.
In one embodiment of the invention, in S4, the cell fusion is performed 2-4 days after the termination of the sprint immunization.
The third object of the invention is to provide an application of the hybridoma cell strain in preparing a dechloridone monoclonal antibody.
The fourth object of the present invention is to provide a dechloridone monoclonal antibody secreted by a hybridoma cell strain with a preservation number of CGMCC No.45115.
In one embodiment of the invention, paraffin oil is injected into the abdominal cavity of an immunized animal, then hybridoma cell strain with the preservation number of CGMCC No.45115 is injected into the abdominal cavity, ascites is collected after injection, and the ascites is purified, so that the dechloridone monoclonal antibody is obtained.
In one embodiment of the invention, 8-10 week old BALB/c mice are intraperitoneally injected with 1mL of paraffin oil, 1X 10 per mouse after 7 days 6 Collecting ascites from the hybridoma cell strain with the preservation number of CGMCC No.45115, purifying the ascites by an octanoic acid-ammonium sulfate method, and preserving the obtained dechloridone monoclonal antibody at-20 ℃.
It is a fifth object of the present invention to provide a composition comprising said hybridoma cell line and/or said dodecanone monoclonal antibody.
A sixth object of the present invention is to provide a kit comprising one or more of the hybridoma cell line, the dechlorinone monoclonal antibody and the composition.
The seventh object of the invention is to provide an application of the hybridoma cell strain, the dechlorine ketone clone antibody, the composition or the kit in detection of dechlorine ketone, especially in analysis and detection of dechlorine ketone residues in food safety detection.
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 the dechloridone 50 A value of 1.1. Mu. g L -1 ) Can realize the detection of the residual quantity of the dechlorine ketone, provides raw materials for the immunodetection of the residual dechlorine ketone in food, and has practical application value.
Preservation of biological materials
The hybridoma cell strain secreting the anti-dechloridone monoclonal antibody belongs to a monoclonal cell strain, the hybridoma cell strain is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms (China) at the 03 month of 2022, the preservation address is the North Chen West Lu No. 1 No. 3 in the Korean region of Beijing city, the preservation number is CGMCC No.45115, and the hybridoma cell strain is classified and named as the monoclonal cell strain.
Drawings
In order that the invention may be more readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings, in which:
FIG. 1 is a schematic representation of subtype identification of a monoclonal antibody of the invention.
FIG. 2 shows affinity assays for monoclonal antibodies of the invention.
FIG. 3 is a standard curve of inhibition of dechlorine by a monoclonal antibody of the invention.
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.
(1) The following examples relate to the following media:
RPMI-1640 medium (mg/L): l-arginine 290, L-asparagine 50, L-aspartic acid 20, L-cystine dihydrochloride 65.15, L-glutamic acid 20, glycine 10, L-histidine 15, L-hydroxyproline 20, L-isoleucine 50, L-leucine 50, L-lysine hydrochloride 40, L-methionine 15, L-phenylalanine 15, L-proline 20, L-serine 30, L-threonine 20, L-tryptophan 5, L-tyrosine 23.19, L-valine 20, p-aminobenzoic acid 1, calcium nitrate 100, anhydrous magnesium sulfate 48.84, anhydrous sodium dihydrogen phosphate 676.13, potassium chloride 400, sodium chloride 6000, glucose 2000, reduced glutathione 1, phenol red 5, L-glutamine 300, biotin 0.2, D-calcium pantothenate 0.25, folic acid 1, i-inositol 35, nicotinamide 1, choline chloride 3, pyridoxine hydrochloride 1, riboflavin 0.2, thiamine hydrochloride 1, vitamin B120.005, sodium bicarbonate 2000.
(2) The configuration of the solutions involved in the following examples 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, uniformly mixing, adjusting the pH value to 9.6, adding the double distilled water to 1000mL, and storing at 4 ℃ for later use;
phosphate Buffer (PBS): 8.0g NaCl,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;
antibody dilution: PBS was added with 0.1% gelatin;
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: 5, mixing to obtain TMB color development liquid, and mixing immediately.
