CN112266330B - Methoxyarenol derivative, immunogen, anti-methoxyadrol specific antibody, preparation method and application thereof - Google Patents
Methoxyarenol derivative, immunogen, anti-methoxyadrol specific antibody, preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a methoxy adrenaline derivative, immunogen, anti-methoxy adrenaline specific antibody, and a preparation method and application thereof. Firstly, coupling a novel methoxy adrenaline derivative with recombinant bovine serum albumin obtained by genetic engineering transformation to prepare a methoxy adrenaline artificial antigen, immunizing an experimental animal with the methoxy adrenaline artificial antigen to obtain an anti-methoxy adrenaline specific antibody, wherein ELISA detection shows that the specific antibody has strong specificity and high sensitivity, and interference experiments show that the specific antibody has no cross reaction with 32 common hormones; the anti-methoxy-adrenaline specific antibody is applied to the preparation of a methoxy-adrenaline detection reagent, which comprises a methoxy-adrenaline homogeneous enzyme immunoassay reagent and a methoxy-adrenaline latex enhanced immunoturbidimetry detection reagent, and the detection reagent can realize high-throughput and rapid detection of the methoxy-adrenaline on a full-automatic biochemical analyzer.
Description
Technical Field
The invention relates to a methoxy adrenaline derivative, immunogen, anti-methoxy adrenaline specific antibody, a preparation method and application thereof, belonging to the technical field of biomedical detection.
Background
Methoxyarenes (MN), 3-methoxy-adrenaline, are primarily intermediates of adrenaline. Catecholamine (CA) is a hormone secreted by adrenal medulla, adrenal neurons, and adrenal chromaffin, and mainly includes epinephrine (E) and Norepinephrine (NE). Adrenaline is mainly converted into MN by adrenal medulla and chromaffin cells under the action of catechol-O-methyltransferase (COMT), and noradrenaline is mainly converted into methoxy noradrenaline (NMN) by tissues other than neurons and chromaffin cells under the action of COMT. The determination of the methoxy adrenaline in the urine can indirectly reflect the secretion of catecholamine in vivo, and has important clinical significance for the diagnosis of adrenal diseases. Urine MN is often jointly detected with urine NMN clinically, mainly applied to: (1) diagnosis of secondary hypertension; (2) An index for evaluating glycemic control in a type 2 diabetic; (3) diagnosis of adrenal medullary hyperplasia; (4) The diagnosis of diseases such as hyperthyroidism, hypothyroidism, acute and chronic hepatitis, liver cirrhosis, chronic renal insufficiency, etc. There are references to: the diagnosis of adrenal medullary hyperfunction by 24h urine of MN and NMN is considered to be the most reliable examination at present.
The laboratory methods for detecting the methoxyepinephrine include a colorimetric method, a radioimmunoassay method, a high performance liquid chromatography method, a liquid tandem mass spectrometry method, an enzyme-linked immunosorbent assay method, an immunoturbidimetry method and the like. Colorimetrically detects the sum of catecholamines and methoxyadrenaline species rather than the level of a single compound; the radioimmunoassay has poor stability and has the problems of radioactive ray radiation, pollution and the like; pretreatment of the high performance liquid chromatography is complex, and target analytes are required to be completely separated on the chromatography, so that the analysis time is long; the liquid tandem mass spectrometry has high and quick detection sensitivity, but has complex structure and high maintenance cost, needs to be operated by specially trained technicians, and is not easy to popularize in basic medical institutions. The ELISA method has high sensitivity and good specificity, but has long detection time and poor accuracy, and is difficult to be used for batch detection.
Therefore, methoxy adrenaline detection products which have wide linear range, high sensitivity, high accuracy, high precision, short detection time, simple sample treatment, high instrument automation degree and can continuously detect multiple samples are lacking in the market at present.
Disclosure of Invention
In order to overcome the disadvantages of the prior art, a first object of the present invention is to provide a methoxy adrenaline derivative which is a newly synthesized compound that does not exist in nature.
The first purpose of the invention is realized by adopting the following technical scheme: a methoxy adrenaline derivative has a structural formula shown in a formula I:
The second purpose of the present invention is to provide a method for synthesizing the above-mentioned methoxy epinephrine derivative, which is different from the conventional synthesis method, has a good synthesis effect, and significantly improves the synthesis efficiency of the methoxy epinephrine derivative.
The second purpose of the invention is realized by adopting the following technical scheme: a method for synthesizing the methoxy adrenaline derivative is shown as the following formula:
the reaction process comprises the following steps:
(A1) Synthesis procedure of Compound 3: dissolving the methoxy adrenaline and the compound 2 in N, N-dimethylformamide, and adding K 2 CO 3 After reaction, compound 3 is obtained by extraction, drying, concentration and purification;
(A2) Synthesizing a methoxy adrenaline derivative: dissolving the compound 3 and benzyltriethylammonium chloride in chloroform to prepare a reaction solution; and dropwise adding the NaOH solution into the reaction solution, adjusting the pH =6 of the solution after reaction, and then extracting, drying, concentrating and purifying to obtain the methoxy adrenaline derivative.
Specifically, the reaction process comprises the following steps:
(a1) Synthesis of Compound 3:
50.0g of methoxyepinephrine, 25.0g of Compound 2 are weighed out and dissolved together in 250mL of N, N-Dimethylformamide (DMF), and 20.0g of K is added 2 CO 3 Preparing a reaction mixed solution; the reaction mixture was stirred at room temperature for 12 hours; after the reaction is finished, adding 250mL of purified water into the reaction mixed solution, then extracting with 200mL of Dichloromethane (DCM), and repeating the extraction step for 3 times; passing the combined organic phase obtained by extraction through Na 2 SO 4 Drying and concentrating; the residue obtained after concentration was purified by silica gel dry column (PE: EA = 5:1) to obtain compound 3.
(a2) Synthesis of methoxy adrenaline derivatives:
5.0g of Compound 3 and 0.75g of benzyltriethylammonium chloride were weighed out and dissolved in 50mL of chloroform (CHCl) 3 ) Preparing a reaction solution; dropwise adding NaOH solution with the concentration of 0.55g/mL into the reaction solution at the temperature of 65 ℃, and stirring for 5.0 hours at the temperature of 65 ℃; adjusting the pH of the obtained residue after the reaction to be =6 by using 1N HCI, carrying out acidification treatment, then extracting by using 150mL Ethyl Acetate (EA), and repeating the extraction step for 3 times; drying and concentrating the combined organic phase obtained by extraction; purifying the residue obtained after concentration by pre-HPLC to obtain the compound, namely the methoxy adrenaline derivative.
It is a third object of the present invention to provide a methoxy adrenergic immunogen.
