CN113968804B - Hapten and artificial antigen of monel sulfone as well as preparation methods and application of hapten and artificial antigen - Google Patents
Hapten and artificial antigen of monel sulfone as well as preparation methods and application of hapten and artificial antigen Download PDFInfo
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Abstract
The invention discloses a hapten and an artificial antigen of monel sulfone as well as preparation methods and application thereof. The structural formula of the monel sulfone hapten is shown as a formula I, and the monel sulfone hapten can be prepared by the following method: oxidizing p-trifluoro-methylthio benzoic acid in glacial acetic acid by using oxyhydrogen oxide to obtain the p-trifluoro-methylthio benzoic acid. The invention provides a monel sulfone hapten and an artificial antigen, and lays a foundation for preparing a high-sensitivity anti-monel sulfone antibody; finally, the antibody with the optimal property is obtained by screening, and the maximum titer can reach 4 multiplied by 10 4 The sensitivity was 2.6. Mu.g/L. The synthesis method of the monel sulfone hapten and the artificial antigen provided by the invention is simple and efficient, the price of the required chemical reagent is low, the sensitivity of the obtained antibody is high, and the monel sulfone hapten and the artificial antigen can be completely used in immunoassay and have important significance in veterinary drug residue detection.
Description
Technical Field
The invention relates to a hapten and an artificial antigen of monel sulfone as well as a preparation method and application thereof, belonging to the field of food safety detection.
Background
Monental (Monepantel) is a newly synthesized aminoacetonitrile derivative class of anti-insect agents, has high activity for the treatment and control of adult-stage nematodes in the gastrointestinal tract of sheep, including haemonchus contortus, aphanizomenon cincticum, trichostrongylus serpentine, trichostrongylus delbrunei and Cupressus brevicaulis, and is effective against a broad spectrum of commercially available anti-parasitic drug-resistant strains. Monelter has a rapid, highly effective and penetrating neuromuscular effect, and can effectively kill nematodes resistant to other classes of drugs by causing excessive contraction of body wall muscles leading to parapharyngeal paralysis, spastic contraction and eventual death. European union 394/2013 states that the marker residue of monetitol is monetitol sulfone with a maximum residual limit in goat milk of 0.17mg/kg.
The currently reported determination method for monel sulfone is mainly a liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method, and other methods are relatively lacking. Although the instrumental analysis method has high sensitivity and specificity, the method needs a complex pretreatment process and professional operators, and the instrument is expensive and is not suitable for rapid screening of a large number of samples. The immunoassay method has the advantages of strong specificity, high sensitivity, low cost, simple and convenient operation, suitability for simultaneous detection of a large number of samples and the like on the basis of antigen-antibody specificity identification, and makes up for the defects of instrument detection. Therefore, it is necessary to establish an immunological detection method.
The molecular design of haptens and antigen synthesis are key steps to obtain highly specific and avidity antibodies. According to the active group of the hapten, a corresponding biological coupling method is selected, the hapten and the carrier protein are coupled to obtain an artificial antigen, and an immune animal obtains an antibody, so that a foundation is laid for establishing an immunoassay method of the monel sulfone.
Disclosure of Invention
The invention aims to provide a monel sulfone hapten and an artificial antigen, and the prepared product is used for researching a monel sulfone immunoassay method and provides a necessary artificial antigen for future research.
The structural formula of the monel sulfone hapten provided by the invention is shown as a formula I:
the monel sulfone hapten can be prepared according to the following preparation method:
oxidizing p-trifluoro-methylthio benzoic acid in glacial acetic acid by using oxyhydrogen oxide to obtain the p-trifluoro-methylthio benzoic acid.
Specifically, the ratio of the p-trifluoromethylthiobenzoic acid, the glacial acetic acid and the hydrogen peroxide is 100mg: 10-20 mL: 20-30 mL;
the reaction temperature is 20-25 ℃, and the reaction time is 20-24 h;
and after the reaction is finished, diluting with pure water, extracting with dichloromethane, washing the extract liquor with pure water after spin-drying, and performing suction filtration to obtain a white solid, namely the monellin hapten.
The invention also provides a monel sulfone artificial antigen which is obtained by coupling the monel sulfone hapten with carrier protein;
the carrier protein is selected from any one of bovine serum albumin, ovalbumin and keyhole limpet hemocyanin.
