Melatonin amino derivative, immunogen, anti-melatonin specific antibody and preparation method and application thereof

Abstract

The invention provides a melatonin amino derivative, an immunogen, an anti-melatonin specific antibody and a preparation method and application thereof. The structural formula of the melatonin amino derivative is as shown in the formula (I): # imgabs0 # formula (I). A melatonin immunogen with high immunogenicity, a corresponding antibody, a melatonin enzyme-labeled conjugate and a melatonin homogeneous enzyme immunoassay reagent are obtained by using the melatonin amino derivative. The melatonin immunogen is strong in specificity and high in immunogenicity, the prepared melatonin antibody is strong in specificity and high in titer, the prepared homogeneous enzyme immunodetection reagent can rapidly and accurately determine the melatonin content in a biological sample, and a plurality of samples can be detected on a full-automatic biochemical analyzer at the same time; the high-flux and rapid detection of the melatonin is realized, and the sensitivity reaches a pg / mL level.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical detection, and specifically relates to a melatonin amino derivative, an immunogen, an anti-melatonin specific antibody, and a preparation method and application thereof. Background Art

[0002] Melatonin (MT) is a hormone related to biological rhythm secreted by the pineal gland in the brain, which controls the circadian rhythmic changes of various physiological activities. Circadian rhythm is the biological clock in the human body, which tells the human body when to sleep and when to wake up, and maintains a regular sleep and wake cycle. It is called the "sleep hormone". The secretion of melatonin is stimulated by darkness and inhibited by light. Melatonin exists in many cells and organs such as lymph, thymus, gastrointestinal tract and skin. It has a significant regulatory effect on the human immune, reproductive, endocrine and central nervous systems. It is also closely related to biological rhythms such as sleep and sedation. It has been clinically applied in many fields such as reproduction, hypnosis, anti-aging, immune regulation and anti-tumor. Melatonin has a broad-spectrum organ protection effect, and has a protective effect on cardiovascular disease in anti-oxidation, anti-inflammatory and hypertension, myocardial ischemia / reperfusion injury. Low levels of melatonin may cause insomnia, immunodeficiency, depression and other emotional disorders. Melatonin is fat-soluble. After being secreted by pineal cells, it enters the peripheral veins through the glandular capillary network and is transported in the blood after binding to albumin. The half-life is about 30-60 minutes. It is mainly metabolized by the liver. About 90% of it is inactivated by 6-hydroxylation catalyzed by liver microsomal hydroxylase to form 6-hydroxymelatonin, of which about 70-80% of 6-hydroxymelatonin combines with sulfate to form melatonin and is excreted in the urine. In addition, about 10% of 6-hydroxyglucuronic acid melatonin is also generated. 6-Hydroxymelatonin sulfate is the main metabolite of melatonin. The concentration of 6-hydroxymelatonin sulfate in urine can reflect the level of melatonin in the blood during the urine sample collection period.

[0003] Changes in melatonin levels can lead to sleep disorders, jet lag, depression, stress, schizophrenia, hypothalamic amenorrhea, anorexia nervosa, immune disorders, and precocious puberty. When melatonin levels in the body decrease, it can cause a variety of diseases and aggravate the clinical symptoms of the disease, resulting in adverse expected results, such as sleep disorders, insulin resistance and reduced levels, diabetic retinopathy, myocardial infarction, hypertension, schizophrenia, depression, cancer, precocious puberty, autism, etc. With the changes in time, season, and physical health, the level of melatonin in the body varies greatly. Judging the level of melatonin in the body by clinical observation of sleep quality alone is subjective and inaccurate. By monitoring the concentration of melatonin metabolites in urine, the level of melatonin in the body can be objectively reflected, and guidance can be given for the treatment of different diseases. Melatonin supplements should be taken for patients with melatonin deficiency. Melatonin supplements are popular as a sleep aid, but taking too much melatonin can cause drowsiness and lower core body temperature, extremely high levels of melatonin can cause headaches and fatigue, and large doses of melatonin may also affect human reproduction. Therefore, after taking melatonin, you need to regularly check the levels of melatonin and its metabolites in your body to avoid overdose.

[0004] At present, the laboratory methods for detecting melatonin and its metabolites include colorimetry, radioimmunoassay, high performance liquid chromatography, liquid phase tandem mass spectrometry, enzyme-linked immunosorbent assay, and immunoturbidimetry. The colorimetric method detects the sum of melatonin and 6-hydroxymelatonin sulfate and other substances rather than the level of a single compound; the radioimmunoassay method has poor stability and has problems such as radiation and pollution; the high performance liquid chromatography method has complex pretreatment and requires the target analyte to be completely separated on the chromatogram, so the analysis time is long; the liquid phase tandem mass spectrometry method has high sensitivity and fast speed, but the structure is complex and the maintenance cost is high. It requires specially trained technicians to operate and is not easy to popularize in primary medical institutions; the enzyme-linked immunosorbent assay method has high sensitivity and good specificity, but the detection time is long and the accuracy is poor, making it difficult to use for batch detection. Therefore, there is a lack of melatonin and its metabolite detection products on the market that have a wide linear range, high sensitivity, high accuracy, high precision, short detection time, simple sample processing, high degree of instrument automation, and can continuously detect multiple samples. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a melatonin amino derivative, an immunogen, an anti-melatonin specific antibody and a preparation method and application thereof, and to establish an immunoassay technology platform for high-throughput and rapid detection of melatonin.

[0006] The technical solution adopted by the present invention to solve its technical problem is:

[0007] 1. Provide a melatonin amino derivative, the structural formula of which is shown in the following formula (I):

[0008]

[0009] 2. Provide a method for preparing the above-mentioned melatonin amino derivative, the synthetic route of the preparation method is as follows:

[0010]

[0011] The specific preparation steps are as follows:

[0012] a. Synthesis of Compound A-2

[0013]

[0014] Compound A-1 (10 g, 61.3 mmol) was dissolved in dimethylformamide (80 mL), and then tert-butyl 3-bromopropylcarbamate (14.6 g, 61.3 mmol) and diisopropylethylamine (18.3 mL) were added to prepare a reaction mixture solution; the reaction mixture solution was stirred at 80°C for 12 hours; purified water (100 mL) was then added to the reaction mixture solution to terminate the reaction, and the solution obtained after the reaction was extracted with ethyl acetate (150 mL×4), and the extraction step was repeated 4 times; the organic phases obtained from each extraction were combined, and the combined organic phases were washed with brine, and then dried and concentrated over Na2SO4 to obtain a crude product; finally, the crude product was purified by a silica gel column (petroleum ether: ethyl acetate = 20:1) to obtain a white solid compound A-2.