The third purpose of the invention is realized by adopting the following technical scheme: a methoxy adrenaline immunogen is formed by connecting a methoxy adrenaline derivative shown in the structural formula I and a carrier protein, and the structural formula is shown in the formula II:
wherein the carrier protein is recombinant bovine serum albumin, and further, the amino acid sequence of the recombinant bovine serum albumin is shown in a sequence table SEQ ID NO:1 is shown.
The amino acid sequence of the recombinant bovine serum albumin (SEQ ID NO: 1) is specifically as follows:
MKWVTFISLLLLFSSAYSRGVFRRDTHKKSEIAHRFKDLGEEHFKGLVLIAFSQYLQQCPFDEHVKKLVNELTEFAKKTCVADESHAGCEKSLHTLFGDELCKKVASLRETYGDMADCCEKQEPERNECFLSHKDDSPDLPKLKPDPNTLCDEFKADEKKFWGKYLYEIARRHPYFYAPELLYYANKKYNGVFQECCQAEDKGACLLPKKIETMREKVLTSSARQRLRCASIQKKFGERALKAWSVARLSQKFPKAEFVEVTKKLVTDLTKVHKECCHGDLLECADDRADLAKYICDNQDTISSKLKKECCDKPLLEKSHCIAEVEKDAIPENLPPLTADFAEDKKDVCKNYQEAKDAFLGSFLYEYSRRHPEYAVSVLLRLAKKEYEATLEECCAKDDPHACYSTVFDKLKKHLVDEPQNLIKQNCDQFEKLGEYGFQNALIVRYTRKKVPQVSTPTLVEVSRSLGKVGTRCCTKKPESERMPCTEDYLSLILNRLCVLHEKTPVSEKKVTKCCTESLVNRRPCFSALTPDETYVPKAFDEKLFTFHADICTLPDTEKKQIKKQTALVELLKHKPKATEEQLKKTVMENFVAFVDKCCAADDKEACFAVEGPKKLVVSTQTALA。
a fourth object of the present invention is to provide a method for preparing the methoxyepinephrine immunogen as described above.
The fourth purpose of the invention is realized by adopting the following technical scheme: a method of preparing a methoxyepinephrine immunogen as described above, comprising the steps of:
(B1) Preparation of carrier protein solution: dissolving the recombinant bovine serum albumin in a phosphate buffer solution to obtain a carrier protein solution;
(B2) Preparation of a solution of a methoxy adrenaline derivative: mixing the methoxy epinephrine derivative shown in the structural formula I with dimethylformamide, ethanol, a potassium phosphate buffer solution, 1-ethyl-3- (-3-dimethylaminopropyl) carbodiimide and N-hydroxy thiosuccinimide, and stirring for dissolving to obtain a methoxy epinephrine derivative solution;
(B3) Synthesis of methoxy-adrenaline immunogen: and (3) adding the methoxy adrenaline derivative solution obtained in the step (B2) into the carrier protein solution obtained in the step (B1), stirring for reaction, and purifying by dialysis to obtain the methoxy adrenaline immunogen.
Specifically, the preparation method of the methoxy adrenaline immunogen comprises the following steps:
(b1) Preparation of the carrier solution: dissolving a carrier protein in 0.35mol/L potassium phosphate buffer (pH = 8.5) to give a carrier protein solution at a final concentration of 5.0 mg/mL;
(b2) Preparation of a solution of a methoxy adrenaline derivative: mixing 250.0mg of the above-mentioned methoxy epinephrine derivative, 7.5mL of dimethylformamide, 7.5mL of ethanol, 15.0mL of potassium phosphate buffer (10.0 mmol/L, pH = 8.0), 150.0mg of 1-ethyl-3- (-3-dimethylaminopropyl) carbodiimide, and 90.0mg of N-hydroxythiosuccinimide, and stirring and dissolving the mixture for reaction for 3 hours to obtain a methoxy epinephrine derivative solution;
(b3) Synthesis of methoxy-adrenaline immunogen: dropwise adding the methoxy adrenaline derivative solution obtained in the step (b 2) into the carrier protein solution obtained in the step (b 1), stirring overnight at-4 ℃, and purifying by dialysis to obtain the methoxy adrenaline immunogen.
The fifth object of the present invention is to provide an anti-methoxyepinephrine-specific antibody.
The fifth purpose of the invention is realized by adopting the following technical scheme: the anti-methoxy-epinephrine-specific antibody is a specific antibody obtained by injecting the methoxy-epinephrine immunogen into a test animal, wherein the test animal is one of rabbits, goats, sheep, mice, rats, guinea pigs and horses.
The sixth object of the present invention is to provide a method for producing the above-mentioned anti-methoxyepinephrine-specific antibody.
The sixth purpose of the invention is realized by adopting the following technical scheme: a method for preparing an anti-methoxy-epinephrine-specific antibody as described above, comprising the steps of:
(C1) Diluting the methoxy adrenaline immunogen with a phosphate buffer solution to obtain a methoxy adrenaline artificial antigen solution, mixing the methoxy adrenaline artificial antigen solution with an equivalent amount of Freund's complete adjuvant, and performing multi-point injection on the experimental animal;
(C2) After 3-6 weeks, mixing the same methoxy adrenaline artificial antigen solution with equivalent Freund incomplete adjuvant, performing multipoint injection on the experimental animal, and then injecting once every 3-6 weeks for 3-10 times in total;
(C3) And (3) taking blood of the experimental animal which is injected in the step (C2), separating and purifying to obtain the anti-methoxy-epinephrine specific antibody.
Specifically, the preparation method of the anti-methoxy-epinephrine-specific antibody comprises the following steps of:
(c1) Diluting the methoxyepinephrine immunogen with 0.15mol/L sodium phosphate buffer (pH = 7.0) to a final concentration of 3.5mg/mL to obtain an artificial antigen solution, mixing the 3.0mL artificial antigen solution with an equivalent amount of Freund's complete adjuvant, and performing multi-point injection on the experimental animal rabbit;
(c2) After 4 weeks, carrying out multi-point injection on the experimental animal rabbit by using 3.0mL of the same artificial antigen solution and an equivalent amount of Freund's incomplete adjuvant, and then carrying out injection once every 5 weeks for 6 times in total;
(c3) And (c 2) taking blood from the experimental animal rabbit which is injected in the step (c 2), separating and purifying to obtain the anti-methoxy-epinephrine specific antibody.
A seventh object of the present invention is to provide the use of an anti-methoxyepinephrine-specific antibody as described above.
The seventh purpose of the invention is realized by adopting the following technical scheme: the anti-methoxy-epinephrine specific antibody is used for preparing a methoxy-epinephrine detection reagent, and the methoxy-epinephrine detection reagent comprises a methoxy-epinephrine homogeneous enzyme immunoassay reagent and a methoxy-epinephrine latex enhanced immunoturbidimetry detection reagent.
