CN106749653A - The preparation method of anti-citrinin monoclonal antibody, citrinin kit and its detection method - Google Patents
The preparation method of anti-citrinin monoclonal antibody, citrinin kit and its detection method Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/14—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from fungi, algea or lichens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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Abstract
The present invention discloses a kind of preparation method of anti-citrinin monoclonal antibody, citrinin ELISA detection kit and its detection method.The preparation of anti-citrinin monoclonal antibody synthesizes citrinin comlete antigen first, then animal is immunized as immunizing antigen using the citrinin comlete antigen, reapplying hybridoma cell technology carries out cell fusion and screening, obtain the hybridoma cell strain of the anti-citrinin monoclonal antibody of stably excreting, by preparing ascites and purifying, anti-citrinin monoclonal antibody is obtained.The preparation method that the present invention provides anti-citrinin monoclonal antibody has the advantages that preparation condition is gentle, simple to operate, process control, there is provided the sensitivity of citrinin ELISA detection kit be 5ng/ml, be with a wide range of applications.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a preparation method of an anti-citrinin monoclonal antibody, a citrinin ELISA detection kit and a detection method thereof.
Background
Citrinin (CIT), also known as CITRININ, is a toxic secondary metabolite produced by fungi and has the molecular formula C13H14O5The chemical structure is (3R, 4S) -4, 6 dihydro-8 hydroxy-3, 4, 5-trimethyl-6-oxo-3H-2-phenylpyrazole-7-carboxylic acid, and the relative molecule is 250.3 Da. Citrinin is a toxin produced by filamentous fungi such as Penicillium, Aspergillus, Monascus, etc., and has severe hepatorenal toxicity, teratogenic, carcinogenic, and mutagenic effects. Research in recent years has found that citrinin can be detected in many food and agricultural products, mainly because of the mildew of agricultural products or food such as corn, rice, bread, milk, apple, pear, etc. Therefore, food safety problems caused by citrinin are receiving more and more attention.
Monascus has a long source of application in China, and can be used for producing various traditional Chinese foods such as monascus wine, fermented red bean curd, red yeast rice and the like by fermenting monascus. Citrinin is a secondary metabolite of fungi, often associated with monascus pigment production, and in most monascus fermented products, citrinin content is up to tens to hundreds of times of eu standards. For food safety reasons, citrinin has been classified as one of the indispensable toxins in europe.
At present, methods for detecting Citrinin (CIT) include colloidal gold test strip detection, enzyme-linked immunosorbent assay (ELISA), uv spectrophotometry, hplc, and hplc. These methods all involve two core immunoreagents, the citrinin antigen and the citrinin antibody. The Citrinin (CIT) belongs to a small substance (with the molecular weight of 250Da) and only has antigenicity but no immunogenicity. Therefore, it is necessary to prepare an antibody by immunizing an animal with a complete antigen prepared by coupling Citrinin (CIT) to a carrier protein.
The ELISA method uses a specific reaction of an antigen and an antibody to link an analyte with an enzyme, and then generates a color reaction between the enzyme and a substrate for quantitative determination. The ELISA method has higher detection sensitivity and specificity, has lower requirements on samples, is simple to process, is suitable for detecting and popularizing and applying batch samples, has wide practical value space, and is widely applied to clinical detection of various mycotoxins in recent years.
At present, no commercial citrinin ELISA detection kit exists in China, only citrinin detection test paper (CN1603823A) for qualitative detection exists, and the main reason is that an anti-citrinin antibody with high affinity and high specificity cannot be prepared, so that the preparation of the citrinin ELISA detection kit by preparing the anti-citrinin antibody with high affinity and high specificity has important significance.
Disclosure of Invention
The preparation method of the citrinin monoclonal antibody provided by the invention has the advantages of mild preparation conditions, simplicity in operation and controllable process, and the citrinin ELISA detection kit provided by the invention has the advantages of high specificity, high sensitivity and good stability.
A preparation method of anti-citrinin monoclonal antibody comprises the following steps:
synthesizing a citrinin complete antigen, wherein the citrinin complete antigen is prepared according to the following method: the citrinin firstly reacts with a coupling reagent 2, 2-dichloro-5- (2-phenylethyl) -4- (trimethylsilyl) -3-furanone and N-hydroxysuccinimide, then is coupled with a macromolecular carrier, dialysis is carried out after the coupling reaction until the measured value of the protein content in dialysate is the same as that of blank dialysate, the non-combined citrinin is removed, and the dialysate is concentrated to obtain a citrinin complete antigen; and immunizing an animal by taking the citrinin complete antigen as an immune antigen, performing cell fusion and screening by using a hybridoma cell technology to obtain a hybridoma cell strain which stably secretes the anti-citrinin monoclonal antibody, and preparing ascites and purifying to obtain the anti-citrinin monoclonal antibody.
