CN113372295A - Phenothiazine hapten, complete antigen, preparation method and application thereof - Google Patents
Phenothiazine hapten, complete antigen, preparation method and application thereof Download PDFInfo
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- CN113372295A CN113372295A CN202110773774.8A CN202110773774A CN113372295A CN 113372295 A CN113372295 A CN 113372295A CN 202110773774 A CN202110773774 A CN 202110773774A CN 113372295 A CN113372295 A CN 113372295A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D279/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom and one sulfur atom as the only ring hetero atoms
- C07D279/10—1,4-Thiazines; Hydrogenated 1,4-thiazines
- C07D279/14—1,4-Thiazines; Hydrogenated 1,4-thiazines condensed with carbocyclic rings or ring systems
- C07D279/18—[b, e]-condensed with two six-membered rings
- C07D279/20—[b, e]-condensed with two six-membered rings with hydrogen atoms directly attached to the ring nitrogen atom
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
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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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
Abstract
The invention provides a phenothiazine hapten, a complete antigen, a preparation method and application thereof. The invention introduces carboxyl on phenothiazine molecules by a diazotization method, couples modified phenothiazine and carrier protein by a mixed anhydride method or a carbodiimide method, and prepares a complete antigen, wherein the structural formula of the complete antigen is shown as a formula II. The invention identifies the synthesized phenothiazine complete antigen and optimizes the identification condition of the high performance liquid chromatography. The phenothiazine complete antigen prepared by the invention has good immune effect, the antibody titer of antiserum obtained after animal immunization can reach 1:3000 through indirect enzyme-linked immunoassay, and the preparation method is simple, low in cost and suitable for large-scale popularization and application.
Description
Technical Field
The invention relates to the technical field of antigen preparation, in particular to a phenothiazine hapten, a complete antigen, a preparation method and application thereof.
Background
Phenothiazine drugs are low in elimination rate in a human body and easy to generate drug residues in the human body, and particularly, after phenothiazine tranquilizers remained in edible animals enter the human body through a food chain, the phenothiazine tranquilizers can have adverse effects on endocrine, movement, circulation and other systems of the human body and liver organs, so that toxic and side effects such as leucopenia, liver dysfunction, agranulocytosis, contact dermatitis, rash and the like are caused, and great risks are brought to the health of the human body. The common instrumental analysis method and the immunological detection method are less in application, but compared with the instrumental analysis method, the immunological detection method is lower in cost and short in detection time, and is suitable for field detection of large-batch samples. Complete antigens are crucial as the basis for immunological detection methods. In order to enrich the immunological detection method aiming at the phenothiazine residue in the food, the invention successfully prepares the phenothiazine complete antigen and lays the foundation for establishing the related immunological detection method later.
Due to the structural particularity of phenothiazine, the difficulty of synthesizing the antigen by the phenothiazine is increased, most of the existing methods aiming at the phenothiazine synthesized antigen are complex in operation, time-consuming and labor-consuming in the preparation process and full of dangerousness. In order to simplify the preparation process and reduce the risk, the invention researches and designs a synthesis route of the complete antigen synthesized by the phenothiazine and designs the identification method of the complete antigen of the phenothiazine so as to ensure the successful synthesis of the complete antigen.
Disclosure of Invention
The invention aims to provide a phenothiazine hapten, a complete antigen, a preparation method and application thereof, and provides the complete antigen for the immunological detection of phenothiazine.
The purpose of the invention is realized as follows: a phenothiazine hapten, the molecular structural formula of which is shown in formula I:
the preparation method of the phenothiazine hapten comprises the following steps:
(a) adjusting the pH value of the p-aminobenzoic acid solution to 1-3 by using an HCL solution at the temperature of 2-6 ℃, and stirring in a vortex mode until the solution is completely dissolved;
(b) mixing the obtained acidic p-aminobenzoic acid solution with sodium nitrite solution to obtain light yellow solution, and refrigerating at 2-6 deg.C for 20-40 min; wherein the molar ratio of the p-aminobenzoic acid to the sodium nitrite is 1: 2-3;
(c) adding phenothiazine into the solution obtained in the step (b), wherein the molar ratio of phenothiazine to p-aminobenzoic acid is 1:1, and then magnetically stirring for 10-14h in a dark room to obtain the phenothiazine hapten.