Preferably, the application of the anti-methoxy-epinephrine-specific antibody, the methoxy-epinephrine homogeneous enzyme immunoassay reagent consists of an R1 reagent and an R2 reagent, wherein the R1 reagent comprises the anti-methoxy-epinephrine-specific antibody and an R1 buffer solution, and the R2 reagent comprises a methoxy-epinephrine glucose-6-phosphate dehydrogenase labeled conjugate and an R2 buffer solution;
the R1 buffer solution contains an enzyme substrate, a coenzyme, bovine serum albumin and a Tris buffer solution, wherein the enzyme substrate is glucose-6-phosphate, and the coenzyme is oxidized nicotinamide adenine dinucleotide;
the methoxy adrenaline glucose-6-phosphate dehydrogenase labeled conjugate is formed by coupling a methoxy adrenaline derivative shown in the structural formula I and glucose-6-phosphate dehydrogenase; the structural formula is shown as formula III:
the R2 buffer solution is a Tris buffer solution containing bovine serum albumin.
Specifically, the preparation method of the methoxy adrenaline homogeneous enzyme immunoassay reagent comprises the following steps:
(D1) Adding 250.0mg bovine serum albumin, 250.0mg glucose-6-phosphate and 50.0mg oxidized nicotinamide adenine dinucleotide into 250mL Tris buffer solution (50 mmol/L, pH = 8.5) in sequence, stirring and dissolving to prepare R1 buffer solution, adding the anti-methoxy-adrenaline specific antibody into the R1 buffer solution according to the volume ratio of 1: 1000, mixing uniformly, and adjusting the pH to 7.6 by using 1.0 mol/L hydrochloric acid to prepare an R1 reagent;
(D2) 250.0mg of bovine serum albumin was added to 250mL of Tris buffer (100 mmol/L, pH = 8.7) and dissolved with stirring to prepare an R2 buffer, and then the methoxy-adrenaline glucose-6-phosphate dehydrogenase labeled conjugate was added to the above R2 buffer at a volume ratio of 1: 1000 and mixed, and then pH was adjusted to 8.0 with 1.0 mol/L hydrochloric acid to prepare a reagent R2.
The preparation method of the methoxy adrenaline glucose-6-phosphate dehydrogenase labeled conjugate comprises the following steps:
(E1) Weighing glucose-6-phosphate dehydrogenase with 20.0 mg activity unit of 200KU, dissolving in 50.0mL sodium phosphate (100 mmol/L, pH = 8.0) buffer solution at room temperature, adding 150.0mg reduced nicotinamide adenine dinucleotide, 75.0 mg glucose-6-phosphate and 0.75 mL carbitol, dropwise adding 2.5 mL dimethyl sulfoxide, stirring and dissolving to obtain glucose-6-phosphate dehydrogenase solution;
(E2) Weighing 15.0 mg of the methoxy adrenaline derivative shown in the structural formula I in an anhydrous state, dissolving the methoxy adrenaline derivative in 500.0 muL of dimethylformamide, cooling the solution to 0 ℃, adding 4.5 muL of tributylamine, 2.5 muL of isobutyl chloroformate and 3.5 muL of L N, N' -dicyclohexylcarbodiimide, and stirring for 45 minutes at 0 ℃ to obtain an activation solution of the methoxy adrenaline derivative;
(E3) And dropwise adding the methoxy adrenaline derivative activating solution into a glucose-6-phosphate dehydrogenase solution, stirring and reacting for 12 hours at the temperature of-4 ℃, and purifying by a G-25 gel chromatography column after the reaction is finished to obtain the methoxy adrenaline glucose-6-phosphate dehydrogenase labeled conjugate.
Preferably, in the application of the anti-methoxyepinephrine specific antibody, the methoxyepinephrine latex enhanced immunoturbidimetry detection reagent consists of an L1 reagent and an L2 reagent;
the L1 reagent consists of the anti-methoxy-epinephrine-specific antibody, a buffer solution with the pH =8.0, bovine serum albumin, sodium chloride, tween-20, glycerol, ethylene diamine tetraacetic acid, a coagulant and a preservative;
the L2 reagent consists of polystyrene latex particles coated by a methoxy adrenaline-bovine serum albumin complex, a buffer solution with the pH =8.0, bovine serum albumin, sodium chloride, tween-20, glycerol, ethylene diamine tetraacetic acid and a preservative;
the methoxy adrenaline-bovine serum albumin complex is formed by coupling the methoxy adrenaline derivative shown in the structural formula I and bovine serum albumin, and the structural formula is shown in a formula IV:
the diameter range of the polystyrene latex particles is 50-250nm;
the buffer solution is one of phosphate buffer solution, glycine buffer solution, MES buffer solution, borate buffer solution, tris-HCl buffer solution or barbital buffer solution;
the coagulant is one of PEG-4000, PEG-6000, PEG-8000 or dextran sodium sulfate;
the preservative is one of sodium azide, thimerosal, phenol or ethylmercuric sodium thiosulfate.
Specifically, the preparation method of the methoxy epinephrine latex enhanced immunoturbidimetric assay reagent comprises the following steps:
(F1) Dissolving 5.0mL of an anti-methoxyepinephrine-specific antibody in 250.0mL of potassium phosphate buffer (50.0 mmol/L pH = 8.0), adding 100.0mg of bovine serum albumin, 25.0mg of sodium chloride, 250.0. Mu.L of Tween-20, 250.0. Mu.L of glycerol, 100.0. Mu.L of ethylenediaminetetraacetic acid, 150.0. Mu.L of PEG-4000, and 5.0mg of sodium azide, stirring the mixture uniformly, and adjusting the pH =7.3 to prepare an L1 reagent;
(F2) 1.5mg of polystyrene latex particles with a carboxyl group on the surface and a diameter of 125nm are added into 15.0mL of MES buffer (50.0 mmol/L, pH = 7.0), then 5.0mg of carbodiimide is added, reaction is carried out at 25 ℃ for 3 hours to prepare latex particle solution, 1.2mg of methoxyepinephrine-bovine serum albumin complex is diluted with 7.5mL of borate buffer (50.0 mmol/L, pH = 9.2) and immediately added into the latex particle solution, reaction is carried out at 41 ℃ for 18 hours, then 3.0mL of glycine buffer (100.0 mmol/L, pH = 8.0) is added and stirred for 3 hours, after the reaction is terminated, the supernatant is centrifuged, the precipitate is washed with 20.0mL of Tris-HCl buffer (50.0 mmol/L, pH = 8.0) for 3 times, then 50.0mL of glycine buffer (50.0 mmol/L, pH = 8.6) is diluted into suspension, and finally, tween 0.0mg, tween 0.0, 0.0.0.0.0, sodium chloride, disodium azide and ethylene diamine tetraacetate (100.0.0.0.0.0.0.0.0.0.0 mg, 250. Mu.0.0.0.0.0.0L and stirring are added to prepare homogeneous suspension and stirring is added to prepare EDTA.