In a preferred embodiment of the preparation method of the anti-citrinin mab provided by the present invention, the specific method for synthesizing the citrinin complete antigen comprises:
(1) activation of citrinin: dissolving 13-17 mg of citrinin, 13-17 mg of 2, 2-dichloro-5-2-phenethyl-4-trimethyl silicon-3-furanone and 5-7 mg of N-hydroxysuccinimide in 0.2ml of anhydrous tetrahydrofuran, slowly dropwise adding 10.1mol/L HCl100 mu L, shaking uniformly at room temperature, centrifuging to obtain a supernatant, and washing with the anhydrous tetrahydrofuran;
(2) citrinin-carrier coupling reaction: after tetrahydrofuran is dried, dissolving the residue in 0.5ml of dimethylformamide, then dropwise adding the solution into 0.5ml of 5% macromolecular carrier solution, adjusting the pH value of the solution to 8.0, and slowly shaking the solution in a constant temperature box at 37 ℃ for 60 min;
(3) separation and purification: after the coupling reaction, the mixture is placed in 0.1mol/L NaHCO3Dialyzing in the solution for 1 hour, dialyzing in a mode of changing the liquid for the first 3 times for 1.5 hours, changing the liquid for the third time for 4.5 hours and changing the liquid for the third time for 18 hours to ensure that the measured value of the protein content in the dialyzate is the same as that of the blank dialyzate, removing the non-combined citrinin, concentrating the dialyzate to obtain the citrinin complete antigen, and storing at the temperature of freeze-drying-20 ℃.
In a preferred embodiment of the method for preparing the anti-citrinin mab, the macromolecular carrier is bovine serum albumin, ovalbumin, keyhole limpet hemocyanin or bovine thyroglobulin.
In a preferred embodiment of the method for preparing the anti-citrinin mab provided by the present invention, the citrinin complete antigen is used as an immune antigen to immunize an animal, and then a hybridoma cell technology is applied to perform cell fusion and screening to obtain a hybridoma cell strain which stably secretes the anti-citrinin mab, and the anti-citrinin mab is obtained by preparing ascites and purifying, which specifically comprises the following steps:
step one, animal immunization: taking the citrinin complete antigen as an immunizing antigen and Freund's adjuvant to immunize a BALB/c mouse for multiple times until the titer detection reaches 1: 10W;
step two, cell fusion: taking spleen cells of an immunized mouse, taking PEG1450 with the volume ratio of 50% as a fusion agent, and mixing the mixture according to the proportion of 10: 1, fusing the cell number with a mouse myeloma cell Sp2/0, selectively culturing the fused cells by using an HAT culture medium, culturing by using an HT culture medium after 7 days, and culturing by using a DMEM culture medium after 14 days;
step three, subcloning the positive hybridoma cell strain: detecting fused cells by adopting a 96-hole enzyme label plate coated with citrinin complete antigen, wherein the coating concentration of the 96-hole enzyme label plate is 200ng/ml, the addition amount of the fused cells is 100 mu l/hole, and when positive cells are detected, performing subcloning until the positive rate reaches 100%;
step four, preparation and purification of antibody
(1) Preparation of antibody: injecting 0.5ml of liquid paraffin into the abdominal cavity of each sensitized BALB/c mouse 7-10 days in advance, then inoculating the positive hybridoma cells established in the step three to the BALB/c mouse injected with the liquid paraffin, collecting ascites after 7-10 days, and centrifuging to obtain supernatant;
(2) purification of the antibody: and (3) diluting the supernatant into a PBS solution according to the ratio of 1:10, gradually adding the supernatant into a chromatographic column balanced by PBS from the upper part of the column at the speed of 0.5ml/min, washing the column by the PBS solution, and eluting the column by a glycine buffer solution with the pH of 2.9 to obtain the purified anti-citrinin monoclonal antibody.
In a preferred embodiment of the method for preparing anti-citrinin mab of the present invention, the animal immunization method of step one comprises: the first immunization, 50 mu g of each immunizing antigen is added with an equal volume of complete Freund adjuvant and injected subcutaneously; after 2 weeks, the second immunization is carried out, and 50 mu g of immune antigen is added with an equal volume of incomplete Freund's adjuvant and injected subcutaneously; after 2 weeks, the third immunization, 25 mug/antigen plus the same volume of incomplete Freund's adjuvant, subcutaneous injection; after 7-10 days, ELISA is used for detecting the titer of the serum of the immune mice, and the titer reaches 1:10W, so that cell fusion can be prepared; 3 days before cell fusion, 25. mu.g of antigen was injected intraperitoneally to boost immunity.
