Homogeneous enzyme immunodetection system based on click chemistry coupling technology as well as preparation method and application of homogeneous enzyme immunodetection system

Abstract

The invention discloses a homogeneous enzyme immunodetection system based on a click chemistry coupling technology as well as a preparation method and application of the homogeneous enzyme immunodetection system. A technology (including micromolecule-protein carrier coupling and micromolecule-labeled enzyme coupling) for coupling a micromolecule hapten and a macromolecular protein by utilizing a specific coupling reaction of an azido functional group and an alkynyl functional group in click chemistry. The coupling reaction does not need to use a special activator, the reaction can be carried out under a very mild condition, and no compound containing two special structures of azido and alkynyl exists in a human body, so that the specificity of a small-molecule hapten and macromolecular protein coupling structure can be remarkably improved, and the sensitivity of the coupling reaction is improved. And the specificity of the micromolecular specific antibody can also be obviously improved. The key technology of a homogeneous enzyme immunodetection platform is improved, and compared with a traditional homogeneous enzyme immunodetection platform, the homogeneous enzyme immunodetection system based on the click chemistry coupling technology has the advantages of being easy and convenient to prepare, rapid in reaction, safe, free of pollution, low in cost, excellent in reagent performance and the like; and large-scale production and application are facilitated.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a homogeneous enzyme immunoassay system based on click chemistry coupling technology, a preparation method thereof, and an application thereof. Background Art

[0002] In 1972, Ruben stein and Ullman developed a homogeneous enzyme immunoassay (HEIA). The classic homogeneous enzyme immunoassay is a detection technology that combines biochemistry and immunology, mainly used to detect trace small molecule substances in biological samples, such as small molecule drugs, drugs, endogenous small molecule substances (hormones, metabolites, organic acids), etc. The basic principle of this technology is: the hapten is combined with an enzyme to form an enzyme-labeled hapten, and the activities of both the hapten and the enzyme are retained. When the enzyme-labeled hapten binds to an antibody, the labeled enzyme is in close contact with the antibody, causing the active center of the enzyme to be affected and its activity to be inhibited. When the antibody, enzyme-labeled hapten in the reagent and the specimen are mixed, the hapten in the sample competes with the enzyme-labeled hapten for binding to the antibody in the reagent. If the content of the hapten in the sample is high, the proportion of the enzyme-labeled hapten bound to the antibody decreases, and the amount of free enzyme-labeled hapten with enzyme activity increases. Therefore, after the reaction, the enzyme activity is in a certain proportional relationship with the hapten in the sample, and the content of the hapten in the sample can be deduced based on the measurement result of the enzyme activity. The whole process of homogeneous enzyme immunoassay is a competitive reaction, and the whole reaction occurs in a liquid homogeneous system. There is no need to separate the bound and free markers through solid phase. The free small molecule antigen in the reaction system can competitively bind to the specific site of the antibody with the "small molecule-enzyme" conjugate. The more free small molecule antigen in the sample, the more antibodies that competitively bind, and the more "small molecule-enzyme" conjugates released by the antibodies. The stronger the signal obtained from the reaction of the "small molecule-enzyme" conjugate with the substrate. By automatically detecting the change in the OD340nm absorbance value with an automatic biochemical analyzer, the content of the sample can be accurately calculated. Since this technology was introduced, after more than 50 years of application and development, it has currently been widely used in many fields such as therapeutic drug monitoring, disease diagnosis, hormone detection, drug testing, food safety, etc.

[0003] The preparation technology of small molecule hapten and macromolecule protein conjugates (including: preparation of small molecule-protein carrier conjugates, preparation of small molecule-labeled enzyme conjugates) is the key technology for the preparation of homogeneous enzyme immunoassay reagents. The synthesis of small molecule hapten and protein conjugates requires retaining the specific sites of small molecules, conjugating small molecules to protein molecules, and at the same time ensuring the biological activity of the conjugated protein. For example, during the conjugation process of "small molecule-labeled enzyme", anhydrous and low temperature conditions must be strictly maintained, and the dosage of the activator also needs to be repeatedly measured to ensure the balance between the biological activity of the conjugate and the number of small molecule linkages. Traditional conjugation methods mainly involve activating small molecule haptens with activators and performing cross-linking reactions with macromolecular protein solutions. This process requires strict control of multiple aspects such as reaction time, temperature, and activator dosage. The desired conjugate can only be obtained after dialysis purification of the reaction products. Since the cross-linking groups provided by commonly used protein molecules are similar, the choice of linking method and the design of conjugate products mainly depend on the functional group structure of small molecule compounds or their derivatives. Small molecule compounds generally with free carboxyl groups or free amino groups, or those with both groups, can be directly conjugated to protein molecules through certain methods; other small molecule compounds without free carboxyl groups or free amino groups need to be appropriately structurally modified to transform them into derivatives with free carboxyl groups or free amino groups before they can be linked to protein molecules. Traditional bioconjugation techniques have many defects such as harsh reaction conditions, complex operations, low yields, high reagent toxicity, low activity of conjugate products, many by-products, and difficult separation and purification.

[0004] Click chemistry, also known as "click chemistry", "link chemistry", "rapid conjugation combinatorial chemistry", was created and proposed by American chemist Sharpless in 2001. It refers to a class of modular, highly selective coupling reactions with simple reaction raw materials, mild reaction conditions, high product yields, and convenient separation and purification, to achieve the efficient and rapid synthesis of functional molecules, and synthesize various biological macromolecules by forming carbon-heteroatom (C-X-C) bonds between various monomer molecule modules. In traditional organic synthetic chemistry, in order to pursue yields and efficiency, the construction of molecular skeletons usually needs to be carried out in organic solvents and under conditions such as heating, acids or bases, and inert gas protection. Many organic reactions also require pre-protection of reactive groups, resulting in a complex and cumbersome synthesis process. In 2002, Sharpless and Meldal independently reported the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. This reaction has been rapidly and widely applied in the synthesis fields of high molecular polymers, drugs, new materials, etc., and has become the representative reaction of click chemistry. The two of them also won the Nobel Prize in Chemistry in 2022 for this.

[0005] Rapamycin, also known as Sirolimus, is derived from Streptomyces hygroscopicus on Easter Island and is a triene macrolide antibiotic immunosuppressant. The effective blood drug concentration range of rapamycin is relatively narrow, and its efficacy and toxic side effects are significantly correlated with the blood drug concentration. Insufficient dosage will lead to the body's rejection of transplanted organs, while excessive dosage will cause serious side effects, including kidney toxicity, liver toxicity and a series of other complications. Due to factors such as the patient's disease state, type of organ transplantation, age, and dosage of medication, there are significant individual differences in drug absorption and metabolism, and it is difficult to directly estimate the blood drug concentration after administration. Therefore, it is of great clinical significance to detect the individual rapamycin blood drug concentration of patients in a timely manner to adjust the dosage or frequency of medication and formulate an individualized treatment plan. The existing methods for detecting rapamycin mainly include high performance liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry (LC / MS / MS), radioimmunoassay and fluorescence polarization method. The pre-treatment of HPLC and LC / MS / MS methods is complex, time-consuming, and requires high requirements for operators; radioimmunoassay is radioactive to operators; the instruments and reagents of fluorescence polarization method mainly rely on imports and the cost is relatively high. In this application, taking the small molecule drug rapamycin as an example, a homogeneous enzyme immunoassay reagent for rapamycin based on click chemistry coupling technology and its preparation and use methods are disclosed.

