Preparation method and application of biotin-labeled small molecule

Abstract

The invention relates to a preparation method and application of biotin-labeled micromolecules, and the method comprises the following steps: mixing the micromolecules with an activator for reaction to obtain an activated micromolecule reagent; wherein the molecular weight of the small molecules is less than 1000Da; mixing and reacting the activated small molecule reagent with a biotinylated marker reagent to prepare a biotin-labeled small molecule reagent; wherein the molecular weight of the biotinylated marker is 1,000 Da to 10,000 Da. According to the technical scheme, the molecular weight of biotinylated small molecules can be improved, subsequent desalination and purification are easy, the purity is high, the impurity content is low, the calibration signal value and the distinction degree are good, and improvement of the precision and the accuracy of clinical detection is facilitated.

Description

Technical Field

[0001] The present application relates to the technical field of biological reagents, and particularly relates to a preparation method and application of a biotin-labeled small molecule. Background Art

[0002] The biotin-avidin system (BAS) is a biological reaction amplification system developed in the 1970s. With the advent of biotin derivatives, BAS has been quickly and widely applied in the field of medical detection. In recent years, a large number of studies have confirmed that the biotin-avidin system can almost bind to various markers that have been successfully studied. The strong binding with high affinity between biotin and avidin, as well as the multi-stage amplification effect, make BAS immunolabeling and related tracer analysis more sensitive. It has become a new technology widely used for qualitative, quantitative detection and localization observation research of trace antigens and antibodies.

[0003] In practical applications, for some small molecule hormones, the common Biotin-LCLC-NHS reagent is usually used for their biotinylation labeling. Since the molecular weight of small molecule hormones is usually less than 1000 Da, after biotinylation labeling with Biotin-LCLC-NHS, the purity is relatively low and the impurity content is relatively high. Furthermore, when it is applied to chemiluminescence detection, the obtained detection signal value and discrimination degree are both relatively poor, which is not conducive to improving the accuracy and precision of detection. Summary of the Invention

[0004] To solve or partially solve the problems existing in the related technologies, the present application provides a preparation method and application of a biotin-labeled small molecule, so that after the small molecule is labeled, the purity after purification is relatively high, the impurity content is relatively low, the calibration signal value and discrimination degree are relatively good, which is conducive to improving the accuracy and precision of clinical detection.

[0005] The first aspect of the present application provides a preparation method of a biotin-labeled small molecule, which includes:

[0006] Mixing and reacting the small molecule with an activator to obtain an activated small molecule reagent; wherein, the molecular weight of the small molecule is less than 1000 Da;

[0007] Mixing and reacting the activated small molecule reagent with a biotinylated labeling reagent to prepare a biotin-labeled small molecule reagent; wherein, the molecular weight of the biotinylated labeling is 1000 Da - 10000 Da.

[0008] In some embodiments, after mixing and reacting the activated small molecule with the biotinylated labeling reagent, it further includes:

[0009] Desalt and purify the biotin-labeled small molecule reagent; preferably, the molar ratio of the activated small molecule to the biotinylated label is greater than 1.

[0010] In some embodiments, the biotin-labeled small molecule reagent is desalted and purified through a desalting column to remove the free activated small molecule.

[0011] In some embodiments, the label is a long-chain molecule; preferably, the long-chain molecule is a hydrophilic long-chain molecule; more preferably, the hydrophilic long-chain molecule is selected from polyethylene glycol or dextran.

[0012] In some embodiments, the small molecule is a derivative or structural analogue of a sex hormone.

[0013] The second aspect of the present application provides a biotin-labeled small molecule reagent prepared according to the preparation method of any of the above embodiments.

[0014] The third aspect of the present application provides an application of the above biotin-labeled small molecule reagent in sex hormone detection, or an application in the preparation of a sex hormone detection kit.

[0015] The fourth aspect of the present application provides a photoactivated chemiluminescence detection kit for detecting sex hormones, which includes reagent 1 and reagent 2. Reagent 1 is the above biotin-labeled small molecule reagent; reagent 2 includes luminescent particles, and the surface of the luminescent particles is coated with an antibody against the sex hormone to be detected.

[0016] In some embodiments, the sex hormone to be detected is testosterone, progesterone or estradiol; the small molecule and the sex hormone to be detected can be recognized and bound by the same antibody coated on the surface of the luminescent particles; preferably, the binding forces of the small molecule and the corresponding sex hormone to be detected with the antibody are different.

[0017] In some embodiments, the photoactivated chemiluminescence detection kit further includes reagent 3, and reagent 3 includes a releasing agent, and the releasing agent is a strong acid, a strong base and / or a displacing agent; preferably, the releasing agent is a citrate buffer; and / or

[0018] The photoactivated chemiluminescence detection kit further includes reagent 4, and reagent 4 includes avidin-coated photosensitive particles; preferably, the concentration of reagent 4 is 40 μg / mL to 50 μg / mL.

[0019] It should be understood that the above general description and the following detailed description are only exemplary and explanatory, and cannot limit the present application. Detailed implementation manners

[0020] To make the present invention easy to understand, the present invention will be described in detail below. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific embodiments described. It should also be understood that the terms used herein are only for describing the specific embodiments and do not represent limitations.