The invention also provides a citrinin ELISA detection kit, which comprises the citrinin-resistant monoclonal antibody prepared by the preparation method of the citrinin-resistant monoclonal antibody.
In a preferred embodiment of the citrinin ELISA detection kit provided by the invention, the kit further comprises a 96-hole enzyme label plate coated by a citrinin complete antigen, a citrinin standard substance, an enzyme-labeled goat anti-mouse antibody, a coating solution, a diluent, a confining solution, a developing solution and a stop solution;
wherein,
the concentrations of the citrinin standard substance are respectively 0, 5, 10, 20, 40 and 80 ng/ml;
the enzyme-labeled goat anti-mouse antibody is a goat anti-mouse antibody labeled by horseradish peroxidase;
the coating solution is 0.01mol/L of carbonate buffer solution with pH9.6, and is used for diluting the antigen coated by the 96-well enzyme label plate to 200 ng/ml;
the diluent is 0.01mol/L PBS solution with pH7.4;
the blocking solution is PBS/T20 containing 5% calf serum;
the color developing liquid is TMB single-component color developing liquid;
the stop solution is 2mol/L H2SO4。
In a preferred embodiment of the citrinin ELISA detection kit provided by the present invention, the citrinin complete antigen coated on the 96-well ELISA plate is a citrinin ovalbumin complete antigen.
In a preferred embodiment of the citrinin ELISA detection kit provided by the present invention, the kit further comprises a washing solution, wherein the washing solution is PBS/T20.
The invention also provides a method for detecting the content of the citrinin by using the citrinin ELISA detection kit, which comprises the following steps
Diluting the citrinin complete antigen in the 96-hole enzyme label plate coated by the citrinin complete antigen to 200ng/ml by using a coating solution, and standing overnight at 4 ℃;
discarding the solution in the pores, washing with washing solution three times for 3min each time,
sealing with a sealing solution, incubating at 37 ℃ for 1h, and washing the 96-well enzyme label plate with a washing solution;
respectively adding a citrinin standard substance and 50 mu l/hole of a sample to be detected into the 96-hole enzyme label plate, then adding 50 mu l/hole of a pure citrinin product, incubating for 1h at 37 ℃, discarding the solution in the hole, and washing with a washing solution;
adding 100 mul/hole of enzyme-labeled goat anti-mouse antibody diluted by 1:2000 times of diluent into the 96-hole enzyme label plate, incubating for 30min at 37 ℃, and washing the plate for 3 times;
adding color development liquid, reacting for 15min at 37 ℃ in a dark place, adding stop solution, and reading OD values of the citrinin standard substance and the sample to be detected when the wavelength is 450 nm;
data processing: OD without citrinin inhibition450nmHas a value of B0OD corresponding to the concentration of other citrinin standards450nmNumerical value as B, expressed as B/B0As ordinate, the concentration of the citrinin standard substance added in each hole is used as abscissa, a standard curve is drawn, and the OD of the sample to be detected is calculated450nmValues of (A) and (B)0Finding out the corresponding concentration according to the standard curve, namely the citrinin concentration of the sample to be detected.
Compared with the prior art, the preparation method of the citrinin monoclonal antibody, the citrinin ELISA detection kit and the detection method thereof provided by the invention have the following beneficial effects:
the preparation method of the citrinin monoclonal antibody provided by the invention adopts 2, 2-dichloro-5-2-phenethyl-4-trimethyl silicon-3-furanone with polymerization as a coupling reagent, so that a cross-linked product has a compact structure, and the substance has a protection effect on functional groups such as carboxyl and the like of citrinin; meanwhile, the method does not form a spacer arm between the citrinin and the macromolecular carrier, eliminates the generation of a bridge antibody, keeps the specificity of the antibody, and has the advantages of mild preparation conditions, simple operation, controllable process and the like;
the citrinin ELISA detection kit provided by the invention has the advantages of simple sample treatment, stable detection result, high accuracy, high sensitivity, low detection cost, wide sample coverage and the like.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without inventive efforts, wherein:
FIG. 1 is a flowchart of the steps of a method for detecting the content of citrinin by using the citrinin ELISA kit according to the present invention;
FIG. 2 is a standard curve 1 of the citrinin ELISA kit of the present invention;
FIG. 3 is a standard curve 2 of the citrinin ELISA kit of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a preparation method of an anti-citrinin monoclonal antibody, wherein the anti-citrinin monoclonal antibody adopts 2, 2-dichloro-5- (2-phenylethyl) -4- (trimethylsilyl) -3-furanone (DPTF) and N-hydroxysuccinimide (NHS) as coupling agents, Bovine Serum Albumin (BSA), Ovalbumin (OVA), Keyhole Limpet Hemocyanin (KLH) or Bovine Thyroglobulin (BTG) as macromolecular carriers to prepare artificial complete antigens, then a hybridoma cell strain which stably secretes the anti-citrinin monoclonal antibody is obtained by cell fusion and screening by using a hybridoma cell technology, and the anti-citrinin monoclonal antibody monomer monoclonal antibody is obtained by preparing ascites and purifying. The invention also provides a citrinin ELISA detection kit for the monomer monoclonal antibody of the citrinin, which is prepared by the method, and the sensitivity of the ELISA detection kit reaches 5 ng/ml.