A phenothiazine complete antigen is prepared from the phenothiazine hapten and a carrier Protein, and the molecular structural formula of the phenothiazine complete antigen is shown as a formula II:
the preparation method of the phenothiazine complete antigen is prepared by a mixed anhydride method, and comprises the following steps:
(a) adding 1, 4-dioxane and N-N-dimethylformamide into a phenothiazine hapten solution in an ice bath environment, shaking and mixing for 1-3min, standing for 5-15min, adding triethylamine, standing for 5-15min, adding isobutyl chloroformate, removing the ice bath, and magnetically stirring for 20-40 min; wherein, phenothiazine hapten is 1, 4-dioxane, N-N-dimethylformamide, triethylamine, isobutyl chloroformate which is 0.1mmoL, 1-2mL, 0.1-0.3nmoL, 0.1-0.2 nmoL;
(b) mixing a carrier protein-sodium borate solution with the reaction solution obtained in the step (a), wherein the carrier protein-sodium borate: and (2) reacting the phenothiazine hapten with 0.1mmoL at the temperature of 2-6 ℃ for 10-14h, filling the reaction solution into a dialysis bag, dialyzing the reaction solution in PBS buffer solution for 60-80h, changing dialysate every 4-8h, and obtaining the phenothiazine complete antigen after the dialysis is finished.
The preparation method of the phenothiazine complete antigen is prepared by a carbodiimide method and comprises the following steps:
(a) adding N, N-dimethylformamide into a phenothiazine hapten solution, then stirring and adding dicyclohexylcarbodiimide and N-hydroxysuccinimide, magnetically stirring overnight, and then centrifuging to obtain a supernatant which is recorded as liquid A; wherein, phenothiazine hapten is N, N-dimethylformamide is dicyclohexylcarbodiimide and N-hydroxysuccinimide is 0.1mmoL, 1-2nmoL, 0.1-0.2mmoL and 0.1-0.2 mmoL;
(b) weighing carrier protein, dissolving the carrier protein in PBS buffer solution, and recording the solution as B solution, wherein the carrier protein-sodium borate: phenothiazine hapten is 12 mg: 0.1 mmoL;
(c) adding the solution A into the solution B, and magnetically stirring for 10-14 h; and then centrifuging, taking supernatant, filling the supernatant into a dialysis bag, dialyzing in a PBS buffer solution for 60-80h, changing dialysate every 4-8h, and obtaining the phenothiazine complete antigen after dialysis.
The carrier protein is bovine serum albumin, and the PBS buffer solution is 0.01mol/L and has a pH value of 7.4.
The identification method of the phenothiazine complete antigen is characterized in that high performance liquid chromatography is adopted for identification, wherein a mobile phase is methanol-water, and a sample solvent is water.
The application of the phenothiazine complete antigen in preparing a polyclonal antibody of a phenothiazine drug.
The phenothiazine complete antigen prepared by the invention has good immune effect, the antibody titer of antiserum obtained after animal immunization can reach 1:3000 through indirect enzyme-linked immunoassay, and the preparation method is simple, low in cost and suitable for large-scale popularization and application.
Drawings
Fig. 1 is a standard graph.
FIG. 2 is a UV spectrum of complete antigen 1.
FIG. 3 is a complete antigen 2 UV spectrum.
FIG. 4 shows the results of complete antigen 1 gel electrophoresis.
FIG. 5 shows the results of complete antigen 2 gel electrophoresis.
Fig. 6 is a BSA chromatogram determined for different mobile phases.
FIG. 7 is a complete antigen 1 chromatogram determined for different mobile phases.
FIG. 8 is a complete antigen 2 chromatogram determined for different mobile phases.
Fig. 9 is a BSA chromatogram for different solvents.