[0015] b. Synthesis of Compound A

[0016]

[0017] Compound A-2 (12 g, 37.5 mmol) was dissolved in anhydrous ethanol (120 mL), and hydrazine hydrate (3.5 g, 53 mmol) was then added to prepare a reaction mixture solution; the reaction mixture solution was stirred at 70°C for 1 hour; after stirring, the reaction mixture solution was cooled to 10°C, and purified water (100 mL) was added to the reaction mixture solution to terminate the reaction, and the solution obtained after the reaction was extracted with dichloromethane (150 mL×4), and the extraction step was repeated 4 times; the organic phases obtained from each extraction were combined, and the combined organic phases were washed with brine, and then dried and concentrated by Na2SO4 to obtain a crude product; finally, the crude product was purified by a silica gel column to obtain a yellow oily compound A.

[0018] c. Synthesis of Compound 2

[0019]

[0020] Compound 1 (25 g, 0.1 mol) and ethyl chloroformate (11 g, 0.1 mol) were dissolved in dichloromethane (250 mL), and then NaOH (4 g, 0.1 mol) was added to prepare a reaction mixture solution, and the reaction mixture solution was stirred under N2 conditions for 12 hours; then the reaction mixture solution was diluted with ethyl acetate (150 mL), and then washed with brine, and dried with sodium sulfate; finally, the solvent was evaporated to obtain solid compound 2.

[0021] d. Synthesis of Compound 3

[0022]

[0023] Compound 2 (13 g, 42.7 mmol) was dissolved in tetrahydrofuran (200 mL), and then aluminum chloride (5.7 g, 47 mmol) and acetyl chloride (3.7 g, 47 mmol) were added to prepare a reaction mixture solution, and the reaction mixture solution was stirred at 25°C for 0.5 hour; then purified water (100 mL) was added to the reaction mixture solution to quench the reaction, and extraction was performed with ethyl acetate; the organic layer obtained by the extraction was concentrated and purified by silica gel column chromatography to obtain compound 3.

[0024] e. Synthesis of Compound 4

[0025]

[0026] Compound 3 (2.0 g, 5.8 mmol) was dissolved in dimethylformamide (20 mL), and then compound A (1.1 g, 5.8 mmol) and K2CO3 (2.4 g) were added to prepare a reaction mixture solution, and the reaction mixture solution was stirred at 80°C for 12 hours; purified water (30 mL) was then added to the reaction mixture solution, and then extracted with ethyl acetate; the organic layer obtained by the extraction was concentrated and purified by flash chromatography to obtain a brown solid compound 4.

[0027] f. Synthesis of amino derivatives of melatonin

[0028]

[0029] Compound 4 (1.2 g, 2.7 mmol) was dissolved in dichloromethane (10 mL), and trifluoroacetic acid (2 mL) was then added to prepare a reaction mixture solution, and the reaction mixture solution was stirred at 25° C. for 12 hours; the reaction mixture solution was then concentrated and purified by flash chromatography to obtain a melatonin amino derivative.

[0030] 3. Provide a melatonin immunogen, which is formed by connecting the above-mentioned melatonin amino derivative with keyhole limpet hemocyanin, and its structural formula is shown in the following formula (II):

[0031]

[0032] 4. Provide a method for preparing the above-mentioned melatonin immunogen, comprising the following steps:

[0033] A1. Weigh the melatonin amino derivative represented by the above structural formula (I), and dissolve it in sodium phosphate buffer to obtain a melatonin amino derivative solution;

[0034] A2. Weigh keyhole limpet hemocyanin, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, N-hydroxysulfosuccinimide, and dimethylformamide, dissolve them in potassium phosphate buffer, and then stir and react at 25° C. for 10 to 60 minutes to obtain a keyhole limpet hemocyanin active solution;

[0035] A3. Pour the keyhole limpet hemocyanin active solution obtained in step A2 directly into the melatonin amino derivative solution obtained in step A1 and mix well to obtain a coupling reaction mixed solution, and then stir the coupling reaction mixed solution at 25° C. for 10 to 60 minutes, and stand at 2 to 8° C. for 3 to 12 hours after the stirring to obtain a melatonin immunogen solution;

[0036] A4. The melatonin immunogen solution obtained in step A3 is purified by G-25 dextran gel chromatography to obtain a melatonin immunogen.

[0037] As a preferred embodiment of the present invention, the method for preparing the melatonin immunogen comprises the following steps:

[0038] a1. Weigh 10.0 mg of the amino derivative of melatonin represented by the above formula (Ⅰ) and dissolve it in 10.0 mL of sodium phosphate buffer (concentration of 0.1 mol / L, pH = 8.0) to obtain a solution of the amino derivative of melatonin;

[0039] a2. Weigh 30.0 mg of keyhole limpet hemocyanin, 3.0 mg of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, 10.0 mg of N-hydroxysulfosuccinimide, and 1.0 mL of dimethylformamide, dissolve in 10.0 mL of potassium phosphate buffer (concentration of 50.0 mmol / L, pH = 6.5), and then stir and react at 25 ° C for 90 minutes to obtain a keyhole limpet hemocyanin active solution;

[0040] a3. Pour the keyhole limpet hemocyanin active solution obtained in step a2 directly into the melatonin amino derivative solution obtained in step a1 and mix well to obtain a coupling reaction mixed solution, then stir the coupling reaction mixed solution at 25°C for 30 minutes, and after stirring, let it stand at 4°C for 8 hours to obtain a melatonin immunogen solution.

[0041] a4. The melatonin immunogen solution obtained in step a3 was purified by G-25 polysaccharide gel chromatography column (pre-equilibrated with 50.0 mmol / L, pH = 8.5 Tris buffer) to obtain the melatonin immunogen after purification, which was stored at -20°C.