[0006] The homogeneous enzyme immunoassay system based on click chemistry coupling technology in this application utilizes the specific coupling reaction between the azide functional group and the alkyne functional group in click chemistry. This coupling reaction does not require the use of special activators and can occur under very mild conditions. Moreover, there are no compounds containing these two special structures of azide and alkyne in the human body. Therefore, the use of this click chemistry coupling technology can significantly improve the specificity of the coupling structure between small molecule hapten and macromolecule protein, and thus also significantly improve the specificity of small molecule specific antibodies. Compared with the homogeneous enzyme immunoassay platform based on traditional coupling technology, the homogeneous enzyme immunoassay system based on click chemistry coupling technology provided by the present invention has the advantages of simple preparation, rapid reaction, safe and pollution-free, low cost, and superior reagent performance, which is conducive to large-scale production and application. Summary of the Invention

[0007] In order to overcome the defects existing in the prior art, the present invention provides a homogeneous enzyme immunoassay system based on click chemistry coupling technology and its preparation method and application. The technical solutions adopted by the present invention to solve its technical problems are as follows:

[0008] Solution 1: Provide a homogeneous enzyme immunoassay system based on click chemistry coupling technology. The analysis system consists of R1 reagent, R2 reagent, calibrator and quality control product. The R1 reagent contains a small molecule-specific antibody and R1 buffer. The R2 reagent contains a glucose-6-phosphate dehydrogenase-labeled small molecule click chemistry conjugate and R2 buffer. The small molecule-specific antibody is an antibody produced after immunizing experimental animals with a small molecule click chemistry conjugate immunogen. The small molecule click chemistry conjugate immunogen is formed by coupling a small molecule click chemistry derivative with a click chemistry carrier, and its structural formula is shown in Formula I:

[0009]

[0010] The small molecule click chemistry derivative is an azide-modified small molecule hapten, and the small molecule hapten is any organic compound with a molecular weight less than 4000 Da. Preferably, the small molecule hapten includes any organic compound with a molecular weight less than 4000 Da among drugs, hormones, drugs, stimulants, metabolites, vitamins, agricultural and veterinary drugs, food additives, environmental pollutants. More preferably, the small molecule hapten is any drug with a molecular weight less than 4000 Da. Further preferably, the small molecule hapten is rapamycin;

[0011] The experimental animals are mammals, and the mammals are one of rabbits, sheep, goats, mice, rats, guinea pigs, donkeys, horses, alpacas or camels;

[0012] The click chemistry carrier is an alkyne-modified immunogenic protein, and the immunogenic protein is one of serum albumin, immunoglobulin, thyroglobulin, fibrinogen, ovalbumin, hemocyanin, toxoid protein, myoglobin, polylysine, polyglutamic acid;

[0013] The R1 buffer contains an enzyme substrate, a coenzyme, bovine serum albumin and Tris buffer. The enzyme substrate is glucose-6-phosphate, and the coenzyme is nicotinamide adenine dinucleotide oxidized form;

[0014] The glucose-6-phosphate dehydrogenase-labeled small molecule click chemistry conjugate is formed by coupling the above small molecule click chemistry derivative with click chemistry recombinant glucose-6-phosphate dehydrogenase, and its structural formula is shown in Formula II:

[0015]

[0016] The click chemistry recombinant glucose-6-phosphate dehydrogenase is alkyne-modified glucose-6-phosphate dehydrogenase; the R2 buffer is Tris buffer containing bovine serum albumin;

[0017] The calibration product is prepared by dissolving a certain amount of pure small molecule hapten in a certain amount of Tris-HCl buffer to prepare a high-concentration stock solution, and diluting this stock solution in Tris-HCl buffer according to the calibration concentration requirements to form standard solutions of different concentrations for use as calibration solutions;

[0018] The quality control product is prepared by dissolving a certain amount of pure small molecule hapten in a certain amount of Tris-HCl buffer to prepare a high-concentration stock solution, and diluting this stock solution in Tris-HCl buffer according to the detection concentration requirements to form standard solutions of different concentrations for use as quality control samples.

[0019] Scheme 2; Provide a preparation method of a homogeneous enzyme immunoassay system based on click chemistry coupling technology as described in Scheme 1, and the preparation method includes the following steps:

[0020] (A1) Preparation of R1 reagent: Add 0.05-1.00% bovine serum albumin, 5-500 mmol / L glucose-6-phosphate, and 5-500 mmol / L nicotinamide adenine dinucleotide oxidized form to 5-500 mmol / L Tris buffer in sequence, stir and dissolve to prepare R1 buffer, then add the anti-small molecule specific antibody described in Scheme 1 to the above R1 buffer in a volume ratio of 1:100-1:10000, mix well, and adjust the pH to 7.0-9.0 with 0.6-12 mol / L hydrochloric acid to prepare R1 reagent;

[0021] (A2) Preparation of R2 reagent: Add 0.05-1.00% bovine serum albumin to 10-1000 mmol / L Tris buffer, stir and dissolve to prepare R2 buffer, then add the small molecule click chemistry conjugate labeled with glucose-6-phosphate dehydrogenase described in Scheme 1 to the above R2 buffer in a volume ratio of 1:100-1:10000, mix well, and adjust the pH to 7.0-9.0 with 0.6-12 mol / L hydrochloric acid to prepare R2 reagent;

[0022] (A3) Preparation of calibration product: Add pure small molecule hapten powder to 6 portions of Tris-HCl buffer with a concentration of 10.0-100.0 mmol / L and pH = 7.0-7.5 respectively, stir and dissolve, and dilute to 6 standard solutions of different concentrations according to a certain ratio, then add 0.1-1.0% sodium chloride, 0.1-1.0% bovine serum albumin, 0.15-3.00% ethylenediaminetetraacetic acid, and 0.01-0.10% sodium azide to each solution respectively, stir evenly to obtain the small molecule hapten calibration product;

[0023] Preparation of quality control product: Pure powder of small molecule hapten is added into 4 portions of Tris-HCl buffer solution with a concentration of 10.0 - 100.0 mmol / L and pH = 7.0 - 7.5 respectively, stirred and dissolved, and then diluted into 4 standard solutions with different concentrations according to the detection concentration requirements. Then, 0.1 - 1.0% sodium chloride, 0.1 - 3.0% bovine serum albumin, 0.15 - 3.00% ethylenediaminetetraacetic acid, and 0.01 - 0.10% sodium azide are added into each solution respectively, and stirred evenly to obtain the small molecule hapten quality control product.

[0024] As an alternative solution, the preparation method of the anti-small molecule specific antibody comprises the following steps:

[0025] (B1) Dilute the small molecule click chemistry conjugate immunogen described in Scheme 1 with PBS buffer solution to 2.0 - 5.0 mg / mL to obtain an artificial antigen solution, and then mix 2.0 - 5.0 mL of the artificial antigen solution with an equal amount of Freund's complete adjuvant and inject it into the experimental animals described in Scheme 1.

[0026] (B2) After 1 - 4 weeks, mix 2.0 - 5.0 mL of the same artificial antigen solution with an equal amount of Freund's incomplete adjuvant and inject it into the above experimental animals once, and then inject it once every 1 - 4 weeks for a total of 3 - 8 times.

[0027] (B3) Collect blood from the above immunized experimental animals, separate and purify the antiserum to obtain the anti-small molecule specific antibody.

[0028] As an alternative solution, the preparation method of the small molecule click chemistry conjugate immunogen has the following reaction formula:

[0029]

[0030] The specific operation steps are as follows:

[0031] (C1) Prepare three fresh buffer solutions required for the click reaction: Weigh 1.75 - 7.0 mg of tetrachlorophthalic anhydride (THPA) and dissolve it in 200 μL of purified water to obtain a tetrachlorophthalic anhydride buffer solution with a concentration of 20 - 80 mM; weigh 8 - 32 mg of CuSO4 and dissolve it in 2.5 mL of purified water to obtain a CuSO4 buffer solution with a concentration of 20 - 80 mM; weigh 6 - 24 mg of sodium ascorbate and dissolve it in 200 μL of purified water to obtain a sodium ascorbate buffer solution with a concentration of 150 - 600 mM; calculate the required amount of each azide-modified small molecule hapten according to its molecular weight, weigh the required amount of the azide-modified small molecule hapten and dissolve it in an appropriate amount of DMSO to prepare an azide-modified small molecule hapten solution with a final concentration of 5 - 20 mM;

[0032] (C2) Perform click chemical coupling reaction between the alkyne-modified immunogenic protein and the azide-modified small molecule hapten: Measure 750 μL of the azide-modified small molecule hapten solution with a concentration of 5 - 20 mM, 375 μL of the tetrachlorophthalic anhydride buffer solution with a concentration of 20 - 80 mM, 375 μL of the CuSO4 buffer solution with a concentration of 20 - 80 mM, and 375 μL of the sodium ascorbate buffer solution with a concentration of 150 - 600 mM prepared in step (C1), and add them sequentially to 4 mL of the alkyne-modified immunogenic protein solution with a concentration of 5 mg / mL (the solvent is 10 mM, pH = 7.4 PBS buffer solution), mix well and incubate at room temperature of 25 °C for 0.5 - 3 hours, then let it stand overnight at 4 °C;

[0033] (C3) Purify the conjugate: After the reaction is completed, transfer the conjugate reaction solution in step (C2) to a dialysis membrane with a MWCO of 6 - 8000, use 1 - 4 L of normal saline as the dialysis solution for dialysis treatment 2 - 6 times, and then dialyze with 10 mM, pH = 7.4 PBS buffer solution for 6 - 24 hours to obtain a small molecule click chemical conjugate immunogen solution.