(3) The detection method involved in the following examples is as follows:
detection of inhibition rate of dechlorine ketoneThe method comprises the following steps: the most appropriate antigen and antibody concentrations in the ic-ELISA were selected by a checkerboard assay. The antigen was diluted to 0.03,0.1,0.3 and 1 μg/mL with Carbonate Buffer (CBS) and the antibody was diluted to 0.03,0.1,0.3 and 1 μg/mL with antibody dilution. After selecting the best working point, diluting the standard substance of the dechlorine ketone to 0,0.01,0.03,0.11,0.33,1,3 and 9 mu g/mL, and calculating IC according to the IC-ELISA operation steps, finally, using OriginPro 8.5 as a graph to obtain the standard inhibition curve of the dechlorine ketone 50 。
Examples
A hybridoma cell strain of an anti-dechloridone monoclonal antibody and a preparation method thereof specifically comprise the following steps:
A. synthesis of decachloroketone hapten
The synthesis route of the decachloroketone hapten KLV is as follows:
0.10g (0.16 mmol) of Fan Rong g of dimethyl sulfoxide was added to 2mL of dimethyl sulfoxide, 6mL of 1mol L -1 Heating NaOH solution to 80 ℃ and stirring for reaction for 1h; after the reaction solution temperature is reduced to room temperature, adding a proper amount of pure water, using 1mol L -1 HCl was adjusted to pH 4, extracted with ethyl acetate, anhydrous Na 2 SO 4 Drying and concentrating; purifying with silica gel column, concentrating, and drying to obtain white solid, namely the decachloroketone hapten KLV.
B. Preparation of complete antigen of dechloridone
The hapten KLV prepared in the step A is coupled with Bovine Serum Albumin (BSA) to obtain complete antigen KLV-BSA, and the complete antigen KLV-BSA is specifically prepared by the following steps:
a. the hapten KLV 4.6mg (0.0075 mmol) obtained in the step A is dissolved in 200 mu L of anhydrous N, N-dimethylformamide, 1.7mg (0.009 mmol) of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) and 1.0mg (0.009 mmol) of N-hydroxysuccinimide (NHS) are added in sequence, and the mixture is stirred at room temperature for reaction for 0.5h, which is called solution A; 10mg (0.00015 mmol) of bovine serum albumin BSA was weighed and 2mL of boric acid buffer solution, designated as solution B, was added; slowly dropwise adding the solution A into the solution B under stirring, and reacting for 2 hours at room temperature to obtain a mixed solution containing KLV-BSA;
b. and (3) dialysis: taking a 10cm dialysis bag, boiling in boiling water for 5min, washing with deionized water at 60 ℃ for 3min, and preserving in deionized water at 4 ℃ for later use; the mixture containing KLV-BSA in step a was placed in a dialysis bag and treated with 0.01mol L -1 PBS is used as a dialyzate, the dialyzate is dialyzed for 3d at 4 ℃, and the dialyzate is replaced for three times a day, so that complete antigen, unconjugated hapten and other small molecular substances are separated, and the complete decachloroketone antigen is obtained.
Complete antigens prepared include KLV-BSA immunogen and KLV-OVA coating antigen, wherein the immunogen KLV-BSA was used for the next step of mouse immunization.
The preparation of the coated original KLV-OVA is the same as that of KLV-BSA and is used in the detection of the subsequent application.
C. Immunization of mice
After the KLV-BSA immunogen was mixed and emulsified with an equal volume of Freund's adjuvant, an injection was obtained and BALB/c mice were injected subcutaneously through the nape of the neck.
First immunization (100. Mu.g/dose) with an equal volume of mixed emulsion of Freund's complete adjuvant and KLV-BSA immunogen as the injection, and 5 booster immunization (50. Mu.g/dose) with an equal volume of mixed emulsion of Freund's incomplete adjuvant and KLV-BSA immunogen as the injection. One month is separated from the first immunization and the first boosting, and 21 days is separated from the multiple boosting. The final immunization was performed by sprinting with KLV-BSA immunogen (25. Mu.g/dose, without adjuvant) diluted to a concentration of 0.5mg/mL with physiological saline; the interval between the sprint immunity and the last boost immunity is 20 days; serum titers and inhibition were detected by indirect competitive enzyme-linked immunosorbent assay (iceelisa).