[0042] 5. Provide an anti-melatonin specific antibody, which is a specific antibody produced by immunizing an experimental animal with the above-mentioned melatonin immunogen, and the experimental animal is one of rabbits, goats, mice, sheep, guinea pigs or horses; the anti-melatonin specific antibody is used to prepare a melatonin immunoassay reagent, and the melatonin immunoassay reagent includes a homogeneous enzyme immunoassay reagent, a latex-enhanced immunoturbidimetric assay reagent, a chemiluminescent immunoassay reagent, an enzyme-linked immunosorbent assay reagent, and an immunochromatographic assay reagent for detecting the melatonin content in a sample, and the immunochromatographic assay reagent includes a fluorescent immunochromatographic assay reagent and a colloidal gold immunochromatographic assay reagent.

[0043] 6. Provide a method for preparing the above-mentioned anti-melatonin specific antibody, comprising the following steps:

[0044] B1. The melatonin immunogen represented by the above formula (II) is diluted with sodium phosphate buffer to obtain an antigen solution, and then the antigen solution is mixed with an equal amount of Freund's complete adjuvant and injected into the experimental animal;

[0045] B2. After 2 to 5 weeks, mix the same antigen solution with an equal amount of Freund's incomplete adjuvant and inject the above experimental animals once, and then inject them once every 2 to 5 weeks, for a total of 5 to 8 injections;

[0046] B3. Take blood from the immunized experimental animals, separate and purify the antiserum, and obtain anti-melatonin specific antibodies.

[0047] As a preferred embodiment of the present invention, the method for preparing the above-mentioned anti-melatonin specific antibody comprises the following steps:

[0048] b1. The melatonin immunogen represented by the above formula (II) was diluted to 3.0 mg / mL with sodium phosphate buffer to obtain an antigen solution, and then 3.0 mL of the antigen solution was mixed with an equal amount of Freund's complete adjuvant and injected into the experimental animals;

[0049] b2. After 3 weeks, 3.0 mL of the same antigen solution mixed with an equal amount of Freund's incomplete adjuvant was injected into the above experimental animals once, and then injected once every 3 weeks, for a total of 6 injections;

[0050] b3. Take blood from the immunized experimental animals, separate and purify the antiserum, and obtain anti-melatonin specific antibodies. VII. Provide a melatonin enzyme-labeled conjugate, which is formed by connecting the above-mentioned melatonin amino derivative with glucose-6-phosphate dehydrogenase, and its structural formula is shown in the following formula (III):

[0051]

[0052] 8. Provide a method for preparing the above-mentioned melatonin enzyme-labeled conjugate, comprising the following steps:

[0053] C1. Weigh the melatonin amino derivative represented by the above structural formula (I), dissolve it in sodium phosphate buffer, then add dimethylformamide and glucose-6-phosphate and stir evenly to obtain a melatonin amino derivative solution;

[0054] C2. Weigh glucose-6-phosphate dehydrogenase and dissolve it in sodium phosphate buffer, lower the solution temperature to 2-8°C, then add N-hydroxysulfosuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide simultaneously, and slowly stir the reaction at 25°C for 5-20 minutes to obtain a glucose-6-phosphate dehydrogenase active solution;

[0055] C3. The temperature of the melatonin amino derivative solution prepared in step C1 and the glucose-6-phosphate dehydrogenase active solution prepared in step C2 are both reduced to 2-8° C., and then the melatonin amino derivative solution is added dropwise to the glucose-6-phosphate dehydrogenase active solution. The entire dropping process lasts for 3-5 minutes, and then the mixed solution is stirred at 2-8° C. for 2-12 hours to obtain a melatonin enzyme-labeled conjugate solution;

[0056] C4. The melatonin enzyme-labeled conjugate solution prepared in step C3 is purified by G-25 dextran gel chromatography column, and after purification, bovine serum albumin and sodium azide are added to obtain a melatonin enzyme-labeled conjugate.

[0057] As a preferred embodiment of the present invention, the method for preparing the above-mentioned melatonin enzyme-labeled conjugate comprises the following steps:

[0058] c1. Weigh 10.0 mg of the amino derivative of melatonin represented by the above formula (I), dissolve it in 10.0 mL of sodium phosphate buffer (concentration of 0.1 mol / L, pH = 8.0), then add 1.0 mL of dimethylformamide and 5.0 mg of glucose-6-phosphate and stir evenly to obtain a solution of the amino derivative of melatonin;

[0059] c2. Weigh 10.0 mg of glucose-6-phosphate dehydrogenase (G6PDH) and dissolve it in 10.0 mL of sodium phosphate buffer (concentration of 50.0 mmol / L, pH = 7.2), lower the solution temperature to 2-8 ° C, then add 5.0 mg of N-hydroxysulfosuccinimide and 10.0 mg of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide at the same time, and slowly stir the reaction at 25 ° C for 10 minutes to obtain a glucose-6-phosphate dehydrogenase active solution;

[0060] c3. The temperature of the melatonin amino derivative solution prepared in step c1 and the glucose-6-phosphate dehydrogenase active solution prepared in step c2 are both reduced to 2-8°C, and then the melatonin amino derivative solution is added dropwise to the glucose-6-phosphate dehydrogenase active solution. The entire dropping process lasts for 4 minutes, and then the mixed solution is stirred at 2-8°C for 8 hours to obtain a melatonin enzyme-labeled conjugate solution.

[0061] c4. The melatonin enzyme-labeled conjugate solution prepared in step c3 was purified by a G-25 dextran gel chromatography column (pre-equilibrated with 50.0 mmol / L, pH = 8.5 Tris buffer), and after purification, 0.3% by mass of bovine serum albumin and 0.05% by mass of sodium azide were added to obtain a melatonin enzyme-labeled conjugate.

[0062] 9. Provide a melatonin homogeneous enzyme immunoassay reagent, including R1 reagent and R2 reagent, wherein the R1 reagent comprises the above-mentioned anti-melatonin specific antibody and a homogeneous enzyme substrate solution, and the homogeneous enzyme substrate solution is prepared from glucose-6-phosphate and Tris buffer; the R2 reagent comprises the above-mentioned melatonin enzyme-labeled conjugate and a coenzyme solution, and the coenzyme solution is prepared from oxidized nicotinamide adenine dinucleotide and Tris buffer; when used, the R1 reagent and the R2 reagent are used in a volume ratio of 1:1 to 4:1, preferably in a volume ratio of 1:1.