[0034] As an alternative scheme, the preparation method of the glucose-6-phosphate dehydrogenase-labeled small molecule click chemical conjugate has the following reaction formula:

[0035]

[0036] The specific operation steps are as follows:

[0037] (D1) Prepare 3 kinds of fresh buffer solutions required for the click reaction: Weigh 1.75 - 7.0 mg of tetrachlorophthalic anhydride (THPA) and dissolve it in 200 μL of purified water to obtain a tetrachlorophthalic anhydride buffer solution with a concentration of 20 - 80 mM; weigh 4 - 16 mg of CuSO4 and dissolve it in 2.5 mL of purified water to obtain a CuSO4 buffer solution with a concentration of 10 - 40 mM; weigh 6 - 24 mg of sodium ascorbate and dissolve it in 200 μL of purified water to obtain a sodium ascorbate buffer solution with a concentration of 150 - 600 mM; calculate the required amount of each azide-modified small molecule hapten according to its molecular weight, weigh the required amount of the azide-modified small molecule hapten and dissolve it in an appropriate amount of DMSO to prepare an azide-modified small molecule hapten solution with a final concentration of 1.25 - 5.0 mM;

[0038] (D2) Perform click chemical coupling reaction between alkynyl-modified glucose-6-phosphate dehydrogenase and azide-modified small molecule hapten: Measure 150 μL of the azide-modified small molecule hapten solution with a concentration of 1.25 - 5.0 mM, 75 μL of the tetrachlorophthalic anhydride buffer solution with a concentration of 20 - 80 mM, 75 μL of the CuSO4 buffer solution with a concentration of 10 - 40 mM, and 75 μL of the sodium ascorbate buffer solution with a concentration of 150 - 600 mM prepared in step (D1), and sequentially add them to 666 μL of the alkynyl-modified glucose-6-phosphate dehydrogenase solution with a concentration of 3 mg / mL (the solvent is 10 mM, PBS buffer solution with pH = 7.4), mix well and incubate at room temperature of 25 °C for 0.5 - 3 hours (the reaction efficiency can be improved by extending the reaction time);

[0039] (D3) Purify the conjugate: After the reaction is completed, filter the coupling reaction solution in step (D2) using a pre-equilibrated G-25 Sephadex gel chromatography column to obtain a glucose-6-phosphate dehydrogenase-labeled small molecule click chemical conjugate solution, and finally add BSA with a mass fraction of 0.1 - 1.0%, NaN3 with a mass fraction of 0.01 - 0.1%, and glycerol with a volume fraction of 25 - 50% for storage.

[0040] As an alternative solution, the preparation method of the azide-modified small molecule hapten comprises the following steps:

[0041] Hydrogen bromide (0.81 g, 10 mmol), sodium azide (0.33 g, 5 mmol), and a small molecule hapten (1 mmol) were co-dissolved in 20 mL of N,N-dimethylformamide (DMF) to obtain a reaction mixture. The reaction mixture was stirred and refluxed for 10 - 50 hours; the reaction mixture was cooled to 25 °C, purified water (50 mL) was added, and then 100 mL of dichloromethane (DCM) was added for extraction; the extract was washed with brine and dried over Na2SO4, the solvent was removed by vacuum drying, and the crude product was purified by silica gel column chromatography (hexane / acetone = 1:1) to obtain an azide-modified small molecule hapten.

[0042] As an alternative, the method for preparing the alkynyl-modified immunogenic protein comprises the following steps:

[0043] Basic reaction principle: Using N-hydroxysuccinimide (NHS) to replace the amino (-NH2) functional group in the immunogenic protein with an alkynyl (-C≡CH) functional group;

[0044] An alkynyl-N-hydroxysuccinimide crosslinker was used for the conjugation reaction with the immunogenic protein. 20 - 200 mg of the immunogenic protein and 10 - 100 mg of the alkynyl-N-hydroxysuccinimide crosslinker were successively added to 3000 μL of a phosphate buffer solution with a concentration of 0.2 M and a pH value of 8.0 to prepare a reaction solution; the reaction solution was incubated at room temperature of 25 °C for 0.5 - 3 hours, and then left to stand overnight at 4 °C; after the reaction, purification was carried out by dialysis, and dialysis was performed 1 - 3 times using a phosphate buffer solution with a pH value of 7.0 - 7.5 to obtain an alkynyl-modified immunogenic protein solution with a final working concentration of 5 mg / mL.

[0045] As an alternative, the method for preparing the alkynyl-modified glucose-6-phosphate dehydrogenase comprises the following steps:

[0046] Basic reaction principle: Using maleimide to replace the sulfhydryl (-SH) functional group in glucose-6-phosphate dehydrogenase with an alkynyl (-C≡CH) functional group;

[0047] (E1) Preparation of a glucose-6-phosphate dehydrogenase solution for maleimide conjugation reaction: Dissolve disodium ethylenediaminetetraacetate in a phosphate buffer solution (PBS) with a concentration of 50 mM until the final concentration of disodium ethylenediaminetetraacetate is 5 - 20 mM, adjust the pH value to 7.4 to obtain a glucose-6-phosphate dehydrogenase buffer solution, and then add 6.0 mg of glucose-6-phosphate dehydrogenase to 1000 μL of the glucose-6-phosphate dehydrogenase buffer solution and stir to dissolve to obtain a glucose-6-phosphate dehydrogenase solution;

[0048] (E2) Preparation of an alkynyl-maleimide crosslinker capable of binding to glucose-6-phosphate dehydrogenase: Weigh 3.0 mg of alkynyl-maleimide and add it to 200 μL of DMF. Stir to dissolve to obtain a solution with a concentration of 15 mg / mL, which is the alkynyl-maleimide crosslinker;

[0049] (E3) Completion of the alkynylation reaction of glucose-6-phosphate dehydrogenase: Dropwise add the alkynyl-maleimide crosslinker prepared in step (E2) into the glucose-6-phosphate dehydrogenase solution prepared in step (E1), while stirring evenly at room temperature of 25 °C, and incubate at 25 °C for 1 - 3 hours;

[0050] (E4) Purification: After the reaction, purification is carried out by dialysis. Dialyze 2 - 6 times using a phosphate buffer with a pH value of 7.0 - 7.5 to obtain an alkynyl-modified glucose-6-phosphate dehydrogenase solution with a final working concentration of 3 mg / mL.

[0051] Scheme Three: Provide an application of the homogeneous enzyme immunoassay system based on click chemistry coupling technology as described in Scheme One. Apply the homogeneous enzyme immunoassay system based on click chemistry coupling technology to assemble into a detection kit product, and use the detection kit product to detect the content of small molecule haptens in biological samples; the biological sample is a liquid physiological sample of a human or an animal, and the liquid physiological sample includes but is not limited to serum, plasma, whole blood, urine, saliva, lymph fluid, tissue fluid, cerebrospinal fluid, amniotic fluid or tear fluid; the small molecule hapten is any organic compound with a molecular weight less than 4000 Da.

[0052] As a preferred scheme, the small molecule hapten includes any organic compound with a molecular weight less than 4000 Da among drugs, hormones, drugs, stimulants, metabolites, vitamins, agricultural and veterinary drugs, food additives, environmental pollutants; more preferably, the small molecule hapten is any drug with a molecular weight less than 4000 Da; further preferably, the small molecule hapten is rapamycin.

[0053] As a specific application scheme, apply the homogeneous enzyme immunoassay system based on click chemistry coupling technology to assemble into a rapamycin detection kit product, and use the rapamycin detection kit product to detect the content of rapamycin in biological samples, and the biological sample is human serum or plasma.