D. Cell fusion
After three days of sprint immunization, cell fusion was performed according to the conventional PEG method, specifically as follows:
a. collecting eyeball and blood, killing a decachloroketone immunized mouse by a cervical dislocation method, immediately putting the mouse into 75% alcohol for sterilization, soaking for about 5min, taking out spleen of the mouse by aseptic operation, moderately grinding the spleen with a rubber head of a syringe, obtaining spleen cell suspension through a 200-mesh cell screen, collecting, centrifuging (1200 rpm,8 min), washing the spleen cells with RPMI-1640 culture medium for three times, and diluting the spleen cells to a certain volume after the last centrifuging, and counting for later use;
b. collecting murine myeloma SP2/0 cells: SP2/0 tumor cells were cultured in 10% FBS (fetal bovine serum) RPMI-1640 medium at 5% CO 7-10 days prior to fusion 2 In an incubator. The number of SP2/0 tumor cells required before fusion reaches (1-4). Times.10 7 Ensuring SP2/0 tumor cells to be in logarithmic growth phase before fusion. During fusion, collecting tumor cells, suspending in RPMI-1640 basic culture solution, and performing cell count;
c. the fusion process was 7min. 1min, 1mL of PEG 1500 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,8 min), discarding supernatant, re-suspending in RPMI-1640 screening medium containing 20% fetal bovine serum and 2% 50 XHAT, adding 200 μl/well to 96-well cell plate, and standing at 37deg.C and 5% CO 2 Culturing in an incubator.
E. Cell screening and cell strain establishment
The fused cells were subjected to a half-change of 2% of 50 XHAT RPMI-1640 medium on day 3 of cell fusion, a full-change of 20% fetal bovine serum and 1% of 100 XHT RPMI-1640 medium on day 5, and cell supernatants were collected on day 7 for selection. Screening is carried out in two steps: the first step is to screen out positive cell holes by using an ICELISA, and the second step is to use the parachloroketone as a standard substance, and to measure the inhibition effect of positive cells by using the ICELISA. Cell holes with better inhibition on the dechloridone standard are selected, subcloning is carried out by adopting a limiting dilution method, and detection is carried out by adopting the same method. Repeating the steps three times to obtain a monoclonal hybridoma cell strain.
Test case
A. Preparation and identification of Decochloroketone monoclonal antibody
8-10 weeks old KLV-BSA was used to immunize BALB/c mice, each of which was intraperitoneally injected with 1mL of sterile paraffin oil; each small after 7 daysIntraperitoneal injection of mice 1X 10 6 Hybridoma cells, ascites was collected from the seventh day, and the ascites was purified by the octanoic acid-ammonium sulfate method. Finally obtaining the purified dechloridone monoclonal antibody, and storing at the temperature of minus 20 ℃.
Immunoglobulin subtype identification is carried out on the dechloridone monoclonal antibody obtained by ascites purification by using a mouse monoclonal antibody subtype identification kit, the subtype is IgG2b type, and the light chain type is kappa type detected by the mouse monoclonal antibody subtype identification kit, as shown in figure 1.
Calculation of affinity constants using indirect ELISA assay: the concentration of the monoclonal antibody corresponding to OD 450nm at different antigen concentrations is calculated by plotting the concentration of the antibody-OD 450nm values, and when the concentration of the monoclonal antibody at the OD 450nm value is 50%, the concentration is substituted into the formula Ka= (n-1)/2 (nAb' -Ab) to calculate the affinity constant Ka (Lmol) -1 ). Ab and Ab 'represent the antibody concentrations (mol L) corresponding to OD 450nm when the antigen concentrations are Ag and Ag', respectively -1 ) n=ag/Ag'. As shown in FIG. 2, the affinity of the dechlorine ketone monoclonal antibody was 9.73X10 8 Lmol -1 。
The results show that the prepared monoclonal antibody of the dechlorine ketone has higher affinity and can be used for the immune analysis detection of the dechlorine ketone and the preparation of an affinity column.
B. Application of dechlorinone monoclonal antibody
The method for preparing the dechlorine ketone monoclonal antibody by using the hybridoma cell strain through in-vivo ascites is applied to an addition recovery test of dechlorine ketone, and specifically comprises the following steps:
(a) Coating: the coated precursor KLV-OVA obtained in the above step B was used in an amount of 0.05mol L -1 The pH 9.6 carbonate buffer was diluted to 0.1g/mL, 100. Mu.L/well, and reacted at 37℃for 2 hours.
