CN114807051A - Hybridoma cell strain of anti-chlordecone monoclonal antibody and application thereof - Google Patents
Hybridoma cell strain of anti-chlordecone monoclonal antibody and application thereof Download PDFInfo
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Abstract
The invention relates to a hybridoma cell strain of a chlordecone-resistant monoclonal antibody and application thereof, and relates to the technical field of food safety immunodetection. The hybridoma cell strain of the anti-chlordecone monoclonal antibody provided by the invention is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms at 03 months 03 and 03 days 2022, and the preservation address is No. 3 of the No. 1 Hospital of Xilu, North Chen of the Chaoyang district, Beijing, and the preservation number is CGMCC No. 45115. The monoclonal antibody secreted by the cell strain provided by the inventionHas better specificity and detection sensitivity (IC) to the chlordeca-ketone 50 The value was 1.1. mu. g L ‑1 ) The method can realize the detection of the residual quantity of the chlordecone, provides raw materials for the immunodetection of the residual quantity of the chlordecone in food, and has practical application value.
Description
Technical Field
The invention relates to the technical field of food safety immunodetection, in particular to a hybridoma cell strain of a chlordecone-resistant monoclonal antibody and application thereof.
Background
Decachlorone (CLD), also known as galbanum officinale (kepo), is an artificially synthesized organochlorine insecticide. Dechloroketone has been banned for its property of being able to persist in the environment with Persistent Organic Pollutants (POPs) because it is poorly soluble in water, has strong hydrophobicity, is particularly easily concentrated in organisms, is difficult to chemically and biologically degrade, and has the possibility of migrating long distances across boundaries. Decolone has been widely used for controlling banana root borers, it is also used in fly larvae insecticides, fungicides, control of potato beetles, rust mites, potato and tobacco cutworms and in ant and cockroach attractants, it is a highly toxic organic substance that has some effect on the central nervous system, urogenital system, immune system of the fetus. Decolone can affect the nervous system of human body, and can cause symptoms such as trembling, weight loss, eyeball spasm, costitis, arthralgia, hepatomegaly, and oligospermia. Experimental research on human bodies and animals shows that after absorbed, the chlordecone is mainly enriched in the liver, is slowly metabolized through bile and is discharged out of the body through excrement, and the chlordecone has obvious carcinogenicity on rats and mice and can enable other organs such as the liver to generate malignant tumors. Therefore, the establishment of a method for quickly and effectively detecting the content of the chlordecone has important significance and market value.
The problem to be solved is to solve aiming at the efficient and sensitive detection method of the chlordecone. Current detection methods include instrumental assays and immunoassays. Instrumental methods such as high performance liquid chromatography (aqua, Peng, Musuo, et al. gas chromatography-tandem mass spectrometry for measuring Decolone [ J ]. Chinese environmental monitoring, 2015 (5): 129) 133; Saint-Hilaire M, integrating C, Bertin T, Lavison-Bompard G, Freerin T, Fournier A, Feidt C, Rycen G, ParinJ. development and identification of an HPLC-MS/MS method with an EChERS extraction analysis slurry and chlorine in organic chemistry 2018, 30; 252: 147) for measuring environmental contamination of soil sample and chemical analysis, GC 10. moisture analysis, 3513. GC 13. environmental analysis for measuring soil sample [ M-III, M.), 21(7): 4928-; alarm SOLER, MARC LEBRUN, Yoan LABROUSSE, et al. solid-phase microextraction and gas chromatography-mass spectrometry for quantitative determination of chlorinated in water, plant and soil samples [ J ] Fruits,2014 (4): 325-339). Due to the complicated pretreatment of the sample, more interferents, limitation of working conditions of the instrument and higher technical requirements on operators, the instrument and the method are not suitable for field detection.
Compared with an instrument detection method, the immunoassay method has the characteristics of low cost, high flux, high sensitivity, low requirement on technical personnel and the like, so that the immunoassay method is suitable for rapid screening of a large number of samples. Therefore, the immunoassay method has important significance for detecting the chlordecone.
The enzyme-linked immunosorbent assay (ELISA) is a low-cost, rapid and portable immunological detection method, has low requirement on the purity of a sample during detection, is simple and convenient to operate, and is suitable for rapidly detecting results on the spot of a large number of samples. Establishing an efficient immunological detection method, and screening a monoclonal antibody with high specificity is an important prerequisite.
