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

Abstract

The invention discloses a homogeneous enzyme immunoassay system based on a click chemistry coupling technology as well as a preparation method and application of the homogeneous enzyme immunoassay 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 alkynyl functional group and an azido 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 any compound containing two special structures, namely alkynyl and azido, does not exist in a human body, so that the specificity of a micromolecule hapten and macromolecular protein coupling structure can be remarkably improved; and the specificity of the micromolecular specific antibody can also be obviously improved. The key technology of a homogeneous enzyme immunoassay platform is improved, and compared with a traditional homogeneous enzyme immunoassay platform, the homogeneous enzyme immunoassay 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 invention belongs to the field of biotechnology, and specifically relates to a homogeneous enzyme immunoassay system based on click chemistry coupling technology and a preparation method and application thereof. Background Art

[0002] In 1972, Rubenstein and Ullman created a homogeneous enzyme immunoassay (HEIA). The classic homogeneous enzyme immunoassay is a detection technology that combines biochemistry and immunity. It is mainly used to detect trace amounts of small molecules in biological samples, such as small molecule drugs, drugs, endogenous small molecules (hormones, metabolites, organic acids), etc. The basic principle of this technology is: hapten combines with enzyme to form enzyme-labeled hapten, retaining the activity of hapten and enzyme. When the enzyme-labeled hapten combines with the antibody, the labeled enzyme is in close contact with the antibody, so that the active center of the enzyme is affected and the activity is inhibited. When the antibody and enzyme-labeled hapten in the reagent are mixed with the specimen, the hapten in the sample and the enzyme-labeled hapten competitively bind to the antibody in the reagent. If the hapten content in the sample is high, the proportion of 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 proportional to the hapten in the sample. The content of the hapten in the sample can be calculated based on the results of the enzyme activity determination. The whole process of homogeneous enzyme immunoassay is a competitive reaction. The whole reaction occurs in a liquid homogeneous system. There is no need to separate the bound and free markers through a solid phase. The free small molecule antigens in the reaction system can compete with the "small molecule-enzyme" conjugate to bind to the specific site of the antibody. The more free small molecule antigens in the sample, the more antibodies that compete for binding, the more "small molecule-enzyme" conjugates released by the antibody, and the stronger the signal obtained by the reaction of the "small molecule-enzyme" conjugate with the substrate. The content of the sample can be accurately calculated by automatically detecting the change in the OD340nm absorbance value through a fully automatic biochemical analyzer. Since its advent, this technology has been widely used in many fields such as therapeutic drug monitoring, disease diagnosis, hormone detection, drug testing, food safety, etc. after more than 50 years of application and development.

[0003] The preparation technology of small molecule hapten and macromolecular protein conjugate (including: preparation of small molecule-protein carrier conjugate, preparation of small molecule-labeled enzyme conjugate) is the key technology for the preparation of homogeneous enzyme immunoassay reagents. The synthesis of small molecule hapten and protein conjugate requires the retention of small molecule specific sites, coupling small molecules to protein molecules, and ensuring the biological activity of the protein after coupling. For example, the "small molecule-labeled enzyme" coupling process must strictly maintain anhydrous and low temperature, and the amount of activator must be repeatedly measured to ensure the balance between the biological activity of the conjugate and the number of small molecule connections. The traditional coupling method mainly uses an activator to activate the small molecule hapten and cross-link with the macromolecular protein solution. This process requires strict control of multiple links such as reaction time, temperature, and activator dosage. The reaction product can only be obtained after purification by dialysis. Since the cross-linking groups that can be provided by commonly used protein molecules are similar, the selection of the connection method and the design of the coupling product mainly depend on the functional group structure on the small molecule compound or its derivative. Generally, small molecule compounds with free carboxyl or free amino groups or both can be directly coupled with protein molecules by certain methods; other small molecule compounds without free carboxyl or free amino groups need to undergo appropriate structural modification to transform them into derivatives with free carboxyl or free amino groups before they can be connected with protein molecules. Traditional bioconjugation technology has many defects, such as harsh reaction conditions, complex operation, low yield, high reagent toxicity, low activity of coupling products, many by-products, and difficulty in separation and purification.

[0004] Click chemistry is also known as "click chemistry", "link chemistry" and "quick-matching combination chemistry". It was created and proposed by American chemist Sharpless in 2001. It refers to a type of modular, highly selective coupling reaction with simple reaction materials, mild reaction conditions, high product yield, and convenient separation and purification, so as to achieve efficient and rapid synthesis of functional molecules, and synthesize various biological macromolecules by connecting various monomer molecular modules through carbon-heteroatom (CXC) bonds. In traditional organic synthetic chemistry, in order to pursue yield and efficiency, the construction of molecular skeletons usually needs to be carried out in organic solvents, and conditions such as heating, acid or base, and inert gas protection are used. Many organic reactions also require pre-protection of active groups, which makes the synthesis process complicated and cumbersome. In 2002, Sharpless and Meldal independently reported the copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. This reaction has been rapidly and widely used in the synthesis fields of polymers, drugs, new materials, etc., becoming a representative reaction of click chemistry. The two also won the 2022 Nobel Prize in Chemistry for this.

[0005] Rapamycin, also known as Sirolimus, is derived from Streptomyces hygroscopicus on Easter Island and is a triene macrolide antibiotic immunosuppressant. Rapamycin has a narrow effective blood concentration range, and its efficacy, toxicity and side effects are significantly correlated with blood concentration. Insufficient doses can cause the body to reject transplanted organs, and excessive doses can 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, organ transplant type, age, and medication dosage, there are large individual differences in drug absorption and metabolism, and it is difficult to directly estimate the blood concentration after administration. Therefore, it is clinically important to detect the individual rapamycin blood concentration of the patient, adjust the dosage or frequency of medication in a timely manner, 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. The HPLC and LC / MS / MS methods have complex pre-treatment, long time consumption, and high requirements for operators; the radioimmunoassay method has radioactive hazards to operators; the fluorescence polarization method instruments and reagents are mainly imported and costly. This application takes the small molecule drug rapamycin as an example and discloses a rapamycin homogeneous enzyme immunoassay reagent based on click chemistry coupling technology and its preparation and use methods.