FIG. 10 is a chromatogram of complete antigen 1 from different solvents.
FIG. 11 is a chromatogram of complete antigen 2 from different solvents.
FIG. 12 is a comparison chromatogram of complete antigen 1 and BSA.
Figure 13 is a comparison chromatogram of complete antigen 2 and BSA.
FIG. 14 is a graph showing the results of three immunization.
FIG. 15 is a graph showing the results of titer determination.
Detailed Description
The technical solution of the present invention will be described in detail with reference to specific examples. The test conditions and procedures not mentioned in the examples of the present invention were carried out according to the conventional methods in the art or the conditions suggested by the manufacturer.
Example 1: hapten modification
Molecular modification is carried out on phenothiazine by a diazotization method, and carboxyl is introduced. The method comprises the following steps:
the method comprises the following steps: adjusting the pH value of the p-aminobenzoic acid solution to 2 by using 1.2moL/L HCL solution at the temperature of 4 ℃, and stirring by vortexing until the solution is completely dissolved.
Step two: 1mL of acidic p-aminobenzoic acid solution was added to 100. mu.L of 0.01moL/L sodium nitrite solution and mixed, the solution turned to pale yellow, and refrigerated at 4 ℃ for 30 min.
Step three: 0.1mmoL phenothiazine was added to the diazotized solution and stirred magnetically for 12h in a dark room.
At this point, the hapten molecule is modified and can synthesize a complete antigen with the carrier protein. The hapten modification route is as follows:
example 2 Synthesis of complete antigen by Mixed anhydride method
The reaction principle is that the carboxyl at the end of hapten molecule and the amino on the carrier protein structure are connected in the form of peptide bond through dehydration condensation reaction. The specific operation steps are as follows:
the method comprises the following steps: under an ice bath environment, 1mL of 1, 4-dioxane and N, N-dimethylformamide are added into the modified hapten respectively, vortex oscillation is carried out for 2min, 26.5 mu L of triethylamine is added after 10min, 16 mu L of isobutyl chloroformate is added after 10min, at the moment, the ice bath is removed, and magnetic stirring is carried out for 30 min.
Step two: mixing 2mL of 0.6mg/mL bovine serum albumin-sodium borate solution with the above solution, reacting at 4 deg.C for 12h, placing into dialysis bag, dialyzing in 0.01M Phosphate Buffer Solution (PBS) for 72h, and changing dialysate every 6 h. After dialysis, the mixture was stored at-20 ℃. This is complete antigen 1, and the synthetic route of complete antigen 1 is shown below.
EXAMPLE 3 Synthesis of complete antigen by carbodiimide method
The method comprises the following steps: adding 200 mu L N N-dimethylformamide into the modified hapten, stirring and adding 20.633mg dicyclohexylcarbodiimide and 11.509mg N-hydroxysuccinimide, magnetically stirring overnight, centrifuging at 10000r/min for 10min, and taking the supernatant as solution A.
Step two: accurately weighed 12mg of BSA was dissolved in 5mL of PBS and recorded as solution B.
Step three: adding the solution A into the solution B, and magnetically stirring for 12 h. Centrifuging at 10000r/min for 10min, collecting supernatant, packaging into dialysis bag, dialyzing in PBS for 72h, and changing dialysate every 6 h. After dialysis, the mixture was stored at-20 ℃. This is complete antigen 2, the synthetic route is shown below:
example 4 identification of complete antigens
1. Coomassie brilliant blue method for determining complete antigen concentration
The method comprises the following steps: a1 mg/mL bovine serum albumin solution was prepared.
Step two: six test tubes were taken and added to the solutions of table 1 below. Respectively adding 4mL of Coomassie brilliant blue solution, mixing uniformly, reacting for 5min at room temperature, and measuring absorbance at 595nm by using a spectrophotometer.