Disclosure of Invention
In order to solve the technical problems, the invention provides a hybridoma cell strain of a chlordecone-resistant monoclonal antibody and application thereof. The antibody secreted by the hybridoma cell strain has higher detection sensitivity to the chlordecone, and can be used for establishing an immunological detection method for the chlordecone.
The first purpose of the invention is to provide a hybridoma cell strain of a monoclonal antibody against chlordecone, wherein the hybridoma cell strain is preserved in China general microbiological culture Collection center at 03 months 03 and 03 days 2022, the preservation address is No. 3 of Xilu No. 1 of Beijing Korean district, and the preservation number is CGMCC No. 45115.
The second purpose of the invention is to provide a preparation method of a hybridoma cell strain of the anti-chlordecone monoclonal antibody, which comprises the following steps:
s1, dissolving cleavan in an organic solvent, adding an alkaline solution, heating to 70-90 ℃, stirring for reaction for 0.5-2h, and adjusting the pH value to 4-5 by using an acidic solution to obtain a chlordecone hapten;
s2, preparing a chlordecone complete antigen by using the chlordecone hapten in S1, and preparing the obtained chlordecone complete antigen into an antigen-containing Freund complete adjuvant and an antigen-containing Freund incomplete adjuvant;
s3, carrying out primary immunization, boosting immunization and sprint immunization on the immunized animal, wherein the primary immunization adopts the antigen-containing Freund complete adjuvant in S2, the boosting immunization adopts the antigen-containing Freund incomplete adjuvant in S2, and the sprint immunization adopts the chlordecone complete antigen in S2;
s4, taking spleen cells and myeloma cells of the immune animals after the immune animals are subjected to the puncture in the S3 for cell fusion, and obtaining the hybridoma cell strain of the anti-chlordecone monoclonal antibody.
In one embodiment of the present invention, in S1, extraction, purification and drying steps are further included.
In one embodiment of the present invention, in S1, the organic solvent is one or more of dimethylsulfoxide, 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 mass ratio of the gram to the basic solvent is 1: 1-3.
In one embodiment of the invention, the mass ratio of the gram to the basic solvent is 1: 2.
in one embodiment of the present invention, in S1, the concentration in grams of the organic solvent is from 0.01 to 0.05 g/mL.
In one embodiment of the present invention, in S1, the concentration in grams of organic solvent is 0.02 g/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 structural formula of the chlordecone hapten is as follows:
in one embodiment of the present invention, in S1, the method for preparing the chlordecone hapten specifically comprises the steps of dissolving 1equ. gram of chlordecone in dimethyl sulfoxide, adding 2equ. sodium hydroxide solution, heating to 80 ℃, and stirring for reaction for 1 hour; adding pure water, regulating pH to 4-5 with hydrochloric acid solution, extracting, purifying, and drying to obtain white solid, i.e. the chlordecone hapten.
In one embodiment of the present invention, in S2, the chlordecone complete antigen is derived from coupling the chlordecone hapten with a carrier protein; the structural formula of the chlordecone complete antigen is as follows:
in one embodiment of the invention, the carrier protein comprises Bovine Serum Albumin (BSA).
In an embodiment of the present invention, in S2, the method for preparing the chlordecone antigen includes the steps of dissolving the chlordecone hapten, N-hydroxysuccinimide (NHS) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI) in a solvent, reacting to obtain a mixed solution, and adding the mixed solution to a carrier protein solution to react to obtain the chlordecone 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 chlordecone hapten in the solvent is 10.0-14.0 g/L.
In one embodiment of the invention, the concentration of the chlordecone hapten in the solvent is 12.0 g/L.
In one embodiment of the invention, the mass ratio of the chlordecone hapten to the carrier protein is 30-80: 1.
in one embodiment of the invention, the mass ratio of the chlordecaketone hapten to the carrier protein is 50: 1.
in one embodiment of the present invention, in S2, the method for preparing the decachlorone complete antigen specifically includes the steps of dissolving the decachlorone hapten KLV in anhydrous N, N-dimethylformamide, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), N-hydroxysuccinimide (NHS), and stirring at room temperature to react, namely, solution a; weighing carrier protein, and adding boric acid buffer solution to obtain 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 chlordecone complete antigen.