[0054] The specific preparation method of the rapamycin detection kit product is as follows:

[0055] (A1) Preparation of rapamycin R1 reagent: 0.25% bovine serum albumin, 50 mmol / L glucose-6-phosphate and 50 mmol / L nicotinamide adenine dinucleotide oxidized form were successively added to 50 mmol / L Tris buffer and stirred to dissolve to prepare R1 buffer. Then, the anti-small molecule specific antibody described in Scheme 1 was added to the above R1 buffer in a volume ratio of 1:1500 and mixed evenly. Then, the pH was adjusted to 8.0 with 6 mol / L hydrochloric acid to prepare R1 reagent;

[0056] (A2) Preparation of rapamycin R2 reagent: 0.25% bovine serum albumin was added to 100 mmol / L Tris buffer and stirred to dissolve to prepare R2 buffer. Then, the rapamycin click chemistry conjugate labeled with glucose-6-phosphate dehydrogenase was added to the above R2 buffer in a volume ratio of 1:1200 and mixed evenly. Then, the pH was adjusted to 7.6 with 6 mol / L hydrochloric acid to prepare R2 reagent;

[0057] (A3) Preparation of rapamycin calibrator: Rapamycin pure powder was added to 6 portions of 50.0 mmol / L Tris-HCl buffer with pH = 7.2 and stirred to dissolve, and diluted into standard solutions with final concentrations of 0.00 ng / mL, 2.50 ng / mL, 5.00 ng / mL, 10.00 ng / mL, 20.00 ng / mL, and 40.00 ng / mL respectively. Then, 0.5% sodium chloride, 1.0% bovine serum albumin, 0.75% ethylenediaminetetraacetic acid, and 0.05% sodium azide were added to each solution and stirred evenly to obtain rapamycin calibrator;

[0058] (A4) Preparation of rapamycin quality control product: Rapamycin pure powder was added to 4 portions of 50.0 mmol / L Tris-HCl buffer with pH = 7.2 and stirred to dissolve, and diluted into standard solutions with final concentrations of 0.00 ng / mL, 2.00 ng / mL, 12.00 ng / mL, and 36.00 ng / mL respectively. Then, 0.5% sodium chloride, 1.0% bovine serum albumin, 0.75% ethylenediaminetetraacetic acid, and 0.05% sodium azide were added to each solution and stirred evenly to obtain rapamycin quality control product.

[0059] The preparation method of the anti-rapamycin specific antibody comprises the following steps:

[0060] (B1) Dilute the rapamycin click chemistry conjugate immunogen with PBS buffer to 3.0 mg / mL to obtain an artificial antigen solution. Then, mix 3.0 mL of the artificial antigen solution with an equal amount of Freund's complete adjuvant and inject it into rabbits;

[0061] After 2 weeks, mix 3.0 mL of the same artificial antigen solution with an equal amount of Freund's incomplete adjuvant, and inject the above rabbits once. Then inject once every 3 weeks for a total of 5 injections.

[0062] Collect blood from the immunized rabbits above, separate and purify the antiserum to obtain anti-rapamycin specific antibodies.

[0063] The preparation method of the rapamycin click chemistry conjugate immunogen is as follows:

[0064]

[0065] The specific operation steps are as follows:

[0066] (C1) Prepare 3 fresh buffer solutions required for the click reaction: Weigh 3.5 mg of tetrachlorophthalic anhydride (THPA) and dissolve it in 200 μL of purified water to obtain a 40 mM tetrachlorophthalic anhydride buffer solution; Weigh 16 mg of CuSO4 and dissolve it in 2.5 mL of purified water to obtain a 40 mM CuSO4 buffer solution; Weigh 12 mg of sodium ascorbate and dissolve it in 200 μL of purified water to obtain a 300 mM sodium ascorbate buffer solution; Calculate the required amount of the azide-modified rapamycin according to the molecular weight of the azide-modified rapamycin, and weigh the required amount of the azide-modified rapamycin and dissolve it in an appropriate amount of DMSO to prepare an azide-modified rapamycin solution with a final concentration of 10 mM.

[0067] (C2) Perform a click chemistry conjugation reaction between the alkyne-modified immunogenic protein and the azide-modified rapamycin: Measure 750 μL of the 10 mM azide-modified rapamycin solution, 375 μL of the 40 mM tetrachlorophthalic anhydride buffer solution, 375 μL of the 40 mM CuSO4 buffer solution, and 375 μL of the 300 mM sodium ascorbate buffer solution prepared in step (C1), and add them sequentially to 4 mL of a 5 mg / mL alkyne-modified immunogenic protein solution (the solvent is 10 mM, pH = 7.4 PBS buffer solution), mix well and incubate at room temperature of 25 °C for 1 hour, then let it stand overnight at 4 °C.

[0068] (C3) Conjugate purification: After the reaction is completed, transfer the conjugation reaction solution in step (C2) to a MWCO 6 - 8000 dialysis membrane, use 3 L of physiological saline as the dialysis solution for dialysis treatment 4 times, and then dialyze with a 10 mM, pH = 7.4 PBS buffer solution for 12 hours to obtain a rapamycin click chemistry conjugate immunogen solution.

[0069] The preparation method of the glucose-6-phosphate dehydrogenase-labeled rapamycin click chemical conjugate has the following reaction formula:

[0070]

[0071] The specific operation steps are as follows:

[0072] (D1) Prepare 3 kinds of fresh buffer solutions required for the click reaction: Weigh 3.5 mg of tetrachlorophthalic anhydride (THPA) and dissolve it in 200 μL of purified water to obtain a tetrachlorophthalic anhydride buffer solution with a concentration of 40 mM; Weigh 8 mg of CuSO4 and dissolve it in 2.5 mL of purified water to obtain a CuSO4 buffer solution with a concentration of 20 mM; Weigh 12 mg of sodium ascorbate and dissolve it in 200 μL of purified water to obtain a sodium ascorbate buffer solution with a concentration of 300 mM; Calculate the required amount of the azide-modified rapamycin according to the molecular weight of the azide-modified rapamycin, and weigh the required amount of the azide-modified rapamycin and dissolve it in an appropriate amount of DMSO to prepare an azide-modified rapamycin solution with a final concentration of 2.5 mM;

[0073] (D2) Perform a click chemical coupling reaction between the alkyne-modified glucose-6-phosphate dehydrogenase and the azide-modified rapamycin: Measure 150 μL of the azide-modified rapamycin solution with a concentration of 2.5 mM, 75 μL of the tetrachlorophthalic anhydride buffer solution with a concentration of 40 mM, 75 μL of the CuSO4 buffer solution with a concentration of 20 mM, and 75 μL of the sodium ascorbate buffer solution with a concentration of 300 mM prepared in step (D1), and add them successively to 666 μL of the alkyne-modified glucose-6-phosphate dehydrogenase solution with a concentration of 3 mg / mL (the solvent is 10 mM, pH = 7.4 PBS buffer solution), mix well and incubate at room temperature of 25 °C for 1 hour (extending the reaction time can improve the reaction efficiency);

[0074] (D3) Purify the conjugate: After the reaction is completed, filter the conjugate reaction solution in step (D2) using a pre-equilibrated G-25 Sephadex gel chromatography column to obtain a glucose-6-phosphate dehydrogenase-labeled rapamycin click chemical conjugate solution, and finally add 0.5% BSA by mass fraction, 0.1% NaN3 by mass fraction, and 50% glycerol by volume fraction for storage.

[0075] The chemical structure of the azide-modified rapamycin (rapamycin click chemical derivative) is shown in formula Ⅲ:

[0076]

[0077] The specific route of the synthesis method of the above azide-modified rapamycin is as follows:

[0078]

[0079] The preparation method of the above azido-modified rapamycin comprises the following steps:

[0080] Hydrogen bromide (0.81 g, 10 mmol), sodium azide (0.33 g, 5 mmol) and rapamycin (0.9 g, 1 mmol) are co-dissolved in 20 mL of N,N-dimethylformamide (DMF) to obtain a reaction mixture, and the reaction mixture is stirred and refluxed for 30 hours; the reaction mixture is cooled to 25 °C, purified water (50 mL) is added, and then 100 mL of dichloromethane (DCM) is added for extraction; the extract is washed with brine and dried over Na2SO4, the solvent is removed by vacuum drying, and the crude product is purified by silica gel chromatography column (hexane / acetone = 1:1) to obtain azido-modified rapamycin.

[0081] The preparation method of the alkynyl-modified bovine serum albumin comprises the following steps:

[0082] An alkynyl-N-hydroxysuccinimide crosslinker is used for a linking reaction with bovine serum albumin. 20 mg of bovine serum albumin and 60 mg of alkynyl-N-hydroxysuccinimide crosslinker are sequentially added to 3000 μL of phosphate buffer solution with a concentration of 0.2 M and a pH value of 8.0 to prepare a reaction solution; the reaction solution is incubated at room temperature of 25 °C for 1 hour, and then left standing overnight at 4 °C; after the reaction is completed, purification is carried out by dialysis, and dialysis is carried out 2 times with phosphate buffer solution with a pH value of 7.2 to obtain an alkynyl-modified bovine serum albumin solution with a final working concentration of 5 mg / mL.

