CN112358549A - Preparation of dimethyl isotrichum monoclonal antibody and enzyme-linked immunosorbent assay method thereof - Google Patents
Preparation of dimethyl isotrichum monoclonal antibody and enzyme-linked immunosorbent assay method thereof Download PDFInfo
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- 125000000118 dimethyl group Chemical group [H]C([H])([H])* 0.000 title claims abstract description 31
- 238000000034 method Methods 0.000 title claims abstract description 31
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
-
- 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
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
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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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
The invention relates to a preparation method of a dimethyl isotrichum alcohol monoclonal antibody and an enzyme-linked immunosorbent assay method thereof, belonging to the technical field of immunochemistry and analytical biology. The invention provides a preparation method of a dimethyl isotrichum monoclonal antibody, which comprises the following steps: firstly synthesizing a dimethyl isotrichol modifier, then preparing 2-MIB modifier-BSA and 2-MIB modifier-OVA, and then preparing the dimethyl isotrichol monoclonal antibody by using the 2-MIB modifier-BSA and 2-MIB modifier-OVA. The invention takes camphor with a molecular structure similar to that of 2-MIB as an initiator for hapten modification, and synthesizes a modifier of the 2-MIB after the camphor is modified, so that the modifier not only keeps the complete molecular structure of the 2-MIB, but also introduces a connecting arm with an end group as carboxyl; attack of the carbonyl group by Grignard reagents, followed by cleavage of the double bondOxidizing to generate carboxyl, and finally connecting the carboxyl of the derivative with carrier protein to prepare immunogen and coating antigen, thereby reducing the production cost. Meanwhile, the monoclonal antibody of anti-2-MIB is successfully prepared by the hybridoma technology; an indirect competitive ELISA, sensitivity (IC), was established to detect 2-MIB50Value) was 52.61 ng/mL.
Description
Technical Field
The invention relates to a preparation method of a dimethyl isotrichum alcohol monoclonal antibody and an enzyme-linked immunosorbent assay method thereof, belonging to the technical field of immunochemistry and analytical biology.
Background
2-MIB is one of the most main and most common smelly substances causing soil odor and musty odor in water, mainly secretion of blue-green algae, actinomycetes and fungi, and human olfaction is extremely sensitive to the substances, so long as the water contains trace 2-MIB, people can feel deterioration of water quality, the content of the 2-MIB directly influences the quality of the water, namely the Olfaction Threshold Concentration (OTC) is extremely low and is about 5-10 ng/L. In the sanitary Standard for Drinking Water (GB 5749-. With the improvement of living standard, the attention degree of people to drinking water is increased, and the requirement on the taste of the drinking water is higher and higher. However, most of China's drinking water sources are surface water, and the probability of existence of 2-MIB is very high. Therefore, the method for detecting the 2-MIB, which is sensitive, accurate, rapid, simple and convenient, is important to guarantee the water quality safety.
Currently, the detection methods for 2-MIB are mainly divided into two categories, namely sensory analysis and instrumental analysis. The sensory analysis method mainly depends on human sensory organs to grade the smell intensity, is greatly influenced by subjective factors, and cannot measure earthy substances lower than the smell threshold; the instrumental analysis method comprises the steps of firstly extracting the 2-MIB by adopting different pretreatment methods such as a liquid-liquid extraction method, a sweeping and trapping method, a solid-phase microextraction method and the like, and then qualitatively or quantitatively analyzing the 2-MIB in a sample by a chromatography method such as a gas chromatography or a gas chromatography-mass spectrometer.
Although the instrument analysis method is accurate, the method has the defects of expensive instrument, complex operation, complex sample pretreatment, time consumption, high detection cost, incapability of being used for on-site rapid detection and the like. Therefore, it is urgently needed to establish an analysis method for detecting 2-MIB, which has high sensitivity, good selectivity and convenient operation.
Compared with the conventional instrument method, the enzyme-linked immunosorbent assay (ELISA) has the advantages of high sensitivity, strong specificity and rapid analysis, and is widely used in the analysis fields of clinic, medicine, food, environment and the like. So far, the literature report of measuring the content of 2-MIB in water by ELISA is rarely seen at home and abroad.
