CN115322187A - Selenium hapten and artificial antigen as well as preparation method and application thereof - Google Patents
Selenium hapten and artificial antigen as well as preparation method and application thereof Download PDFInfo
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- CN115322187A CN115322187A CN202210952698.1A CN202210952698A CN115322187A CN 115322187 A CN115322187 A CN 115322187A CN 202210952698 A CN202210952698 A CN 202210952698A CN 115322187 A CN115322187 A CN 115322187A
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- selenium
- hapten
- artificial antigen
- ethyl acetate
- kohlrabi
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- 239000011669 selenium Substances 0.000 title claims abstract description 72
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 title claims abstract description 65
- 229910052711 selenium Inorganic materials 0.000 title claims abstract description 62
- 239000000427 antigen Substances 0.000 title claims abstract description 27
- 102000036639 antigens Human genes 0.000 title claims abstract description 27
- 108091007433 antigens Proteins 0.000 title claims abstract description 27
- 238000002360 preparation method Methods 0.000 title claims description 8
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- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical group CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 24
- 238000006243 chemical reaction Methods 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 15
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- 150000001875 compounds Chemical class 0.000 claims description 6
- 239000002244 precipitate Substances 0.000 claims description 6
- -1 succinimidyl ester Chemical class 0.000 claims description 6
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- 108010058846 Ovalbumin Proteins 0.000 claims description 5
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- YOETUEMZNOLGDB-UHFFFAOYSA-N 2-methylpropyl carbonochloridate Chemical compound CC(C)COC(Cl)=O YOETUEMZNOLGDB-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
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- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- GNNALEGJVYVIIH-UHFFFAOYSA-N benzene-1,2-diamine;hydrochloride Chemical compound Cl.NC1=CC=CC=C1N GNNALEGJVYVIIH-UHFFFAOYSA-N 0.000 description 2
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- JPJALAQPGMAKDF-UHFFFAOYSA-N selenium dioxide Chemical compound O=[Se]=O JPJALAQPGMAKDF-UHFFFAOYSA-N 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- HSTOKWSFWGCZMH-UHFFFAOYSA-N 3,3'-diaminobenzidine Chemical compound C1=C(N)C(N)=CC=C1C1=CC=C(N)C(N)=C1 HSTOKWSFWGCZMH-UHFFFAOYSA-N 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 1
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- JNRBLFVQPTZFAG-UHFFFAOYSA-L copper;selenite Chemical compound [Cu+2].[O-][Se]([O-])=O JNRBLFVQPTZFAG-UHFFFAOYSA-L 0.000 description 1
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- KZNICNPSHKQLFF-UHFFFAOYSA-N dihydromaleimide Natural products O=C1CCC(=O)N1 KZNICNPSHKQLFF-UHFFFAOYSA-N 0.000 description 1
- RNGFNLJMTFPHBS-UHFFFAOYSA-L dipotassium;selenite Chemical compound [K+].[K+].[O-][Se]([O-])=O RNGFNLJMTFPHBS-UHFFFAOYSA-L 0.000 description 1
- BVTBRVFYZUCAKH-UHFFFAOYSA-L disodium selenite Chemical compound [Na+].[Na+].[O-][Se]([O-])=O BVTBRVFYZUCAKH-UHFFFAOYSA-L 0.000 description 1
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- PEUPCBAALXHYHP-UHFFFAOYSA-L zinc;selenite Chemical compound [Zn+2].[O-][Se]([O-])=O PEUPCBAALXHYHP-UHFFFAOYSA-L 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D421/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having selenium, tellurium, or halogen atoms as ring hetero atoms
- C07D421/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having selenium, tellurium, or halogen atoms as ring hetero atoms containing two hetero rings
- C07D421/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having selenium, tellurium, or halogen atoms as ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
-
- 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
-
- 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
-
- 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
- C07K14/77—Ovalbumin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
-
- 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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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Urology & Nephrology (AREA)
- Gastroenterology & Hepatology (AREA)
- Hematology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biomedical Technology (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
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- Peptides Or Proteins (AREA)
Abstract
The invention discloses a selenium hapten, which is characterized in that: the structural formula is as follows:
the structural formula is selenium hapten. Coupling the above selenium hapten with protein carrier via amido bond to obtainThe obtained selenium element artificial antigen.
Description
Technical Field
The invention belongs to the technical field of biochemical engineering, and particularly relates to a selenium hapten, a preparation method for preparing the selenium antigen by using the hapten and application of the selenium antigen.