[0006] The homogeneous enzyme immunoassay system based on click chemistry coupling technology of the present application utilizes the specific coupling reaction of the alkynyl functional group and the azido functional group in click chemistry. The coupling reaction does not require the use of a special activator and can react under very mild conditions. In addition, there are no compounds containing the two special structures of alkynyl and azide in the human body. Therefore, the use of the click chemistry coupling technology can significantly improve the specificity of the coupling structure of small molecule haptens and macromolecular proteins, and thus 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, safety 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 of 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 solution adopted by the present invention to solve its technical problems is 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 anti-small molecule specific antibody and R1 buffer, the R2 reagent contains small molecule click chemistry conjugate labeled with glucose-6-phosphate dehydrogenase and R2 buffer; the anti-small molecule specific antibody is an antibody produced by immunizing experimental animals with 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 alkyne-modified small molecule hapten, and the small molecule hapten is any organic compound with a molecular weight of less than 4000Da; preferably, the small molecule hapten includes any organic compound with a molecular weight of less than 4000Da among drugs, hormones, drugs, stimulants, metabolites, vitamins, agricultural and veterinary drugs, food additives, and environmental pollutants; more preferably, the small molecule hapten is any drug with a molecular weight of less than 4000Da; further preferably, the small molecule hapten is rapamycin;

[0011] The experimental animal is a mammal, and the mammal is one of rabbit, sheep, goat, mouse, rat, guinea pig, donkey, horse, alpaca or camel;

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

[0013] The R1 buffer contains 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;

[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 an azide-modified glucose-6-phosphate dehydrogenase; the R2 buffer is a Tris buffer containing bovine serum albumin;

[0017] The calibration material 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 storage solution, and diluting the storage solution in Tris-HCl buffer according to the calibration concentration requirements to form standard solutions of different concentrations, which are used 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 storage solution, and diluting the storage 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] Solution 2: A method for preparing a homogeneous enzyme immunoassay system based on click chemistry coupling technology as described in Solution 1 is provided, wherein the preparation method comprises the following steps:

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

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

[0022] (A3) Preparation of calibrators: Add small molecule hapten pure powder to 6 portions of Tris-HCl buffer with a concentration of 10.0-100.0 mmol / L and pH = 7.0-7.5, stir to 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 by mass fraction to each solution, stir evenly, and the small molecule hapten calibrators are obtained;

[0023] (A4) Preparation of quality control products: Add small molecule hapten pure powder to 4 portions of Tris-HCl buffer with a concentration of 10.0-100.0 mmol / L and pH = 7.0-7.5, stir to dissolve, and dilute to 4 standard solutions of different concentrations according to the detection concentration requirements. Then, add 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 by mass fraction to each solution, stir evenly, and the small molecule hapten quality control product is obtained.

[0024] As an optional scheme, the method for preparing the anti-small molecule specific antibody comprises the following steps:

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

[0026] (B2) 1-4 weeks later, 2.0-5.0 mL of the same artificial antigen solution mixed with an equal amount of Freund's incomplete adjuvant is injected into the above experimental animals once, and then injected once every 1-4 weeks, for a total of 3-8 injections;

[0027] (B3) Blood is collected from the immunized experimental animals, and antiserum is separated and purified to obtain anti-small molecule specific antibodies.

[0028] As an optional solution, the preparation method of the small molecule click chemistry conjugate immunogen is as follows:

[0029]

[0030] The specific steps are as follows:

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

[0032] (C2) using an azido-modified immunogenic protein and an alkynyl-modified small molecule hapten to perform a click chemistry coupling reaction: taking 750 μL of a 5-20 mM alkynyl-modified small molecule hapten solution prepared in step (C1), 375 μL of a 20-80 mM tetrachlorobiphenyl dicarboxylic anhydride buffer solution, 375 μL of a 20-80 mM CuSO4 buffer solution, and 375 μL of a 150-600 mM sodium ascorbate buffer solution, and sequentially adding them to 4 mL of a 5 mg / mL azido-modified immunogenic protein solution (solvent: 10 mM, pH=7.4 PBS buffer), mixing and incubating at room temperature 25°C for 0.5-3 hours, and then standing at 4°C overnight;

[0033] (C3) Conjugate purification: After the reaction is completed, the conjugated reaction solution in step (C2) is transferred to a MWCO 6-8000 dialysis membrane, dialyzed 2-6 times using 1-4 L of normal saline as the dialysate, and then dialyzed for 6-24 hours using a 10 mM, pH = 7.4 PBS buffer to obtain a small molecule click chemistry conjugate immunogen solution.

[0034] As an optional solution, the preparation method of the small molecule click chemistry conjugate labeled with glucose-6-phosphate dehydrogenase is as follows:

[0035]

[0036] The specific steps are as follows:

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

[0038] (D2) using azido-modified glucose-6-phosphate dehydrogenase and alkynyl-modified small molecule hapten to carry out click chemistry coupling reaction: 150 μL of 1.25-5.0 mM alkynyl-modified small molecule hapten solution prepared in step (D1), 75 μL of 20-80 mM tetrachlorobiphenyl dicarboxylic anhydride buffer solution, 75 μL of 10-40 mM CuSO4 buffer solution, and 75 μL of 150-600 mM sodium ascorbate buffer solution were measured, and added to 666 μL of 3 mg / mL azido-modified glucose-6-phosphate dehydrogenase solution (solvent is 10 mM, pH=7.4 PBS buffer), mixed and incubated at room temperature 25°C for 0.5-3 hours (extending the reaction time can improve the reaction efficiency);

[0039] (D3) Conjugate purification: After the reaction is completed, the coupling reaction solution in step (D2) is filtered using a pre-equilibrated G-25 polysaccharide gel chromatography column to obtain a small molecule click chemistry conjugate solution labeled with glucose-6-phosphate dehydrogenase, and finally 0.1-1.0% BSA by mass, 0.01-0.1% NaN3 by mass, and 25-50% glycerol by volume are added for storage.

[0040] As an optional scheme, the method for preparing the alkyne-modified small molecule hapten comprises the following steps:

[0041] K2CO3 (1.4 g, 10 mmol), 3-bromo-propyne (0.6 g, 5 mmol) and a small molecule hapten (1 mmol) were dissolved in 20 mL of N,N-dimethylformamide (DMF) to obtain a reaction mixture, and the reaction mixture was stirred and refluxed for 15-60 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 chromatography (hexane / acetone=1:1) to obtain an alkynyl-modified small molecule hapten.