The preparation method of the methoxy adrenaline-bovine serum albumin complex comprises the following steps:
10.0mg of bovine serum albumin was diluted with 7.5mL of sodium phosphate buffer (100.0 mmol/L, pH = 7.5), then 100.0mg of the above-mentioned methoxy epinephrine derivative represented by the formula I was added, 50.0mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide was further added, and the reaction was carried out at 0 ℃ for 10 hours, followed by dialysis against 100.0mL of phosphate buffer (100.0 mmol/L, pH = 7.5) at-4 ℃ for 12 hours to obtain a methoxy epinephrine-bovine serum albumin complex.
Compared with the prior art, the invention has the beneficial effects that:
1. the methoxy adrenaline derivative and the synthetic method thereof designed by the invention are targeted new design and research and do not exist in the prior art;
2. the anti-methoxy-epinephrine specific antibody prepared from the methoxy-epinephrine derivatives has strong specificity and high sensitivity, and does not have any cross reaction with 32 common hormones and hormone metabolites, so that the anti-methoxy-epinephrine specific antibody can be used for preparing a methoxy-epinephrine detection reagent with higher accuracy, precision, sensitivity and specificity;
3. the two methoxy adrenaline detection reagents can realize high-flux and rapid detection of the methoxy adrenaline on a full-automatic biochemical analyzer, can simultaneously detect a plurality of samples, have the advantages of simple and convenient operation, high sensitivity, strong specificity, accurate result and the like, can effectively reduce the detection cost of the methoxy adrenaline, and are favorable for clinical popularization and use.
Drawings
FIG. 1 is a standard curve for ELISA detection of methoxyepinephrine in example 4;
FIG. 2 is a calibration curve of the homogeneous enzyme immunoassay reagent for synephrine of example 8;
FIG. 3 is a calibration curve of the methoxyepinephrine latex-enhanced immunoturbidimetric assay of example 10.
Detailed Description
The present invention will be further described with reference to the accompanying drawings, which are simplified schematic drawings illustrating only the basic structure of the present invention and showing only the constitution related to the present invention, and detailed embodiments. Unless otherwise specified, reagents, instruments, equipment, consumables used in the following examples are all available from a regular distributor.
Example 1: synthesis of methoxy adrenaline derivatives
Synthesis of methoxy adrenergic derivatives by the following synthetic route:
the specific synthetic steps are as follows:
(1) Synthesis of Compound 3:
50.0g of methoxyepinephrine, 25.0g of Compound 2 are weighed out and dissolved together in 250mL of N, N-Dimethylformamide (DMF), and 20.0g of K is added 2 CO 3 Preparing a reaction mixed solution; the reaction mixture was stirred at room temperature for 12 hours; after the reaction is finished, adding 250mL of purified water into the reaction mixed solution, then extracting with 200mL of Dichloromethane (DCM), and repeating the extraction step for 3 times; passing the combined organic phase obtained by extraction through Na 2 SO 4 Drying and concentrating; the residue obtained after concentration is passed throughPurification on a silica gel drying column (PE: EA = 5:1) gave 26.0g of compound 3 in 52% yield.
(2) Synthesis of methoxy adrenaline derivatives:
5.0g of Compound 3 and 0.75g of benzyltriethylammonium chloride were weighed out and dissolved in 50mL of chloroform (CHCl) 3 ) Preparing a reaction solution; dropwise adding NaOH solution with the concentration of 0.55g/mL into the reaction solution at the temperature of 65 ℃, and stirring for 5.0 hours at the temperature of 65 ℃; adjusting the pH of the obtained residue after the reaction to be =6 by using 1N HCI, carrying out acidification treatment, then extracting by using 150mL Ethyl Acetate (EA), and repeating the extraction step for 3 times; drying and concentrating the combined organic phase obtained by extraction; the residue obtained after the concentration was purified by pre-HPLC to obtain 0.88g of a compound as a white solid, i.e., a methoxyepinephrine derivative, in a yield of 17.6%.
Example 2: preparation of Methoxyarenol immunogens
The preparation method of the methoxy adrenaline immunogen comprises the following specific steps:
(1) Preparation of the carrier solution: dissolving a carrier protein in 0.35mol/L potassium phosphate buffer (pH = 8.5) to give a carrier protein solution at a final concentration of 5.0 mg/mL;
(2) Preparation of a solution of a methoxy adrenaline derivative: mixing 250.0mg of the above-mentioned methoxy epinephrine derivative, 7.5mL of dimethylformamide, 7.5mL of ethanol, 15.0mL of potassium phosphate buffer (10.0 mmol/L, pH = 8.0), 150.0mg of 1-ethyl-3- (-3-dimethylaminopropyl) carbodiimide, and 90.0mg of N-hydroxythiosuccinimide, and stirring and dissolving the mixture for reaction for 3 hours to obtain a methoxy epinephrine derivative solution;
(3) Synthesis of methoxy-adrenaline immunogen: and (3) dropwise adding the methoxy adrenaline derivative solution obtained in the step (2) into the carrier protein solution obtained in the step (1), stirring overnight at-4 ℃, and purifying by dialysis to obtain the methoxy adrenaline immunogen.
Example 3: preparation of anti-methoxy-adrenaline specific antibody
The preparation method of the anti-methoxy adrenaline specific antibody comprises the following specific steps:
(1) Diluting the methoxyepinephrine immunogen with 0.15mol/L sodium phosphate buffer (pH = 7.0) to a final concentration of 3.5mg/mL to obtain an artificial antigen solution, mixing the 3.0mL artificial antigen solution with an equivalent amount of Freund's complete adjuvant, and performing multi-point injection on the experimental animal rabbit;
(2) After 4 weeks, 3.0mL of the same artificial antigen solution and the same amount of Freund's incomplete adjuvant are injected into the rabbit of the experimental animal in a multi-point way, and then the injection is performed once every 5 weeks for 6 times in total;
(3) And (3) taking blood from the experimental animal rabbit injected in the step (2), separating and purifying to obtain the anti-methoxy-epinephrine specific antibody.
Example 4: ELISA method for testing performance of anti-methoxyepinephrine-specific antibody
1. Establishment of an ELISA detection standard curve for methoxyadrenalin:
(1) Preparation of a standard substance:
a stock solution of 1mg/mL was prepared by dissolving a pure powder of methoxyepinephrine (purchased from Sigma) in a methanol solution. The stock solutions were diluted sequentially with ELISA buffer to 160.0ng/mL, 80.0ng/mL, 40.0ng/mL, 20.0ng/mL, 10.0ng/mL, 0.0ng/mL of standard solution. Wherein, the ELISA buffer solution is prepared by 50.0mmol/L Tris buffer solution, 1.5 percent of NaCl by mass fraction and 0.25 percent of BSA by volume fraction.