In one embodiment of the present 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 present invention, the volume ratio of the anhydrous N, N-dimethylformamide to the boric acid buffer solution is 1: 10.
in one embodiment of the present invention, in S2, the antigen-containing freund 'S complete adjuvant is an emulsion of freund' S complete adjuvant mixed with an equal volume of decachloroketone complete antigen.
In one embodiment of the present invention, in S2, the antigen-containing freund 'S incomplete adjuvant is an emulsion of freund' S incomplete adjuvant mixed with an equal volume of decachloroketone complete antigen.
In one embodiment of the present invention, the whole immunization process comprises 1 first immunization, 4-6 boosts and 1 sprint immunization in S3.
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 booster immunization is 28-31 days, the interval between the booster immunization is 20-22 days, and the interval between the booster immunization and the sprint immunization is 18-21 days in the whole immunization process.
In one embodiment of the invention, in S3, the first immunization dose is 95-105 μ g/mouse, the boosting dose is 45-55 μ g/mouse, and the sprint immunization dose is 20-30 μ g/mouse in the whole immunization process.
In an embodiment of the present invention, in S3, in the boosting process, the immunized animal is sampled, and the immune animal with high content of the chlordecone antibody in the serum is screened by detecting serum titer and inhibition rate through an indirect competitive enzyme-linked immunosorbent assay (iclisa).
In one embodiment of the invention, the blood collection is performed 6-8 days after the completion of the boosting process.
In one embodiment of the invention, in S3, the immunizing is by back subcutaneous injection into the immunized animal.
In one embodiment of the present invention, in S4, the cell fusion is performed by culturing the fused cells in HAT medium, detecting positive cell wells by indirect ELISA, further determining the inhibitory effect of the positive cell wells by indirect competitive ELISA, and subcloning the positive cell wells with the best inhibition by limiting dilution to 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 present invention, in S4, the cell fusion is performed by a polyethylene glycol (PEG4000) method.
In one embodiment of the present invention, in S4, the cell fusion is performed 2 to 4 days after the completion of the spike immunization.
The third purpose of the invention is to provide an application of the hybridoma cell strain in preparation of a chlordecone monoclonal antibody.
The fourth purpose of the invention is to provide a chlordecone monoclonal antibody, wherein the chlordecone monoclonal antibody is secreted by a hybridoma cell strain with the preservation number of CGMCC No. 45115.
In one embodiment of the invention, paraffin oil is injected into the abdominal cavity of an immune animal, then a 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 to obtain the chlordecone monoclonal antibody.
In one embodiment of the present invention, BALB/c mice 8-10 weeks old were injected with 1mL paraffin oil intraperitoneally, and 7 days later, injected with 1X 10 paraffin oil intraperitoneally 6 The hybridoma cell strain with the preservation number of CGMCC No.45115 collects ascites from day 7, purifies the ascites by an octanoic acid-ammonium sulfate method, and stores the obtained chlordecone monoclonal antibody at-20 ℃.
The fifth purpose of the invention is to provide a composition, which contains the hybridoma cell strain and/or the chlordecone monoclonal antibody.
The sixth purpose of the invention is to provide a kit, wherein the kit contains one or more of the hybridoma cell strain, the chlordecone monoclonal antibody and the composition.
The seventh purpose of the invention is to provide an application of the hybridoma cell strain, the chlordecone, the composition or the kit in detecting chlordecone, especially in analyzing and detecting chlordecone residue 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) on the chlordecone 50 The value was 1.1. mu. g L -1 ) The method can realize the detection of the residual quantity of the chlordecone, provides raw materials for the immunodetection of the residual quantity of the chlordecone in food, and has practical application value.
Biological material preservation
A hybridoma cell strain secreting the monoclonal antibody against the chlordecone belongs to a monoclonal cell strain, is preserved in China general microbiological culture collection center at 03 months 03 and 2022, has the preservation address of No. 3 Xilu-Beijing West Chen-Yang district, the preservation number of CGMCC No.45115 and is named as a monoclonal cell strain in classification.
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 present disclosure taken in conjunction with the accompanying drawings, in which:
FIG. 1 is a diagram showing the subtype identification of the monoclonal antibody of the present invention.
FIG. 2 is an affinity assay for the monoclonal antibody of the invention.
FIG. 3 is a standard curve of inhibition of the monoclonal antibody of the present invention against chlordecone.
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.
(1) The media involved in the following examples are as follows:
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, calcium D-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, and sodium bicarbonate 2000.