Disclosure of Invention
The invention aims to provide a preparation method of a 2-MIB monoclonal antibody and an enzyme-linked immunosorbent assay method thereof aiming at the defects of the prior art. The specific scheme is as follows:
the preparation method of the monoclonal antibody of dimethyl isotrichum comprises the following steps:
step 1: synthesizing a dimethyl isotrichum modifier, wherein the structure of the dimethyl isotrichum modifier is shown in a structural formula 1:
(structural formula 1)
Step 2: preparing 2-MIB modifier-BSA and 2-MIB modifier-OVA by using the dimethyl isotrichum modifier obtained in the step (1), wherein the 2-MIB modifier is dimethyl isotrichum modifier, the BSA is bovine serum albumin, and the OVA is ovalbumin;
and step 3: preparing a dimethyl isotretinoin monoclonal antibody by using the 2-MIB modifier-BSA and the 2-MIB modifier-OVA obtained in the step 2;
preferably, the step 1 specifically comprises:
1) preparing a reactant from allyl bromide and camphor under the action of a Grignard reagent, wherein the structure of the reactant is shown as a structural formula 2:
(structural formula 2)
2) Reacting the reactant obtained in the step 1) with NaIO4 and KMnO4 to obtain a dimethyl isotrichol modifier;
preferably, the reaction conditions of the allyl bromide and camphor are as follows: the temperature is-70 ℃, and the reaction time is 5-6 h;
preferably, the step 2 specifically comprises:
dissolving dimethyl isotrichol modifier, N-hydroxysuccinimide and N, N '-dicyclohexylcarbodiimide in N, N' -dimethylformamide, centrifuging after the reaction is finished, taking supernate, respectively adding the supernate into bovine serum albumin and ovalbumin solutions, and respectively collecting supernate containing 2-MIB modifier-BSA and 2-MIB modifier-OVA;
preferably, the dosage of the bovine serum albumin or the ovalbumin is 50 mg, and the reaction condition is stirring for 24 hours at 4 ℃;
preferably, the step 3 specifically comprises:
1) immunization: 2-MIB modifier-BSA immune mice, and 2-MIB modifier-OVA as the coating antigen for indirect competitive ELISA detection of antiserum titer;
2) fusing and screening: fusing a mouse spleen cell and a myeloma cell under the action of polyethylene glycol to obtain a positive hybridoma cell, and subcloning the positive hybridoma cell for 2-3 times by using a limiting dilution method;
3) mass production of monoclonal antibodies: inoculating hybridoma cells to a mouse to produce a dimethyl isotrichum monoclonal antibody;
preferably, the 1) immunization operation is specifically: when in the first immunization, a BALB/C mouse is immunized by the 2-MIB modifier-BSA; the second immunization after two weeks, Freund's incomplete adjuvant is adopted; after two weeks, the third immunization is carried out, and the titer of the antiserum is detected by indirect competitive ELISA using 2-MIB modifier-OVA as the coating antigen; taking blood for the fourth immunization two weeks later;
preferably, the 2) fusion operation is specifically: mixing splenocytes and myeloma cells according to the proportion of 5: 1-10: 1, fusing under the action of polyethylene glycol, and suspending hybridoma cells by HAT culture solution;
preferably, the 2) screening operation is specifically: screening by using an indirect competitive ELISA method, and subcloning cells with positive holes developing colors for 2-3 times by using a limiting dilution method;
preferably, the 3) monoclonal antibody mass production operation is specifically: injecting Freund's incomplete adjuvant into abdominal cavity to sensitize BALB/C mouse, inoculating hybridoma cell in abdominal cavity 7-10 days later, collecting ascites before death of mouse, centrifuging, and collecting clarified ascites to obtain monoclonal antibody;
the enzyme-linked immunosorbent assay method of the monoclonal antibody of dimethyl isotrichum alcohol is characterized by using the prepared monoclonal antibody of dimethyl isotrichum alcohol to perform performance characterization and establish the enzyme-linked immunosorbent assay method for measuring the content of 2-MIB in water.
Dimethyl isobornyl alcohol (2-methylisoborneol, 2-MIB,) is a saturated cyclic tertiary alcohol substance, is mainly a secondary metabolite of blue algae and actinomycetes, has certain volatility, and is one of the most common odor substances causing odor in drinking water. Because the olfaction threshold concentration is extremely low, strong earthy mildew can be generated by about 5-10 ng/L, the detection of smelly substances can reflect the water quality condition in time, and the safety of the water quality is improved.