Primary reagents and consumables
The agent citrinin was purchased from ACROSS corporation;
2, 2-dichloro-5- (2-phenylethyl) -4- (trimethylsilyl) -3-furanone, N-hydroxysuccinimide, anhydrous tetrahydrofuran, dimethylformamide, Bovine Serum Albumin (BSA), Ovalbumin (OVA), Keyhole Limpet Hemocyanin (KLH), Bovine Thyroglobulin (BTG) were purchased from SIGMA;
animals 6 weeks old, female, BALB/c mice, purchased from the Experimental animals center of cooperative university of medicine;
SP2/0 myeloma cells were purchased from the institute of basic medicine, national academy of medical sciences;
HiTrap Protein G affinity chromatography columns were purchased from GE;
freund's adjuvant, 50% polyethylene glycol (PEG)1450 solution, HAT, HT and goat anti-mouse IgG labeled with horseradish peroxidase (HRP), Tetramethylbenzidine (TMB) were Sigma products;
the DMEM medium is a Gibco product;
newborn bovine serum was purchased from Hangzhou Sijiqing bioengineering, Inc.
EXAMPLE one preparation of citrinin-bovine serum Albumin (CIT-BSA) complete antigen
(1) Activation of citrinin: 13.2mg of citrinin (formula C)13H13O5Molecular weight 250.3Da) and 14.1mg of 2, 2-dichloro-5- (2-phenylethyl) -4- (trimethylsilyl) -3-furanone (formula C)15H18SiCl2O2Molecular weight 335) and 5.8mg of N-hydroxysuccinimide (formula C)4H5NO3Molecular weight 115) is dissolved in 0.2ml of anhydrous tetrahydrofuran, 100 mul of 0.1mol/L HCl is slowly dropped into the anhydrous tetrahydrofuran, the mixture is shaken for 60min at the shaking speed of 150r/min at the temperature of 30 ℃, then centrifuged for 15min at the centrifugation speed of 4000r/min, supernatant is taken out and put in a test tube, the test tube is washed for 2-3 times by the anhydrous tetrahydrofuran, the supernatant is combined and dried in the test tube, and the drying mode is as follows: at 25 deg.C, the wind speed is 0.5m3Blowing the glass by nitrogen gas/s.
(2) Citrinin-carrier coupling reaction: after the tetrahydrofuran was dried, the residue was dissolved in 0.5ml of dimethylformamide, and this solution was then added dropwise to 0.5ml of 5% BSA solution by dropwise addition and adjusted to pH 8.0, and slowly shaken in a 37 ℃ incubator for 60 min.
(3) Separation and purification: after the coupling reaction, 0.1mol/L NaHCO is used3Dialyzing the solution for 1 hr, then dialyzing in such a way that the time interval of the first 3 times of fluid exchange is 1.5 hr, the time interval of the third middle fluid exchange is 4.5 hr, and the time interval of the third last fluid exchange is 18 hr to make the measured value of protein content in the dialysate identical to that of the blank dialysate, and removing the proteinAnd concentrating the combined citrinin and dialysate to obtain citrinin-bovine serum albumin (CIT-BSA) complete antigen prepared by coupling reaction, and freeze-drying at-20 deg.C for storage.
EXAMPLE II preparation of citrinin-ovalbumin (CIT-OVA) complete antigen
Preparation of citrinin ovalbumin (CIT-OVA) complete antigen substantially the same as that of the preparation of citrinin-bovine serum albumin (CIT-BSA) complete antigen described in example one, except that in the citrinin-carrier coupling reaction step, a solution of Bovine Serum Albumin (BSA) is not added, but a solution of Ovalbumin (OVA) is added.