TABLE 1 test tube solution composition
| Serial number | Bovine serum albumin solution/mL | water/ |
| 1 | 0.0 | 1.0 |
| 2 | 0.2 | 0.8 |
| 3 | 0.4 | 0.6 |
| 4 | 0.6 | 0.4 |
| 5 | 0.8 | 0.2 |
| 6 | 1.0 | 0.0 |
Step three: the protein concentration was plotted as the abscissa and the absorbance as the ordinate to prepare a standard curve.
Step four: complete antigen 1 and 2 were diluted 10-fold, and 1.0mL of the diluted complete antigen was dissolved in test tubes 7 and 8, respectively, and absorbance was measured in the same manner and substituted into a standard curve to calculate the complete antigen concentration. The absorbance at 595nm of solutions of six different protein concentrations was measured spectrophotometrically and the results were as follows:
TABLE 2 Absorbance obtained for different concentrations of protein
And (4) according to the obtained data, taking the protein concentration as an abscissa and the absorbance as an ordinate, making a standard curve, and calculating a standard equation. As shown in fig. 1. The regression equation is calculated to be y-0.0563 x +2.8882, and the correlation coefficient is 0.9753.
The complete antigens 1 and 2 were diluted 10-fold, the absorbance was measured, and the protein concentration of the complete antigen was calculated according to the equation.
TABLE 3 Absorbance of complete antigen
The absorbance of complete antigen 1 was measured to be 2.890A, and according to the standard equation, X was calculated to be 0.1421mg/mL, and the concentration of complete antigen 1 was 1.421 mg/mL. Complete antigen 2 had an absorbance of 2.889A, and the concentration in complete antigen 2 was 1.25mg/mL, calculated according to the standard equation to give X of 0.125 mg/mL.
2. Ultraviolet spectrophotometry method for identification
And (3) measuring the maximum absorption peak of the synthesized complete antigen by an ultraviolet spectrophotometer, comparing the maximum absorption peak with the maximum absorption peaks of the carrier protein BSA and the hapten phenothiazine, and observing whether the difference exists so as to evaluate whether the complete antigen is synthesized successfully.
The method comprises the following steps: preparing 1mg/mL bovine serum albumin solution by using ultrapure water as a solvent; preparing 0.3mg/mL phenothiazine solution by using methanol as a solvent; the complete antigen was diluted with ultrapure water to a concentration consistent with that of bovine serum albumin solution.
Step two: the bovine serum albumin solution and the complete antigen solution were scanned sequentially at 200-400nm using ultrapure water baseline calibration. The phenothiazine solution was scanned at 200-400nm using methanol for baseline correction. The results are shown in FIGS. 2 and 3
The bovine serum albumin is prepared into 1mg/mL by ultrapure water for measurement, and the result shows that the characteristic absorption peak of the bovine serum albumin is between 278 and 280 nm. A phenothiazine solution having a concentration of 0.3mg/mL was prepared using methanol as a solvent, and the phenothiazine solution was diluted several times to a final concentration of 0.003mg/mL after measurement of disordered absorption peaks, showing that there were two characteristic absorption peaks of phenothiazine at 253nm and 318nm, respectively, and that the maximum absorption peak at 318 nm. Complete antigen 1 was pipetted at 300. mu.L and water was added to 5mL, indicating characteristic absorption peaks at 247nm and 334nm, and a maximum absorption peak at 334 nm. The complete antigen 2 absorbs 300 mu L of water to 5mL, the absorption peak is not obvious when the detection is carried out, and the absorption peak is not obvious when the detection is carried out by absorbing 600 mu L of water to 5mL, and the result shows that the complete antigen 2 only has one characteristic absorption peak at 251 nm. In FIG. 2, the maximum absorption peaks of complete antigen 1 relative to phenothiazine and BSA are obviously different, which can preliminarily prove the success of complete antigen 1 coupling. In FIG. 3, the maximum absorption peaks of complete antigen 2 relative to phenothiazine and BSA are distinct, which can preliminarily prove the success of complete antigen 2 coupling.