[0083] The preparation method of the alkynyl-modified glucose-6-phosphate dehydrogenase comprises the following steps:

[0084] (E1) Prepare a glucose-6-phosphate dehydrogenase solution for maleimide coupling reaction: Dissolve disodium ethylenediaminetetraacetate in phosphate buffer solution (PBS) with a concentration of 50 mM until the final concentration of disodium ethylenediaminetetraacetate is 10 mM, adjust the pH value to 7.4 to obtain a glucose-6-phosphate dehydrogenase buffer solution, and then add 6.0 mg of glucose-6-phosphate dehydrogenase to 1000 μL of glucose-6-phosphate dehydrogenase buffer solution, stir and dissolve to obtain a glucose-6-phosphate dehydrogenase solution;

[0085] (E2) Prepare an alkynyl-maleimide crosslinker that can bind to glucose-6-phosphate dehydrogenase: Weigh 3.0 mg of alkynyl-maleimide and add it to 200 μL of DMF, stir and dissolve to obtain a solution with a concentration of 15 mg / mL, which is the alkynyl-maleimide crosslinker;

[0086] (E3) Complete the alkynylation reaction of glucose-6-phosphate dehydrogenase: Dropwise add the alkynyl-maleimide crosslinker prepared in step (E2) into the glucose-6-phosphate dehydrogenase solution prepared in step (E1), stir evenly at room temperature (25 °C) simultaneously, and incubate at 25 °C for 2 hours;

[0087] (E4) Purification: After the reaction, purification is carried out by dialysis. Dialyze 4 times using a phosphate buffer solution with a pH value of 7.3 to obtain an alkynyl-modified glucose-6-phosphate dehydrogenase solution with a final working concentration of 3 mg / mL.

[0088] The beneficial effects of the present invention are as follows:

[0089] The homogeneous enzyme immunoassay system based on click chemistry coupling technology of the present invention utilizes the specific coupling reaction between the azide functional group and the alkynyl functional group in click chemistry. This coupling reaction does not require the use of special activators and can occur under very mild conditions. Moreover, there are no compounds containing these two special structures of azide and alkynyl in the human body. Therefore, using this click chemistry coupling technology can significantly improve the specificity of the conjugate structure of small molecule hapten and macromolecule protein, and thus also significantly improve the specificity of small molecule specific antibodies. Compared with the homogeneous enzyme immunoassay platform based on traditional coupling technology, the homogeneous enzyme immunoassay system based on click chemistry coupling technology provided by the present invention has the advantages of simple preparation, rapid reaction, safe and pollution-free, low cost, and excellent reagent performance, which is conducive to large-scale production and application. Description of the Drawings

[0090] Figure 1 Reaction standard curve graph of the rapamycin homogeneous enzyme immunoassay of the present invention. Detailed Embodiments

[0091] The following further describes the present invention in combination with specific embodiments. The following embodiments are only used to illustrate the technical solutions of the present invention, but not to limit the scope of the present invention. The technical means used in the embodiments are all conventional means well-known to those of ordinary skill in the art unless otherwise specified. The reagents, instruments, equipment, and consumables used can be obtained from regular channel suppliers, and other materials with the same or similar properties and functions can be used to implement the present invention.

[0092] Example 1. Synthesis of Rapamycin Click Chemistry Derivative

[0093] The synthesis steps of the rapamycin click chemistry derivative (azide-modified rapamycin) are as follows:

[0094] Hydrogen bromide (0.81 g, 10 mmol), sodium azide (0.33 g, 5 mmol) and rapamycin (0.9 g, 1 mmol) were co-dissolved in 20 mL of N,N-dimethylformamide (DMF) to obtain a reaction mixture, and the reaction mixture was stirred and refluxed for 30 hours; the reaction mixture was cooled to 25 °C, purified water (50 mL) was added, and then 100 mL of dichloromethane (DCM) was added for extraction; the extract was washed with brine and dried over Na2SO4, the solvent was removed by vacuum drying, and the crude product was purified by silica gel column chromatography (hexane / acetone = 1:1) to obtain azide-modified rapamycin.

[0095] Example 2. Preparation of alkynyl-modified bovine serum albumin

[0096] A method for preparing alkynyl-modified bovine serum albumin comprises the following steps:

[0097] A linking reaction was carried out using an alkynyl-N-hydroxysuccinimide crosslinker and bovine serum albumin. 20 mg of bovine serum albumin and 60 mg of alkynyl-N-hydroxysuccinimide crosslinker were sequentially added to 3000 μL of phosphate buffer with a concentration of 0.2 M and a pH value of 8.0 to prepare a reaction solution; the reaction solution was incubated at room temperature of 25 °C for 1 hour, and then left standing overnight at 4 °C; after the reaction was completed, purification was carried out by dialysis, and dialysis was carried out 2 times using phosphate buffer with a pH value of 7.2 to obtain an alkynyl-modified bovine serum albumin solution with a final working concentration of 5 mg / mL.

[0098] Example 3. Preparation of a rapamycin click chemistry conjugate immunogen

[0099] The preparation method of the rapamycin click chemistry conjugate immunogen is as follows:

[0100] (1) Prepare 3 fresh buffer solutions required for the click reaction: Weigh 3.5 mg of tetrachlorophthalic anhydride (THPA) and dissolve it in 200 μL of purified water to obtain a tetrachlorophthalic anhydride buffer solution with a concentration of 40 mM; weigh 16 mg of CuSO4 and dissolve it in 2.5 mL of purified water to obtain a CuSO4 buffer solution with a concentration of 40 mM; weigh 12 mg of sodium ascorbate and dissolve it in 200 μL of purified water to obtain a sodium ascorbate buffer solution with a concentration of 300 mM; calculate the required amount of the azide-modified rapamycin according to the molecular weight of the azide-modified rapamycin, and weigh the required amount of the azide-modified rapamycin and dissolve it in an appropriate amount of DMSO to prepare an azide-modified rapamycin solution with a final concentration of 10 mM;

[0101] (2) Perform a click chemistry coupling reaction between the alkyne-modified immunogenic protein and azide-modified rapamycin: Measure 750 μL of the azide-modified rapamycin solution with a concentration of 10 mM, 375 μL of the tetrachlorophthalic anhydride buffer solution with a concentration of 40 mM, 375 μL of the CuSO4 buffer solution with a concentration of 40 mM, and 375 μL of the sodium ascorbate buffer solution with a concentration of 300 mM prepared in step (1), and sequentially add them to 4 mL of the alkyne-modified immunogenic protein solution with a concentration of 5 mg / mL (the solvent is PBS buffer with 10 mM and pH = 7.4). Mix well and incubate at room temperature of 25 °C for 1 hour, then let it stand overnight at 4 °C;

[0102] (3) Purify the conjugate: After the reaction, transfer the coupling reaction solution in step (2) to a MWCO 6 - 8000 dialysis membrane, perform dialysis treatment 4 times using 3 L of physiological saline as the dialysis solution, and then dialyze with PBS buffer with a concentration of 10 mM and pH = 7.4 for 12 hours to obtain the rapamycin click chemistry conjugate immunogen solution.

[0103] Example 4. Preparation of anti-rapamycin specific antibody

[0104] The preparation method of the anti-rapamycin specific antibody includes the following steps:

[0105] (1) Dilute the rapamycin click chemistry conjugate immunogen with PBS buffer to 3.0 mg / mL to obtain an artificial antigen solution, and then mix 3.0 mL of the artificial antigen solution with an equal volume of Freund's complete adjuvant and inject it into a rabbit;

[0106] (2) After 2 weeks, mix 3.0 mL of the same artificial antigen solution with an equal volume of Freund's incomplete adjuvant and inject it into the above rabbit once, and then inject it once every 3 weeks for a total of 5 injections;

[0107] (3) Take blood from the immunized rabbit above, separate and purify the antiserum to obtain the anti-rapamycin specific antibody.