The invention discloses a preparation method of a 2-MIB monoclonal antibody and an enzyme-linked immunosorbent assay (ELISA) method thereof. The method is characterized in that camphor with a molecular structure similar to that of 2-MIB is used as an initiator for hapten modification, and the camphor is modified to synthesize a modifier of the 2-MIB, so that the modifier not only retains the complete molecular structure of the 2-MIB, but also introduces a connecting arm with an end group as carboxyl; crosslinking carboxyl on the connecting arm with carrier protein to prepare immunogen and coating antigen; immunizing a mouse by using the immunogen, preparing a monoclonal antibody for resisting 2-MIB by using a hybridoma technology, and establishing indirect competitive ELISA (enzyme-linked immuno sorbent assay) by using the 2-MIB as a detection target object. The invention successfully synthesizes the 2-MIB modifier, and then uses13The C NMR (nuclear magnetic resonance) chart and the HRMS (high resolution mass spectrometry) chart represent the 2-MIB modifier, and the results show that the 2-MIB modifier is successfully prepared. Finally, through multiple immunizations of mice and fusion and screening of hybridoma cells, monoclonal antibody for resisting 2-MIB is obtained, and ELISA, Ling for measuring 2-MIB is establishedSensitivity (IC)50Value) was 52.61 ng/mL.
The invention has the advantages that:
1) 2-MIB is expensive, and if 2-MIB is directly used for coupling carrier protein to prepare immunogen and coating antigen, research cost is greatly increased. The invention uses cheap camphor as the starting material of hapten modification, firstly attacks carbonyl through Grignard reagent, then oxidizes double bonds to generate carboxyl, and finally connects the carboxyl of the derivative with carrier protein to prepare immunogen and coating antigen. Not only reduces the production cost, but also keeps the complete molecular structure of the 2-MIB.
2) The monoclonal antibody of 2-MIB is successfully prepared by hybridoma technology.
3) Based on the self-made antibody, the indirect competitive ELISA for detecting 2-MIB is successfully established.
Drawings
FIG. 1 shows the molecular structures of Dimethylisotrichol and Camphor.
FIG. 2 is a flow chart of the preparation of dimethylisotretinoin modifications, immunogens and coating antigens.
FIG. 32-MIB modification13C NMR chart.
Fig. 42-HRMS diagram of MIB modification.
FIG. 5 inhibition of cell supernatants.
FIG. 6 shows a standard curve of ELISA targeting 2-MIB based on monoclonal antibody.
Detailed Description
The present invention is described in detail below by way of examples, it being necessary to point out here that this example is intended to illustrate the invention further only, but not to be construed as limiting the scope of the invention, which is to be given the full breadth of the appended claims, and that those skilled in the art may make insubstantial modifications and adaptations of the invention in light of the above teachings.
Example 1:
1. preparation of 2-MIB modifiers, immunogens and coated antigens
(1) A round bottom flask with a branch opening is taken, 40 mmol of magnesium and 2 particles of iodine are added, and then air is pumped out and nitrogen is introduced for protection. 20 mL of tetrahydrofuran was added to the flask via syringe and heated to reflux. Then, 30 mmol of allyl bromide was dissolved in a small amount of tetrahydrofuran, slowly injected into the flask via a syringe, and the reaction was slowly stirred. When the reaction mixture was almost colorless, 15 mmol of camphor was dissolved in 15 mL of tetrahydrofuran, injected into the prepared Grignard reagent by syringe, and reacted at-70 ℃ for 5 hours. After the reaction, water was added to quench the reaction, and the target product was extracted with ethyl acetate. And finally purifying the target substance by a silica gel column, wherein the eluent is ethyl acetate and petroleum ether.
(2) Taking a clean round-bottom flask, and sequentially adding 5.0 mmol of the mixture
82.4 mmol of NaIO41.72 mmol of KMnO 4100 mL of water and 17 mL of diethyl ether. After the feeding is finished, stirring at normal temperature for overnight reaction. After the reaction is finished, the target product is purified by a silica gel column.
(3) Weighing 12.9 mg of the extract at room temperature
7.7 mg NHS and 12.4 mg DCC in 500. mu.L DMF, mixed well and stirred slowly for 24 h. After the reaction was complete, the mixture was centrifuged at 7000 r/min for 10 min at 4 ℃. The supernatant was taken and added dropwise to a BSA/OVA solution (50 mg BSA/OVA in 2 mL PBS). The mixture was then stirred at 4 ℃ for 24 h. Finally, centrifugation is carried out at 4 ℃ for 10 min, and the supernatant containing 2-MIB derivative-BSA or 2-MIB derivative-OVA is collected and added to a volume of 0.01 mol L-1Dialyzed against PBS for 3 days. After lyophilization, the conjugates were stored at-20 ℃ until use.