(2) Standard curves were prepared using ELISA assay for methoxyepinephrine:
the anti-methoxy-adrenaline specific antibody prepared in example 3 was diluted with potassium phosphate buffer (50.0 mmol/L, pH = 8.0) to a final concentration of 1: 10000, coated at 100 μ L/well on a 96-well enzyme-linked plate, and left at 4 ℃ for 18 hours; after the 96 Kong Mei conjugate plates coated with the anti-methoxyepinephrine-specific antibody were washed 3 times with potassium phosphate buffer, 200. Mu.L/well of 0.5% volume fraction BSA solution was added and left at 4 ℃ for 12 hours. Then, the mixture was washed 3 times with potassium phosphate buffer, and 20. Mu.L/well of a standard solution was added. Then adding HRP-methoxy adrenaline conjugate with working concentration of 100 mu L/hole; after incubation for 30min at room temperature, the plate is washed 5 times by potassium phosphate buffer; then 100. Mu.L of TMB substrate was added to each well and incubated at room temperature for 30min. Then, 100. Mu.L of a stop solution (2.0 mol/L sulfuric acid) was added to each well. The absorbance at 450nm was measured using a microplate reader. The standard curve was prepared by calibration based on the absorbance at 450nm corresponding to each standard solution, and the results are shown in FIG. 1.
Detecting the content of the methoxy adrenaline in the sample to be detected:
(1) Preparing a sample to be tested:
the preparation method comprises the following steps: a stock solution of 1.0mg/mL was prepared by dissolving a pure powder of methoxyepinephrine (purchased from Sigma) in a methanol solution, and the stock solution was diluted in blank urine to final concentrations of 0.0ng/mL, 20.0ng/mL, 80.0ng/mL, and 160.0ng/mL, thereby forming blank, low, medium, and high concentration urine samples, respectively. The blank urine is urine of a healthy human without the methoxyl adrenaline.
(2) The test method comprises the following steps:
and (3) using the ELISA test method for the methoxyadrenaline, replacing the blank, low, medium and high concentration urine samples with standard solutions, and testing the absorbance values of the blank, low, medium and high concentration urine samples at 450 nm.
(3) And (3) testing results:
the amount of methoxyepinephrine in each sample was calculated and 3 replicate well assays were performed on each sample in comparison to the standard curve for the ELISA assay of methoxyepinephrine shown in figure 1, and the recovery was calculated based on the actual amount of methoxyepinephrine in the sample as described above and is shown in table 1.
TABLE 1 results of ELISA detection of Methoxyprenol
| Urine sample | Blank space | Low value | Median value | High value |
| Sample concentration (ng/mL) | 0.00 | 20.00 | 80.00 | 160.00 |
| |
0.00 | 20.76 | 80.99 | 162.20 |
| |
0.00 | 20.51 | 79.27 | 159.78 |
| Measurement 3 | 0.00 | 19.96 | 81.03 | 161.00 |
| Mean value (ng/mL) | 0.00 | 20.41 | 80.43 | 160.99 |
| Recovery (%) | - | 102.1 | 100.5 | 100.6 |
From the results in table 1, it can be seen that: the recovery rates of the methoxy adrenaline in samples with different concentrations are high and are 97-103% by using the ELISA detection method of the methoxy adrenaline specific antibody, which indicates that the anti-methoxy adrenaline specific antibody can be used for detecting the methoxy adrenaline in the samples, and has high sensitivity and high accuracy of detection results.
Example 5:32 common hormones and hormone metabolites interference experiment
32 common hormones and hormone metabolites are selected as interferents to carry out an interference experiment, the interferents are prepared into a sample to be detected with the concentration of 100.0ng/mL, the concentration of the corresponding interferents is detected by adopting the ELISA detection method of the embodiment 4, and the names and detection results of the 32 common hormones and hormone metabolites are detailed in a table 2.
TABLE 2 names of common interferents and test results
| Serial number | Name of interfering substance | Equivalent to methoxy adrenaline Concentration (ng/mL) | Serial number | Name of interfering substance | Concentration equivalent to that of methoxyproterenol (ng/mL) |
| 1 | Cortisol | 0.00 | 17 | Aldosterone | 0.00 |
| 2 | Androstenedione | 0.00 | 18 | Androsterone | 0.00 |
| 3 | Corticosterone | 0.00 | 19 | Corticosterone | 0.00 |
| 4 | Deoxycorticosterone | 0.00 | 20 | Dehydroepiandrosterone | 0.00 |
| 5 | Dehydroepiandrosterone sulfate | 0.00 | 21 | Dihydrotestosterone | 0.00 |
| 6 | Estradiol | 0.00 | 22 | Estriol | 0.00 |
| 7 | Estrone | 0.00 | 23 | The cholanol ketone | 0.00 |
| 8 | 17-hydroxypregnanolone | 0.00 | 24 | 17-hydroxyprogesterone | 0.00 |
| 9 | Pregnenolone | 0.00 | 25 | Progesterone | 0.00 |
| 10 | Testosterone | 0.00 | 26 | Pregnant triol | 0.00 |
| 11 | Pregnanediol | 0.00 | 27 | 17 alpha-hydroxyprogesterone | 0.00 |
| 12 | Androstenedione | 0.00 | 28 | 17-ketosteroids | 0.00 |
| 13 | 17-hydroxycorticosteroids | 0.00 | 29 | Adrenalin | 0.00 |
| 14 | Norepinephrine | 0.00 | 30 | Dopamine | 0.00 |
| 15 | Homovanillic acid | 0.00 | 31 | Dihydroxy mandelic acid | 0.00 |
| 16 | Vanillymandelic acid | 0.00 | 32 | Methoxy norepinephrine | 0.00 |
The measurement results show that: the concentrations of the 32 common hormones and hormone metabolites equivalent to the concentration of the methoxyproterenol are all 0.00ng/mL. Therefore, the anti-methoxy-epinephrine specific antibody provided by the invention has strong specificity and does not have any cross reaction with common interferents.
Example 6: preparation of methoxy adrenaline homogeneous enzyme immunoassay reagent
The preparation method of the methoxy adrenaline homogeneous enzyme immunoassay reagent comprises the following specific steps:
(1) Adding 250.0mg bovine serum albumin, 250.0mg glucose-6-phosphate and 50.0mg oxidized nicotinamide adenine dinucleotide into 250mL Tris buffer solution (50 mmol/L, pH = 8.5) in sequence, stirring and dissolving to prepare R1 buffer solution, adding the anti-methoxy-adrenaline specific antibody into the R1 buffer solution according to the volume ratio of 1: 1000, mixing uniformly, and adjusting the pH to 7.6 by using 1.0 mol/L hydrochloric acid to prepare an R1 reagent;
(2) 250.0mg of bovine serum albumin was added to 250mL of Tris buffer (100 mmol/L, pH = 8.7) and stirred to dissolve it to prepare an R2 buffer, and then a methoxy-adrenaline glucose-6-phosphate dehydrogenase labeled conjugate was added to the above R2 buffer at a volume ratio of 1: 1000 and mixed, and then pH was adjusted to 8.0 with 1.0 mol/L hydrochloric acid to prepare an R2 reagent.