[0042] As an optional scheme, the method for preparing the azido-modified immunogenic protein comprises the following steps:

[0043] Reaction basic principle: Use N-hydroxysuccinimide (NHS) reaction to replace the amino (-NH2) functional group in the immunogenic protein with the azido (-N3) functional group;

[0044] A azido-N-hydroxysuccinimide crosslinker is used to carry out a connection reaction with the immunogenic protein, and 20-200 mg of the immunogenic protein and 10-100 mg of the azido-N-hydroxysuccinimide crosslinker are sequentially added to 3000 μL of a 0.2M phosphate buffer solution with a pH value of 8.0 to prepare a reaction solution; the reaction solution is incubated at room temperature of 25°C for 0.5-3 hours, and then allowed to stand at 4°C overnight; after the reaction is completed, it is purified by dialysis, and dialyzed 1-3 times using a phosphate buffer solution with a pH value of 7.0-7.5 to obtain an azido-modified immunogenic protein solution with a final working concentration of 5 mg / mL.

[0045] As an optional scheme, the preparation method of the azido-modified glucose-6-phosphate dehydrogenase comprises the following steps:

[0046] Reaction basic principle: Use maleimide reaction to replace the sulfhydryl (-SH) functional group in glucose-6-phosphate dehydrogenase with the azido (-N3) functional group;

[0047] (E1) preparing a glucose-6-phosphate dehydrogenase solution for maleimide coupling reaction: dissolving ethylenediaminetetraacetic acid disodium in phosphate buffered saline (PBS) with a concentration of 50 mM to a final concentration of ethylenediaminetetraacetic acid disodium of 5-20 mM, adjusting the pH value to 7.4 to obtain a glucose-6-phosphate dehydrogenase buffer, and then adding 6.0 mg of glucose-6-phosphate dehydrogenase to 1000 μL of the glucose-6-phosphate dehydrogenase buffer, stirring to dissolve, and obtaining a glucose-6-phosphate dehydrogenase solution;

[0048] (E2) preparing an azido-maleimide crosslinker that can bind to glucose-6-phosphate dehydrogenase: weighing 3.0 mg of azido-maleimide and adding it to 200 μL of DMF, stirring and dissolving to obtain a solution with a concentration of 15 mg / mL, which is the azido-maleimide crosslinker;

[0049] (E3) completing the azidation reaction of glucose-6-phosphate dehydrogenase: adding the azido-maleimide crosslinker prepared in step (E2) dropwise to the glucose-6-phosphate dehydrogenase solution prepared in step (E1), stirring evenly at room temperature 25° C., and incubating at 25° C. for 1-3 hours;

[0050] (E4) Purification: After the reaction is completed, the enzyme is purified by dialysis using a phosphate buffer solution having a pH value of 7.0-7.5 for 2-6 times to obtain a azido-modified glucose-6-phosphate dehydrogenase solution having a final working concentration of 3 mg / mL.

[0051] Scheme 3: Provide an application of a homogeneous enzyme immunoassay system based on click chemistry coupling technology as described in Scheme 1, apply the homogeneous enzyme immunoassay system based on click chemistry coupling technology to assemble into a small molecule detection kit product, and use the small molecule detection kit product for detecting the content of small molecule haptens in biological samples; the biological sample is a liquid physiological sample of human or animal, and the liquid physiological sample includes but is not limited to serum, plasma, whole blood, urine, saliva, lymph, tissue fluid, cerebrospinal fluid, amniotic fluid or tears; the small molecule hapten is any organic compound with a molecular weight of less than 4000Da.

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

[0053] As a specific application scheme, the homogeneous enzyme immunoassay system based on click chemistry coupling technology is used to assemble a rapamycin detection kit product, and the rapamycin detection kit product is used to detect the rapamycin content in a biological sample, 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 sequentially added to 50 mmol / L Tris buffer and stirred to dissolve to prepare R1 buffer, then anti-rapamycin specific antibody was added to the R1 buffer at a volume ratio of 1:1200 and mixed, and 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, and then glucose-6-phosphate dehydrogenase labeled rapamycin click chemistry conjugate was added to the R2 buffer at a volume ratio of 1:1000 and mixed, and then the pH was adjusted to 7.6 with 6 mol / L hydrochloric acid to prepare R2 reagent;

[0057] (A3) Preparation of rapamycin calibrator: add rapamycin pure powder to 6 portions of 50.0 mmol / L Tris-HCl buffer at pH 7.2, stir to dissolve, dilute to 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, then add 0.5% sodium chloride, 1.0% bovine serum albumin, 0.75% ethylenediaminetetraacetic acid, and 0.05% sodium azide to each solution, stir evenly, and the rapamycin calibrator is obtained;

[0058] (A4) Preparation of rapamycin quality control: Add pure rapamycin powder to 4 portions of 50.0 mmol / L Tris-HCl buffer (pH=7.2), stir to dissolve, and dilute to 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, add 0.5% sodium chloride, 1.0% bovine serum albumin, 0.75% ethylenediaminetetraacetic acid, and 0.05% sodium azide by mass to each solution, respectively, and stir evenly to obtain the rapamycin quality control.

[0059] The method for preparing the anti-rapamycin specific antibody comprises the following steps:

[0060] (B1) diluting the rapamycin click chemistry conjugate immunogen to 3.0 mg / mL with PBS buffer to obtain an artificial antigen solution, and then mixing 3.0 mL of the artificial antigen solution with an equal amount of Freund's complete adjuvant and injecting the mixture into rabbits;

[0061] (B2) 2 weeks later, 3.0 mL of the same artificial antigen solution mixed with an equal amount of Freund's incomplete adjuvant was injected into the rabbits once, and then injected once every 3 weeks, for a total of 5 injections;

[0062] (B3) Blood was collected from the immunized rabbits, and antiserum was separated and purified to obtain anti-rapamycin specific antibodies.