(b) Washing: the plate solution was poured off and washed 3 times with wash solution for 3min each.
(c) Closing: 200. Mu.L/well of blocking solution was added thereto and reacted at 37℃for 2 hours. And (5) drying for standby after washing.
(d) Sample adding: adding 100 mu LPBS to the positive control wells; 100 mu L of 0.3-50 mu g L concentration is added into the detection hole -1 And a standard solution of dechloroketone. Then, the dechlorine ketone monoclonal antibody is used0.01mol L -1 PBS was diluted to 0.1g/mL and added to each dilution of the coated wells, 100. Mu.L/well, and reacted at 37℃for 30min; after extensive washing, add 1:3000 dilution of HRP-goat anti-mouse IgG, 100. Mu.L/well, reaction at 37℃for 30min.
(e) 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 ℃.
(f) Termination and measurement: 50. Mu.L of stop solution was added to each well to terminate the reaction, and the OD of each well was measured with a microplate reader 450 Values.
Sensitivity to decachlorone was detected by means of iceelisa, as shown in figure 3, according to standard equation y=0.11702+0.8059 (1+x/1.1366) 1.8534 (R 2 =0.9961), IC was calculated 50 1.1 mu g L -1 。
Determination of IC of Tectone monoclonal antibody by ICELISA 50 1.1 mu g L -1 The detection method has high sensitivity to the dechlorine ketone and can be used for dechlorine ketone immunoassay detection.
The cross of the monoclonal antibody to the dechlorine ketone is 100%, the cross rate of the dechlorine ketone analogue is less than 6%, and the cross rate is= (IC of the dechlorine ketone) 50 IC of analog 50 ) X 100%, IC of decachloroketone analogue 50 The values and crossover rates are shown in table 1. The monoclonal antibody obtained by the invention has higher sensitivity to the dechlorine ketone and IC 50 The value is 1.1 mug/mL, which shows that the monoclonal antibody has high sensitivity and specificity.
TABLE 1
Compounds of formula (I) IC 50 (μg/L) Cross Rate (%)
Ten chloroketones 1.1 100
Six-six 50 2.2
Sudan (pill) for treating cancer 20 5.5
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 (6)

1. The hybridoma cell strain of the anti-dechloridone monoclonal antibody is characterized in that the hybridoma cell strain is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) on the year 2022, and the preservation address is 1 # 3 and the preservation number is CGMCC No.45115 in North Xiyun of the Korean region of Beijing city.
2. Use of the hybridoma cell line of claim 1 for the preparation of a monoclonal antibody to decachloroketone.
3. A dodecanone monoclonal antibody, wherein said dodecanone monoclonal antibody is secreted by the hybridoma cell line of claim 1.
4. A composition comprising the dechlorinone monoclonal antibody of claim 3.
5. A kit comprising one or more of the monoclonal antibody of claim 3 and the composition of claim 4.
6. Use of the cloning antibody of dechlorine ketone as defined in claim 3, the composition as defined in claim 4 or the kit as defined in claim 5 for the preparation of a preparation for detecting dechlorine ketone.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5334528A (en) * 1989-10-30 1994-08-02 The Regents Of The University Of California Monoclonal antibodies to cyclodiene insecticides and method for detecting the same
CN102190724A (en) * 2011-03-31 2011-09-21 上海大学 Chlordecone antigen and antibody and preparation method thereof
CN110616195A (en) * 2019-10-11 2019-12-27 江南大学 Metformin monoclonal antibody hybridoma cell strain and application thereof
CN113717949A (en) * 2021-09-22 2021-11-30 江南大学 Hybridoma cell strain capable of secreting ketoconazole monoclonal antibody and application thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5334528A (en) * 1989-10-30 1994-08-02 The Regents Of The University Of California Monoclonal antibodies to cyclodiene insecticides and method for detecting the same
CN102190724A (en) * 2011-03-31 2011-09-21 上海大学 Chlordecone antigen and antibody and preparation method thereof
CN110616195A (en) * 2019-10-11 2019-12-27 江南大学 Metformin monoclonal antibody hybridoma cell strain and application thereof
CN113717949A (en) * 2021-09-22 2021-11-30 江南大学 Hybridoma cell strain capable of secreting ketoconazole monoclonal antibody and application thereof

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