[0108] Example 5. Preparation of alkyne-modified glucose-6-phosphate dehydrogenase

[0109] The preparation method of the alkyne-modified glucose-6-phosphate dehydrogenase includes the following steps:

[0110] (1) Preparation of glucose-6-phosphate dehydrogenase solution for maleimide coupling reaction: Dissolve disodium ethylenediaminetetraacetate in phosphate buffer (PBS) with a concentration of 50 mM until the final concentration of disodium ethylenediaminetetraacetate is 10 mM, adjust the pH value to 7.4 to obtain glucose-6-phosphate dehydrogenase buffer solution, and then add 6.0 mg of glucose-6-phosphate dehydrogenase to 1000 μL of glucose-6-phosphate dehydrogenase buffer solution, stir to dissolve to obtain glucose-6-phosphate dehydrogenase solution;

[0111] (2) Preparation of alkyne-maleimide crosslinker that can bind to glucose-6-phosphate dehydrogenase: Weigh 3.0 mg of alkyne-maleimide and add it to 200 μL of DMF, stir to dissolve to obtain a solution with a concentration of 15 mg / mL, which is the alkyne-maleimide crosslinker;

[0112] (3) Completion of the alkynylation reaction of glucose-6-phosphate dehydrogenase: Dropwise add the alkyne-maleimide crosslinker prepared in step (2) into the glucose-6-phosphate dehydrogenase solution prepared in step (1), stir evenly at room temperature of 25 °C at the same time, and incubate at 25 °C for 2 hours;

[0113] (4) Purification: After the reaction, purification is carried out by dialysis method, and dialysis is carried out 4 times with phosphate buffer with a pH value of 7.3 to obtain an alkyne-modified glucose-6-phosphate dehydrogenase solution with a final working concentration of 3 mg / mL.

[0114] Example 6. Preparation of click chemistry conjugate of rapamycin labeled with glucose-6-phosphate dehydrogenase

[0115] The preparation method of the click chemistry conjugate of rapamycin labeled with glucose-6-phosphate dehydrogenase is as follows:

[0116] (1) Prepare 3 kinds of fresh buffer solutions required for click reaction: Weigh 3.5 mg of tetrachlorophthalic anhydride (THPA) and dissolve it in 200 μL of purified water to obtain a tetrachlorophthalic anhydride buffer solution with a concentration of 40 mM; Weigh 8 mg of CuSO4 and dissolve it in 2.5 mL of purified water to obtain a CuSO4 buffer solution with a concentration of 20 mM; Weigh 12 mg of sodium ascorbate and dissolve it in 200 μL of purified water to obtain a sodium ascorbate buffer solution with a concentration of 300 mM; Calculate the required dosage of the azide-modified rapamycin according to the molecular weight of the azide-modified rapamycin, weigh the required dosage of the azide-modified rapamycin and dissolve it in an appropriate amount of DMSO to prepare an azide-modified rapamycin solution with a final concentration of 2.5 mM;

[0117] (2) Click chemistry coupling reaction of alkynyl-modified glucose-6-phosphate dehydrogenase with azide-modified rapamycin: Measure 150 μL of the azide-modified rapamycin solution with a concentration of 2.5 mM, 75 μL of the tetrachlorophthalic anhydride buffer solution with a concentration of 40 mM, 75 μL of the CuSO4 buffer solution with a concentration of 20 mM, and 75 μL of the sodium ascorbate buffer solution with a concentration of 300 mM prepared in step (1), and add them successively to 666 μL of the alkynyl-modified glucose-6-phosphate dehydrogenase solution with a concentration of 3 mg / mL (the solvent is 10 mM, PBS buffer solution with pH = 7.4), mix well and incubate at room temperature of 25 °C for 1 hour (the reaction efficiency can be improved by extending the reaction time);

[0118] (3) Purification of the conjugate: After the reaction is completed, filter the coupling reaction solution in step (2) using a pre-equilibrated G-25 Sephadex gel chromatography column to obtain a glucose-6-phosphate dehydrogenase-labeled rapamycin click chemistry conjugate solution, and finally add 0.5% BSA by mass fraction, 0.1% NaN3 by mass fraction and 50% glycerol by volume fraction for storage.

[0119] Example 7. Preparation of a rapamycin detection kit product

[0120] The specific preparation method of the rapamycin detection kit product is as follows:

[0121] (1) Preparation of rapamycin R1 reagent: Add 0.25% bovine serum albumin, 50 mmol / L glucose-6-phosphate and 50 mmol / L oxidized nicotinamide adenine dinucleotide to 50 mmol / L Tris buffer solution and stir to dissolve to prepare R1 buffer solution, then add the anti-small molecule specific antibody described in Scheme 1 to the above R1 buffer solution in a volume ratio of 1:1500, mix well, and adjust the pH to 8.0 with 6 mol / L hydrochloric acid to prepare R1 reagent;

[0122] (2) Preparation of rapamycin R2 reagent: Add 0.25% bovine serum albumin to 100 mmol / L Tris buffer solution and stir to dissolve to prepare R2 buffer solution, then add the glucose-6-phosphate dehydrogenase-labeled rapamycin click chemistry conjugate to the above R2 buffer solution in a volume ratio of 1:1200, mix well, and adjust the pH to 7.6 with 6 mol / L hydrochloric acid to prepare R2 reagent;

[0123] (3) Preparation of rapamycin calibrator: Rapamycin pure powder was added to 6 portions of Tris-HCl buffer with a concentration of 50.0 mmol / L and pH = 7.2, stirred and dissolved, and diluted into standard solutions with final concentrations of 0.00 ng / mL, 2.50 ng / mL, 5.00 ng / mL, 10.00 ng / mL, 20.00 ng / mL, and 40.00 ng / mL respectively. Then, 0.5% sodium chloride, 1.0% bovine serum albumin, 0.75% ethylenediaminetetraacetic acid, and 0.05% sodium azide were added to each solution respectively, and stirred evenly to obtain the rapamycin calibrator;

[0124] (4) Preparation of rapamycin quality control product: Rapamycin pure powder was added to 4 portions of Tris-HCl buffer with a concentration of 50.0 mmol / L and pH = 7.2, stirred and dissolved, and diluted into standard solutions with final concentrations of 0.00 ng / mL, 2.00 ng / mL, 12.00 ng / mL, and 36.00 ng / mL respectively. Then, 0.5% sodium chloride, 1.0% bovine serum albumin, 0.75% ethylenediaminetetraacetic acid, and 0.05% sodium azide were added to each solution respectively, and stirred evenly to obtain the rapamycin quality control product.

[0125] Example 8. Performance verification of rapamycin detection kit product

[0126] The rapamycin detection kit product prepared in Example 7 was subjected to performance verification. The main performance indicators detected were precision, recovery rate, inter-day precision, linear range, analytical sensitivity, specificity, etc. The performance indicators were based on the relevant requirements of GB / T 26124-2011 "In Vitro Diagnostic Reagents (Kits) for Clinical Chemistry" and "National Health Industry Standard of the People's Republic of China: Linear Evaluation of Quantitative Determination Methods", and with reference to the relevant CLSI standards. The methods and results of reagent performance verification are as follows:

[0127] (1) Preparation of rapamycin homogeneous enzyme immunoassay standard curve: First, set the reaction parameters of Hitachi 7180 automatic biochemical analyzer (Table 1), then add rapamycin R1 reagent to the automatic biochemical analyzer, and then add rapamycin calibrator, mix well, incubate at 37°C for 5 minutes, finally add rapamycin R2 reagent, mix well, after incubating at 37°C for 1.5 minutes, read the absorbance A1 at a wavelength of 340 nm, and read the absorbance A2 after incubating at a constant temperature for 3.5 minutes, calculate △A = A2 - A1, and the automatic biochemical analyzer automatically calculates the reaction rate at different calibrator concentrations (Table 2) to obtain the reaction standard curve, as shown in the appendix Figure 1 as shown.

[0128] Table 1. Reaction parameters of Hitachi 7180 automatic biochemical analyzer

[0129]

[0130] Table 2. Calibration absorbance change values (reaction rates) of calibration products at different concentrations

[0131]

[0132] (2) Precision and recovery verification: Using the standard curve of rapamycin homogeneous enzyme immunoassay prepared in the above step (1), the Hitachi 7180 automatic biochemical analyzer was used to repeatedly measure the blank, low-value, medium-value, and high-value quality control products 10 times, and the test data are shown in Table 3.

[0133] Table 3. Experimental results of precision and recovery verification

[0134]

[0135]

[0136] The results show that: the coefficient of variation (CV) of the rapamycin content in different concentration quality control products measured by the rapamycin detection kit product prepared in the present invention is lower than 5.0%, and the recovery rates are all between 95% and 105%, meeting the requirements of clinical test standards. It shows that the homogeneous enzyme immunoassay system based on click chemistry coupling technology of the present invention has high precision in detecting the content of small molecule haptens in biological samples and accurate results.