3. SDS-PAGE electrophoretic identification
The molecular weight of protein substances is different, so that the mobility of the protein substances is different under the electrophoresis condition, and the SDS-PAGE electrophoresis method utilizes concentrated gel to compress all protein molecules on the same horizontal line and utilizes separation gel to separate the protein molecules. The specific experimental process is as follows:
the method comprises the following steps: 10mL of separation gel with the concentration of 10% is prepared, 4.1mL of ultrapure water, 2.5mL of 4x separation gel buffer solution, 3.3mL of 30% acrylamide, 100 muL of 10% ammonium persulfate and 5 muL of tetramethylbutanediamine are respectively added, the separation gel is added into a gel plate of an electrophoresis tank and added to 3/4 parts of the volume of the gel plate, the gel is sealed by the ultrapure water after the addition is finished, the mixture is kept stand for 30min, and after a clear interface appears between the separation gel and a water layer, the gel is shown to be polymerized.
Step two: 4mL of 5% concentrated gel is prepared, and 2.28mL of over-water, 1mL of 4 Xconcentrated gel buffer, 0.68mL of 30% acrylamide, 40 μ L of 10% ammonium persulfate and 4 μ L of tetramethylbutanediamine are added respectively. Adding the concentrated gel into the gel plate to avoid generating bubbles until the gel reaches the top end of the glass plate, inserting the comb, standing for 10-20min, and carefully pulling out the comb after the gel is polymerized to avoid damaging the sample adding hole.
Step three: proteins are denatured and their concentration is matched to the carrier protein concentration based on the measured complete antigen concentration. And uniformly mixing the sample loading buffer solution and the sample according to the volume ratio of 1:4, and then placing the mixture in a boiling water bath for heating for 3-5 min. Cooling to room temperature, centrifuging at 2000r/min in a centrifuge for 30s, and collecting supernatant.
Step four: adding the supernatant into a gel sample adding hole, adding 10 mu L/hole of carrier protein and complete antigen, adding 5 mu L/hole of protein Marker, adding 5 times diluted electrophoresis buffer solution, and starting electrophoresis. The voltage of the concentrated gel is 80v, after 30min, the band runs to the separation gel, the voltage electrophoresis is changed to 120v, and the process is finished for 1 h.
Step five: and cutting the separation gel after electrophoresis, placing the separation gel in Coomassie brilliant blue quick staining solution for staining for 15min, placing the separation gel in a destaining solution for destaining until the separation gel is transparent, and taking a picture in a gel imaging system for storage.
The size of the molecular weight of the protein determines the migration size of the protein under the electrophoresis condition, and the larger the molecular weight is, the smaller the migration speed is. The complete antigen is prepared by coupling phenothiazine micromolecule on carrier protein, so the molecular weight of the complete antigen is larger than that of the carrier protein, and theoretically, the band of the complete antigen is slower than that of the carrier protein or has obvious tailing phenomenon compared with the carrier protein. As shown in fig. 4 and 5. In FIG. 4, the left band is the result of BSA, and the right band is the result of complete antigen 1. The result shows that the synthesized complete antigen 1 has an obvious tailing phenomenon compared with BSA, the migration speed of the conjugate protein is less than that of BSA, the molecular weight of the conjugate protein is larger than that of BSA, and further, the successful coupling of the BSA and the hapten phenothiazine is shown. The left band in FIG. 5 is the result for BSA, and the right band is the result for complete antigen 2. The result shows that the synthesized complete antigen 2 has an obvious tailing phenomenon compared with BSA (bovine serum albumin), the migration speed of the conjugate protein is less than that of the BSA, the molecular weight of the conjugate protein is larger than that of the BSA, and further the complete antigen 2 is successfully synthesized.
4. High performance liquid chromatography
Chromatographic conditions are as follows: column temperature: 30 ℃; detection wavelength: 230 nm; flow rate: 1.0 mL/min.
(1) Selection of mobile phase
Mobile phase: methanol A, water B or phosphate buffer solution; gradient elution: 0-6min, wherein the amount of A is 55-80%; 6-15min, the amount of A is 80%; 15-16min, the amount of A is 80-90%; 16-21min, the amount of A is 90%; 21-24min, the amount of A is 90-95%; 24-35min, the amount of A is 55%.