2. Preparation of monoclonal antibodies
(1) Immunization: dissolving the immunogen in physiological saline, mixing with equal volume of Freund's complete adjuvant, and subcutaneously immunizing BALB/C mouse at multiple points with the dose of the immunogen of 100 μ g per mouse; the second immunization is carried out after two weeks, the Freund's complete adjuvant is changed into the Freund's incomplete adjuvant, and the rest steps are the same; after two weeks, carrying out third immunization, repeating the second immunization operation, and taking blood from the tail part one week later, and carrying out ELISA (enzyme-linked immunosorbent assay) to detect the titer of the antiserum; taking blood for the fourth immunization after two weeks, repeating the third immunization step, and taking blood to measure titer; the last abdominal cavity is boosted two weeks after blood collection without adjuvant.
(2) Fusing: three days after abdominal cavity boosting immunization, collecting spleen of mouse, mixing spleen cells and myeloma cells (SP 2/0) at ratio of 5:1, fusing under the action of 50% polyethylene glycol 4000, suspending hybridoma cells with HAT culture solution, adding 96-well culture plate containing feeder cells, standing at 37 deg.C and 5% CO2Culturing in an incubator.
(3) Screening: after about 2 weeks of fusion, negative wells are screened by indirect ELISA, while positive wells with clear color are subcloned 2 times by limiting dilution for timely detection.
(4) Mass production of monoclonal antibodies: injecting Freund's incomplete adjuvant into abdominal cavity to sensitize BALB/C mouse, inoculating hybridoma cell in abdominal cavity 7 days later, observing ascites of mouse, and collecting ascites when ascites is as much as possible and before death. Centrifuging for several times to obtain clarified ascites as monoclonal antibody, mixing monoclonal antibody with glycerol and phosphate buffer solution (PB) at a ratio of 4:5:1, and storing at-20 deg.C.
Example 2:
1. preparation of 2-MIB modifiers, immunogens and coated antigens
(1) A round bottom flask with a branch opening is taken, 40 mmol of magnesium and 2 particles of iodine are added, and then air is pumped out and nitrogen is introduced for protection. 20 mL of tetrahydrofuran was added to the flask via syringe and heated to reflux. Then, 30 mmol of allyl bromide was dissolved in a small amount of tetrahydrofuran, slowly injected into the flask via a syringe, and the reaction was slowly stirred. When the reaction mixture was almost colorless, 15 mmol of camphor was dissolved in 15 mL of tetrahydrofuran, injected into the prepared Grignard reagent by syringe, and reacted at-70 ℃ for 6 hours. After the reaction, water was added to quench the reaction, and the target product was extracted with ethyl acetate. And finally purifying the target substance by a silica gel column, wherein the eluent is ethyl acetate and petroleum ether.
(2) Taking a clean round-bottom flask, and sequentially adding 5.0 mmol of the mixture
82.4 mmol of NaIO41.72 mmol of KMnO 4100 mL of water and 17 mL of diethyl ether. After the feeding is finished, stirring at normal temperature for overnight reaction. After the reaction is finished, the target product is purified by a silica gel column.
(3) Weighing 12.9 mg of the extract at room temperature
7.7 mg NHS and 12.4 mg DCC in 500. mu.L DMF, mixed well and stirred slowly for 24 h. After the reaction was complete, the mixture was centrifuged at 7000 r/min for 10 min at 4 ℃. The supernatant was taken and added dropwise to a BSA/OVA solution (50 mg BSA/OVA in 2 mL PBS). The mixture was then stirred at 4 ℃ for 24 h. Finally, centrifugation is carried out at 4 ℃ for 10 min, and the supernatant containing 2-MIB derivative-BSA or 2-MIB derivative-OVA is collected and added to a volume of 0.01 mol L-1Dialyzed against PBS for 3 days. After lyophilization, the conjugates were stored at-20 ℃ until use.
2. Preparation of monoclonal antibodies
(1) Immunization: dissolving the immunogen in physiological saline, mixing with equal volume of Freund's complete adjuvant, and subcutaneously immunizing BALB/C mouse at multiple points with the dose of the immunogen of 100 μ g per mouse; the second immunization is carried out after two weeks, the Freund's complete adjuvant is changed into the Freund's incomplete adjuvant, and the rest steps are the same; after two weeks, carrying out third immunization, repeating the second immunization operation, and taking blood from the tail part one week later, and carrying out ELISA (enzyme-linked immunosorbent assay) to detect the titer of the antiserum; taking blood for the fourth immunization after two weeks, repeating the third immunization step, and taking blood to measure titer; the last abdominal cavity is boosted two weeks after blood collection without adjuvant.