[0137] (3) Inter-day precision verification: The instrument should be calibrated before performing the precision verification experiment. Use the quality control product for routine quality control to ensure that it is in control before performing the inter-day precision experiment. If the in-house quality control is out of control or other operation problems occur, the data of this batch must be discarded and another batch of measurements must be added. If multiple test items are detected on the same analysis system, the inter-day precision verification experiment should be carried out for each test item separately. Record the experimental data for statistical analysis, and the qualified standard is that the coefficient of variation (CV) does not exceed 5.0%. The test results are shown in Table 4.

[0138] Table 4. Experimental results of inter-day precision verification

[0139]

[0140]

[0141] The experimental results show that: the coefficients of variation (CV) of the inter-day precision verification of the low-value quality control product, medium-value quality control product, and high-value quality control product detected by the rapamycin detection kit product prepared in the present invention are all lower than 5.0%, meeting the requirements of clinical test standards.

[0142] (4) Linear range verification: Take high-value and low-value quality control products of rapamycin, mix them in proportion to form 10 different concentrations, measure each concentration 3 times, and conduct statistical analysis on the results. Gradually reduce the data points based on the experimental results until a linear relationship is shown, and the widest linear range can be obtained. All quality control products should be randomly measured in one run or several runs with very short intervals, and all detections should be completed within one day.

[0143] The experimental results show that: For the rapamycin detection kit product prepared by the present invention, the correlation coefficient r = 0.9997 in the range of 0.00 - 40.00 ng / mL, and r ≥ 0.990, indicating good linear correlation within this range. In the linear range of 0.00 - 10.00 ng / mL, the absolute deviation is within ±1.00 ng / mL, and in the linear range of 10.00 - 40.00 ng / mL, the relative deviation is within ±5.0%, meeting the requirements of clinical test standards.

[0144] (5) Analytical sensitivity verification: Use purified water as the test sample, measure the change value of the reagent blank absorbance (A2 - A1), and at the same time measure the change value of the absorbance of a calibrated sample with a rapamycin concentration of 3.00 ng / mL. Calculate the absolute value of the difference between the change value of the absorbance of the calibrated sample and the change value of the reagent blank absorbance, and convert it to the analytical sensitivity value (|ΔA|) of the 3.00 ng / mL rapamycin calibrated sample (Table 5).

[0145] Table 5. Analytical sensitivity verification data

[0146]

[0147] Standard requirements for performance indicators: When measuring a rapamycin calibrated sample with a concentration of 3.00 ng / mL, the allowable range of the analytical sensitivity value (|ΔA|) is 0.0100 - 0.3000. It can be seen from the experimental results that the analytical sensitivity of the rapamycin detection kit product prepared by the present invention meets the requirements of clinical test standards.

[0148] (6) Verification of cross-reaction (specificity) of common drugs: Select 100 common drugs for cross-reaction verification. Prepare solutions with a concentration of 50.0 ng / mL of the pure powders of 100 common drugs as the test drug samples respectively, and use the rapamycin homogeneous enzyme immunoassay standard curve prepared in the above step (1). Detect the concentration of each test drug sample with a Hitachi 7180 automatic biochemical analyzer. The names of 100 common drugs and the detection results are shown in Table 6.

[0149] Table 6. Detection results of cross-reaction of 100 common drugs

[0150]

[0151]

[0152]

[0153] The test results show that the actual test values of the above 100 common drugs are all 0.00 ng / mL. Thus, it can be seen that the rapamycin test kit product prepared by the present invention has strong specificity, has no cross-reaction with 100 common drugs, and can specifically detect the content of rapamycin in biological samples.

[0154] Enlightened by the ideal embodiments of the present invention as described above, through the above description, those skilled in the art can make various changes and modifications without departing from the technical idea of the present invention. The technical scope of the present invention is not limited to the content in the specification, and must be determined according to the scope of the claims.

Claims

1. A homogeneous enzyme immunoassay system based on click chemistry coupling technology, characterized in that, The analysis system consists of R1 reagent, R2 reagent, calibrator and quality control product. The R1 reagent contains a small molecule-specific antibody and R1 buffer, and the R2 reagent contains a glucose-6-phosphate dehydrogenase-labeled small molecule click chemistry conjugate and R2 buffer; the small molecule-specific antibody is an antibody produced by immunizing experimental animals with a small molecule click chemistry conjugate immunogen; the small molecule click chemistry conjugate immunogen is formed by coupling a small molecule click chemistry derivative with a click chemistry carrier, and its structural formula is as shown in Formula I: Formula Ⅰ; The small molecule click chemistry derivative is an azide-modified small molecule hapten, and the small molecule hapten is any organic compound with a molecular weight less than 4000 Da; preferably, the small molecule hapten includes any organic compound with a molecular weight less than 4000 Da among drugs, hormones, drugs, stimulants, metabolites, vitamins, agricultural and veterinary drugs, food additives, environmental pollutants; more preferably, the small molecule hapten is any drug with a molecular weight less than 4000 Da; further preferably, the small molecule hapten is rapamycin; The experimental animals are mammals, and the mammals are one of rabbits, sheep, goats, mice, rats, guinea pigs, donkeys, horses, alpacas or camels; The click chemistry carrier is an alkyne-modified immunogenic protein, and the immunogenic protein is one of serum albumin, immunoglobulin, thyroglobulin, fibrinogen, ovalbumin, hemocyanin, toxoid protein, myoglobin, polylysine, polyglutamic acid; The R1 buffer contains an enzyme substrate, coenzyme, bovine serum albumin and Tris buffer. The enzyme substrate is glucose-6-phosphate, and the coenzyme is nicotinamide adenine dinucleotide oxidized form; The glucose-6-phosphate dehydrogenase-labeled small molecule click chemistry conjugate is formed by coupling the above small molecule click chemistry derivative with click chemistry recombinant glucose-6-phosphate dehydrogenase, and its structural formula is as shown in Formula II: Formula II; The click chemistry recombinant glucose-6-phosphate dehydrogenase is an alkyne-modified glucose-6-phosphate dehydrogenase; the R2 buffer is a Tris buffer containing bovine serum albumin; The calibrator is prepared by dissolving a certain amount of small molecule hapten pure product in a certain amount of Tris-HCl buffer to prepare a high-concentration stock solution, and diluting this stock solution in Tris-HCl buffer according to the calibration concentration requirements to form standard solutions with different concentrations for use as calibration solutions; The quality control product is prepared by dissolving a certain amount of small molecule hapten pure product in a certain amount of Tris-HCl buffer to prepare a high-concentration stock solution, and diluting this stock solution in Tris-HCl buffer according to the detection concentration requirements to form standard solutions with different concentrations for use as quality control samples.

2. A preparation method of a homogeneous enzyme immunoassay system based on click chemistry coupling technology as described in claim 1, characterized in that, The preparation method includes the following steps: Preparation of (A1) Reagent R1: Bovine serum albumin, glucose-6-phosphate, and oxidized nicotinamide adenine dinucleotide were successively added to Tris buffer and stirred until dissolved to form R1 buffer. Then, the anti-small molecule specific antibody described in claim 1 was added to the above R1 buffer in a volume ratio of 1:100 - 1:10,000 and mixed evenly. Subsequently, the pH was adjusted with hydrochloric acid to prepare Reagent R1; Preparation of (A2) Reagent R2: Bovine serum albumin was added to Tris buffer and stirred until dissolved to form R2 buffer. Then, the small molecule click chemistry conjugate labeled with glucose-6-phosphate dehydrogenase described in claim 1 was added to the above R2 buffer in a volume ratio of 1:100 - 1:10,000 and mixed evenly. Subsequently, the pH was adjusted with hydrochloric acid to prepare Reagent R2; Preparation of (A3) Calibrator: The pure powder of small molecule hapten was respectively added to 6 portions of Tris-HCl buffer, stirred until dissolved, and diluted into 6 standard solutions with different concentrations according to a certain ratio. Then, sodium chloride, bovine serum albumin, ethylenediaminetetraacetic acid, and sodium azide were respectively added to each solution and stirred evenly to obtain the small molecule hapten calibrator; Preparation of (A4) Quality Control Substance: The pure powder of small molecule hapten was respectively added to 4 portions of Tris-HCl buffer, stirred until dissolved, and diluted into 4 standard solutions with different concentrations according to the detection concentration requirements. Then, sodium chloride, bovine serum albumin, ethylenediaminetetraacetic acid, and sodium azide were respectively added to each solution and stirred evenly to obtain the small molecule hapten quality control substance.