Technical Field
Selenium (selenium), a chemical element with chemical symbol Se and atomic number 34, has strong effects of resisting oxidation, resisting cancer, protecting liver, protecting cardiac muscle, repairing cells, improving human immunity and the like relative to non-metallic trace elements with atomic mass of 78.96 which are necessary for animals and plants. Selenium is an indispensable trace element in human bodies, and due to the unbalanced distribution of selenium in the earth crust and the over-propaganda effect of merchants, modern people can blindly supplement selenium, but the selenium poisoning phenomenon is caused once the selenium is supplemented excessively. In addition, the wastewater from day-to-night industrial activities such as coal mining, metallurgy, etc. contains a large amount of inorganic selenate, which poses serious threat to the surrounding environment if not handled properly, and even more worrisome is a potential threat to human health if aquatic animals and plants are transferred to the human body through the food chain. Furthermore, selenium is a popular trace element in recent decades, and a great deal of scientific research is invested in the biological, pharmacological and physiological roles of selenium in almost every country. Among them, the detection of trace selenium is undoubtedly the biggest challenge for every scientist, and based on the above three points, it is increasingly urgent to establish a method for rapidly, sensitively and specifically detecting selenium.
At present, the monitoring method for selenium still relies on high-end precise instruments and devices, such as atomic absorption method, inductively coupled plasma emission spectrometer, atomic emission spectrometer, mass spectrometer, and the like. Although these methods have high sensitivity and strong specificity, they are cumbersome to operate, expensive and require specialized laboratory technicians, and are not well suited for screening large numbers of samples. Especially with respect to the sample processing methodology and its rigors, slight carelessness can lead to signal peaks that are interfered with by matrix peaks. The immunological detection method has unique advantages in semi-quantitative detection of analytes, and has the advantages of low cost, high sensitivity, strong specificity, more samples which can be analyzed at the same time at one time, capacity of making up the defects of an instrument method, and more importantly, simple sample processing method and operation, and strong resistance to matrixes in the detection process.
To conclude, the crucial factor influencing the immunological detection method is the problem of the specific binding affinity of the antigen and antibody, and especially for small molecules, the modification level of the hapten directly determines the sensitivity and specificity of the antibody. Therefore, the design and synthesis of haptens are the prerequisite for the generation of specific antibodies and the establishment of immunological detection methods, and are the most critical and important steps.
Disclosure of Invention
The invention aims to provide a selenium hapten, a selenium artificial antigen, a method for preparing the selenium hapten and a method for preparing the selenium artificial antigen.
The artificial antigen of the selenium element is an artificial antigen obtained on the basis of the hapten of the selenium element.
A selenium hapten, which has the following structure:
the structural formula is selenium hapten.
The reaction equation of the preparation method of the selenium hapten is as follows:
drawings
FIG. 1 is a hapten for selenium 1 H NMR identification chart.
FIG. 2 is a hapten for selenium 13 C NMR identification data.
FIG. 3 is a diagram of ultraviolet identification of conjugates.
FIG. 4 is Se 4+ ELISA competitive inhibition curve.
FIG. 5 is a structural formula of hapten of selenium element.
Detailed Description
The present invention is described in further detail by the following examples, and it is apparent that the described examples are only a part of the examples of the present invention, and not all of the examples. All other embodiments, which can be obtained by a person skilled in the art without inventive step based on the embodiments of the present invention, are within the scope of protection of the present invention.
Example 1: 152.1mg of 3, 4-diaminobenzoic acid was added to 4mL of 2M hydrochloric acid, mixed in a round-bottomed flask, heated at 80 ℃ and, after complete dissolution, 2 times the molar amount of S was addede 4+ And immediately generating a large amount of green precipitates, stopping heating, continuously reacting until the temperature reaches room temperature, centrifuging to take out solids, washing the solids with pre-frozen double distilled water for 2-3 times, and freeze-drying to obtain the green solid p-carboxylated kohlrabi selenium brain. The yield thereof was found to be 96.2%.
In the present embodiment, the starting material of the reaction is 3,4-diaminobenzoic acid, but is not limited to 3,4-diaminobenzoic acid, and in addition, compounds of 2,3-diaminobenzoic acid or two hydrochloride forms thereof are within the scope of the present patent.
Se in the present embodiment 4+ Is prepared from the elementary substance of selenium in HClO 4 /HNO 3 Medium oxidation, reduction in HCl, but its Se 4+ The standard solution, selenite such as potassium selenite, sodium selenite, zinc selenite, copper selenite, or its water and compound, selenium dioxide, etc. can provide Se 4+ Are all within the scope of this patent.
In this embodiment, 2 times the molar amount of Se is used 4+ Reaction of Se participating in the reaction of this patent 4+ The amount of the substance is more than or equal to 1, and the protection range is kept.