(2) Fusing: three days after abdominal cavity boosting immunization, spleen of mouse is taken, spleen cells and myeloma cells (SP 2/0) are mixed according to the proportion of 10:1, the mixture is fused under the action of 50% polyethylene glycol 4000, hybridoma cells are suspended by HAT culture solution, 96-hole culture plates containing feeder cells in advance are added, and the mixture is placed at 37 ℃ and 5% CO2Culturing in an incubator.
(3) Screening: after about 2 weeks of fusion, the cells are screened by an indirect ELISA method, and the cells with colorless or nearly colorless negative holes and definite color development of positive holes are subcloned for 2-3 times by a limiting dilution method and detected in time.
(4) Mass production of monoclonal antibodies: injecting Freund's incomplete adjuvant into abdominal cavity to sensitize BALB/C mouse, inoculating hybridoma cell into abdominal cavity after 10 days, observing ascites of mouse, and collecting ascites when ascites is as much as possible and before death. Centrifuging for several times to obtain clarified ascites as monoclonal antibody, mixing monoclonal antibody with glycerol and phosphate buffer solution (PB) at a ratio of 4:5:1, and storing at-20 deg.C.
Example 3:
1. preparation of 2-MIB modifiers, immunogens and coated antigens
(1) A round bottom flask with a branch opening is taken, 40 mmol of magnesium and 2 particles of iodine are added, and then air is pumped out and nitrogen is introduced for protection. 20 mL of tetrahydrofuran was added to the flask via syringe and heated to reflux. Then, 30 mmol of allyl bromide was dissolved in a small amount of tetrahydrofuran, slowly injected into the flask via a syringe, and the reaction was slowly stirred. When the reaction mixture is almost colorless, 15 mmol of camphor is dissolved in 15 mL of tetrahydrofuran, injected into the prepared Grignard reagent through a syringe, and reacted for 5-6 h at-70 ℃. After the reaction, water was added to quench the reaction, and the target product was extracted with ethyl acetate. And finally purifying the target substance by a silica gel column, wherein the eluent is ethyl acetate and petroleum ether.
(2) Taking a clean round-bottom flask, and sequentially adding 5.0 mmol of the mixture
82.4 mmol of NaIO41.72 mmol of KMnO 4100 mL of water and 17 mL of diethyl ether. After the feeding is finished, stirring at normal temperature for overnight reaction. After the reaction is finished, the target product is purified by a silica gel column.
(3) Weighing 12.9 mg of the extract at room temperature
7.7 mg NHS and 12.4 mg DCC in 500. mu.L DMF, mixed well and stirred slowly for 24 h. After the reaction was complete, the mixture was centrifuged at 7000 r/min for 10 min at 4 ℃. The supernatant was taken and added dropwise to a BSA/OVA solution (50 mg BSA/OVA in 2 mL PBS). The mixture was then stirred at 4 ℃ for 24 h. Finally, centrifugation is carried out at 4 ℃ for 10 min, and the supernatant containing 2-MIB derivative-BSA or 2-MIB derivative-OVA is collected and added to a volume of 0.01 mol L-1Dialyzed against PBS for 3 days. After lyophilization, the conjugates were stored at-20 ℃ until use.
2. Preparation of monoclonal antibodies
(1) Immunization: dissolving the immunogen in physiological saline, mixing with equal volume of Freund's complete adjuvant, and subcutaneously immunizing BALB/C mouse at multiple points with the dose of the immunogen of 100 μ g per mouse; the second immunization is carried out after two weeks, the Freund's complete adjuvant is changed into the Freund's incomplete adjuvant, and the rest steps are the same; after two weeks, carrying out third immunization, repeating the second immunization operation, and taking blood from the tail part one week later, and carrying out ELISA (enzyme-linked immunosorbent assay) to detect the titer of the antiserum; taking blood for the fourth immunization after two weeks, repeating the third immunization step, and taking blood to measure titer; the last abdominal cavity is boosted two weeks after blood collection without adjuvant.
(2) Fusing: three days after abdominal cavity boosting immunization, collecting spleen of mouse, mixing spleen cells and myeloma cells (SP 2/0) at ratio of 8:1, fusing under the action of 50% polyethylene glycol 4000, suspending hybridoma cells with HAT culture solution, adding 96-well culture plate containing feeder cells, standing at 37 deg.C and 5% CO2Culturing in an incubator.
(3) Screening: after about 2 weeks of fusion, negative wells are screened by indirect ELISA, while positive wells with clear color are subcloned 3 times by limiting dilution for timely detection.