EXAMPLE III preparation of citrinin-keyhole limpet hemocyanin (CIT-KLH) complete antigen
Preparation of citrinin-keyhole limpet hemocyanin (CIT-KLH) complete antigen the same procedure as that used for the preparation of citrinin-bovine serum albumin (CIT-BSA) complete antigen in example one, except that in the citrinin-carrier coupling reaction step, instead of a solution of Bovine Serum Albumin (BSA), a solution of Keyhole Limpet Hemocyanin (KLH) was added.
EXAMPLE four preparation of citrinin-bovine thyroglobulin (CIT-BTG) complete antigen
Preparation of citrinin-bovine thyroglobulin (CIT-BTG) complete antigen the same procedure as that for the preparation of citrinin-bovine serum albumin (CIT-BSA) complete antigen in example one, except that in the citrinin-carrier coupling reaction step, instead of a Bovine Serum Albumin (BSA) solution, a Bovine Thyroglobulin (BTG) solution was added.
EXAMPLE V preparation of anti-citrinin mAb
Step one, animal immunization
Female BALB/c mice of 6 weeks old are selected, and the citrinin-bovine serum albumin (CIT-BSA) complete antigen prepared in the first embodiment is used as an immunizing antigen and Freund's adjuvant to immunize the mice until the titer detection reaches about 1: 10W.
Specifically, for the first immunization, 50 mu g of immune antigen is added with equal volume of complete Freund's adjuvant, and subcutaneous injection is adopted; after 2 weeks, a second immunization, with 50. mu.g/immunization antigen plus an equal volume of incomplete Freund's adjuvant, was injected subcutaneously and its titer was measured as 1: 30000; after 2 weeks, a third immunization, using 25. mu.g/immunization antigen plus an equal volume of incomplete Freund's adjuvant, was injected subcutaneously and the titer was measured as 1: 100000; after 7-10 days, ELISA is used for detecting the serum titer of the immune mice, and the titer reaches 1:10W, so that cell fusion can be prepared. If the titer is lower, the fourth immunization is continued after 2 weeks, and the immunization dose and the immunization mode are consistent with those of the third immunization until the titer detection reaches about 1: 10W. 3 days before cell fusion, 25. mu.g of antigen was injected intraperitoneally to boost immunity.
Step two, cell fusion
Taking spleen cells of the immunized mice in the step one, taking 50 percent (volume) PEG1450 as a fusion agent, and carrying out conventional fusion with mouse myeloma cells Sp2/0 according to the number of 10: 1 cells, wherein the specific number of the spleen cells is 1 × 108The number of mouse myeloma cells Sp2/0 is 1 × 107A plurality of; the fused cells are selectively cultured by HAT medium, then HT medium after 7 days and DMEM medium after 14 days.
Step three, subcloning of positive hybridoma cell strain
The 96-well microplate was coated with the citrinin ovalbumin (CIT-OVA) complete antigen prepared in example two. The coating concentration of the citrinin ovalbumin (CIT-OVA) complete antigen obtained in the step is 200 ng/ml; and (3) adding 100ul of cells fused in the second step into the wells of the 96-well enzyme label plate for detection, performing subcloning when positive cells are detected, and performing multiple subcloning to reach 100% of positive rate to obtain 17 positive hybridoma cell strains, wherein the generated antibody can react with the envelope antigen enzyme to be positive.
Step four, preparation and purification of antibody
(1) Preparing antibody by injecting 0.5ml liquid paraffin into abdominal cavity of each sensitized BALB/c mouse 7-10 days in advance, and inoculating 17 hybridoma cells established in step three to BALB/c mouse injected with liquid paraffin, wherein the number of the inoculated hybridoma cells is 1 × 106Collecting ascites after 7-10 days and centrifuging to obtain supernatant;
(2) purification of the antibody: diluting the supernatant in 0.01mol/L PBS solution with pH7.4 at a ratio of 1:10, gradually adding into a chromatographic column balanced by PBS from the upper part of the column at a speed of 0.5ml/min, washing with PBS solution, and eluting with glycine buffer solution with pH 2.9; the chromatographic column is a HiTrap Protein G affinity chromatographic column;
(3) detection of antibody by ELISA detection method:
after obtaining the purified antibody, detecting the titer of the antibody by an ELISA detection method, wherein the titer of the 17-strain antibody is 1: 6W-1: between 20W, 17 antibodies are detected by a competitive ELISA method, wherein only 5 antibodies have a reaction gradient, and one antibody reaches the maximum sensitivity of 5ng/ml and can completely exceed the standard of a domestic kit.