0.3mg/mL bovine serum albumin solution and complete antigen solution were prepared using water as the solvent. Methanol-water and methanol-phosphate buffer are selected as mobile phases respectively, and the best mobile phase is selected by observing a chromatogram.
In FIG. 6, the upper and lower panels are peak diagrams of BSA measured with methanol-phosphate buffer and methanol-water as mobile phases, respectively, and the two panels are identical except for the mobile phase. It is obvious that the peak time measured with methanol-water as mobile phase is different from that measured with methanol-buffer as mobile phase, the lower graph shows 24.147min, and the upper graph is not obvious. The lower graph shows a better peak shape, the upper graph shows a random peak shape, and it is assumed that the buffer contains too much impurities, and thus a corresponding absorption peak cannot be obtained.
In FIG. 7, the upper and lower panels are the peak diagrams of complete antigen 1 measured with methanol-phosphate buffer solution and methanol-water as mobile phases, respectively, the absorption peak time measured in the lower panel is 25.106min, but no obvious absorption peak or even no peak appears in the upper panel.
In FIG. 8, the upper and lower panels are the peak diagrams of complete antigen 2 measured with methanol-phosphate buffer and methanol-water as mobile phases, respectively. The lower graph shows that the absorption peak time is 32.166min, but the upper graph shows no obvious absorption peak and the peak is disordered.
It is obvious from comparison that the three substances are more suitable to use methanol-water as the mobile phase, so the choice of using methanol-buffer solution as the mobile phase is abandoned.
(2) Selection of solvents
Considering that methanol denatures proteins, water and phosphate buffer were used as solvents to prepare stock solutions.
In FIG. 9, the lower panel shows water as a solvent and the upper panel shows a buffer as a solvent, and it can be seen that buffer as a solvent BSA did not have a significant absorption peak, but water as a solvent BSA had an absorption peak, and therefore, it is assumed that buffer had an influence on the measurement of BSA.
In FIG. 10, the lower panel shows water as the solvent and the upper panel shows the buffer as the solvent, and it can be seen that the complete antigen 1 was randomly detected by using the buffer as the solvent.
In FIG. 11, the lower panel shows water as the solvent and the upper panel shows buffer as the solvent, and it can be seen that two absorption peaks were detected by using buffer as the solvent, and by comparison, the first absorption peak was BSA and the second was complete antigen 2.
In contrast to the complete antigen 1, BSA was unstable when using buffer as a solvent, and the effect of the buffer was very significant, but the complete antigen 2 rather showed a better absorption peak in the buffer. The absorption peak conditions of the three substances are comprehensively considered, and water is more suitable to be used as a solvent.
(3) Identification of coupling conditions
And (3) comparing the absorption peak conditions of the complete antigen and the carrier protein by taking methanol-water as a mobile phase and water as a solvent.
In FIG. 12, the upper panel shows the absorption peak of BSA, and the peak-off time of the maximum absorption peak is 24.147 min. The lower panel shows the absorption peak of complete antigen 1, and the peak-off time of the maximum absorption peak is 25.106 min. In FIG. 13, the upper panel shows the absorption peak of BSA, and the peak-off time of the maximum absorption peak is 24.147 min. The lower panel shows the absorption peak of complete antigen 2, and the peak-off time of the maximum absorption peak is 32.166 min. The two substances have different peak emergence times and different peak shapes.
Through comparative observation, the complete antigen and BSA have different absorption peaks, and the successful synthesis of the complete antigen 1 and the complete antigen 2 can be obtained.
5 detection of immune Effect of complete antigen
Animal immunization is one of the most effective means for testing the success of antigen synthesis. The titer of the polyclonal antibody obtained by animal immunization is detected by an indirect enzyme-linked immunization method. The high or low titer represents the effectiveness of the complete antigen.