The acid in the embodiment is hydrochloric acid, and the molar concentration is 1M, but not limited to hydrochloric acid, dilute sulfuric acid and other non-oxidizing inorganic acids, organic acids trifluoroacetic acid and the like, and all fall within the protection scope of the present patent. In addition, the molar concentration of the acid is 0.1-6M, which is the protection scope of the patent.
113.5mg of the product was mixed with 115mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 80.5mg of N-hydroxysuccinimide (NHS) in 2mL dry Dimethylformamide (DMF), reacted at room temperature under nitrogen for 6 hours and monitored by TLC: and the developing solvent is ethyl acetate, after complete reaction, the mixture is subjected to vacuum rotary evaporation, washed with cold water for 2-3 times, and the precipitate is freeze-dried to obtain the light brown solid powder kohlrabi selenium brain succinimidyl ester. 75.0% yield.
The above amidation reaction using condensing agent EDC/NHS has equivalent weight of 1.2eq/1.4eq, wherein the reaction equivalent weight of EDC and NHS is between 1-5.
In addition to the conventional EDC/NHS reaction system, condensing agents such as N, N-diisopropylethylamine/benzotriazole-N, N' -tetramethyluronium hexafluorophosphate (EIPEA/HBTU), isobutyl chloroformate/triethylamine (IBCF/TEA) used in the reaction are protected by the present patent.
The reaction system of this embodiment is anhydrous DMF, and in addition, anhydrous DMOS, acetone, acetonitrile, dichloromethane, trichloromethane and other organic solvents as the reaction system are all within the scope of this patent.
In the scheme, the initial hapten kohlrabi selenium brain succinimide ester is an active ester type, and belongs to the protection scope of the patent.
After combining 64.8mg of the above activated ester with 39.3mg of 6-aminocaproic acid via amide bond for 6 hours, TLC monitoring with ethyl acetate as a developing solvent, after monitoring the reaction to completion, removing unreacted white precipitate (6-aminocaproic acid), adding 45.8mg of EDC and 34.5mg of NHS without isolating the product, monitoring under the same conditions, after completion of the reaction, rotary evaporating DMF, dissolving the residual product with methanol/dichloromethane (1) and passing through silica gel column, washing with dichloromethane, and evaporating the solvent to obtain the white-like solid hapten. TLC monitoring, developing agent ethyl acetate, rf value 0.9, yield 67.3%, three-step total yield 48.6%, nuclear magnetic identification data: 1 h NMR (400mhz, dmso-d 6) δ 8.79 (s, 1H), 8.34 (d, J =0.6hz, 1h), 7.92 (dd, J =10.4,1.1hz, 2h), 3.31 (dd, J =12.6,6.7hz, 2h), 2.81 (s, 4H), 2.61-2.45 (m, 2H), 1.86-1.24 (m, 6H); carbon spectrum identification data: 13 C NMR(101MHz,DMSO-d6)δ170.75,169.46,165.82,160.70,159.77,135.26,128.36,123.37,122.37,30.62,28.94,25.97,25.91,25.70,24.48。
the embodiments of the present invention use 6-aminocaproic acid as the bridge between the carrier and the protein, but are not limited to compounds having 6 carbon atoms and 3-10 carbon atoms as the bridge.
The linking arm used in the scheme is a fatty carbon chain, and besides, hydrophilic bridge bonds represented by polyethylene glycol also belong to the protection scope of the patent.
The 6-amino-acidified kohlrabi selenium brain intermediate and the final hapten-activated ester product of the protocol are both claimed.
Example 2: dissolving 3mg of the hapten in 200 mu L of anhydrous DMF, dropwise adding 8mg of protein carrier PBS solution into the solution to react for 6 hours at room temperature, dialyzing the solution for 3 days, changing the solution for 6 times, and standing the solution at 4 ℃ for later use.
The selenium artificial antigen prepared on the basis of the selenium hapten also belongs to the protection scope of the invention.
Kohlrabi selenium brain succinimidyl ester without a connecting arm is prepared into an antigen which is also in the protection scope of the patent.
The selenium element artificial antigen is obtained by coupling the selenium element hapten formula I and carrier protein. In an embodiment of the invention, the carrier proteins are in particular Bovine Serum Albumin (BSA) and chicken Ovalbumin (OVA). However, not only these two carrier proteins, but also the aminated nanospheres or biochemical materials, etc. are all within the scope of protection of this patent.