[0063] 10. Provide a method for preparing the above-mentioned melatonin homogeneous enzyme immunoassay reagent, comprising the following steps:

[0064] D1. Preparation of R1 reagent: dissolving glucose-6-phosphate with Tris buffer to prepare a homogeneous enzyme substrate solution; then adding the above-mentioned anti-melatonin specific antibody to the homogeneous enzyme substrate solution, stirring evenly to obtain R1 reagent, wherein the volume ratio of the anti-melatonin specific antibody to the homogeneous enzyme substrate solution is 1:100 to 1:10000; preferably, the volume ratio of the anti-melatonin specific antibody to the homogeneous enzyme substrate solution is 1:1300;

[0065] D2. Preparation of R2 reagent: dissolve oxidized nicotinamide adenine dinucleotide in Tris buffer to prepare a coenzyme solution; then add the above-mentioned melatonin enzyme-labeled conjugate to the coenzyme solution, stir evenly to obtain R2 reagent, the volume ratio of the melatonin enzyme-labeled conjugate to the coenzyme solution is 1:100 to 1:10000; preferably, the volume ratio of the melatonin enzyme-labeled conjugate to the coenzyme solution is 1:1300.

[0066] As a preferred embodiment of the present invention, the preparation method of the above-mentioned melatonin homogeneous enzyme immunoassay reagent comprises the following steps:

[0067] d1. Preparation of R1 reagent: 10.0 g of glucose-6-phosphate was dissolved in 1.0 L of Tris buffer (80.0 mmol / L, pH=8.2) to prepare a homogeneous enzyme substrate solution; the above-mentioned anti-melatonin specific antibody was added to the homogeneous enzyme substrate solution, and stirred evenly to obtain R1 reagent, wherein the volume ratio of the anti-melatonin specific antibody to the homogeneous enzyme substrate solution was 1:1300;

[0068] d2. Preparation of R2 reagent: 10.0 g of oxidized nicotinamide adenine dinucleotide was dissolved in 1.0 L Tris buffer (100.0 mmol / L, pH = 8.5) to prepare a coenzyme solution; the above-mentioned melatonin enzyme-labeled conjugate was added to the coenzyme solution, and stirred evenly to obtain R2 reagent, wherein the volume ratio of the melatonin enzyme-labeled conjugate to the coenzyme solution was 1:1300.

[0069] The beneficial effects of the present invention are as follows: a new melatonin amino derivative is obtained, the melatonin amino derivative can be coupled with free -COOH in the carrier protein through the terminal -NH2 group, and the melatonin amino derivative is used to prepare a melatonin immunogen with strong immunogenicity, so as to immunize experimental animals to obtain anti-melatonin specific antibodies with high titer and strong specificity; at the same time, the melatonin amino derivative can be coupled with free -COOH in glucose-6-phosphate dehydrogenase through the terminal -NH2 group, and the melatonin enzyme-labeled conjugate is prepared using the melatonin amino derivative. The melatonin homogeneous enzyme immunoassay reagent prepared by using the anti-melatonin specific antibody and the enzyme-labeled conjugate can realize high-throughput and rapid detection of melatonin on a fully automatic biochemical analyzer, and the sensitivity reaches the pg / mL level.

[0070] Another beneficial effect of the present invention is that before the homogeneous enzyme immunoassay reagent is used, in order to prevent the glucose-6-phosphate dehydrogenation-hapten enzyme-labeled conjugate from reacting with the substrate (glucose-6-phosphate) and coenzyme (oxidized nicotinamide adenine dinucleotide) of the enzyme, the enzyme-labeled conjugate and the substrate must be placed separately and cannot be mixed. Therefore, the traditional homogeneous enzyme immunoassay reagent is to mix the substrate, coenzyme and hapten-specific antibody together to prepare the R1 reagent, and the glucose-6-phosphate dehydrogenation-hapten enzyme-labeled conjugate is added separately to the R2 reagent. In other words, the substrate and the coenzyme are mixed together before the traditional homogeneous enzyme immunoassay reagent is used. However, if the operator accidentally mixes a small amount of R2 reagent into the R1 reagent before use, or there is a carryover contamination during the operation of the full-automatic biochemical analyzer, the enzyme and the substrate will react in advance, and when the substrate and coenzyme are sufficient, the reaction will continue, resulting in a large amount of substrate consumption, and the oxidized coenzyme will be reduced to a large amount of reduced coenzyme, thereby causing the detection reagent background signal to continue to increase, seriously affecting the accuracy of the test result, and even causing the reagent to be completely scrapped. The present invention improves the formula of the traditional homogeneous enzyme immunoassay reagent, and the coenzyme is added to the R2 reagent, while only the antibody and the substrate are added to the R1 reagent, and no coenzyme is added, so that even if a small amount of R2 reagent is mixed into the R1 reagent before use, the reaction will also be terminated immediately after the coenzyme is consumed. Therefore, the present invention can effectively avoid the background reaction signal increase caused by cross contamination between the R1 reagent and the R2 reagent before the reagent is used, avoids reagent waste, and improves detection accuracy.

[0071] The melatonin homogeneous enzyme immunoassay reagent of the present invention has the advantages of simple operation, high sensitivity, strong specificity, accurate results, etc., and can also effectively reduce the cost of melatonin detection, which is conducive to clinical promotion and use. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 is the melatonin ELISA detection reaction standard curve of the present invention;

[0073] Figure 2 is the standard curve of the melatonin homogeneous enzyme immunoassay reaction of the present invention;

[0074] Figure 3 It is a melatonin amino derivative represented by formula (I) 1 H NMR spectral scanning analysis spectrum;

[0075] Figure 4 This is the LC-MS analysis spectrum of the melatonin amino derivative represented by formula (I). DETAILED DESCRIPTION

[0076] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments. These drawings are simplified schematic diagrams, which only illustrate the basic structure of the present invention in a schematic manner, so they only show the composition related to the present invention. Unless otherwise specified, the reagents, instruments, equipment, and consumables used in the following examples can be purchased from regular channels.