[0063] The preparation method of the rapamycin click chemistry conjugate immunogen has the following reaction formula:

[0064]

[0065] The specific steps are as follows:

[0066] (C1) preparing three fresh buffer solutions required for the click reaction: weighing 3.5 mg of tetrachlorobiphenyl dicarboxylic anhydride (THPA) and dissolving it in 200 μL of purified water to obtain a 40 mM tetrachlorobiphenyl dicarboxylic anhydride buffer solution; weighing 16 mg of CuSO4 and dissolving it in 2.5 mL of purified water to obtain a 40 mM CuSO4 buffer solution; weighing 12 mg of sodium ascorbate and dissolving it in 200 μL of purified water to obtain a 300 mM sodium ascorbate buffer solution; calculating the required amount of alkynyl-modified rapamycin according to its molecular weight, weighing the required amount of alkynyl-modified rapamycin and dissolving it in an appropriate amount of DMSO to prepare an alkynyl-modified rapamycin solution with a final concentration of 10 mM;

[0067] (C2) Click chemistry coupling reaction using azido-modified bovine serum albumin and alkynyl-modified rapamycin: 750 μL of 10 mM alkynyl-modified rapamycin solution prepared in step (C1), 375 μL of 40 mM tetrachlorobiphenyl dicarboxylic anhydride buffer solution, 375 μL of 40 mM CuSO4 buffer solution, and 375 μL of 300 mM sodium ascorbate buffer solution were measured and added to 4 mL of 5 mg / mL azido-modified bovine serum albumin solution (solvent: 10 mM, pH=7.4 PBS buffer), mixed and incubated at room temperature 25°C for 1 hour, and then allowed to stand at 4°C overnight;

[0068] (C3) Conjugate purification: After the reaction is completed, the conjugated reaction solution in step (C2) is transferred to a MWCO 6-8000 dialysis membrane, dialyzed four times using 3 L of normal saline as the dialysate, and then dialyzed for 12 hours using a 10 mM, pH = 7.4 PBS buffer to obtain a rapamycin click chemistry conjugate immunogen solution.

[0069] The preparation method of the glucose-6-phosphate dehydrogenase labeled rapamycin click chemistry conjugate is as follows:

[0070]

[0071] The specific steps are as follows:

[0072] (D1) preparing three fresh buffer solutions required for the click reaction: weighing 3.5 mg of tetrachlorobiphenyl dicarboxylic anhydride (THPA) and dissolving it in 200 μL of purified water to obtain a 40 mM tetrachlorobiphenyl dicarboxylic anhydride buffer solution; weighing 8 mg of CuSO4 and dissolving it in 2.5 mL of purified water to obtain a 20 mM CuSO4 buffer solution; weighing 12 mg of sodium ascorbate and dissolving it in 200 μL of purified water to obtain a 300 mM sodium ascorbate buffer solution; calculating the required amount of alkynyl-modified rapamycin according to its molecular weight, weighing the required amount of alkynyl-modified rapamycin and dissolving it in an appropriate amount of DMSO to prepare an alkynyl-modified rapamycin solution with a final concentration of 2.5 mM;

[0073] (D2) Click chemistry coupling reaction using azido-modified glucose-6-phosphate dehydrogenase and alkynyl-modified rapamycin: 150 μL of 2.5 mM alkynyl-modified rapamycin solution prepared in step (D1), 75 μL of 40 mM tetrachlorobiphenyl dicarboxylic anhydride buffer solution, 75 μL of 20 mM CuSO4 buffer solution, and 75 μL of 300 mM sodium ascorbate buffer solution were measured and added to 666 μL of 3 mg / mL azido-modified glucose-6-phosphate dehydrogenase solution (solvent: 10 mM, pH=7.4 PBS buffer), mixed and incubated at room temperature 25°C for 1 hour (extending the reaction time can improve the reaction efficiency);

[0074] (D3) Conjugate purification: After the reaction is completed, the coupling reaction solution in step (D2) is filtered using a pre-equilibrated G-25 polysaccharide gel chromatography column to obtain a glucose-6-phosphate dehydrogenase-labeled rapamycin click chemistry conjugate solution, and finally 0.5% by mass of BSA, 0.1% by mass of NaN3 and 50% by volume of glycerol are added for storage.

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

[0076]

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

[0078]

[0079] The method for preparing the above-mentioned alkynyl-modified rapamycin comprises the following steps:

[0080] K2CO3 (1.4 g, 10 mmol), 3-bromo-propyne (0.6 g, 5 mmol) and rapamycin (0.9 g, 1 mmol) were dissolved in 20 mL of 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 DCM (100 mL) 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 chromatography (hexane / acetone=1:1) to obtain alkynyl-modified rapamycin.

[0081] The method for preparing the azido-modified bovine serum albumin comprises the following steps:

[0082] A azido-N-hydroxysuccinimide crosslinker was used to carry out a connection reaction with bovine serum albumin. 20 mg of bovine serum albumin and 60 mg of azido-N-hydroxysuccinimide crosslinker were added to 3000 μL of 0.2 M phosphate buffer at a pH of 8.0 to prepare a reaction solution. The reaction solution was incubated at room temperature of 25°C for 1 hour and then allowed to stand at 4°C overnight. After the reaction was completed, it was purified by dialysis and dialyzed twice with a phosphate buffer at a pH of 7.2 to obtain an azido-modified bovine serum albumin solution with a final working concentration of 5 mg / mL.

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

[0084] (E1) preparing a glucose-6-phosphate dehydrogenase solution for maleimide coupling reaction: dissolving ethylenediaminetetraacetic acid disodium in phosphate buffered saline (PBS) with a concentration of 50 mM to a final concentration of ethylenediaminetetraacetic acid disodium of 10 mM, adjusting the pH value to 7.4 to obtain a glucose-6-phosphate dehydrogenase buffer, and then adding 6.0 mg of glucose-6-phosphate dehydrogenase to 1000 μL of the glucose-6-phosphate dehydrogenase buffer, stirring to dissolve, and obtaining a glucose-6-phosphate dehydrogenase solution;

[0085] (E2) preparing an azido-maleimide crosslinker that can bind to glucose-6-phosphate dehydrogenase: weighing 3.0 mg of azido-maleimide and adding it to 200 μL of DMF, stirring and dissolving to obtain a solution with a concentration of 15 mg / mL, which is the azido-maleimide crosslinker;

[0086] (E3) completing the azidation reaction of glucose-6-phosphate dehydrogenase: adding the azido-maleimide crosslinker prepared in step (E2) dropwise to the glucose-6-phosphate dehydrogenase solution prepared in step (E1), stirring evenly at room temperature 25° C., and incubating at 25° C. for 2 hours;

[0087] (E4) Purification: After the reaction, the enzyme was purified by dialysis using a phosphate buffer solution with a pH of 7.3 for 4 times to obtain a 3 mg / mL azido-modified glucose-6-phosphate dehydrogenase solution.