(2) The solutions referred to in the following examples were prepared as follows:
carbonate Buffer (CBS): weighing Na 2 CO 3 1.59 g，NaHCO 3 2.93 g of the mixture is respectively dissolved in a small amount of double distilled water and then mixed, the double distilled water is added to about 800mL and mixed evenly, the pH value is adjusted to 9.6, the double distilled water is added to the volume of 1000mL, and the mixture is stored for later use at 4 ℃;
phosphate Buffered Saline (PBS): 8.0g NaCl, 0.2g KCl, 0.2g KH 2 PO 4 ，2.9g Na 2 HPO 4 ·12H 2 Dissolving 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;
PBST: PBS containing 0.05% tween-20;
antibody dilution: adding 0.1% gelatin into PBS;
TMB color development liquid: solution A: na (Na) 2 HPO 4 ·12H 2 18.43g 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 proportion of 1: 5, mixing to obtain the TMB color developing solution which is mixed at present.
(3) The detection methods referred to in the following examples are as follows:
the method for detecting the inhibition rate of the chlordecone comprises the following steps: the most suitable 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. mu.g/mL with Carbonate Buffer (CBS) and the antibody was diluted to 0.03, 0.1, 0.3 and 1. mu.g/mL with antibody diluent. After selecting the optimal working point, the chlordecone standard is diluted to equal concentrations of 0, 0.01, 0.03, 0.11, 0.33, 1, 3 and 9 mug/mL, according to the operation steps of IC-ELISA, and finally the standard inhibition curve of chlordecone is obtained by drawing with originPro 8.5, and IC is calculated 50 。
Examples
A hybridoma cell strain of a monoclonal antibody against chlordecone and a preparation method thereof specifically comprise the following steps:
A. synthesis of Dechlorone hapten
The synthesis route of the chlordeca-ketone hapten KLV is as follows:
0.10g (0.16mmol) of cremaster are dissolved in 2mL of dimethyl sulfoxide, and6mL of 1mol L -1 Heating the NaOH solution to 80 ℃, and stirring for reaction for 1 h; cooling the reaction solution to room temperature, adding a proper amount of pure water, and adding 1mol L of pure water -1 Adjusting pH to 4 with HCl, extracting with ethyl acetate, anhydrous Na 2 SO 4 Drying and concentrating; purifying by a silica gel column, concentrating and drying to obtain a white solid, namely the chlordecone hapten KLV.
B. Preparation of Dechlorone complete antigen
And B, coupling the hapten KLV prepared in the step A with Bovine Serum Albumin (BSA) to obtain a complete antigen KLV-BSA, wherein the specific preparation method of the complete antigen KLV-BSA is as follows:
a. weighing 4.6mg (0.0075mmol) of hapten KLV prepared in the step A, dissolving in 200 mu L of anhydrous N, N-dimethylformamide, sequentially adding 1.7mg (0.009mmol) of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) and 1.0mg (0.009mmol) of N-hydroxysuccinimide (NHS), and stirring at room temperature for 0.5h to obtain solution A; weighing 10mg (0.00015mmol) of bovine serum albumin BSA, and adding 2mL of boric acid buffer solution to obtain solution B; slowly dropwise adding the solution A into the solution B under stirring, and reacting at room temperature for 2h to obtain a mixed solution containing KLV-BSA;
b. and (3) dialysis: boiling 10cm dialysis bag in boiling water for 5min, washing with 60 deg.C deionized water for 3min, and storing in 4 deg.C deionized water; placing the mixed solution containing KLV-BSA in the step a into a dialysis bag, and using 0.01mol L -1 And (3) taking PBS as dialysate, dialyzing for 3d at 4 ℃, and replacing the dialysate three times per day to separate the complete antigen, the uncoupled hapten and other small molecular substances to obtain the chlordecone complete antigen.
The complete antigen prepared comprises KLV-BSA immunogen and KLV-OVA coating antigen, wherein the immunogen KLV-BSA is used for the next step of mouse immunization.
The original KLV-OVA coating is prepared by the same method as KLV-BSA and is used for the subsequent application and detection.
C. Immunization of mice
KLV-BSA immunogen is mixed with equal volume of Freund's adjuvant and emulsified to obtain injection, through the neck and back subcutaneous injection BALB/c mouse.