[0077] Example 1. Synthesis of amino derivatives of melatonin

[0078] The chemical structure of the amino derivative of melatonin is shown in formula (I):

[0079]

[0080] The preparation method of the above-mentioned melatonin amino derivative has the following synthetic route:

[0081]

[0082] The specific preparation steps are as follows:

[0083] a. Synthesis of Compound A-2

[0084]

[0085] Compound A-1 (10 g, 61.3 mmol) was dissolved in dimethylformamide (80 mL), and then tert-butyl 3-bromopropylcarbamate (14.6 g, 61.3 mmol) and diisopropylethylamine (18.3 mL) were added to prepare a reaction mixture solution; the reaction mixture solution was stirred at 80°C for 12 hours; purified water (100 mL) was then added to the reaction mixture solution to terminate the reaction, and the solution obtained after the reaction was extracted with ethyl acetate (150 mL×4), and the extraction step was repeated 4 times; the organic phases obtained from each extraction were combined, and the combined organic phases were washed with brine, and then dried and concentrated over Na2SO4 to obtain a crude product; finally, the crude product was purified by a silica gel column (petroleum ether: ethyl acetate = 20:1) to obtain a white solid compound A-2 (12 g, yield 61.1%).

[0086] b. Synthesis of Compound A

[0087]

[0088] Compound A-2 (12 g, 37.5 mmol) was dissolved in anhydrous ethanol (120 mL), and hydrazine hydrate (3.5 g, 53 mmol) was then added to prepare a reaction mixture solution; the reaction mixture solution was stirred at 70°C for 1 hour; after stirring, the reaction mixture solution was cooled to 10°C, and purified water (100 mL) was added to the reaction mixture solution to terminate the reaction, and the solution obtained after the reaction was extracted with dichloromethane (150 mL×4), and the extraction step was repeated 4 times; the organic phases obtained from each extraction were combined, and the combined organic phases were washed with brine, and then dried and concentrated by Na2SO4 to obtain a crude product; finally, the crude product was purified by a silica gel column to obtain a yellow oily compound A (5 g, yield 70.2%).

[0089] c. Synthesis of Compound 2

[0090]

[0091] Compound 1 (25 g, 0.1 mol) and ethyl chloroformate (11 g, 0.1 mol) were dissolved in dichloromethane (250 mL), and then NaOH (4 g, 0.1 mol) was added to prepare a reaction mixture solution, and the reaction mixture solution was stirred under N2 conditions for 12 hours; then the reaction mixture solution was diluted with ethyl acetate (150 mL), and then washed with brine, and dried with sodium sulfate; finally, the solvent was evaporated to obtain solid compound 2 (13 g).

[0092] d. Synthesis of Compound 3

[0093]

[0094] Compound 2 (13 g, 42.7 mmol) was dissolved in tetrahydrofuran (200 mL), and then aluminum chloride (5.7 g, 47 mmol) and acetyl chloride (3.7 g, 47 mmol) were added to prepare a reaction mixture solution, and the reaction mixture solution was stirred at 25°C for 0.5 hours; then purified water (100 mL) was added to the reaction mixture solution to quench the reaction, and extraction was performed with ethyl acetate; the organic layer obtained by the extraction was concentrated and purified by silica gel column chromatography to obtain compound 3 (7.0 g).

[0095] e. Synthesis of Compound 4

[0096]

[0097] Compound 3 (2.0 g, 5.8 mmol) was dissolved in dimethylformamide (20 mL), and then compound A (1.1 g, 5.8 mmol) and K2CO3 (2.4 g) were added to prepare a reaction mixture solution, and the reaction mixture solution was stirred at 80°C for 12 hours; purified water (30 mL) was then added to the reaction mixture solution, and then extracted with ethyl acetate; the organic layer obtained by the extraction was concentrated and purified by flash chromatography to obtain a brown solid compound 4 (1.2 g).

[0098] f. Synthesis of amino derivatives of melatonin

[0099]

[0100] Compound 4 (1.2 g, 2.7 mmol) was dissolved in dichloromethane (10 mL), and trifluoroacetic acid (2 mL) was added to prepare a reaction mixture solution. The reaction mixture solution was stirred at 25° C. for 12 hours; the reaction mixture solution was then concentrated and purified by flash chromatography to obtain a melatonin amino derivative (0.6 g).

[0101] The structure of the above melatonin amino derivatives was identified:

[0102] (1) 1 H NMR spectroscopic analysis instrument used Varian mercury plus 400MHz; TMS was used as internal standard. (2) LC-MS: Agilent 1200A, chromatographic column: C18; chromatographic column size: 4.6×50mm; mobile phase: B (ACN), A (0.05% FA aqueous solution). The identification results are detailed in the attached figure of the specification ( Figure 3 , Figure 4 ).

[0103] Example 2. Synthesis of melatonin immunogen

[0104] The melatonin immunogen in this embodiment is formed by linking the melatonin amino derivative shown in formula (I) with keyhole limpet hemocyanin, and its structural formula is shown in the following formula (II):

[0105]

[0106] The specific steps of the synthesis method of the melatonin immunogen are as follows:

[0107] a1. Weigh 10.0 mg of the amino derivative of melatonin represented by the above formula (Ⅰ) and dissolve it in 10.0 mL of sodium phosphate buffer (concentration of 0.1 mol / L, pH = 8.0) to obtain a solution of the amino derivative of melatonin;

[0108] a2. Weigh 30.0 mg of keyhole limpet hemocyanin, 3.0 mg of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, 10.0 mg of N-hydroxysulfosuccinimide, and 1.0 mL of dimethylformamide, dissolve in 10.0 mL of potassium phosphate buffer (concentration of 50.0 mmol / L, pH = 6.5), and then stir and react at 25 ° C for 90 minutes to obtain a keyhole limpet hemocyanin active solution;

[0109] a3. Pour the keyhole limpet hemocyanin active solution obtained in step a2 directly into the melatonin amino derivative solution obtained in step a1 and mix well to obtain a coupling reaction mixed solution, then stir the coupling reaction mixed solution at 25°C for 30 minutes, and after stirring, let it stand at 4°C for 8 hours to obtain a melatonin immunogen solution.