[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 of the alkynyl functional group and the azido functional group in click chemistry, and the coupling reaction does not require the use of a special activator, and can react under very mild conditions, and there is no compound containing the two special structures of alkynyl and azido in the human body, so the use of the click chemistry coupling technology can significantly improve the specificity of the coupling structure of small molecule haptens and macromolecular proteins, 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, safety and pollution-free, low cost, superior reagent performance, etc., which is conducive to large-scale production and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] Figure 1 The reaction standard curve diagram of the rapamycin homogeneous enzyme immunoassay of the present invention. DETAILED DESCRIPTION

[0091] The present invention will be further described below in conjunction with specific embodiments. The following examples are only used to illustrate the technical solution of the present invention, but are not intended to limit the scope of the present invention. The technical means used in the embodiments are conventional means known to those of ordinary skill in the art unless otherwise specified, and the reagents, instruments, equipment, and consumables used can be purchased from regular channels, 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 derivatives

[0093] The synthesis steps of rapamycin click chemistry derivatives (alkyne-modified rapamycin) are as follows:

[0094] K2CO3 (1.4 g, 10 mmol), 3-bromo-propyne (0.6 g, 5 mmol) and rapamycin (0.9 g, 1 mmol) were dissolved in 20 mL of 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 DCM (100 mL) 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 chromatography (hexane / acetone=1:1) to obtain 0.5 g of alkynyl-modified rapamycin.

[0095] Example 2. Preparation of Azide-Modified Bovine Serum Albumin

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

[0097] A azido-N-hydroxysuccinimide crosslinker was used to carry out a connection reaction with bovine serum albumin. 20 mg of bovine serum albumin and 60 mg of azido-N-hydroxysuccinimide crosslinker were added to 3000 μL of 0.2 M phosphate buffer at a pH of 8.0 to prepare a reaction solution. The reaction solution was incubated at room temperature of 25°C for 1 hour and then allowed to stand at 4°C overnight. After the reaction was completed, it was purified by dialysis and dialyzed twice with a phosphate buffer at a pH of 7.2 to obtain an azido-modified bovine serum albumin solution with a final working concentration of 5 mg / mL.

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

[0099] The preparation method of rapamycin click chemistry conjugate immunogen, the specific steps are as follows:

[0100] (1) Prepare three fresh buffer solutions required for the click reaction: weigh 3.5 mg of tetrachlorobiphenyl dicarboxylic anhydride (THPA) and dissolve it in 200 μL of purified water to obtain a 40 mM tetrachlorobiphenyl dicarboxylic 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 alkynyl-modified rapamycin based on its molecular weight, weigh the required amount of alkynyl-modified rapamycin and dissolve it in an appropriate amount of DMSO to prepare an alkynyl-modified rapamycin solution with a final concentration of 10 mM;

[0101] (2) Click chemistry coupling reaction using azido-modified bovine serum albumin and alkynyl-modified rapamycin: 750 μL of 10 mM alkynyl-modified rapamycin solution prepared in step (1), 375 μL of 40 mM tetrachlorobiphenyl dicarboxylic anhydride buffer solution, 375 μL of 40 mM CuSO4 buffer solution, and 375 μL of 300 mM sodium ascorbate buffer solution were added to 4 mL of 5 mg / mL azido-modified bovine serum albumin solution (solvent: 10 mM, pH = 7.4 PBS buffer), mixed and incubated at room temperature 25°C for 1 hour, and then allowed to stand at 4°C overnight;

[0102] (3) Conjugate purification: After the reaction is completed, the conjugated reaction solution in step (2) is transferred to a MWCO 6-8000 dialysis membrane and dialyzed four times using 3 L of normal saline as the dialysate. The solution is then dialyzed for 12 hours using a 10 mM, pH 7.4 PBS buffer to obtain a rapamycin click chemistry conjugate immunogen solution.

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

[0104] The method for preparing an anti-rapamycin specific antibody comprises the following steps:

[0105] (1) diluting the rapamycin click chemistry conjugate immunogen to 3.0 mg / mL with PBS buffer to obtain an artificial antigen solution, and then mixing 3.0 mL of the artificial antigen solution with an equal amount of Freund's complete adjuvant and injecting the mixture into rabbits;

[0106] (2) Two weeks later, 3.0 mL of the same artificial antigen solution mixed with an equal amount of Freund's incomplete adjuvant was injected into the rabbits once, and then once every three weeks, for a total of five injections;

[0107] (3) Blood was collected from the immunized rabbits, and the antiserum was separated and purified to obtain anti-rapamycin specific antibodies.

[0108] Example 5. Preparation of Azide-Modified Glucose-6-Phosphate Dehydrogenase

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

[0110] (1) preparing a glucose-6-phosphate dehydrogenase solution for maleimide coupling reaction: dissolving disodium ethylenediaminetetraacetate in 50 mM phosphate buffer (PBS) to a final concentration of 10 mM ethylenediaminetetraacetate disodium, adjusting the pH value to 7.4 to obtain a glucose-6-phosphate dehydrogenase buffer, and then adding 6.0 mg of glucose-6-phosphate dehydrogenase to 1000 μL of the glucose-6-phosphate dehydrogenase buffer, stirring to dissolve, and obtaining a glucose-6-phosphate dehydrogenase solution;

[0111] (2) preparing an azido-maleimide crosslinker that can bind to glucose-6-phosphate dehydrogenase: weighing 3.0 mg of azido-maleimide and adding it to 200 μL of DMF, stirring and dissolving to obtain a solution with a concentration of 15 mg / mL, i.e., the azido-maleimide crosslinker;

[0112] (3) completing the azidation reaction of glucose-6-phosphate dehydrogenase: adding the azido-maleimide crosslinker prepared in step (2) dropwise to the glucose-6-phosphate dehydrogenase solution prepared in step (1), stirring evenly at room temperature 25° C., and incubating at 25° C. for 2 hours;

[0113] (4) Purification: After the reaction, the enzyme was purified by dialysis using a phosphate buffer solution with a pH of 7.3 for 4 times to obtain a 3 mg / mL azido-modified glucose-6-phosphate dehydrogenase solution.