(1) Animal immunization
The method comprises the steps of immunizing a New Zealand big ear white rabbit by adopting a subcutaneous injection method, accurately measuring 0.1mL of complete antigen, dissolving the complete antigen in a phosphate buffer solution, mixing the complete antigen with equal volume of Freund complete adjuvant for primary immunization, immunizing respectively on 22 th, 37 th, 52 th and 67 th days after primary immunization by adopting a method of mixing Freund incomplete adjuvant and complete antigen with equal volume, collecting blood at the edge of a rabbit ear by veins on 47 th and 62 th days, taking heart blood on 78 th day, standing and layering at 4 ℃, taking supernatant, and centrifuging for 15min at 3000r/min to obtain antiserum.
(2) Antibody purification
The prepared rabbit antiserum is purified by a saturated ammonium sulfate salting-out precipitation method. 3mL of each of the prepared serum and the physiological saline was taken, and after the two were sufficiently mixed, 6mL of a saturated ammonium sulfate solution was slowly dropped thereinto while shaking, and the mixture was left in a refrigerator at 4 ℃ overnight. Centrifuging at low temperature of 5000rpm for 20min, and collecting precipitate; adding 3mL of physiological saline and 1.5mL of saturated ammonium sulfate into the centrifuged supernatant, adjusting the concentration to 33%, continuing centrifugation, mixing the precipitates after the two times of centrifugation, adding 2mL of 0.01moL of phosphate buffer solution into the precipitates, fully and uniformly mixing the precipitates, putting the precipitates into a dialysis bag, dialyzing for 72 hours at 4 ℃, finally determining by using a Neisseria reagent, and when ammonium ions cannot be detected, indicating that the antiserum is completely purified, subpackaging, and freezing and storing at-86 ℃.
(3) Indirect enzyme-linked immunosorbent assay for measuring antibody titer
(a) Coating: diluting the antigen-coated solution with coating buffer solution to 10 μ g/mL, coating on 96-weLL enzyme-labeled plate at 150 μ L/weLL for 3h at 37 deg.C, washing the plate with washing solution (PBST) for 3 times, and then washing the plate at 300 μ L/weLL for 1min each time, and then patting the plate dry.
(b) And (3) sealing: add blocking solution, 150. mu.L/weLL, incubate at 37 ℃ for 1.5h, and wash plates with PBST as above.
(c) Sample adding: phosphate buffer was used as a blank control, and the other rows were sequentially subjected to 100. mu.L/weLL of antiserum (1: 100, 1:500, 1:1000, 1:3000, 1:5000) diluted in gradient, and three parallel experiments were performed, incubated at 37 ℃ for 1h, washed with PBST for 3 times, and patted dry.
(d) Adding an enzyme-labeled secondary antibody: diluting enzyme-labeled secondary antibody with blocking solution at a ratio of 1:1000, incubating at 150. mu.L/weLL in 37 ℃ incubator for 1h, washing the plate with PBST for 3 times, and patting dry.
(e) Substrate color development: add o-phenylenediamine color reagent (ready to use), 150. mu.L/weLL, and react for 20min at 37 ℃ in the dark.
(f) And (4) terminating: the reaction was stopped by adding 2mol/L H2 solution in 2SO4 and 50. mu.L/weLL.
(g) Measuring an OD value: the dilution of the serum at 490nm when the absorbance reached 1.0 was measured by a microplate reader after the reaction was terminated as the serum titer.
In an immune animal test, the titer of antiserum can show a rising trend along with the multiple immunization of animals, but the immunity of organisms to the artificially synthesized antigen is generated after the animals are immunized for a long time, the immune response is weakened, and the titer of the antiserum is reduced. Therefore, venous blood was collected from the edge of rabbit ears on day 10 after each immunization. As shown in FIG. 14, the antiserum titer gradually increased with the increase of the number of immunizations until the absorbance of the antiserum measured after the third immunization reached 1.124. As is clear from FIG. 15, the phenothiazine antibody titer was 1:3000, which satisfied the experimental requirements, indicating that the phenothiazine polyclonal antiserum contained antibodies specific for RB, thus further confirming that the synthetic artificial antigen was correct.