EXAMPLE VI citrinin ELISA test kit
The citrinin ELISA detection kit comprises a 96-hole enzyme label plate coated by citrinin complete antigen, an anti-citrinin monoclonal antibody, a citrinin standard substance, an enzyme-labeled goat anti-mouse antibody, a coating solution, a washing solution, a confining solution, a diluent, a developing solution and a stop solution;
wherein,
the citrinin complete antigen coated by the 96-hole enzyme label plate can be citrinin-bovine serum albumin (CIT-BSA) complete antigen, citrinin ovalbumin (CIT-OVA) complete antigen, citrinin-keyhole limpet hemocyanin (CIT-KLH) complete antigen or citrinin-bovine thyroglobulin (CIT-BTG) complete antigen, and the citrinin ovalbumin (CIT-OVA) complete antigen is selected by the kit;
the anti-citrinin monoclonal antibody is the anti-citrinin monoclonal antibody prepared in example five;
the concentrations of the citrinin standard substance are respectively 0, 5, 10, 20, 40 and 80 ng/ml;
the enzyme-labeled goat anti-mouse antibody is a goat anti-mouse antibody labeled by horseradish peroxidase (HRP);
the coating solution is 0.01mol/L of carbonate buffer solution with pH9.6, and is used for diluting the antigen coated by the 96-hole enzyme label plate to 200 ng/ml;
the washing solution is PBS/T20;
the blocking solution is PBS/T20 containing 5% calf serum;
the diluent is 0.01mol/L PBS solution with pH7.4;
the color developing liquid is TMB single-component color developing liquid;
the stop solution is 2mol/L H2SO4。
Please refer to fig. 1, which is a flowchart illustrating the steps of the method for detecting citrinin content by using the citrinin ELISA test kit of the present invention.
Example seven, method for detecting citrinin content by citrinin ELISA detection kit
Step S1, using coating liquid to dilute the complete antigen in the 96-hole enzyme label plate coated by the citrinin ovalbumin (CIT-OVA) complete antigen to 200ng/ml, and staying overnight at 4 ℃;
step S2, discarding the solution in the hole, washing three times with washing solution, each time for 3min,
step S3, sealing with a sealing solution, incubating for 1h at 37 ℃, and then washing the 96-well enzyme label plate with a washing solution;
step S4, respectively adding a citrinin standard substance and 50 mu l/hole of a sample to be detected into the 96-hole enzyme label plate, then adding 50 ul/hole of a pure citrinin product, incubating for 1h at 37 ℃, discarding the solution in the hole, and washing with a washing solution, wherein the concentrations of the citrinin standard substance are 0, 5, 10, 20, 40 and 80ng/ml respectively;
step S5, adding 100 mul/hole of enzyme-labeled goat anti-mouse antibody diluted by 1:2000 times of diluent into the 96-hole enzyme label plate, incubating for 30min at 37 ℃, and washing the plate for 3 times;
step S6, adding a color development solution, reacting for 15min in a dark place at 37 ℃, adding a stop solution, and reading the absorbance (OD) values of the citrinin standard substance and the sample to be detected when the wavelength is 450 nm;
step S7, data processing: OD without citrinin inhibition (citrinin concentration ═ 0)450nmHas a value of B0OD corresponding to the concentration of other citrinin standards450nmThe value is B, and the relative absorbance value B/B0As ordinate, the concentration of the citrinin standard substance added in each hole is used as abscissa, a standard curve is drawn, and the OD of the sample to be detected is calculated450nmValues of (A) and (B)0Finding out the corresponding concentration according to the standard curve, namely the citrinin concentration of the sample to be detected.
EXAMPLE VIII evaluation of the accuracy of the citrinin ELISA test kit
1) Establishment of a Standard Curve
Wherein, when the concentration of the standard substance is 0ng/ml, the corresponding OD average value is B0The OD average value for the other standards was B, and the relative absorbance value y1 was calculated by the following formula, y1 ═ B/B0(ii) a The y1 value is plotted as ordinate and the concentration of the standard is plotted as abscissa to form a calibration curve 1, which is shown in detail in FIG. 2.
Making a standard curve 2 by Logit-log linear regression according to the relation between the concentration and the OD value of the standard substance;
the calculation method of y2 is as follows:
p is B/B0，y2＝ln(p/1-p)。
The y2 value is plotted as ordinate and the concentration of the standard is plotted as abscissa to form a calibration curve 2, which is shown in detail in FIG. 3.
From standard curve 1 and standard curve 2, one can obtain: coefficient of correlation R20.9993; IC50 ═ 18.4; the inhibition rate was 85.9%.