The preparation method of the selenium hapten specifically comprises the following steps: coupling the selenium hapten (shown in a formula I) with carrier protein through amido bond to obtain the selenium artificial antigen. Wherein, the coupling ratio of the hapten to the BSA carrier protein is calculated by an ultraviolet spectrophotometer to be 32.9:1, coupling ratio to OVA carrier protein of 9.9:1 furthermore, not limited to these two coupling ratios, 3:1 to 45:1 also belong to the scope of protection of this patent.
Antibodies prepared by using the selenium antigen also belong to the protection scope of the patent. The antibody can be a polyclonal antibody, a monoclonal antibody or an antibody serum.
The application of the selenium hapten, the artificial antigen and the prepared antibody in qualitative or quantitative detection of the selenium hapten is also the protection scope of the patent.
The selenium hapten/artificial antigen provided by the invention has the advantages of simple synthetic method, high purity and great reference value for preparation of a selenium hapten antibody and immunological detection application.
EXAMPLE 3 immunization of mice with selenium hapten monoclonal antibodies
The protocols, materials, reagents used in the following examples are all conventional or commercially available unless otherwise specified.
Firstly, the method comprises the following steps: immunization of animals
6 balb/c mice were immunized with 100. Mu.g of each BSA conjugate obtained in example 2, antigen was dissolved in physiological saline and Freund's complete adjuvant was mixed to a water-in-oil state, and then they were immunized by subcutaneous multiple immunization, three weeks later, freund's complete adjuvant was changed to Freund's incomplete adjuvant, after mixing uniformly, they were immunized by subcutaneous multiple immunization three times, three weeks apart were provided at intervals, and blood was collected 7 days after each immunization and monitored. 21 days after the fourth immunization, the fusion was carried out three days after tail vein injection of 100. Mu.L of BSA conjugate (without any adjuvant).
II, secondly: cell fusion and cloning
Conventionally, spleens of mice were aseptically removed under a clean bench, counted, and logarithmically grown myeloma cells (sp 2/0AG 14) were administered at a rate of 1:1, then slightly shaking to make the mixed cells into a uniform and loose state, slowly adding PEG 20001mL into the cells at 37 ℃, slowly stirring while adding, finishing adding within 1 minute, then continuously keeping at 37 ℃ for fusing for 1 minute, culturing by HAT medium and placing in a 37 ℃ incubator for 10 days. And (3) replacing the HAT liquid with HT medium, monitoring the cell supernatant and inhibition by indirect ELISA, carrying out limiting dilution on the inhibited colonies, continuously culturing for 10 days in an incubator at 37 ℃ for subcloning, and continuously monitoring the cell supernatant by ELISA. The same procedure for limiting dilution was then repeated until dilution was such that monoclonal colonies appeared.
Thirdly, the steps of: monoclonal antibody preparation and purification thereof
Performing amplification culture on the hybridoma obtained after subcloning and establishing strains, and measuring the titer of cell supernatant to be as high as 1:9000, culturing ascites by using mouse abdominal cavity, collecting ascites after 10-15 days, purifying ascites with protein A/G mixed column to obtain high purity antibody with titer as high as 1:810000. purified antibody BCA assay was quantitated and then an equal amount of glycerol was added and stored at-20.
Antibody subtypes other than igG 2a, and other antibody subtypes prepared by using the antigen, such as igG2b, igG g1, igG g2, igG g3, etc., are within the scope of protection of the present patent
Example 4: establishment of Indirect ELISA method
Coating: 100ngmL -1 The OVA conjugate of (3) was placed in a 96-well microplate at 4 ℃ overnight in 100. Mu.L per well.
And (3) sealing: the coating solution was discarded, and 330. Mu.L of 1% gelatin solution was added thereto at 37 ℃ for 2 hours.
Competition: 0,3.90625,7.8125,15.625,31.25,62.5,125,250,500, and 1000pmol mL -1 Concentration of Se 4+ The resulting mixture was mixed with 1mg of an aqueous solution of o-phenylenediamine hydrochloride in advance at 4 ℃ for 15 minutes, and then diluted with 1% gelatin +0.1M PBS to obtain 50. Mu.L of a mixture of 50. Mu.L of the mixture and 50. Mu.L of 1:120000 antibody was mixed and then given 4 ℃ for 40 minutes with gentle horizontal shaking for 10 seconds.
In addition to o-phenylenediamine hydrochloride, other compounds that are analogs such as 3,4-diaminobenzoic acid, 2,3-diaminobenzoic acid, 3,3' -diaminobenzidine, or hydrochloride forms thereof are within the scope of this patent.
Secondary antibody: PBS-Twen-20 plates were washed four times, 1:10000 dilutions of goat anti-mouse enzyme-labeled secondary antibody were incubated at 37 ℃ for 40 min in each well (100. Mu.L).