[0110] a4. The melatonin immunogen solution obtained in step a3 was purified by G-25 polysaccharide gel chromatography column (pre-equilibrated with 50.0 mmol / L, pH = 8.5 Tris buffer) to obtain the melatonin immunogen after purification, which was stored at -20°C.

[0111] Example 3. Preparation of anti-melatonin specific antibodies

[0112] The melatonin immunogen prepared in Example 2 was inoculated into experimental rabbits using Freund's adjuvant immunization method, and antiserum was obtained after booster immunization. The specific steps are as follows:

[0113] b1. The melatonin immunogen represented by the above formula (II) was diluted to 3.0 mg / mL with sodium phosphate buffer to obtain an antigen solution, and then 3.0 mL of the antigen solution was mixed with an equal amount of Freund's complete adjuvant and injected into the experimental animals;

[0114] b2. After 3 weeks, 3.0 mL of the same antigen solution mixed with an equal amount of Freund's incomplete adjuvant was injected into the above experimental animals once, and then injected once every 3 weeks, for a total of 6 injections;

[0115] b3. Collect blood from the immunized experimental animals, separate and purify the antiserum, and obtain anti-melatonin specific antibodies.

[0116] Example 4. Testing the sensitivity of anti-melatonin specific antibodies using the melatonin ELISA detection method

[0117] 1. Establishment of standard curve for melatonin ELISA

[0118] (1) Preparation of calibrators

[0119] Melatonin powder (purchased from Sigma) was dissolved in methanol solution to prepare a 1.0 mg / mL stock solution. The stock solution was diluted with ELISA buffer to 240.00 pg / mL, 120.00 pg / mL, 60.00 pg / mL, 30.00 pg / mL, 15.00 pg / mL, and 0.00 pg / mL standard solutions. The ELISA buffer contained 50.0 mmol / L Tris, 150.0 mmol / L NaCl, and 0.25% bovine serum albumin.

[0120] (2) Preparation of standard curve using ELISA test method for melatonin

[0121] The anti-melatonin antibody prepared in Example 3 was diluted to a final concentration of 1:5000 with sodium phosphate buffer, and 100.0 μL / well was coated on a 96-well ELISA plate and placed at 0°C for 12 hours; the 96-well ELISA plate coated with the anti-melatonin antibody was washed 3 times with sodium phosphate, and 200.0 μL / well of 0.5% bovine serum albumin solution was added, and the plate was sealed and placed at 0°C for 24 hours. Then, the plate was washed 3 times with sodium phosphate buffer, and 20 μL / well of calibrator was added. Then, 100.0 μL / well of working concentration of horseradish peroxidase (HRP)-melatonin conjugate was added; after incubation at 25°C for 30 minutes, the plate was washed 5 times with sodium phosphate; then, 100.0 μL of TMB substrate was added to each well, and the plate was incubated at 25°C for 30 minutes. Then, 100.0 μL of stop solution (2.0 mol / L sulfuric acid) was added to each well. The absorbance value at 450 nm was detected. Calibrate the standard curve according to the absorbance value at 450nm corresponding to each calibrant. The results are shown in the attached figure. Figure 1 shown.

[0122] The method for preparing the horseradish peroxidase-melatonin conjugate comprises the following steps:

[0123] (a) 5.0 mg of horseradish peroxidase was weighed and dissolved in 5.0 mL of phosphate buffer (pH=8.0) at 25° C., and then 5.0 mg of N,N′-dicyclohexylcarbodiimide was added for activation to prepare a horseradish peroxidase solution;

[0124] (b) Weigh 1.0 mg of the melatonin amino derivative described in Example 1, add it to 1.0 mL of isopropanol to dissolve it, and carry out coupling reaction with the horseradish peroxidase solution prepared in step (a). After the reaction is completed, dialysis is performed to purify the horseradish peroxidase-melatonin conjugate.

[0125] 2. Detection of melatonin content in the sample to be tested

[0126] (1) Prepare the sample to be tested

[0127] Preparation method: Melatonin powder (purchased from Sigma) was dissolved in methanol solution to prepare a 1.0 ng / mL melatonin stock solution, and the melatonin stock solution was diluted in artificial urine without melatonin to final concentrations of 0.00, 20.00, 80.00, and 200.00 pg / mL, respectively, to form blank, low, medium, and high concentration urine samples.

[0128] (2) Test method

[0129] The above-mentioned ELISA test method for melatonin was used, and the blank, low, medium and high concentration urine samples were used instead of the calibrator. The absorbance values ​​of the blank, low, medium and high concentration urine samples at 450 nm were detected using an enzyme marker.

[0130] (3) Test results

[0131] Comparison Figure 1 The standard curve of melatonin ELISA test was used to calculate the melatonin content in each sample, and 3 replicate wells were tested for each sample. The recovery rate was calculated based on the actual melatonin content in the above samples. The results are shown in Table 1.

[0132] Table 1 ELISA test results of melatonin

[0133]

[0134] From the results in Table 1, it can be seen that the recovery rates of melatonin in urine samples of different concentrations detected by the melatonin ELISA detection reagent of the present invention are all in the range of 100.00% to 103.00%, indicating that the anti-melatonin specific antibody of the present invention has a high reaction sensitivity and can be used for the detection of melatonin content in clinical samples.

[0135] Example 5. Preparation of melatonin enzyme-labeled conjugate

[0136] The melatonin enzyme-labeled conjugate in this embodiment is formed by connecting the melatonin amino derivative shown in formula (I) with glucose-6-phosphate dehydrogenase (G6PDH), and its structural formula is shown in the following formula (III):

[0137]

[0138] The specific steps of the synthesis method of the melatonin enzyme-labeled conjugate are as follows:

[0139] c1. Weigh 10.0 mg of the amino derivative of melatonin represented by the above formula (I), dissolve it in 10.0 mL of sodium phosphate buffer (concentration of 0.1 mol / L, pH = 8.0), then add 1.0 mL of dimethylformamide and 5.0 mg of glucose-6-phosphate and stir evenly to obtain a solution of the amino derivative of melatonin;

[0140] c2. Weigh 10.0 mg of glucose-6-phosphate dehydrogenase (G6PDH) and dissolve it in 10.0 mL of sodium phosphate buffer (concentration of 50.0 mmol / L, pH = 7.2), lower the solution temperature to 2-8 ° C, then add 5.0 mg of N-hydroxysulfosuccinimide and 10.0 mg of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide at the same time, and slowly stir the reaction at 25 ° C for 10 minutes to obtain a glucose-6-phosphate dehydrogenase active solution;

[0141] c3. The temperature of the melatonin amino derivative solution prepared in step c1 and the glucose-6-phosphate dehydrogenase active solution prepared in step c2 are both reduced to 2-8°C, and then the melatonin amino derivative solution is added dropwise to the glucose-6-phosphate dehydrogenase active solution. The entire dropping process lasts for 4 minutes, and then the mixed solution is stirred at 2-8°C for 8 hours to obtain a melatonin enzyme-labeled conjugate solution.