(4) Mass production of monoclonal antibodies: injecting Freund's incomplete adjuvant into abdominal cavity to sensitize BALB/C mouse, inoculating hybridoma cell in abdominal cavity 7-10 days later, observing ascites of mouse, and collecting ascites when ascites is as much as possible and before death. Centrifuging for several times to obtain clarified ascites as monoclonal antibody, mixing monoclonal antibody with glycerol and phosphate buffer solution (PB) at a ratio of 4:5:1, and storing at-20 deg.C.
Example 4:
enzyme-linked immunosorbent assay method for establishing dimethyl isotrichum alcohol monoclonal antibody
(1) Solution preparation
(a) Sodium carbonate-sodium bicarbonate buffer
2.606g of Na were weighed2CO3•10H2O，3.434g NaHCO3Uniformly mixing and dissolving the mixture by using 800 mL of ultrapure water, adjusting the pH value, adding water to 1L, and preparing a sodium carbonate-sodium bicarbonate buffer solution with the pH = 9.6 and 0.05 mol/L;
(b) phosphate buffer (stock solution, PBSx10)
21.961g of Na were weighed2HPO4•12H2O，6.031g NaH2PO4•2H2O, 87.666g NaCl, and 800 mL ultrapure water are mixed and heated to dissolve; adjusting pH = 7.4 with 1mol/L NaOH; adding ultrapure water to 1L to prepare 0.1mol/L phosphate buffer solution (stock solution) containing 0.15 mol/L NaCl and having pH = 7.4;
(c) blocking solution
Weighing casein, heating and dissolving the casein in 0.01 mol/L PBS to prepare 0.5-2% casein solution;
(d) phosphate buffer-Tween stock (0.1 mol/L phosphate buffered stock with 1% Tween20, PBSTx10, pH = 7.4).
(e) Substrate solution (20 mL pure water, 1 mL sodium acetate buffer, 200. mu.L Tetramethylbenzidine (TMB) (1%); 20. mu.L hydrogen peroxide (5%))
Sodium acetate buffer solution
Weighing 3.450g CH3COONa•3H2O, dissolved in 100 mL of ultrapure water and then diluted with 1mol/L citric acid (21.031 g C was weighed out)6H8O7•H2O dissolved in 100 mL water) to adjust pH = 5.8, and then adding water to a 250 mL volumetric flask to prepare 0.1mol/L sodium acetate buffer;
② TMB, weighing 0.0717g TMB, dissolving with 7.17 mL dimethyl sulfoxide, mixing to prepare 1%, v/v;
(f)H2SO4solution: remove 25 mL of concentrated H2SO4Dissolved in 475 mL of ultrapure water to prepare 5% H2SO4A solution;
(g) weighing 0.0028 g of 2-MIB standard substance, dissolving in 2.80 mL of pure water to prepare 1 mg/mL of 2-MIB stock solution for later use;
(2) main instrument
CO2A constant-temperature incubator: HF 151 UV; an enzyme-labeling instrument: ZS-3; high-capacity low-speed centrifuge: LXJ-II; inverting the light microscope: COIC;
(3) indirect competitive ELISA procedure
(a) Adding coating antigen with a certain concentration into an enzyme label plate, wherein each hole is 200 mu L, and keeping the temperature in a refrigerator at 4 ℃ overnight;
(b) wash the plate three times with PBST buffer (PBST stock, 1:10 dilution, 350 μ Ι/well);
(c) adding casein for blocking, keeping the temperature and the moisture at room temperature for 1 hour, wherein each hole is 280 mu L;
(d) washing the plate three times with PBST buffer;
(e) sequentially and respectively adding 100 mu L of standard solution and 100 mu L of antibody with certain dilution into each hole of the enzyme label plate, preserving heat and preserving moisture at room temperature, and standing for 1 hour;
(f) washing the plate three times with PBST buffer;
(g) adding enzyme-labeled secondary antibody (goat anti-mouse IgG-horseradish peroxidase) 200 μ L per well, keeping the temperature and moisture at room temperature, and standing for 1 hour;
(h) washing the plate three times with PBST buffer;
(i) adding substrate solution (newly prepared), keeping the temperature and humidity at room temperature for reaction in a dark place with 200 mu L of each hole, and oscillating on a micro oscillator for about 15-25 minutes;
(j) 5% of H is added2SO4The enzyme reaction was stopped in 80. mu.L of solution per well;
(k) and (5) measuring the absorbance by using a microplate reader, making a standard curve, and analyzing and discussing the result.
(4) Standard Curve of ELISA
The logarithm of the 2-MIB concentration is plotted as the abscissa against the relative signal B/B0x100% as ordinate to make a standard curve (B)0: the standard concentration is the absorbance corresponding to 0 ng/mL; b: absorbance for other standard concentrations).