2) Samples with known contents were tested by the ELISA test kit for citrinin prepared in example 6 of the present invention according to the test method of example 7, and the recovery rate (recovery rate ═ added amount/test amount × 100%) was calculated, and the results are shown in the following table:
the preparation method of the citrinin monoclonal antibody, the citrinin ELISA detection kit and the detection method thereof provided by the invention have the following beneficial effects:
the preparation method of the citrinin monoclonal antibody provided by the invention adopts 2, 2-dichloro-5-2-phenethyl-4-trimethyl silicon-3-furanone with polymerization as a coupling reagent, so that a cross-linked product has a compact structure, and the substance has a protection effect on functional groups such as carboxyl and the like of citrinin; meanwhile, the method does not form a spacer arm between the citrinin and the macromolecular carrier, eliminates the generation of a bridge antibody, keeps the specificity of the antibody, and has the advantages of mild preparation conditions, simple operation, controllable process and the like;
the citrinin ELISA detection kit provided by the invention has the advantages of simple sample treatment, stable detection result, high accuracy, high sensitivity, low detection cost, wide sample coverage and the like.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A preparation method of anti-citrinin monoclonal antibody is characterized by comprising the following steps:
synthesizing a citrinin complete antigen, wherein the citrinin complete antigen is prepared according to the following method: the citrinin firstly reacts with a coupling reagent 2, 2-dichloro-5- (2-phenylethyl) -4- (trimethylsilyl) -3-furanone and N-hydroxysuccinimide, then is coupled with a macromolecular carrier, dialysis is carried out after the coupling reaction until the measured value of the protein content in dialysate is the same as that of blank dialysate, the non-combined citrinin is removed, and the dialysate is concentrated to obtain a citrinin complete antigen;
and immunizing an animal by taking the citrinin complete antigen as an immune antigen, performing cell fusion and screening by using a hybridoma cell technology to obtain a hybridoma cell strain which stably secretes the anti-citrinin monoclonal antibody, and preparing ascites and purifying to obtain the anti-citrinin monoclonal antibody.
2. The method for preparing citrinin-resistant monoclonal antibody according to claim 1, wherein the specific method for synthesizing citrinin complete antigen comprises:
(1) activation of citrinin: dissolving 13-17 mg of citrinin, 13-17 mg of 2, 2-dichloro-5-2-phenethyl-4-trimethyl silicon-3-furanone and 5-7 mg of N-hydroxysuccinimide in 0.2ml of anhydrous tetrahydrofuran, slowly dropwise adding 10.1mol/L HCl100 mu L, shaking uniformly at room temperature, centrifuging to obtain a supernatant, and washing with the anhydrous tetrahydrofuran;
(2) citrinin-carrier coupling reaction: after tetrahydrofuran is dried, dissolving the residue in 0.5ml of dimethylformamide, then dropwise adding the solution into 0.5ml of 5% macromolecular carrier solution, adjusting the pH value of the solution to 8.0, and slowly shaking the solution in a constant temperature box at 37 ℃ for 60 min;
(3) separation and purification: after the coupling reaction, the mixture is placed in 0.1mol/L NaHCO3Dialyzing in the solution for 1 hour, dialyzing in a mode of changing the liquid for the first 3 times for 1.5 hours, changing the liquid for the third time for 4.5 hours and changing the liquid for the third time for 18 hours to ensure that the measured value of the protein content in the dialyzate is the same as that of the blank dialyzate, removing the non-combined citrinin, concentrating the dialyzate to obtain the citrinin complete antigen, and storing at the temperature of freeze-drying-20 ℃.
3. The method of claim 2, wherein the macromolecular carrier is bovine serum albumin, ovalbumin, keyhole limpet hemocyanin, or bovine thyroglobulin.
4. The method for preparing citrinin mab of claim 1, wherein the citrinin complete antigen is used as an immune antigen to immunize an animal, and then a hybridoma cell technology is used to perform cell fusion and screening to obtain a hybridoma cell line stably secreting the citrinin mab, and ascites is prepared and purified to obtain the citrinin mab, which specifically comprises the following steps:
step one, animal immunization: taking the citrinin complete antigen as an immunizing antigen and Freund's adjuvant to immunize a BALB/c mouse for multiple times until the titer detection reaches 1: 10W;
step two, cell fusion: taking spleen cells of an immunized mouse, taking PEG1450 with the volume ratio of 50% as a fusion agent, and mixing the mixture according to the proportion of 10: 1, fusing the cell number with a mouse myeloma cell Sp2/0, selectively culturing the fused cells by using an HAT culture medium, culturing by using an HT culture medium after 7 days, and culturing by using a DMEM culture medium after 14 days;
step three, subcloning the positive hybridoma cell strain: detecting fused cells by adopting a 96-hole enzyme label plate coated with citrinin complete antigen, wherein the coating concentration of the 96-hole enzyme label plate is 200ng/ml, the addition amount of the fused cells is 100 mu l/hole, and when positive cells are detected, performing subcloning until the positive rate reaches 100%;
step four, preparation and purification of antibody
(1) Preparation of antibody: injecting 0.5ml of liquid paraffin into the abdominal cavity of each sensitized BALB/c mouse 7-10 days in advance, then inoculating the positive hybridoma cells established in the step three to the BALB/c mouse injected with the liquid paraffin, collecting ascites after 7-10 days, and centrifuging to obtain supernatant;
(2) purification of the antibody: and (3) diluting the supernatant into a PBS solution according to the ratio of 1:10, gradually adding the supernatant into a chromatographic column balanced by PBS from the upper part of the column at the speed of 0.5ml/min, washing the column by the PBS solution, and eluting the column by a glycine buffer solution with the pH of 2.9 to obtain the purified anti-citrinin monoclonal antibody.