The preparation method of the methoxy adrenaline glucose-6-phosphate dehydrogenase labeled conjugate comprises the following steps:
(1) Weighing glucose-6-phosphate dehydrogenase with 20.0 mg activity unit of 200KU, dissolving in 50.0mL sodium phosphate (100 mmol/L, pH = 8.0) buffer solution at room temperature, adding 150.0mg reduced nicotinamide adenine dinucleotide, 75.0 mg glucose-6-phosphate and 0.75 mL carbitol, dropwise adding 2.5 mL dimethyl sulfoxide, stirring and dissolving to obtain glucose-6-phosphate dehydrogenase solution;
(2) Weighing 15.0 mg of the methoxyadrenaline derivative synthesized in example 1 in an anhydrous state, dissolving the methoxyadrenaline derivative in 500.0 muL of dimethylformamide, cooling the solution to 0 ℃, adding 4.5 muL of tributylamine, 2.5 muL of isobutyl chloroformate and 3.5 muL of L N, N' -dicyclohexylcarbodiimide, and stirring for 45 minutes at 0 ℃ to obtain a methoxyadrenaline derivative activation solution;
(3) And dropwise adding the methoxy adrenaline derivative activating solution into a glucose-6-phosphate dehydrogenase solution, stirring and reacting for 12 hours at the temperature of-4 ℃, and purifying by a G-25 gel chromatography column after the reaction is finished to obtain the methoxy adrenaline glucose-6-phosphate dehydrogenase labeled conjugate.
Example 7: preparation of methoxy adrenaline calibration material and quality control material
(1) Preparation of a calibrator: adding the pure methoxyepinephrine powder into 6 parts of Tris-HCl buffer solution with the concentration of 50.0mmol/L and the pH =7.2 respectively, stirring and dissolving the mixture until the final concentration is 0.00ng/mL, 33.30ng/mL, 83.20ng/mL, 208.00ng/mL, 520.00ng/mL and 1300.00ng/mL respectively, then adding sodium chloride with the mass fraction of 0.5%, bovine serum albumin with the mass fraction of 1.0%, ethylenediamine tetraacetic acid with the mass fraction of 0.75% and sodium azide with the mass fraction of 0.05% into each solution, and stirring the mixture uniformly to obtain the methoxyepinephrine calibrator (with the concentration of 6).
(2) Preparing a quality control product: respectively adding the pure methoxyepinephrine powder into 3 parts of Tris-HCl buffer solution with the concentration of 50.0mmol/L and the pH =7.2, stirring and dissolving until the final concentrations are respectively 0.00ng/mL, 100.00ng/mL and 400.00ng/mL, then respectively adding 0.5% sodium chloride, 1.0% bovine serum albumin, 0.75% ethylene diamine tetraacetic acid and 0.05% sodium azide into each solution, and uniformly stirring to obtain the methoxyepinephrine quality control product (with 3 concentrations).
Example 8: calibration curve making and quality control experiment of methoxy adrenaline homogeneous enzyme immunoassay reagent
1. Preparing a calibration curve for the homogeneous enzyme immunoassay of the methoxy adrenaline:
placing an R1 reagent, an R2 reagent and a calibrator into a Mirey BS480 full-automatic biochemical analyzer, and then setting reaction parameters of the biochemical analyzer, wherein the specific parameters are detailed in a table 3; in the actual operation process, the volume ratio of the R1 reagent and the R2 reagent needs to be continuously adjusted, the light measuring point is adjusted at the same time, and finally, a homogeneous enzyme immunoassay calibration curve is automatically obtained by a biochemical analyzer, as shown in figure 2.
TABLE 3 Merrill BS480 full-automatic biochemical analyzer reaction parameter settings
| Name of item | Methoxyproterenol |
| R1 reagent | 160.0µL |
| R2 reagent | 40.0µL |
| Sample size | 10.0µL |
| Calibration method | Two-point end point method |
| Dominant wavelength | 340nm |
| Sub-wavelength | 405nm |
| Reaction time | 10 minutes |
| Incubation time | 8 minutes |
| Reaction direction | Rise up |
| Results | ng/mL |
| Accuracy of results | 0.01 |
| Fitting method | Line graph |
| Concentration of calibrator | 0.00ng/mL、33.30ng/mL、83.20ng/mL、208.00ng/mL、520.00ng/mL、1300.00ng/mL |
2. Quality control quality detection experiment:
the quality control products are measured by using the homogeneous enzyme immunoassay method for the methoxy adrenaline, the content of the methoxy adrenaline in each quality control product is calculated according to the homogeneous enzyme immunoassay calibration curve prepared in the step 1, each quality control product is repeatedly measured for 10 times, and the detection results and data analysis are detailed in a table 4.
TABLE 4 Methoxyproterenol homogeneous enzyme immunoassay reagent test results and data analysis
| Quality control product | Blank space | Low value of | High value |
| Concentration (ng/mL) | 0.00 | 100.00 | 400.00 |
| |
0.00 | 106.0 | 420.10 |
| |
0.00 | 95.40 | 388.70 |
| Test 3 | 0.00 | 98.10 | 392.40 |
| Test 4 | 0.00 | 99.60 | 391.70 |
| Test 5 | 0.00 | 95.10 | 423.30 |
| Test 6 | 0.00 | 98.00 | 402.70 |
| Test 7 | 0.00 | 103.50 | 398.00 |
| Test 8 | 0.00 | 98.70 | 390.60 |
| Test 9 | 0.00 | 97.30 | 397.20 |
| Test 10 | 0.00 | 97.70 | 398.40 |
| Mean value (ng/mL) | 0.00 | 98.94 | 400.31 |
| Standard Deviation (SD) | / | 3.40 | 12.07 |
| Precision (CV%) | / | 3.44 | 3.01 |
| Recovery (%) | / | 98.94 | 100.08 |
The experimental results show that: the CV values of the measured contents of the methoxy adrenaline in the quality control products with different concentrations are all lower than 5%, and the recovery rates are all between 95% and 105%, which shows that the precision of the methoxy adrenaline homogeneous enzyme immunoassay reagent for measuring the content of the methoxy adrenaline in a biological sample is higher, and the result is accurate.