The first immunization (100. mu.g/mouse) was performed using an equal volume of mixed emulsion of Freund's complete adjuvant and KLV-BSA immunogen as an injection, and the 5 booster immunizations (50. mu.g/mouse) were performed using an equal volume of mixed emulsion of Freund's incomplete adjuvant and KLV-BSA immunogen as an injection. The interval between the first immunization and the first boosting immunization is one month, and the interval between the boosting immunization is 21 days. The last time, the vaccine is pricked and immunized by KLV-BSA immunogen (25 mug/mouse without adjuvant) which is diluted to the concentration of 0.5mg/mL by physiological saline; the interval between the sprint immunization and the last boosting immunization is 20 days; the serum titer and inhibition rate were measured by indirect competitive enzyme-linked immunosorbent assay (iclelisa).
D. Cell fusion
After three days of spurting immunization, cell fusion is carried out according to a conventional PEG method, and the specific steps are as follows:
a. taking eyeballs and blood, immediately placing the mice immunized by chlordecone 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;
b. 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 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;
c. the fusion process is 7 min. 1min, 1mL of PEG 1500 is added to the cells 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 5 min. Centrifuging (800rpm, 8min), discarding supernatant, resuspending in RPMI-1640 screening medium containing 20% fetal calf serum, 2% 50 × HAT, adding to 96-well cell plate at 200 μ L/well, standing at 37 deg.C and 5% CO 2 Culturing in an incubator.
E. Cell screening and cell line establishment
On day 3 of cell fusion, half-exchange of the fused cells with 2% RPMI-1640 screening medium at 50 XHAT, and on day 5, total-exchange 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: the first step is to screen out positive cell holes by using an ICELISA, the second step is to select p-chlordone as a standard substance, and the inhibition effect of the positive cells is measured by using the ICELISA. And selecting cell wells with good inhibition to the chlordecone standard substance, subcloning by adopting a limiting dilution method, and detecting by using the same method. Repeating the steps for three times to obtain the monoclonal hybridoma cell strain.
Test example
A. Preparation and identification of chlordecone monoclonal antibody
Taking KLV-BSA for immunizing BALB/c mice with age of 8-10 weeks, and injecting 1mL 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 was collected from the seventh day, and the ascites was purified by the caprylic-ammonium sulfate method. Finally, the purified monoclonal antibody of the chlordeca-ketone is obtained and stored at the temperature of-20 ℃.
The monoclonal antibody of the chlordecone obtained by purifying ascites is subjected to immunoglobulin subtype identification by using a mouse monoclonal antibody subtype identification kit, the subtype is IgG2b type, and the light chain type detected by the mouse monoclonal antibody subtype identification kit is kappa type, as shown in figure 1.
The calculation method of the affinity constant by indirect ELISA measurement is as follows: the antibody concentration at OD 450nm was calculated by plotting the antibody concentration-OD 450nm values, and the affinity constant Ka (Lmol) was calculated by substituting the concentration of the monoclonal antibody at OD 450nm value of 50% into the formula Ka ═ n-1)/2 (nAb' -Ab) -1 ). Ab and Ab 'represent the concentrations of antibody at OD 450nm (mol L) when the antigen concentrations are Ag and Ag', respectively -1 ) And n is Ag/Ag'. As shown in FIG. 2, the monoclonal antibody to chlordecone has an affinity of 9.73X 10 8 Lmol -1 。
The results show that the prepared chlordecone monoclonal antibody has higher affinity and can be used for chlordecone immunoassay detection and preparation of an affinity column.
B. Application of chlordecone monoclonal antibody
The application of the chlordecone monoclonal antibody prepared from the hybridoma cell strain through in-vivo ascites in the chlordecone addition and recovery test specifically comprises the following steps:
(a) coating: 0.05mol L of the coating original KLV-OVA obtained in the step B is used -1 The mixture was diluted to 0.1g/mL in carbonate buffer at pH 9.6, 100. mu.L/well and reacted at 37 ℃ for 2 h.
(b) Washing: the plate solution was decanted and washed 3 times for 3min each with washing solution.
(c) And (3) sealing: add 200. mu.L/well blocking solution and react at 37 ℃ for 2 h. And drying after washing for later use.