Results and analysis
Dimethylisotrichol is a small molecule compound with only one active site-hydroxyl. If the 2-MIB is directly modified, hydroxyl groups are consumed, and the structural integrity of the hapten is affected. In general, to obtain specific antibodies, the structural integrity of the hapten is maintained as much as possible during the immunogen preparation process. Therefore, the invention selects the cheap camphor as the starting material for modification. The first step is to react camphor with Grignard reagent to obtain an intermediate; and oxidizing the intermediate to obtain a modified 2-MIB. FIGS. 3 and 4 show the 2-MIB modification, respectively13C NMR chart and HRMS chart. The data was analyzed as follows:13C NMR (101 MHz, CDCl3) δ 178.59, 79.54, 52.50, 49.16, 46.55, 44.93, 42.97, 30.66, 26.82, 21.36, 21.02, 10.45, wherein δ 178.59 corresponds to the value of the chemical shift of C in the carboxyl group; [ M + Na ] of 2-MIB derivatives]+Is 235.1412, [ M + Na ] in the map]+Is 235.1294. The result shows that the invention successfully introduces the connecting arm with the end group of carboxyl into the camphor structure and keeps the complete molecular structure of 2-MIB; the 2-MIB modifier is activated by DCC/NHS and then coupled with BSA/OVA to prepare the immunogen and the coating antigen of the 2-MIB.
FIG. 5 shows the inhibition of cell supernatants, the cell fusion and selective culture of hybridoma cells according to the invention, and the inhibition of antibodies in the cell supernatants, measured by indirect competitive ELISA, at a concentration of 3. mu.g mL-12-MIB solution of (2). The results are shown in FIG. 5 (photographs taken before the reaction was stopped, thus showing a blue signal), with 2 wells of cell supernatant having a significant inhibitory effect. The two wells were then expanded into 24-well plates for subcloning experiments.
A standard curve is drawn on the basis of the optimal conditions by optimizing three experimental conditions, namely the concentration of the blocking solution, the type of the analyte diluent and the dilution ratio of the enzyme-labeled secondary antibody (see figure 6). Here, an ELISA based on a monoclonal antibody and using 2-MIB as a detection object was established. An indirect competition ELISA standard curve which is established based on the monoclonal antibody and takes 2-MIB as a detection target has the sensitivity (expressed by an IC50 value) of 52.61 ng/mL.
Claims (11)
1. The preparation method of the dimethyl isotrichum monoclonal antibody is characterized by comprising the following steps:
step 1: synthesizing a dimethyl isotrichum modifier, wherein the structure of the dimethyl isotrichum modifier is shown in a structural formula 1:
(structural formula 1)
Step 2: preparing 2-MIB modifier-BSA and 2-MIB modifier-OVA by using the dimethyl isotrichum modifier obtained in the step (1), wherein the 2-MIB modifier is dimethyl isotrichum modifier, the BSA is bovine serum albumin, and the OVA is ovalbumin;
and step 3: and (3) preparing the dimethyl isotrichum monoclonal antibody by using the 2-MIB modifier-BSA and the 2-MIB modifier-OVA obtained in the step (2).
2. The method for preparing a monoclonal antibody against dimethyl isotrichum as claimed in claim 1, wherein the step 1 comprises:
1) preparing a reactant from allyl bromide and camphor under the action of a Grignard reagent, wherein the structure of the reactant is shown as a structural formula 2:
(structural formula 2)
Reacting the reactant obtained in the step 1) with NaIO4 and KMnO4 to obtain the dimethyl isotrichol modifier.
3. The method of claim 2, wherein the allyl bromide and camphor are reacted under the following conditions: the temperature is-70 ℃, and the reaction time is 5-6 h.
4. The method for preparing the monoclonal antibody against dimethyl isotrichum as claimed in claim 1, wherein the step 2 comprises:
dissolving dimethyl isotrichol modifier, N-hydroxysuccinimide and N, N '-dicyclohexylcarbodiimide in N, N' -dimethylformamide, centrifuging after the reaction is finished, taking supernate, respectively adding the supernate into bovine serum albumin and ovalbumin solutions, and respectively collecting supernate containing 2-MIB modifier-BSA and 2-MIB modifier-OVA.
5. The method of claim 4, wherein the amount of bovine serum albumin or ovalbumin is 50 mg, and the reaction is carried out at 4 ℃ for 24 hours under stirring.