The coupling molar ratio of the monel sulfone hapten to the carrier protein is 5-30: 1, preferably 8.8:1;
the monel sulfone artificial antigen may be used as immunogen or coating antigen.
The carrier protein can be coupled on the carboxyl carbon of the hapten by adopting an active ester method to obtain the monel sulfone artificial antigen, and the specific steps are as follows: dissolving the monel sulfone hapten, N-hydroxysuccinimide (NHS), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) in N, N-Dimethylformamide (DMF), and stirring at room temperature overnight to obtain solution A; dissolving the carrier protein in a carbonate buffer solution to obtain a solution B; dropwise adding the solution A into the solution B, and stirring at room temperature for reaction overnight to obtain a mixed solution of the conjugate; the mixture was dialyzed in PBS for three days to obtain the monel sulfone artificial antigen.
Said monel sulfone hapten or said monel sulfone artificial antigen having the following applications:
1) Preparing an anti-monellin antibody;
2) The food product was tested for the residue of monel sulfone.
The invention also provides a monoclonal antibody of the monel sulfone, which is obtained by immunizing experimental animals (female BALB/c mice) with the monel sulfone artificial antigen through cell fusion, screening and in vitro induction.
The monoclonal antibody of the monel sulfone can be used for preparing a monel sulfone detection reagent or a kit.
The monoclonal antibody to monellan sulfone has the following applications:
1) Immunodetection of monel sulfone;
2) Preparing a monel sulfone immunochromatographic test strip;
3) And (3) preparing a monel sulfone colloidal gold test strip.
The invention provides a monel sulfone hapten and an artificial antigen, and lays a foundation for preparing a high-sensitivity anti-monel sulfone antibody; finally, screening to obtain antibody with optimal property, and the maximum titer can reach 4 x 10 4 The sensitivity was 2.6. Mu.g/L. The synthesis method of the monel sulfone hapten and the artificial antigen provided by the invention is simple and efficient, the price of the required chemical reagent is low, the sensitivity of the obtained antibody is high, and the monel sulfone hapten and the artificial antigen can be completely used in immunoassay and have important significance in veterinary drug residue detection.
Drawings
FIG. 1 is a mass spectrometric identification of a monel sulfone hapten.
FIG. 2 is a MALDI-TOF identification chart of BSA.
FIG. 3 is a MALDI-TOF identification chart of a monel sulfone artificial antigen.
FIG. 4 is a standard graph of the detection of monetoffee using a monoclonal antibody.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 preparation and characterization of monellan sulfone haptens
100mg of p-trifluoromethylthiobenzoic acid was weighed out and dissolved in 10mL of glacial acetic acid, and 20mL of 37% hydrogen peroxide was added dropwise with stirring and reacted at room temperature of 25 ℃ for 24 hours. Diluting the reaction solution with pure water, extracting with dichloromethane, spin-drying the extract, washing with pure water, and vacuum-filtering to obtain 86mg of white solid, which is the monel sulfone hapten, and identifying by mass spectrometry.
The reaction equation is as follows:
the mass spectrum identification result is shown in fig. 1, the molecular weight of 4- ((trifluoromethyl) sulfonyl) benzoic acid is 254.18, the obtained parent ion fragment in the negative ion mode is m/z =253.18, and the characteristic parent ion fragment with m/z =253.0 exists in a mass spectrum, which indicates that the monellin-teral sulfone hapten is successfully synthesized.
Example 2 preparation and characterization of Moneratel sulfone Artificial antigen
The preparation method of the immunogen and the coating antigen is characterized in that the type of the carrier protein is used, the carrier protein of the immunogen mainly adopts KLH, the carrier protein of the coating antigen mainly adopts BSA and OVA, and the coupling method is an active ester method.
Weighing 20mg of prepared monel sulfone hapten (MOPS), 26.2mg of N-hydroxysuccinimide (NHS) and 43.6mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), dissolving in 1.5mL of N, N-Dimethylformamide (DMF), and stirring at room temperature for 6 hours to obtain solution A; dissolving 50mg BSA (50 mg OVA or 20mg KLH) with 0.05M CB to obtain solution B; slowly dropping the solution A into the solution B drop by drop, stirring at room temperature for overnight reaction to obtain a mixed solution of the conjugate; the mixture was dialyzed in 0.01M PBS for three days to obtain the monel sulfone artificial antigen.