Example 9: preparation of methoxy adrenaline latex enhanced immunoturbidimetry detection reagent
The preparation method of the methoxy epinephrine latex enhanced immunoturbidimetric assay reagent comprises the following steps:
(F1) Dissolving 5.0mL of an anti-methoxyepinephrine-specific antibody in 250.0mL of potassium phosphate buffer (50.0 mmol/L pH = 8.0), adding 100.0mg of bovine serum albumin, 25.0mg of sodium chloride, 250.0. Mu.L of Tween-20, 250.0. Mu.L of glycerol, 100.0. Mu.L of ethylenediaminetetraacetic acid, 150.0. Mu.L of PEG-4000, and 5.0mg of sodium azide, stirring the mixture uniformly, and adjusting the pH =7.3 to prepare an L1 reagent;
(F2) 1.5mg of polystyrene latex particles having a diameter of 125nm and surface carboxyl groups are added to 15.0mL of MES buffer (50.0 mmol/L, pH = 7.0), 5.0mg of carbodiimide is then added, the mixture is reacted at 25 ℃ for 3 hours to prepare a latex particle solution, 1.2mg of the methoxyepinephrine-bovine serum albumin complex is diluted with 7.5mL of borate buffer (50.0 mmol/L, pH = 9.2), the resulting solution is immediately added to the latex particle solution, the resulting solution is reacted at 41 ℃ for 18 hours, 3.0mL of glycine buffer (100.0 mmol/L, pH = 8.0) is then added and stirred for 3 hours, the supernatant is removed by centrifugation after the reaction is terminated, the precipitate is washed with 20.0mL of 20.0 mmol/L of HCl buffer (50.0 mmol/L, pH = 8.0) for 3 times, the precipitate is then diluted with 50.0mL of glycine buffer (50.0 mmol/L, pH = 8.6) to prepare a latex suspension, and Tris buffer (100.0 mg of Tween, 25.0, 0.0.0 mg of albumin, 0.0.0.0, 250. Mu.0.0.0 mg of sodium chloride, 250. Mu.0.0.0.0.0L of EDTA, and a reagent of disodium chloride is added and stirred to prepare a uniform suspension.
The preparation method of the methoxy adrenaline-bovine serum albumin complex comprises the following steps:
10.0mg of bovine serum albumin was diluted with 7.5mL of sodium phosphate buffer (100.0 mmol/L, pH = 7.5), then 100.0mg of the methoxyepinephrine derivative synthesized in example 1 was added, 50.0mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide was further added, and the mixture was reacted at 0 ℃ for 10 hours, followed by dialysis at-4 ℃ for 12 hours with 100.0mL of phosphate buffer (100.0 mmol/L, pH = 7.5), to obtain a methoxyepinephrine-bovine serum albumin complex.
Example 10: preparation of calibration curve of methoxy adrenaline latex enhanced immunoturbidimetry detection reagent and quality control experiment
1. Preparing a calibration curve of the methoxy epinephrine latex enhanced immunoturbidimetric assay reagent:
putting an L1 reagent, an L2 reagent and a calibrator into an Olympus AU480 full-automatic biochemical analyzer, and then setting reaction parameters of the biochemical analyzer, wherein the detailed parameters are shown in a table 5; in the actual operation process, the volume ratio of the L1 reagent and the L2 reagent needs to be continuously adjusted, the light measuring point is adjusted at the same time, and finally, a latex enhanced immunoturbidimetric assay calibration curve is automatically obtained by a biochemical analyzer, as shown in FIG. 3.
TABLE 5 fully automatic Biochemical Analyzer reaction parameters of Orlinbas AU480
| Name of item | Methoxyproterenol |
| L1 reagent | 160.0µL |
| L2 reagent | 40.0µL |
| Sample size | 10.0µL |
| Calibration method | Two-point end point method |
| Dominant wavelength | 570nm |
| Sub-wavelength | 412nm |
| Reaction time | 10 minutes |
| Incubation time | 5 minutes |
| Reaction direction | Descend |
| Results | ng/mL |
| Accuracy of results | 0.01 |
| Fitting method | Logit-log 4P |
| Concentration of calibrator | 0.00ng/mL、33.30ng/mL、83.20ng/mL、208.00ng/mL、520.00ng/mL、1300.00ng/mL |
2. Quality control quality detection experiment:
the latex enhanced turbidimetric immunoassay method is utilized to measure the quality control products, the content of the methoxyl adrenaline in each quality control product is calculated according to the latex enhanced turbidimetric immunoassay calibration curve prepared in the step 1, each quality control product is repeatedly measured for 10 times, and the detection results and data analysis are detailed in table 6.
TABLE 6 Methoxyarenol latex-enhanced immunoturbidimetric reagent detection results and data analysis
| Quality control product | Blank space | Low value | High value |
| Concentration (ng/mL) | 0.00 | 100.00 | 400.00 |
| |
0.00 | 100.35 | 396.09 |
| |
0.00 | 100.87 | 398.45 |
| Test 3 | 0.00 | 99.78 | 402.26 |
| Test 4 | 0.00 | 100.25 | 399.43 |
| Test 5 | 0.00 | 100.00 | 400.18 |
| Test 6 | 0.00 | 100.50 | 403.94 |
| Test 7 | 0.00 | 100.06 | 401.50 |
| Test 8 | 0.00 | 99.98 | 397.82 |
| Test 9 | 0.00 | 109.42 | 398.75 |
| Test 10 | 0.00 | 99.66 | 401.00 |
| Mean value (ng/mL) | 0.00 | 101.09 | 399.94 |
| Standard Deviation (SD) | / | 2.95 | 2.31 |
| Precision (CV%) | / | 2.92 | 0.58 |
| Recovery (%) | / | 101.09 | 99.99 |
The experimental results show that: the CV values for measuring the content of the methoxy adrenaline in the quality control products with different concentrations are all lower than 5%, and the recovery rates are all between 95% and 105%, which shows that the precision of the methoxy adrenaline latex enhanced immunoturbidimetric assay reagent for measuring the content of the methoxy adrenaline in a biological sample is higher, and the result is accurate.
Various other changes and modifications to the above-described embodiments and concepts will become apparent to those skilled in the art, and all such changes and modifications are intended to be included within the scope of the present invention as defined in the appended claims.