[0142] c4. The melatonin enzyme-labeled conjugate solution prepared in step c3 was purified by a G-25 dextran gel chromatography column (pre-equilibrated with 50.0 mmol / L, pH = 8.5 Tris buffer), and after purification, 0.3% by mass of bovine serum albumin and 0.05% by mass of sodium azide were added to obtain a melatonin enzyme-labeled conjugate.

[0143] Example 6. Preparation of homogeneous enzyme immunoassay reagent for melatonin

[0144] The specific steps of the preparation method of the melatonin homogeneous enzyme immunoassay reagent are as follows:

[0145] d1. Preparation of R1 reagent: 10.0 g of glucose-6-phosphate was dissolved in 1.0 L of Tris buffer (80.0 mmol / L, pH=8.2) to prepare a homogeneous enzyme substrate solution; the above-mentioned anti-melatonin specific antibody was added to the homogeneous enzyme substrate solution, and stirred evenly to obtain R1 reagent, wherein the volume ratio of the anti-melatonin specific antibody to the homogeneous enzyme substrate solution was 1:1300;

[0146] d2. Preparation of R2 reagent: 10.0 g of oxidized nicotinamide adenine dinucleotide was dissolved in 1.0 L Tris buffer (100.0 mmol / L, pH = 8.5) to prepare a coenzyme solution; the above-mentioned melatonin enzyme-labeled conjugate was added to the coenzyme solution, and stirred evenly to obtain R2 reagent, wherein the volume ratio of the melatonin enzyme-labeled conjugate to the coenzyme solution was 1:1300.

[0147] Example 7. Preparation of standard curve for homogeneous enzyme immunoassay of melatonin

[0148] (1) Set the reaction parameters of the fully automatic biochemical analyzer (Table 2).

[0149] (2) The operation steps are as follows: first add R1 reagent, then add the calibrator, mix well, incubate at 37°C for 5 minutes, and finally add R2 reagent, mix well, keep constant temperature at 37°C for 1.5 minutes, read the absorbance A1 at 340nm, read the absorbance A2 after 3.5 minutes of constant temperature, calculate △A = A2-A1, and use the automatic biochemical analyzer to automatically calculate the reaction rate at different calibrator concentrations to obtain the reaction standard curve, as shown in the attached figure. Figure 2 shown.

[0150] Table 2 Reaction parameters of fully automatic biochemical analyzer

[0151]

[0152] Table 3 Calibration absorbance data of calibrants at different concentrations

[0153]

[0154] Example 8. Performance verification of homogeneous enzyme immunoassay for melatonin

[0155] The performance of the melatonin homogeneous enzyme immunoassay reagent prepared in Example 6 was verified, and the main performance indicators detected were repeatability (precision), recovery rate, specificity, stability, etc. The experimental parameters were set according to the method described in Example 7, and the results of the reagent performance verification were as follows:

[0156] (1) Verification of repeatability and recovery rate of melatonin homogeneous enzyme immunoassay reagent

[0157] Using the melatonin homogeneous enzyme immunoassay standard curve prepared in Example 7, the low, medium and high concentration quality control samples were tested 10 times. The quality control samples were: melatonin storage solution was diluted in artificial urine without melatonin to concentrations of 20.00 pg / mL, 80.00 pg / mL and 200.00 pg / mL, respectively. The test data are detailed in Table 4.

[0158] Table 4 Repeatability and recovery verification test data

[0159]

[0160] The test results show that the melatonin homogeneous enzyme immunoassay reagent of the present invention has good repeatability and high precision in detecting the melatonin content in urine samples, with a CV lower than 3.00% and a recovery rate within the range of 100.00% to 103.00%, which meets the requirements of clinical test standards.

[0161] (2) Specificity verification of melatonin homogeneous enzyme immunoassay reagent

[0162] 40 common hormones and hormone metabolites were selected as interferors for cross-reaction detection. The interferors were dissolved in artificial urine to prepare an interferor solution with a concentration of 1.0 ng / mL. The concentration of each interferor solution was detected using the melatonin homogeneous enzyme immunoassay standard curve prepared in Example 7. The names of the 40 common hormones and hormone metabolites and the test results are detailed in Table 5.

[0163] Table 5 Names and test results of 40 common hormones and hormone metabolites

[0164]

[0165]

[0166] The test results show that the detection values ​​of the above 40 common hormones and hormone metabolites (equivalent to the concentration of melatonin) are all 0.00 pg / mL. It can be seen that the melatonin homogeneous enzyme immunoassay reagent of the present invention has strong specificity and has no cross reaction with common interferences.

[0167] (3) Stability verification of melatonin homogeneous enzyme immunoassay reagent

[0168] The melatonin homogeneous enzyme immunoassay reagent prepared in Example 6 was sealed and stored in a dry environment at 25° C. and protected from light for 24 months, and then the performance was verified again. The performance indicators of the test were repeatability (precision) and recovery rate. The experimental parameters were set according to the method described in Example 7. The reagent performance verification results are detailed in Table 6.

[0169] Table 6 Reagent stability verification test data

[0170]

[0171] The test results show that after being stored for 2 years under appropriate conditions, the melatonin homogeneous enzyme immunoassay reagent of the present invention has stable performance, the repeatability (CV%) of detecting the melatonin content in urine samples is less than 3.00%, and the recovery rate is in the range of 100.00% to 103.00%, which still meets the requirements of clinical test standards.