The binding ratio of BSA and hapten is determined by Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass spectrometry (MALDI-TOF-MS). The results are shown in FIGS. 2 and 3.
Binding ratio = { M (conjugate) -M (protein) }/M (hapten).
The molecular weight of BSA is 64771.06, the molecular weight of hapten as shown in formula I is 254.18, the molecular weight of the coupled substance of the highest peak value analysis of mass spectrum is 68674.30, and the binding ratio of BSA to hapten is 8.8 by calculation: 1, i.e. monel sulfone artificial antigen, 8.8 haptens are coupled on average to one BSA molecule.
Example 3 preparation of monoclonal antibodies to monelitaer sulfone
The artificial antigen MOPS-KLH prepared in example 2 was used to immunize 6 BALB/c female mice aged 6 to 8 weeks. Each immunogen was diluted to 1mg/mL with 0.01M PBS and emulsified with an equal amount of Freund's adjuvant to form a water-in-oil structure. Except for the first immunization, freund complete adjuvant is adopted, the other booster immunization is uniformly adopted, the immunization dose is 100 mu g/mouse, the injection dose is 0.2 mL/mouse, and the neck and the back of the mouse are inoculated at multiple points. Booster immunizations were performed 4 weeks later and were given 1 time every 3 weeks for 2 total immunizations, followed by multiple subcutaneous injections at the back and neck. And then performing booster immunization every three weeks, wherein the Freund complete adjuvant is replaced by the Freund incomplete adjuvant during booster immunization, the immunization dose is the same as the first immunization dose, and the booster immunization times are 2 times. After 7-10 days of two times of enhanced immunization, collecting orbital blood of each mouse, centrifuging, collecting supernatant, measuring, selecting mice with good antiserum titer and inhibition, collecting splenocyte, fusing with myeloma cell, performing three times of single cell colony subcloning, performing strain determination and amplification culture, obtaining antibody by adopting an in vivo induction mode, purifying by using a Protein A immunoaffinity column, and finally storing at-20 ℃ for later use.
Example 4 determination of the Monellar sulfone antisera
1. Detection of antiserum titer by indirect ELISA method
The method comprises the following specific steps:
(1) Coating: the coating antigen was diluted to 0.1. Mu.g/mL with 0.05M carbonate buffer and added to a 96-well clear plate (100. Mu.L/well), incubated overnight at 4 ℃ for 16 hours, and the plate was washed 3 times with PBST buffer.
(2) And (3) sealing: add blocking solution 2% skim milk (150. Mu.L/well), incubate at 37 ℃ for 1.5 hours, discard blocking solution, wash 1 time with PBST buffer, and pat dry.
(3) Adding the antibody to be detected: to each well 50 μ L of 0.01M PBS was added, followed by 50 μ L of diluted antiserum from 1: starting at 5000, dilutions were started with 2 gradients in 0.01M PBS for a total of 4 gradients. The reaction was incubated at 37 ℃ for 30 minutes, washed 3 times with PBST buffer and patted dry. At the same time, the non-immunized mouse antiserum was set as a negative control.
(4) Adding an enzyme-labeled secondary antibody: adding an HRP-labeled goat anti-mouse IGg antibody diluted by an enzyme-labeled secondary antibody diluent according to the volume ratio of 1.
(5) Color development: horseradish peroxidase substrate 3,3',5,5' -tetramethylbenzidine solution and 30% by mass of hydrogen peroxide were mixed in a volume ratio of 1:1, added to microwells (100. Mu.L/well), and developed in an incubator at 37 ℃ for 15 minutes.
(6) And (4) terminating: 2M concentrated sulfuric acid (50. Mu.L/well) was added.
(7) Reading: by OD 450nm And measuring the OD value of the well by wavelength, and taking the corresponding dilution of the antibody with the negative OD value not more than 0.15 and the maximum OD value between 1.5 and 2.0 as the antibody titer. The measured titer of the immunized mice is 0.5 multiplied by 10 4 Above, at most 4 × 10 4 . The result shows that the mouse is immunized by the synthetic hapten coupling carrier protein, and the immunization effect can be better obtained.