6. The method for preparing the monoclonal antibody against dimethyl isotrichum as claimed in claim 1, wherein the step 3 comprises:
1) immunization: 2-MIB modifier-BSA immune mice, and 2-MIB modifier-OVA as the coating antigen for indirect competitive ELISA detection of antiserum titer;
2) fusing and screening: fusing a mouse spleen cell and a myeloma cell under the action of polyethylene glycol to obtain a positive hybridoma cell, and subcloning the positive hybridoma cell for 2-3 times by using a limiting dilution method;
3) mass production of monoclonal antibodies: the mouse is inoculated with hybridoma cell to produce monoclonal antibody of dimethyl isotrichum.
7. The method of claim 6, wherein the 1) immunization comprises: when in the first immunization, a BALB/C mouse is immunized by the 2-MIB modifier-BSA; the second immunization after two weeks, Freund's incomplete adjuvant is adopted; after two weeks, the third immunization is carried out, and the titer of the antiserum is detected by indirect competitive ELISA using 2-MIB modifier-OVA as the coating antigen; the fourth immunization was performed two weeks after the blood draw.
8. The method of claim 6, wherein the 2) fusion process comprises: mixing splenocytes and myeloma cells according to the proportion of 5: 1-10: 1, fusing under the action of polyethylene glycol, and suspending hybridoma cells by HAT culture solution.
9. The method of claim 6, wherein the 2) screening step comprises: and (3) screening by using an indirect competitive ELISA method, and subcloning the cells with definite color development in the positive hole for 2-3 times by using a limiting dilution method.
10. The method of claim 6, wherein the 3) monoclonal antibody is produced in large quantities by: injecting Freund's incomplete adjuvant to sensitize BALB/C mouse, inoculating hybridoma cell in abdominal cavity 7-10 days later, collecting ascites before death of mouse, centrifuging, and collecting clarified ascites to obtain monoclonal antibody.
11. The ELISA method for analyzing the content of 2-MIB in water is characterized in that the ELISA method for analyzing the content of 2-MIB in water is established by using the monoclonal antibody of dimethyl isotrichum prepared in the claim 1 to perform performance characterization.
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| CN113501877A (en) * | 2021-08-23 | 2021-10-15 | 苏州科铭生物技术有限公司 | Preparation method of anti-5-fluorouracil monoclonal antibody and enzyme-linked immunoassay method of 5-fluorouracil |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH08242883A (en) * | 1995-03-10 | 1996-09-24 | Ebara Corp | Monoclonal antibody against 2-mthylisoborneol, its production and measurement of 2-methylisoborneol with the same |
| US6444433B1 (en) * | 2000-01-24 | 2002-09-03 | Board Of Supervisors Of Louisiana State University And Agriculture And Mechanical College | Detection of 2-methylisoborneol by monoclonal antibody production |
| JP2003270246A (en) * | 2002-03-20 | 2003-09-25 | Takashi Kusui | Method for measuring 2-methylisoborneol |
| CN109655517A (en) * | 2019-01-29 | 2019-04-19 | 杭州电子科技大学 | The detection mode of the different borneol of 2- methyl in a kind of aquatic products |
-
2020
- 2020-09-28 CN CN202011037704.8A patent/CN112358549A/en not_active Withdrawn
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH08242883A (en) * | 1995-03-10 | 1996-09-24 | Ebara Corp | Monoclonal antibody against 2-mthylisoborneol, its production and measurement of 2-methylisoborneol with the same |
| US6444433B1 (en) * | 2000-01-24 | 2002-09-03 | Board Of Supervisors Of Louisiana State University And Agriculture And Mechanical College | Detection of 2-methylisoborneol by monoclonal antibody production |
| JP2003270246A (en) * | 2002-03-20 | 2003-09-25 | Takashi Kusui | Method for measuring 2-methylisoborneol |
| CN109655517A (en) * | 2019-01-29 | 2019-04-19 | 杭州电子科技大学 | The detection mode of the different borneol of 2- methyl in a kind of aquatic products |
Non-Patent Citations (2)
| Title |
|---|
| LESLIE C. PLHAK等: "High-Affinity Monoclonal Antibodies for Detection of the Microbial Metabolite, 2-Methylisoborneol", 《JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY》, vol. 51, no. 13, pages 3731 * |
| 刘畅·: "氯霉素和二甲基异茨醇单克隆抗体的制备及酶联免疫吸附分析方法的建立", 《中国优秀硕士学位论文全文数据库工程科技Ⅰ辑》, no. 4 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113501877A (en) * | 2021-08-23 | 2021-10-15 | 苏州科铭生物技术有限公司 | Preparation method of anti-5-fluorouracil monoclonal antibody and enzyme-linked immunoassay method of 5-fluorouracil |
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