[0172] Based on the above-mentioned ideal embodiments of the present invention, and through the above-mentioned description, relevant technicians can make various changes and modifications without departing from the technical concept of the present invention. The technical scope of the present invention is not limited to the contents of the specification, but must be determined according to the scope of the claims.

Claims

1. A melatonin amino derivative, characterized in that: Its structural formula is shown in the following formula (I): Formula (I).

2. A method for preparing the melatonin amino derivative according to claim 1, characterized in that: The synthetic route of the preparation method is as follows: 。 3. A melatonin immunogen, characterized in that: The melatonin amino derivative according to claim 1 is connected with keyhole limpet hemocyanin, and its structural formula is shown in the following formula (II): Formula (II).

4. A method for preparing a melatonin immunogen as claimed in claim 3, characterized in that: The following steps are involved: A1. Weigh the melatonin amino derivative according to claim 1 and dissolve it in sodium phosphate buffer to obtain a melatonin amino derivative solution; A2. Weigh keyhole limpet hemocyanin, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, N-hydroxysulfosuccinimide, and dimethylformamide, dissolve them in potassium phosphate buffer, and then stir and react at 25°C for 10 to 60 minutes to obtain a keyhole limpet hemocyanin active solution; A3. Pour the keyhole limpet hemocyanin active solution obtained in step A2 directly into the melatonin amino derivative solution obtained in step A1 and mix well to obtain a coupling reaction mixed solution, and then stir the coupling reaction mixed solution at 25° C. for 10 to 60 minutes, and after stirring, let it stand at 2 to 8° C. for 3 to 12 hours to obtain a melatonin immunogen solution; A4. The melatonin immunogen solution obtained in step A3 is purified by G-25 dextran gel chromatography to obtain a melatonin immunogen.

5. An anti-melatonin specific antibody, characterized in that: The anti-melatonin specific antibody is a specific antibody produced by immunizing an experimental animal with the melatonin immunogen according to claim 3, and the experimental animal is one of rabbits, goats, mice, sheep, guinea pigs or horses; the anti-melatonin specific antibody is used to prepare a melatonin immunoassay reagent, and the melatonin immunoassay reagent includes a homogeneous enzyme immunoassay reagent, a latex-enhanced immunoturbidimetric assay reagent, a chemiluminescent immunoassay reagent, an enzyme-linked immunosorbent assay reagent, and an immunochromatographic assay reagent for detecting the melatonin content in a sample, and the immunochromatographic assay reagent includes a fluorescent immunochromatographic assay reagent and a colloidal gold immunochromatographic assay reagent.

6. A method for preparing an anti-melatonin specific antibody as claimed in claim 5, characterized in that: The following steps are involved: B1. diluting the melatonin immunogen according to claim 3 with sodium phosphate buffer to obtain an antigen solution, and then mixing the antigen solution with an equal amount of Freund's complete adjuvant and injecting the experimental animal; B2. After 2 to 5 weeks, mix the same antigen solution with an equal amount of Freund's incomplete adjuvant and inject the above experimental animals once, and then inject them once every 2 to 5 weeks, for a total of 5 to 8 injections; B3. Collect blood from the immunized experimental animals, separate and purify the antiserum, and obtain anti-melatonin specific antibodies.

7. A melatonin enzyme-labeled conjugate, characterized in that: The melatonin amino derivative according to claim 1 is connected with glucose-6-phosphate dehydrogenase, and its structural formula is shown in the following formula (III): Formula (III).

8. A method for preparing a melatonin enzyme-labeled conjugate according to claim 7, characterized in that: The following steps are involved: C1. Weigh the melatonin amino derivative according to claim 1, dissolve it in sodium phosphate buffer, then add dimethylformamide and glucose-6-phosphate and stir evenly to obtain a melatonin amino derivative solution; C2. Weigh glucose-6-phosphate dehydrogenase and dissolve it in sodium phosphate buffer. Lower the solution temperature to 2-8°C, then add N-hydroxysulfosuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide simultaneously, and slowly stir the reaction at 25°C for 5-20 minutes to obtain a glucose-6-phosphate dehydrogenase active solution. C3. The temperature of the melatonin amino derivative solution prepared in step C1 and the glucose-6-phosphate dehydrogenase active solution prepared in step C2 are both reduced to 2-8° C., and then the melatonin amino derivative solution is added dropwise to the glucose-6-phosphate dehydrogenase active solution. The entire dropping process lasts for 3-5 minutes, and then the mixed solution is stirred at 2-8° C. for 2-12 hours to obtain a melatonin enzyme-labeled conjugate solution; C4. The melatonin enzyme-labeled conjugate solution prepared in step C3 is purified by G-25 dextran gel chromatography column, and after purification, bovine serum albumin and sodium azide are added to obtain a melatonin enzyme-labeled conjugate.

9. A melatonin homogeneous enzyme immunoassay reagent, characterized in that: The invention comprises an R1 reagent and an R2 reagent, wherein the R1 reagent comprises the anti-melatonin specific antibody according to claim 5 and a homogeneous enzyme substrate solution, wherein the homogeneous enzyme substrate solution is prepared from glucose-6-phosphate and Tris buffer; and the R2 reagent comprises the melatonin enzyme-labeled conjugate according to claim 7 and a coenzyme solution, wherein the coenzyme solution is prepared from oxidized nicotinamide adenine dinucleotide and Tris buffer.

10. A method for preparing the melatonin homogeneous enzyme immunoassay reagent according to claim 9, characterized in that: The following steps are involved: Preparation of D1.R1 reagent: Dissolve glucose-6-phosphate in Tris buffer to prepare a homogeneous enzyme substrate solution; Then, adding the anti-melatonin specific antibody of claim 5 to the homogeneous enzyme substrate solution, stirring evenly to obtain the R1 reagent, wherein the volume ratio of the anti-melatonin specific antibody to the homogeneous enzyme substrate solution is 1:100 to 1:10000; D2. Preparation of R2 reagent: dissolving oxidized nicotinamide adenine dinucleotide in Tris buffer to prepare a coenzyme solution; then adding the melatonin enzyme-labeled conjugate described in claim 7 to the coenzyme solution, stirring evenly to obtain R2 reagent, wherein the volume ratio of the melatonin enzyme-labeled conjugate to the coenzyme solution is 1:100~1:10000.

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