Claims (8)
1. A phenothiazine hapten is characterized in that the molecular structural formula is shown as a formula I:
2. A method of producing a phenothiazine hapten as claimed in claim 1 comprising the steps of:
(a) adjusting the pH value of a p-aminobenzoic acid solution to 1-3 by using an HCl solution at the temperature of 2-6 ℃, and stirring in a vortex mode until the solution is completely dissolved;
(b) mixing the obtained acidic p-aminobenzoic acid solution with sodium nitrite solution to obtain light yellow solution, and refrigerating at 2-6 deg.C for 20-40 min; wherein the molar ratio of the p-aminobenzoic acid to the sodium nitrite is 1: 2.5;
(c) adding phenothiazine into the solution obtained in the step (b), wherein the molar ratio of phenothiazine to p-aminobenzoic acid is 1:1, and magnetically stirring for 10-14h in a dark room to obtain the phenothiazine hapten.
3. A phenothiazine complete antigen, which is prepared from the phenothiazine hapten as claimed in claim 1 and a carrier Protein, and the molecular structural formula of the phenothiazine complete antigen is shown as a formula II:
4. A process for the preparation of a phenothiazine complete antigen as claimed in claim 3, which comprises the step of preparing by the mixed anhydride method:
(a) adding 1, 4-dioxane and N-N-dimethylformamide into a phenothiazine hapten solution in an ice bath environment, shaking and mixing for 1-3min, standing for 5-15min, adding triethylamine, standing for 5-15min, adding isobutyl chloroformate, removing the ice bath, and magnetically stirring for 20-40 min; wherein, phenothiazine hapten: 1, 4-dioxane: N-N-dimethylformamide: triethylamine: isobutyl chloroformate =0.1 mmoL: 1-2 mL: 0.1-0.3 nmoL: 0.1-0.2 nmoL;
(b) mixing a carrier protein-sodium borate solution with the reaction solution obtained in the step (a), wherein the carrier protein-sodium borate: phenothiazine hapten =12 mg: 0.1mmoL, then the reaction is carried out for 10-14h at the temperature of 2-6 ℃, the reaction is carried out in a dialysis bag, the dialysis is carried out for 60-80h in PBS buffer solution, dialysate is changed every 4-8h, and the phenothiazine complete antigen is obtained after the dialysis is finished.
5. A process for the preparation of a phenothiazine complete antigen as claimed in claim 3, wherein said phenothiazine complete antigen is prepared by a carbodiimide method comprising:
(a) adding N, N-dimethylformamide into a phenothiazine hapten solution, then stirring and adding dicyclohexylcarbodiimide and N-hydroxysuccinimide, magnetically stirring overnight, and then centrifuging to obtain a supernatant which is recorded as liquid A; wherein, phenothiazine hapten is N, N-dimethylformamide is dicyclohexylcarbodiimide and N-hydroxysuccinimide is =0.1 mmoL, 1-2nmoL, 0.1-0.2mmoL and 0.1-0.2 mmoL;
(b) weighing carrier protein, dissolving the carrier protein in PBS buffer solution, and recording the solution as B solution, wherein the carrier protein-sodium borate: phenothiazine hapten =12 mg: 0.1 mmoL;
(c) adding the solution A into the solution B, and magnetically stirring for 10-14 h; and then centrifuging, taking supernatant, filling the supernatant into a dialysis bag, dialyzing in a PBS buffer solution for 60-80h, changing dialysate every 4-8h, and obtaining the phenothiazine complete antigen after dialysis.
6. The method according to claim 4 or 5, wherein the carrier protein is bovine serum albumin, and the PBS buffer is 0.01mol/L, pH 7.4.
7. The method for identifying a phenothiazine complete antigen as claimed in claim 3, wherein the identification is carried out by high performance liquid chromatography, wherein the mobile phase is methanol-water, and the sample solvent is water.
8. Use of a phenothiazine holoantigen as claimed in claim 3 in the preparation of a polyclonal antibody to a phenothiazine drug.
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