5. The method for preparing anti-citrinin mab of claim 4, wherein the animal immunization method of step one comprises: the first immunization, 50 mu g of each immunizing antigen is added with an equal volume of complete Freund adjuvant and injected subcutaneously; after 2 weeks, the second immunization is carried out, and 50 mu g of immune antigen is added with an equal volume of incomplete Freund's adjuvant and injected subcutaneously; after 2 weeks, the third immunization, 25 mug/antigen plus the same volume of incomplete Freund's adjuvant, subcutaneous injection; after 7-10 days, ELISA is used for detecting the titer of the serum of the immune mice, and the titer reaches 1:10W, so that cell fusion can be prepared; 3 days before cell fusion, 25. mu.g of antigen was injected intraperitoneally to boost immunity.
6. An ELISA kit for detecting citrinin, comprising the anti-citrinin monoclonal antibody prepared by the method for preparing the anti-citrinin monoclonal antibody according to any one of claims 1 to 5.
7. The citrinin ELISA detection kit of claim 6, further comprising a 96-well enzyme-labeled plate coated with citrinin complete antigen, a citrinin standard, an enzyme-labeled goat anti-mouse antibody, a coating solution, a diluent, a blocking solution, a developing solution and a stop solution;
wherein,
the concentrations of the citrinin standard substance are respectively 0, 5, 10, 20, 40 and 80 ng/ml;
the enzyme-labeled goat anti-mouse antibody is a goat anti-mouse antibody labeled by horseradish peroxidase;
the coating solution is 0.01mol/L of carbonate buffer solution with pH9.6, and is used for diluting the citrinin complete antigen coated on the 96-hole enzyme label plate to 200 ng/ml;
the diluent is 0.01mol/L PBS solution with pH7.4;
the blocking solution is PBS/T20 containing 5% calf serum;
the color developing liquid is TMB single-component color developing liquid;
the stop solution is 2mol/L H2SO4。
8. The citrinin ELISA test kit of claim 7, wherein the citrinin complete antigen coated on the 96-well ELISA plate is citrinin ovalbumin complete antigen.
9. The citrinin ELISA test kit of claim 7, further comprising a washing solution, wherein said washing solution is PBS/T20.
10. The method for detecting the content of citrinin by using the citrinin ELISA test kit according to any one of claims 7 to 9, comprising:
diluting the citrinin complete antigen in the 96-hole enzyme label plate coated by the citrinin complete antigen to 200ng/ml by using a coating solution, and standing overnight at 4 ℃;
discarding the solution in the pores, washing with washing solution three times for 3min each time,
sealing with a sealing solution, incubating at 37 ℃ for 1h, and washing the 96-well enzyme label plate with a washing solution;
respectively adding a citrinin standard substance and 50 ul/hole of a sample to be detected into the 96-hole enzyme label plate, then adding 50 ul/hole of a pure citrinin product, incubating at 37 ℃ for 1h, removing solution in the hole, and washing with a washing solution;
adding 100ul of enzyme-labeled goat anti-mouse antibody per well diluted by 1:2000 times of diluent into the 96-well enzyme-labeled plate, incubating for 30min at 37 ℃, and washing the plate for 3 times;
adding color development liquid, reacting at 37 ℃ in a dark place for 15min, adding stop solution, and reading the absorbance OD value of the citrinin standard substance and the sample to be detected when the wavelength is 450 nm;
data processing: OD without citrinin inhibition450nmHas a value of B0OD corresponding to the concentration of other citrinin standards450nmThe value is B, and the relative absorbance is B/B0The value is the ordinate, the concentration value of the citrinin standard substance added in each hole is used as the abscissa, a standard curve is drawn, and the OD of the sample to be detected is calculated450nmValues of (A) and (B)0Finding out the corresponding concentration according to the standard curve, namely the citrinin concentration of the sample to be detected.
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Application publication date: 20170531 |