(d) Sample adding: add 100. mu. LPBS to the positive control wells; adding 100 mu L of 0.3-50 mu g L into the detection hole -1 A standard solution of chlordecone. Then, 0.01mol L of chlordecone monoclonal antibody is used -1 Diluting PBS to 0.1g/mL, adding into each diluted coated well, reacting at 37 deg.C for 30min at 100 μ L/well; after thorough washing, 1: 3000 diluted HRP-goat anti-mouse IgG, 100. mu.L/well, reacted at 37 ℃ for 30 min.
(e) 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 15 min.
(f) 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 reader 450 The value is obtained.
The sensitivity to chlordecone was measured using an ICELISA, as shown in FIG. 3, according to the standard equation y 0.11702+0.8059(1+ x/1.1366) 1.8534 (R 2 0.9961), IC was calculated 50 Is 1.1 mu g L -1 。
Determination of IC of Dechlorone monoclonal antibody by ICELISA 50 Is 1.1 mu g L -1 The method has high sensitivity to the chlordeca-ketone, and can be used for chlordeca-ketone immunoassay detection.
The monoclonal antibody has 100% cross-over to the chlordecone and cross-over to the chlordecone analogueThe cross rate is less than 6 percent, and the cross rate is (IC of decachloroketone) 50 IC of/analogue 50 ) X 100%, IC of Decolone analog 50 The values and the crossover rates are shown in table 1. The monoclonal antibody obtained by the invention has higher sensitivity and IC on the chlordeca-ketone 50 The value is 1.1 mug/mL, which indicates that the monoclonal antibody has high sensitivity and specificity.
TABLE 1
| Compound (I) | IC 50 (μg/L) | Crossover ratio (%) |
| Dechloroketone | 1.1 | 100 |
| Six | 50 | 2.2 |
| Endosulfan | 20 | 5.5 |
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 (10)
1. A hybridoma cell strain of a monoclonal antibody against chlordecone is characterized in that the hybridoma cell strain is preserved in China general microbiological culture Collection center at 03 months and 03 days in 2022, the preservation address is No. 3 of Xilu No. 1 of Beijing Korean district, and the preservation number is CGMCC No. 45115.
2. The preparation method of the hybridoma cell strain of the anti-chlordecone monoclonal antibody comprises the following steps:
s1, dissolving cleavan in an organic solvent, adding an alkaline solution, heating to 70-90 ℃, stirring for reaction for 0.5-2h, and adjusting the pH value to 4-5 by using an acidic solution to obtain a chlordecone hapten;
s2, preparing a chlordecone complete antigen by using the chlordecone hapten in S1, and preparing the obtained chlordecone complete antigen into an antigen-containing Freund complete adjuvant and an antigen-containing Freund incomplete adjuvant;
s3, carrying out primary immunization, boosting immunization and sprint immunization on the immunized animal, wherein the primary immunization adopts the antigen-containing Freund complete adjuvant in S2, the boosting immunization adopts the antigen-containing Freund incomplete adjuvant in S2, and the sprint immunization adopts the chlordecone complete antigen in S2;
s4, taking spleen cells and myeloma cells of the immune animals after the immune animals are subjected to the puncture in the S3 for cell fusion, and obtaining the hybridoma cell strain of the anti-chlordecone monoclonal antibody.
3. The method of claim 2, wherein in S1, the chlordecone hapten is of the formula:
4. the method of claim 2, wherein in S2, the decachlorone complete antigen is obtained by coupling the decachlorone hapten with a carrier protein; the structural formula of the chlordecone complete antigen is as follows:
5. the method according to claim 2, wherein in S2, the method for producing the decachlorone complete antigen comprises the steps of dissolving the decachlorone hapten, N-hydroxysuccinimide, and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in a solvent, reacting to obtain a mixed solution, and adding the mixed solution to a carrier protein solution to react to obtain the decachlorone complete antigen.
6. The use of the hybridoma cell line of claim 1 for the preparation of a monoclonal antibody to chlordecone.
7. A chlordecone monoclonal antibody, which is secreted by the hybridoma cell line of claim 1.
8. A composition comprising the hybridoma cell line of claim 1 and/or the monoclonal antibody against chlordecone of claim 7.
9. A kit comprising one or more of the hybridoma cell line of claim 1, the chlordecone monoclonal antibody of claim 7, and the composition of claim 8.
10. Use of the hybridoma cell line of claim 1, the chlordecone antibody of claim 7, the composition of claim 8, or the kit of claim 9 for detecting chlordecone.
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Citations (4)
| 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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Patent Citations (4)
| 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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