Sequence listing
<110> Hunan Su Yang medical science and technology Limited
<120> methoxy adrenaline derivative, immunogen, anti-methoxy adrenaline specific antibody, preparation method and application thereof
<130> 2020.09.14
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
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Met Lys Trp Val Thr Phe Ile Ser Leu Leu Leu Leu Phe Ser Ser Ala
1 5 10 15
Tyr Ser Arg Gly Val Phe Arg Arg Asp Thr His Lys Lys Ser Glu Ile
20 25 30
Ala His Arg Phe Lys Asp Leu Gly Glu Glu His Phe Lys Gly Leu Val
35 40 45
Leu Ile Ala Phe Ser Gln Tyr Leu Gln Gln Cys Pro Phe Asp Glu His
50 55 60
Val Lys Lys Leu Val Asn Glu Leu Thr Glu Phe Ala Lys Lys Thr Cys
65 70 75 80
Val Ala Asp Glu Ser His Ala Gly Cys Glu Lys Ser Leu His Thr Leu
85 90 95
Phe Gly Asp Glu Leu Cys Lys Lys Val Ala Ser Leu Arg Glu Thr Tyr
100 105 110
Gly Asp Met Ala Asp Cys Cys Glu Lys Gln Glu Pro Glu Arg Asn Glu
115 120 125
Cys Phe Leu Ser His Lys Asp Asp Ser Pro Asp Leu Pro Lys Leu Lys
130 135 140
Pro Asp Pro Asn Thr Leu Cys Asp Glu Phe Lys Ala Asp Glu Lys Lys
145 150 155 160
Phe Trp Gly Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe
165 170 175
Tyr Ala Pro Glu Leu Leu Tyr Tyr Ala Asn Lys Lys Tyr Asn Gly Val
180 185 190
Phe Gln Glu Cys Cys Gln Ala Glu Asp Lys Gly Ala Cys Leu Leu Pro
195 200 205
Lys Lys Ile Glu Thr Met Arg Glu Lys Val Leu Thr Ser Ser Ala Arg
210 215 220
Gln Arg Leu Arg Cys Ala Ser Ile Gln Lys Lys Phe Gly Glu Arg Ala
225 230 235 240
Leu Lys Ala Trp Ser Val Ala Arg Leu Ser Gln Lys Phe Pro Lys Ala
245 250 255
Glu Phe Val Glu Val Thr Lys Lys Leu Val Thr Asp Leu Thr Lys Val
260 265 270
His Lys Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp Arg
275 280 285
Ala Asp Leu Ala Lys Tyr Ile Cys Asp Asn Gln Asp Thr Ile Ser Ser
290 295 300
Lys Leu Lys Lys Glu Cys Cys Asp Lys Pro Leu Leu Glu Lys Ser His
305 310 315 320
Cys Ile Ala Glu Val Glu Lys Asp Ala Ile Pro Glu Asn Leu Pro Pro
325 330 335
Leu Thr Ala Asp Phe Ala Glu Asp Lys Lys Asp Val Cys Lys Asn Tyr
340 345 350
Gln Glu Ala Lys Asp Ala Phe Leu Gly Ser Phe Leu Tyr Glu Tyr Ser
355 360 365
Arg Arg His Pro Glu Tyr Ala Val Ser Val Leu Leu Arg Leu Ala Lys
370 375 380
Lys Glu Tyr Glu Ala Thr Leu Glu Glu Cys Cys Ala Lys Asp Asp Pro
385 390 395 400
His Ala Cys Tyr Ser Thr Val Phe Asp Lys Leu Lys Lys His Leu Val
405 410 415
Asp Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Asp Gln Phe Glu Lys
420 425 430
Leu Gly Glu Tyr Gly Phe Gln Asn Ala Leu Ile Val Arg Tyr Thr Arg
435 440 445
Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg
450 455 460
Ser Leu Gly Lys Val Gly Thr Arg Cys Cys Thr Lys Lys Pro Glu Ser
465 470 475 480
Glu Arg Met Pro Cys Thr Glu Asp Tyr Leu Ser Leu Ile Leu Asn Arg
485 490 495
Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Glu Lys Lys Val Thr
500 505 510
Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala
515 520 525
Leu Thr Pro Asp Glu Thr Tyr Val Pro Lys Ala Phe Asp Glu Lys Leu
530 535 540
Phe Thr Phe His Ala Asp Ile Cys Thr Leu Pro Asp Thr Glu Lys Lys
545 550 555 560
Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Leu Lys His Lys Pro
565 570 575
Lys Ala Thr Glu Glu Gln Leu Lys Lys Thr Val Met Glu Asn Phe Val
580 585 590
Ala Phe Val Asp Lys Cys Cys Ala Ala Asp Asp Lys Glu Ala Cys Phe
595 600 605
Ala Val Glu Gly Pro Lys Lys Leu Val Val Ser Thr Gln Thr Ala Leu
610 615 620
Ala
625
Claims (1)
1. A methoxy adrenaline detection reagent prepared by applying an anti-methoxy adrenaline specific antibody is characterized in that the methoxy adrenaline detection reagent is a methoxy adrenaline homogeneous enzyme immunoassay reagent or a methoxy adrenaline latex enhanced immunoturbidimetry detection reagent;
the methoxy adrenaline homogeneous enzyme immunoassay reagent consists of an R1 reagent and an R2 reagent, wherein the R1 reagent comprises the anti-methoxy adrenaline specific antibody and an R1 buffer solution, and the R2 reagent comprises a methoxy adrenaline glucose-6-phosphate dehydrogenase labeled conjugate and an R2 buffer solution;
the R1 buffer solution contains an enzyme substrate, a coenzyme, bovine serum albumin and a Tris buffer solution, wherein the enzyme substrate is glucose-6-phosphate, and the coenzyme is oxidized nicotinamide adenine dinucleotide;
the methoxy adrenaline glucose-6-phosphate dehydrogenase marker conjugate is formed by coupling a methoxy adrenaline derivative and glucose-6-phosphate dehydrogenase; the structural formula is shown as formula III:
the R2 buffer solution is a Tris buffer solution containing bovine serum albumin;
the methoxy adrenaline latex enhanced immunoturbidimetry detection reagent consists of an L1 reagent and an L2 reagent;
the L1 reagent consists of the anti-methoxy-epinephrine-specific antibody, a buffer solution with the pH =8.0, bovine serum albumin, sodium chloride, tween-20, glycerol, ethylene diamine tetraacetic acid, a coagulant and a preservative;
the L2 reagent consists of polystyrene latex particles coated by a methoxy epinephrine-bovine serum albumin complex, a buffer solution with the pH =8.0, bovine serum albumin, sodium chloride, tween-20, glycerol, ethylene diamine tetraacetic acid and a preservative;
the methoxy adrenaline-bovine serum albumin complex is formed by coupling a methoxy adrenaline derivative and bovine serum albumin, and the structural formula of the methoxy adrenaline-bovine serum albumin complex is shown as a formula IV:
the diameter range of the polystyrene latex particles is 50-250nm;
the buffer solution is one of phosphate buffer solution, glycine buffer solution, MES buffer solution, borate buffer solution, tris-HCl buffer solution or barbital buffer solution;
the coagulant is one of PEG-4000, PEG-6000, PEG-8000 or dextran sodium sulfate;
the preservative is one of sodium azide, thimerosal, phenol or thimerosal;
the anti-methoxy-epinephrine specific antibody is a specific antibody obtained by injecting a methoxy-epinephrine immunogen into an experimental animal, wherein the experimental animal is one of a rabbit, a goat, a sheep, a mouse, a rat, a guinea pig or a horse;
the methoxy adrenaline immunogen is formed by connecting a methoxy adrenaline derivative and carrier protein, and the structural formula of the methoxy adrenaline immunogen is shown as a formula II:
wherein the carrier protein is recombinant bovine serum albumin, and the amino acid sequence of the recombinant bovine serum albumin is shown in a sequence table SEQ ID NO:1 is shown in the specification;
the structural formula of the methoxy adrenaline derivative is shown as a formula I:
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