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

[0115] The preparation method of glucose-6-phosphate dehydrogenase labeled rapamycin click chemistry conjugate, the specific steps are as follows:

[0116] (1) Prepare three fresh buffer solutions required for the click reaction: weigh 3.5 mg of tetrachlorobiphenyl dicarboxylic anhydride (THPA) and dissolve it in 200 μL of purified water to obtain a 40 mM tetrachlorobiphenyl dicarboxylic anhydride buffer solution; weigh 8 mg of CuSO4 and dissolve it in 2.5 mL of purified water to obtain a 20 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 alkynyl-modified rapamycin based on its molecular weight, weigh the required amount of alkynyl-modified rapamycin and dissolve it in an appropriate amount of DMSO to prepare an alkynyl-modified rapamycin solution with a final concentration of 2.5 mM;

[0117] (2) Click chemistry coupling reaction using azido-modified glucose-6-phosphate dehydrogenase and alkynyl-modified rapamycin: 150 μL of 2.5 mM alkynyl-modified rapamycin solution prepared in step (1), 75 μL of 40 mM tetrachlorobiphenyl dicarboxylic anhydride buffer solution, 75 μL of 20 mM CuSO4 buffer solution, and 75 μL of 300 mM sodium ascorbate buffer solution were added to 666 μL of 3 mg / mL azido-modified glucose-6-phosphate dehydrogenase solution (solvent: 10 mM, pH = 7.4 PBS buffer), mixed well, and incubated at room temperature 25°C for 1 hour (extending the reaction time can improve the reaction efficiency);

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

[0119] Example 7. Preparation of 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: 0.25% bovine serum albumin, 50 mmol / L glucose-6-phosphate and 50 mmol / L nicotinamide adenine dinucleotide oxidized form were sequentially added to 50 mmol / L Tris buffer and stirred to dissolve to prepare R1 buffer, then anti-rapamycin specific antibody was added to the R1 buffer at a volume ratio of 1:1200 and mixed, and then the pH was adjusted to 8.0 with 6 mol / L hydrochloric acid to prepare R1 reagent;

[0122] (2) 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, and then glucose-6-phosphate dehydrogenase labeled rapamycin click chemistry conjugate was added to the above R2 buffer at a volume ratio of 1:1000 and mixed, and then the pH was adjusted to 7.6 with 6 mol / L hydrochloric acid to prepare R2 reagent;

[0123] (3) Preparation of rapamycin calibrator: add rapamycin pure powder to 6 portions of 50.0 mmol / L Tris-HCl buffer at pH 7.2, stir to dissolve, and dilute to 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, add 0.5% sodium chloride, 1.0% bovine serum albumin, 0.75% ethylenediaminetetraacetic acid, and 0.05% sodium azide by mass to each solution, stir evenly, and the result is the rapamycin calibrator.

[0124] (4) Preparation of rapamycin quality control: Add pure rapamycin powder to 4 portions of 50.0 mmol / L Tris-HCl buffer at pH 7.2, stir to dissolve, and dilute to 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, add 0.5% sodium chloride, 1.0% bovine serum albumin, 0.75% ethylenediaminetetraacetic acid, and 0.05% sodium azide by mass to each solution, stir evenly, and the result is rapamycin quality control.

[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, and the main performance indicators tested were precision, recovery rate, daytime precision, linear range, analytical sensitivity, specificity, etc. The performance indicators were based on the relevant requirements of GB / T26124-2011 "Clinical Chemistry In Vitro Diagnostic Reagents (Kits)" and "Health Industry Standards of the People's Republic of China: Linearity Evaluation of Quantitative Determination Methods", and with reference to relevant CLSI standards, the method and results of the reagent performance verification are as follows:

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

[0128] Table 1. Hitachi 7180 fully automatic biochemical analyzer reaction parameters

[0129]

[0130] Table 2. Absorbance changes (reaction rates) of calibration products at different concentrations

[0131]

[0132] (2) Verification of precision and recovery: Using the rapamycin homogeneous enzyme immunoassay standard curve prepared in step (1) above, the blank, low value, middle value, and high value quality control products were repeatedly measured 10 times using a Hitachi 7180 fully automatic biochemical analyzer. The test data are shown in Table 3.

[0133] Table 3. Results of precision and recovery verification experiments

[0134]

[0135]

[0136] The results show that the coefficient of variation (CV) of the rapamycin content in the quality control products of different concentrations prepared by the rapamycin detection kit of the present invention is lower than 5.0%, and the recovery rate is between 95% and 105%, which meets the requirements of clinical test standards. This 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 the results are accurate.

[0137] (3) Inter-day precision verification: Before conducting precision verification experiments, the instrument should be calibrated and routine quality control should be performed using quality control products to ensure that inter-day precision experiments can be performed only after quality control is completed. If the indoor quality control is out of control or other operational problems occur, the data for this batch must be discarded and another batch must be measured. If multiple items are tested on the same analytical system, each test item must undergo an inter-day precision verification experiment separately. Record the experimental data for statistical analysis, with the coefficient of variation (CV) not exceeding 5.0% as the qualified standard. The test results are shown in Table 4.

[0138] Table 4. Results of the inter-day precision verification experiment

[0139]

[0140] The experimental results show that the coefficient of variation (CV) of the day-to-day precision verification of the rapamycin detection kit product prepared by the present invention for detecting low-value quality control products, mid-value quality control products, and high-value quality control products is lower than 5.0%, which meets the requirements of clinical inspection standards.

[0141] (4) Linear range verification: Take the high-value quality control product and the low-value quality control product of rapamycin, mix them into 10 different concentrations according to the proportion, measure each concentration 3 times, and perform statistical analysis on the results. According to the experimental results, gradually reduce the data points 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 tests should be completed within one day.

[0142] The experimental results show that the rapamycin detection kit product prepared by the present invention has a correlation coefficient of r=0.9996 and r≥0.990 in the range of 0.00-40.00 ng / mL, indicating that the linear correlation is good 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%, which meets the requirements of clinical inspection standards.

[0143] (5) Analytical sensitivity verification: Using purified water as the test sample, the absorbance change value of the reagent blank (A2-A1) was measured, and the absorbance change value of the fixed-value sample with a rapamycin concentration of 3.00 ng / mL was measured at the same time. The absolute value of the difference between the absorbance change value of the fixed-value sample and the absorbance change value of the reagent blank was calculated and converted into the analytical sensitivity value (|ΔA|) of the 3.00 ng / mL rapamycin fixed-value sample (Table 5).