3. The preparation method of the homogeneous enzyme immunoassay system based on click chemistry coupling technology according to claim 2, wherein, The preparation method of the anti-small molecule specific antibody described above comprises the following steps: (B1) Dilute the small molecule click chemistry conjugate immunogen described in claim 1 with PBS buffer to obtain an artificial antigen solution. Then, mix 2.0 - 5.0 mL of the artificial antigen solution with an equal volume of Freund's complete adjuvant and inject it into the experimental animal described in claim 1; (B2) After 1 - 4 weeks, mix the same artificial antigen solution with an equal volume of Freund's incomplete adjuvant and inject the above experimental animal once. Thereafter, inject once every 1 - 4 weeks for a total of 3 - 8 injections; (B3) Take blood from the immunized above experimental animal, separate and purify the antiserum to obtain the anti-small molecule specific antibody.

4. The preparation method of the homogeneous enzyme immunoassay system based on click chemistry coupling technology according to claim 3, wherein, The preparation method of the small molecule click chemistry conjugate immunogen described above has the following reaction formula: ； The specific operation steps are as follows: (C1) Prepare 3 fresh buffer solutions required for the click reaction: Weigh tetrachlorophthalic anhydride and dissolve it in purified water to obtain a tetrachlorophthalic anhydride buffer solution; weigh CuSO4 and dissolve it in purified water to obtain a CuSO4 buffer solution; weigh sodium ascorbate and dissolve it in purified water to obtain a sodium ascorbate buffer solution; calculate the required dosage of each azide-modified small molecule hapten according to the molecular weight of the azide-modified small molecule hapten, and weigh the required dosage of the azide-modified small molecule hapten and dissolve it in an appropriate amount of DMSO to prepare an azide-modified small molecule hapten solution; (C2) Perform a click chemical coupling reaction between an alkyne-modified immunogenic protein and an azide-modified small molecule hapten: Measure the azide-modified small molecule hapten solution, tetrachlorophthalic anhydride buffer solution, CuSO4 buffer solution, and sodium ascorbate buffer solution prepared in step (C1), and sequentially add them to the alkyne-modified immunogenic protein solution with PBS buffer as the solvent. Mix well and incubate at room temperature for 0.5 - 3 hours, then let it stand overnight at 4°C; (C3) Purify the conjugate: After the reaction is completed, transfer the conjugate reaction solution in step (C2) to a MWCO 6 - 8000 dialysis membrane, and perform dialysis treatment 2 - 6 times using physiological saline as the dialysis solution, and then dialyze with PBS buffer for 6 - 18 hours to obtain a small molecule click chemical conjugate immunogen solution.

5. The preparation method of the homogeneous enzyme immunoassay system based on click chemistry coupling technology according to claim 2, wherein, The preparation method of the glucose-6-phosphate dehydrogenase-labeled small molecule click chemical conjugate has the following reaction formula: ； The specific operation steps are as follows: (D1) Prepare 3 fresh buffer solutions required for the click reaction: Weigh tetrachlorophthalic anhydride and dissolve it in purified water to obtain a tetrachlorophthalic anhydride buffer solution; weigh CuSO4 and dissolve it in purified water to obtain a CuSO4 buffer solution; weigh sodium ascorbate and dissolve it in purified water to obtain a sodium ascorbate buffer solution; calculate the required amount of the azide-modified small molecule hapten according to the molecular weight of each azide-modified small molecule hapten, and weigh the required amount of the azide-modified small molecule hapten and dissolve it in an appropriate amount of DMSO to prepare an azide-modified small molecule hapten solution; (D2) Perform a click chemical coupling reaction between an alkyne-modified glucose-6-phosphate dehydrogenase and an azide-modified small molecule hapten: Measure the azide-modified small molecule hapten solution, tetrachlorophthalic anhydride buffer solution, CuSO4 buffer solution, and sodium ascorbate buffer solution prepared in step (D1), and sequentially add them to the alkyne-modified glucose-6-phosphate dehydrogenase solution with PBS buffer as the solvent. Mix well and incubate at room temperature for 0.5 - 3 hours; (D3) Purify the conjugate: After the reaction is completed, filter the conjugate reaction solution in step (D2) using a pre-equilibrated G-25 Sephadex gel chromatography column to obtain a glucose-6-phosphate dehydrogenase-labeled small molecule click chemical conjugate solution, and finally add BSA, NaN3, and glycerol for storage.

6. The preparation method of the homogeneous enzyme immunoassay system based on click chemistry coupling technology according to claim 4 or 5, characterized in that, The preparation method of the azide-modified small molecule hapten includes the following steps: Dissolve hydrogen bromide, sodium azide, and the small molecule hapten together in N,N-dimethylformamide to obtain a reaction mixture, and stir and reflux the reaction mixture for 30 hours; cool the reaction mixture to 25°C, add an appropriate amount of purified water, and then add dichloromethane for extraction; wash the extract with brine and dry it over Na2SO4, remove the solvent by vacuum drying, and purify the crude product through a silica gel chromatography column to obtain the azide-modified small molecule hapten.

7. The preparation method of the homogeneous enzyme immunoassay system based on click chemistry coupling technology according to claim 4, wherein, The preparation method of the alkyne-modified immunogenic protein includes the following steps: Use an alkynyl-N-hydroxysuccinimide crosslinker to carry out a coupling reaction with an immunogenic protein. Add the immunogenic protein and the alkynyl-N-hydroxysuccinimide crosslinker to phosphate buffer in sequence to prepare a reaction solution; incubate the reaction solution at room temperature for 0.5 - 3 hours, and then let it stand overnight at 4°C; after the reaction is completed, purify it by dialysis, and dialyze with phosphate buffer 1 - 4 times to obtain an alkynyl-modified immunogenic protein solution.

8. The preparation method of the homogeneous enzyme immunoassay system based on click chemistry coupling technology according to claim 5, wherein, The preparation method of the alkynyl-modified glucose-6-phosphate dehydrogenase comprises the following steps: (E1) Prepare a glucose-6-phosphate dehydrogenase solution for maleimide coupling reaction: Dissolve disodium ethylenediaminetetraacetate in phosphate buffer, adjust the pH value to obtain a glucose-6-phosphate dehydrogenase buffer, and then add glucose-6-phosphate dehydrogenase to the glucose-6-phosphate dehydrogenase buffer, stir to dissolve to obtain a glucose-6-phosphate dehydrogenase solution; (E2) Prepare an alkynyl-maleimide crosslinker that can bind to glucose-6-phosphate dehydrogenase: Weigh alkynyl-maleimide and add it to DMF, stir to dissolve to obtain the alkynyl-maleimide crosslinker; (E3) Complete the alkynylation reaction of glucose-6-phosphate dehydrogenase: Dropwise add the alkynyl-maleimide crosslinker prepared in step (E2) into the glucose-6-phosphate dehydrogenase solution prepared in step (E1), stir evenly at room temperature at the same time, and incubate at 25°C for 1 - 4 hours; (E4) Purification: After the reaction is completed, purify it by dialysis, and dialyze with phosphate buffer 2 - 6 times to obtain an alkynyl-modified glucose-6-phosphate dehydrogenase solution.

9. Use of a homogeneous enzyme immunoassay system based on click chemistry coupling technology as described in claim 1, characterized in that, Apply the homogeneous enzyme immunoassay system based on click chemistry coupling technology to assemble into a detection kit product, and use the detection kit product to detect the content of small molecule haptens in biological samples; the biological sample is a liquid physiological sample of a human or an animal, and the liquid physiological sample includes but is not limited to serum, plasma, whole blood, urine, saliva, lymph fluid, tissue fluid, cerebrospinal fluid, amniotic fluid or tears; the small molecule hapten is any organic compound with a molecular weight less than 4000 Da.

10. Use of the homogeneous enzyme immunoassay system based on click chemistry coupling technology according to claim 9, characterized in that, The small molecule hapten includes any organic compound with a molecular weight less than 4000 Da among drugs, hormones, drugs, stimulants, metabolites, vitamins, agricultural and veterinary drugs, food additives, environmental pollutants; preferably, the small molecule hapten is any drug with a molecular weight less than 4000 Da; more preferably, the small molecule hapten is rapamycin.

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