2. Minimum detection limit, half-maximal inhibition, and detection of specificity
The specific operation steps are as follows:
(1) The indirect ELISA method is used for determining MOPS-OVA as the coating antigen, MOPS-KLH immune mice are used for cell fusion to produce antibody, and OD is used 450 The value is about 1.5The corresponding antigen and antibody concentrations are the optimal working concentrations.
(2) Coating: the coating antigen was diluted 80000 times with coating buffer, 100. Mu.L/well, and incubated in a 37 ℃ incubator for 2h.
(3) Washing and sealing: the procedure was the same as for the indirect ELISA method described above.
(4) Preparing a standard solution: the monel sulfone standard was prepared into a stock solution of 2mg/mL using acetonitrile solution, and then diluted to a gradient of 0, 0.1, 0.3, 0.9, 2.7, 8.1, 24.3, 72.9. Mu.g/L monel sulfone standard solution by a double ratio using 0.01mol/L PBS solution of pH7.4 before loading.
(5) Sample adding: each well was added with 50. Mu.L of each concentration standard diluted at a double rate, and then 50. Mu.L of the antibody at the optimum dilution rate per well was added thereto, and the reaction was carried out at 37 ℃ for 30min. After thorough washing, 1: HRP-goat anti-mouse IgG at 5000 dilution, 100. Mu.L/well, reacted at 37 ℃ for 30min.
(6) And (3) color development reaction: the ELISA plate was removed, washed thoroughly, and 100. Mu.L of TMB developing solution was added to each well, and the reaction was carried out at 37 ℃ in the dark for 15min.
7) 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.
8) Data processing: the OD corresponding to each concentration is determined by taking the concentration of each standard as the abscissa 450nm Values are plotted as ordinate against a four parameter log fit using Origin software, as shown in FIG. 4, by calculating IC 50 The value (half maximal inhibitory concentration) determines whether the antibody has recognition for monetoffee.
The results showed that the detection sensitivity of the obtained monoclonal antibody to monetoffee was 2.6. Mu.g/L.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (6)
1. A Moneratel sulfone artificial antigen is obtained by coupling a Moneratel sulfone hapten shown in formula I with a carrier protein;
the carrier protein is selected from any one of bovine serum albumin, ovalbumin and keyhole limpet hemocyanin;
formula I.
2. A method for preparing the monel sulfone artificial antigen as set forth in claim 1, comprising the steps of:
coupling the carrier protein to carboxyl carbon of the monel sulfone hapten shown in the formula I by adopting an active ester method;
formula I.
3. Use of a monel sulfone hapten of formula I or of an artificial monel sulfone antigen according to claim 1) in 1) or 2) below:
1) Preparing an anti-monel sulfone antibody;
2) Detecting the residue of monetitol sulfone in the food product;
formula I.
4. A monoclonal antibody to monel sulfone prepared from the monel sulfone artificial antigen of claim 1.
5. A monel sulfone detection reagent or kit prepared from a monoclonal antibody to monel sulfone of claim 4.
6. Use of a monoclonal antibody against monel sulfone according to claim 4 in any one of the following 1) to 2):
1) Preparing an immunochromatographic test strip of the monel sulfone;
2) And (3) preparing a monel sulfone colloidal gold test strip.
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| WO2010005783A1 (en) * | 2008-07-08 | 2010-01-14 | Boehringer Ingelheim International Gmbh | Pyrrolidinyl and piperidinyl compounds useful as nhe-1 inhibitors |
| CN102083822A (en) * | 2008-04-11 | 2011-06-01 | 遗传技术研究公司 | Pyridyl inhibitors of HEDGEHOG signalling |
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| CN102083822A (en) * | 2008-04-11 | 2011-06-01 | 遗传技术研究公司 | Pyridyl inhibitors of HEDGEHOG signalling |
| WO2010005783A1 (en) * | 2008-07-08 | 2010-01-14 | Boehringer Ingelheim International Gmbh | Pyrrolidinyl and piperidinyl compounds useful as nhe-1 inhibitors |
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| Title |
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| 兽用抗寄生虫药莫奈太尔的研究进展;梁先明 等;《中国兽药杂志》;20140820;第61-65页 * |
| 高效液相色谱-串联质谱法同时测定乳制品中莫奈太尔和莫奈太尔砜;吴映璇 等;《理化检验(化学分册)》;20181231;第883-887页 * |
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