Color development and reading: after washing the plate four times, 100. Mu.L of TMB per well was developed for 8 minutes, and the plate was read at 450nm with a microplate reader after the development was stopped with 2M sulfuric acid.
Calculating and judging: average absorbance of each standard, divided by 0pmol mL -1 Multiplying the absorbance by 100% to obtain the corresponding absorbance percentage of each standard product, namely B/B 0 . Then taking the ratio of the absorbance of each standard as the ordinate and Se with different concentrations 4+ The log value of the test sample is plotted on the abscissa, so that a competitive inhibition rate curve of the indirect ELISA can be obtained, and the monitoring range of the curve is 15.625-250pmol mL -1 Semi-inhibition ratio IC 50 60pmol mL -1 Lowest detection line IC 10 5.86pmol mL -1 . The results are shown in FIG. 4.
Claims (7)
1. A selenium hapten, comprising: the structural formula is as follows:
the structural formula is selenium hapten.
2. An artificial antigen of selenium element, which is characterized in that: coupling the seleno hapten of claim 1 with a carrier protein to obtain the seleno artificial antigen.
3. The elemental selenium artificial antigen of claim 2, wherein: the carrier protein is one of Bovine Serum Albumin (BSA), chicken Ovalbumin (OVA), keyhole Limpet Hemocyanin (KLH), human Serum Albumin (HSA), bovine thyroid albumin (BTG), mouse Serum Albumin (MSA), rabbit Serum Albumin (RSA) or Polylysine (PDL).
4. The elemental selenium artificial antigen of claim 2 or 3, wherein: coupling the compound of claim 1 with a protein carrier through an amide bond to obtain the selenium artificial antigen, wherein the compound of claim 1 reacts with the protein carrier in a ratio of 3:1 to 45: 1.
5. A method for preparing selenium hapten is characterized in that: the method comprises the following steps:
a) Adding 152.1mg of 3, 4-diaminobenzoic acid into 4mL of 2M hydrochloric acid, mixing, adding into a round-bottom flask, heating at 80 ℃, adding 2 times of molar amount of Se after completely dissolving 4+ When green precipitates appear, stopping heating, continuing to react until the temperature reaches room temperature, centrifuging to take out solids, washing the solids with pre-frozen double distilled water for 2-3 times, and freeze-drying to obtain green solid pair of carboxylated kohlrabi selenium brains;
b) 113.5mg of carboxylated kohlrabi selenol obtained in the step A is taken and mixed with 115mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 80.5mg of N-hydroxysuccinimide (NHS) added into anhydrous 2mL of Dimethylformamide (DMF), and the mixture is reacted for 6 hours at room temperature under the protection of nitrogen and monitored by TLC: the developing agent is ethyl acetate, after complete reaction, the ethyl acetate is subjected to vacuum rotary evaporation, the ethyl acetate is washed by cold water for 2 to 3 times, and the precipitate is freeze-dried to obtain light brown solid powder kohlrabi selenium brain succinimidyl ester;
c) 64.8mg of kohlrabi selenium brain succinimidyl ester obtained in step B were combined with 39.3mg of 6-aminocaproic acid by amide bond for 6 hours, monitored by TLC, the developing reagent was ethyl acetate, after monitoring the reaction was complete, the unreacted white precipitate (6-aminocaproic acid) was discarded, and without isolation of the product 45.8mg of EDC and 34.5mg of NHS were added and monitored under the same conditions. After the reaction was complete, DMF was rotary evaporated, the residual product was dissolved with methanol/dichloromethane (1. TLC monitoring, developing agent ethyl acetate, rf value of 0.9, and high performance liquid phase identification of the purity.
6. A preparation method for preparing artificial antigen of selenium element is characterized in that: dissolving 3mg of the white solid prepared by the method of claim 5 in 200 μ L of anhydrous DMF, then adding 8mg of BSA/OVA 0.01M PBS solution dropwise to react for 6 hours at room temperature, then dialyzing with 0.01M PBS for 3 days, changing the solution for 6 times, and finally standing at 4 ℃ for later use, thus obtaining the selenium artificial antigen.
7. An immunoassay kit comprising the seleno-hapten or the seleno-artificial antigen according to claims 1 to 6 or prepared therefrom.
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| WO2022012084A1 (en) * | 2020-07-13 | 2022-01-20 | 海南大学 | Arecoline antigen, and preparation method therefor and application thereof |
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| Title |
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| 潘宝龙;张秀琴;王永志;: "硒标免疫层析技术的实验构建及评价", 检验医学与临床, no. 08, 28 August 2007 (2007-08-28), pages 712 - 713 * |
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