[0144] Table 5. Analytical sensitivity validation data

[0145]

[0146] Performance index standard requirements: for the determination of rapamycin fixed value samples with a concentration of 3.00 ng / mL, the allowable range of analytical sensitivity value (|ΔA|) is 0.0100 to 0.3000. The experimental results show that the analytical sensitivity of the rapamycin detection kit product prepared by the present invention meets the clinical inspection standard requirements.

[0147] (6) Verification of common drug cross-reaction (specificity): 100 common drugs were selected for cross-reaction verification. The pure powders of the 100 common drugs were prepared into solutions with a concentration of 50.0 ng / mL as the drug samples to be tested. The rapamycin homogeneous enzyme immunoassay standard curve prepared in step (1) above was used to detect the concentration of each drug sample to be tested using a Hitachi 7180 fully automatic biochemical analyzer. The names of the 100 common drugs and the test results are shown in Table 6.

[0148] Table 6. Results of cross-reaction tests for 100 common drugs

[0149]

[0150]

[0151] The test results show that the actual detection values ​​of the above 100 common drugs are all 0.00 ng / mL. It can be seen that the rapamycin detection kit product prepared by the present invention has strong specificity, has no cross reaction with the 100 common drugs, and can specifically detect the content of rapamycin in biological samples.

[0152] 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 homogeneous enzyme immunoassay system based on click chemistry coupling technology, characterized in that: The analysis system is composed of R1 reagent, R2 reagent, calibrator and quality control product. The R1 reagent contains anti-small molecule specific antibody and R1 buffer, and the R2 reagent contains small molecule click chemistry conjugate labeled with glucose-6-phosphate dehydrogenase and R2 buffer; the anti-small molecule specific antibody is an antibody produced by immunizing experimental animals with 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: Formula I; The small molecule click chemistry derivative is an alkyne-modified small molecule hapten, and the small molecule hapten is any organic compound with a molecular weight of less than 4000Da; preferably, the small molecule hapten includes any organic compound with a molecular weight of less than 4000Da among drugs, hormones, drugs, stimulants, metabolites, vitamins, agricultural and veterinary drugs, food additives, and environmental pollutants; more preferably, the small molecule hapten is any drug with a molecular weight of less than 4000Da; further preferably, the small molecule hapten is rapamycin; The experimental animal is a mammal, and the mammal is one of rabbit, sheep, goat, mouse, rat, guinea pig, donkey, horse, alpaca or camel; The click chemistry carrier is an azido-modified immunogenic protein, and the immunogenic protein is one of serum albumin, immunoglobulin, thyroglobulin, fibrinogen, ovalbumin, hemocyanin, toxoid protein, myoglobin, poly-lysine, and poly-glutamic acid; The R1 buffer contains 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 shown in Formula II: Formula II; The click chemistry recombinant glucose-6-phosphate dehydrogenase is an azide-modified glucose-6-phosphate dehydrogenase; the R2 buffer is a Tris buffer containing bovine serum albumin; The calibration material 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 storage solution, and diluting the storage solution in Tris-HCl buffer according to the calibration concentration requirements to form standard solutions of different concentrations, which are used as calibration solutions; 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 storage solution, and diluting the storage solution in Tris-HCl buffer according to the detection concentration requirements to form standard solutions of different concentrations for use as quality control samples.

2. A method for preparing a homogeneous enzyme immunoassay system based on click chemistry coupling technology as claimed in claim 1, characterized in that: The preparation method comprises the following steps: (A1) Preparation of R1 reagent: bovine serum albumin, glucose-6-phosphate and oxidized nicotinamide adenine dinucleotide are sequentially added to a Tris buffer and stirred to dissolve to prepare an R1 buffer, and then the anti-small molecule specific antibody described in claim 1 is added to the R1 buffer at a volume ratio of 1:100-1:10000 and mixed evenly, and then the pH is adjusted with hydrochloric acid to prepare an R1 reagent; (A2) Preparation of R2 reagent: adding bovine serum albumin to Tris buffer and stirring to dissolve to prepare R2 buffer, then adding the small molecule click chemistry conjugate labeled with glucose-6-phosphate dehydrogenase described in claim 1 to the R2 buffer at a volume ratio of 1:100-1:10000 and mixing, and then adjusting the pH with hydrochloric acid to prepare R2 reagent; (A3) Preparation of calibrators: Add small molecule hapten pure powder to 6 portions of Tris-HCl buffer, stir to dissolve, and dilute to 6 standard solutions of different concentrations according to a certain ratio. Then, add sodium chloride, bovine serum albumin, ethylenediaminetetraacetic acid, and sodium azide to each solution, stir evenly, and the result is the small molecule hapten calibrator. (A4) Preparation of quality control products: Add small molecule hapten pure powder to 4 portions of Tris-HCl buffer, stir to dissolve, and dilute to 4 standard solutions of different concentrations according to the test concentration requirements. Then, add sodium chloride, bovine serum albumin, ethylenediaminetetraacetic acid, and sodium azide to each solution, stir evenly, and the small molecule hapten quality control product is obtained.

3. The method for preparing a homogeneous enzyme immunoassay system based on click chemistry coupling technology according to claim 2, characterized in that: The method for preparing the anti-small molecule specific antibody comprises the following steps: (B1) diluting the small molecule click chemistry conjugate immunogen described in claim 1 with PBS buffer to obtain an artificial antigen solution, then mixing the artificial antigen solution with an equal amount of Freund's complete adjuvant and injecting the experimental animal described in claim 1; (B2) 1-4 weeks later, the same artificial antigen solution is mixed with an equal amount of Freund's incomplete adjuvant and injected into the above experimental animals once, and then injected once every 1-4 weeks, for a total of 3-8 injections; (B3) Blood is collected from the immunized experimental animals, and the antiserum is separated and purified to obtain anti-small molecule specific antibodies.

4. The method for preparing a homogeneous enzyme immunoassay system based on click chemistry coupling technology according to claim 3, characterized in that: The preparation method of the small molecule click chemistry conjugate immunogen is as follows: ； The specific steps are as follows: (C1) preparing three fresh buffer solutions required for the click reaction: weighing tetrachlorobiphenyl anhydride and dissolving it in purified water to obtain a tetrachlorobiphenyl anhydride buffer solution; weighing CuSO4 and dissolving it in purified water to obtain a CuSO4 buffer solution; weighing sodium ascorbate and dissolving it in purified water to obtain a sodium ascorbate buffer solution; calculating the required amount of each alkynyl-modified small molecule hapten according to the molecular weight of the alkynyl-modified small molecule hapten, weighing the required amount of the alkynyl-modified small molecule hapten and dissolving it in an appropriate amount of DMSO to prepare an alkynyl-modified small molecule hapten solution; (C2) using the azido-modified immunogenic protein and the alkyne-modified small molecule hapten to perform click chemistry coupling reaction: taking the alkyne-modified small molecule hapten solution prepared in step (C1), tetrachlorobiphenyl dicarboxylic anhydride buffer solution, CuSO4 buffer solution, and sodium ascorbate buffer solution, and sequentially adding them to the azido-modified immunogenic protein solution in PBS buffer as solvent, mixing and incubating at room temperature for 0.5-3 hours, and then standing at 4°C overnight; (C3) Conjugate purification: After the reaction is completed, the conjugated reaction solution in step (C2) is transferred to a MWCO 6-8000 dialysis membrane, dialyzed 2-6 times using normal saline as the dialysate, and then dialyzed with PBS buffer for 6-18 hours to obtain a small molecule click chemistry conjugate immunogen solution.

5. The method for preparing a homogeneous enzyme immunoassay system based on click chemistry coupling technology according to claim 2, characterized in that: The preparation method of the glucose-6-phosphate dehydrogenase labeled small molecule click chemistry conjugate is as follows: ； The specific steps are as follows: (D1) preparing three fresh buffer solutions required for the click reaction: weighing tetrachlorobiphenyl dicarboxylic anhydride and dissolving it in purified water to obtain a tetrachlorobiphenyl dicarboxylic anhydride buffer solution; weighing CuSO4 and dissolving it in purified water to obtain a CuSO4 buffer solution; weighing sodium ascorbate and dissolving it in purified water to obtain a sodium ascorbate buffer solution; calculating the required amount of each alkynyl-modified small molecule hapten according to the molecular weight of the alkynyl-modified small molecule hapten, weighing the required amount of the alkynyl-modified small molecule hapten and dissolving it in an appropriate amount of DMSO to prepare an alkynyl-modified small molecule hapten solution; (D2) using the azido-modified glucose-6-phosphate dehydrogenase and the alkynyl-modified small molecule hapten to perform a click chemistry coupling reaction: taking the alkynyl-modified small molecule hapten solution prepared in step (D1), the tetrachlorobiphenyl dicarboxylic anhydride buffer solution, the CuSO4 buffer solution, and the sodium ascorbate buffer solution, and sequentially adding them to the azido-modified glucose-6-phosphate dehydrogenase solution in the PBS buffer solution, mixing well, and incubating at room temperature for 0.5-3 hours; (D3) Conjugate purification: After the reaction is completed, the coupling reaction solution in step (D2) is filtered using a pre-equilibrated G-25 polysaccharide gel chromatography column to obtain a small molecule click chemistry conjugate solution labeled with glucose-6-phosphate dehydrogenase, and finally BSA, NaN3 and glycerol are added for storage.

6. The method for preparing a homogeneous enzyme immunoassay system based on click chemistry coupling technology according to claim 4 or 5, characterized in that: The method for preparing the alkyne-modified small molecule hapten comprises the following steps: K2CO3, 3-bromo-propyne and a small molecule hapten are dissolved together in N,N-dimethylformamide to obtain a reaction mixture, and the reaction mixture is stirred and refluxed for 10-50 hours; the reaction mixture is cooled to 25°C, an appropriate amount of purified water is added, and then dichloromethane 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 a silica gel chromatography column to obtain an alkynyl-modified small molecule hapten.

7. The method for preparing a homogeneous enzyme immunoassay system based on click chemistry coupling technology according to claim 4, characterized in that: The method for preparing the azido-modified immunogenic protein comprises the following steps: A azido-N-hydroxysuccinimide crosslinker is used to carry out a linking reaction with the immunogenic protein, and the immunogenic protein and the azido-N-hydroxysuccinimide crosslinker are added to a phosphate buffer in sequence to prepare a reaction solution; the reaction solution is incubated at room temperature for 0.5-3 hours, and then allowed to stand at 4°C overnight; after the reaction is completed, it is purified by dialysis, and dialyzed 1-4 times using a phosphate buffer to obtain an azido-modified immunogenic protein solution.

8. The method for preparing a homogeneous enzyme immunoassay system based on click chemistry coupling technology according to claim 5, characterized in that: The preparation method of the azido-modified glucose-6-phosphate dehydrogenase comprises the following steps: (E1) preparing a glucose-6-phosphate dehydrogenase solution for maleimide coupling reaction: dissolving disodium ethylenediaminetetraacetate in a phosphate buffer, adjusting the pH value to obtain a glucose-6-phosphate dehydrogenase buffer, and then adding glucose-6-phosphate dehydrogenase to the glucose-6-phosphate dehydrogenase buffer, stirring and dissolving, to obtain a glucose-6-phosphate dehydrogenase solution; (E2) preparing an azido-maleimide crosslinker that can bind to glucose-6-phosphate dehydrogenase: weighing azido-maleimide, adding it to DMF, stirring and dissolving it to obtain the azido-maleimide crosslinker; (E3) completing the azidation reaction of glucose-6-phosphate dehydrogenase: adding the azido-maleimide crosslinker prepared in step (E2) dropwise to the glucose-6-phosphate dehydrogenase solution prepared in step (E1), stirring evenly at room temperature, and incubating at 25° C. for 1-4 hours; (E4) Purification: After the reaction is completed, the solution is purified by dialysis using phosphate buffer for 2-6 times to obtain an azido-modified glucose-6-phosphate dehydrogenase solution.

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

10. The 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 4000Da among drugs, hormones, drugs, stimulants, metabolites, vitamins, veterinary drugs, food additives, and environmental pollutants; preferably, the small molecule hapten is any drug with a molecular weight less than 4000Da; further preferably, the small molecule hapten is rapamycin.

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