[0021] Where a numerical range is provided, it should be understood that each intermediate value between the upper and lower limits of the range and any other specified or intermediate value in the specified range is encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also encompassed within the present invention, subject to any specifically excluded limits in the specified range. Where the specified range includes one or both of the limits, ranges excluding either or both of the included limits are also included in the present invention.

[0022] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention, the preferred methods and materials are now described.

[0023] Term Explanation

[0024] As used in this application, the term "photosensitive particle" refers to a particle containing a sensitizer that can be activated by energy or an active compound and can generate active intermediates such as reactive oxygen species that can react with luminescent particles. In some specific embodiments of this application, the photosensitive particle is a polymer particle filled with a photosensitizer, and the photosensitizer can be a photosensitizer known in the art, preferably a compound that is relatively light-stable and does not effectively react with singlet oxygen, such as compounds like methylene blue, rose bengal, porphyrin, phthalocyanine, and chlorophyll, as well as derivatives of these compounds having substituents of 1 - 50 atoms, where the substituents are used to make these compounds more lipophilic or more hydrophilic and / or as a linking group for attachment to a specific binding partner member. Examples of other photosensitizers known to those skilled in the art can also be used in this application.

[0025] As used in this application, the term "luminescent particle" refers to a particle containing a compound that can react with reactive oxygen species to generate a detectable signal. The photosensitive particle is induced to be activated by energy or an active compound and releases reactive oxygen species in a high-energy state, and the reactive oxygen species in the high-energy state are captured by the nearby luminescent particles, thereby transferring energy to activate the luminescent particles. In some specific embodiments of this application, the luminescent particle comprises a luminescent composition and a carrier, and the luminescent composition is filled in the carrier and / or coated on the surface of the carrier.

[0026] This application provides a method for preparing a biotin-labeled small molecule, which includes:

[0027] S110. Mix a small molecule with an activator and react to obtain an activated small molecule reagent. Herein, the molecular weight of the small molecule is less than 1000 Da.

[0028] S120. Mix the activated small molecule reagent with a biotinylated labeling reagent and react to prepare a biotin-labeled small molecule reagent. Herein, the molecular weight of the biotinylated labeling agent is 1000 Da - 10000 Da.

[0029] In this application, the prepared biotin-labeled small molecule reagent can be used to detect sex hormones, such as testosterone, estradiol or progesterone. In some embodiments, according to the type of sex hormone to be detected, the small molecule can be a corresponding sex hormone derivative, such as a testosterone derivative, an estradiol derivative or a progesterone derivative. Accordingly, the molecular weight of the sex hormone derivative is less than 1000 Da.

[0030] Before mixing the small molecule with the activator in proportion in S110, in some embodiments, dissolve the small molecule in a solvent to an appropriate concentration to obtain a small molecule solution; prepare the activator in water to an appropriate concentration to obtain an activator solution; dissolve the biotinylated labeling agent in a solvent to an appropriate concentration to obtain a biotinylated labeling reagent.

[0031] For example, the activator can be an EDC reagent (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide). For example, the solvent can be a DMSO reagent. Of course, the above-mentioned activator and solvent can also be other liquids with similar properties. Herein, only examples are given and no limitations are made.

[0032] To obtain a sufficient amount of biotin-labeled small molecule, in some embodiments, the molar ratio of the activated small molecule to the biotinylated labeling agent is greater than 1. That is to say, the theoretical value of the molar ratio of the activated small molecule to the biotinylated labeling agent is 1. However, in the actual operation of S120, the addition amount of the activated small molecule can be slightly more than that of the biotinylated labeling agent.

[0033] Further, in some embodiments, after mixing and reacting the activated small molecule with the biotinylated labeling reagent in the above step S120, it further includes: desalting and purifying the biotin-labeled small molecule reagent. By desalting and purifying, the excess activated small molecule can be removed.

[0034] In some embodiments, a desalting column is used to desalt and purify the biotin-labeled small molecule reagent to remove the free activated small molecules. The molecular weight of the biotinylated label is 1000 Da to 10000 D, and the molecular weight of the biotin-labeled small molecule must be greater than 1000 Da. Among them, the retention efficiency of the desalting column for salts and small molecules (<1000 Da) is ≥95%. By using a high molecular weight label to label the small molecule, the molecular weight of the biotin-labeled small molecule is made greater than 1000 Da, and then the molecular weight after the small molecule is labeled can be increased. After desalting and purification, a labeled sex hormone derivative reagent with higher purity and less impurity content can be easily obtained, which is beneficial to improving the accuracy and precision of clinical detection.

[0035] It should be noted that the commonly used biotinylation reagent in the past was usually Biotin-LCLC-NHS (N-succinimidyl 6-biotinamidohexanoic acid) reagent. After using Biotin-LCLC-NHS reagent to biotinylate the small molecule, the molecular weight after labeling is still relatively small, usually less than 1000 Da, and the subsequent desalting is difficult. As a result, the small molecule labeled with Biotin-LCLC-NHS reagent has more impurity content, lower purity, and both the detection signal value and the discrimination degree are poor, which is not conducive to improving the accuracy and precision of detection. In this application, by using a high molecular weight label to label the small molecule, the molecular weight is relatively high, the purification is easier, the purity is higher, the impurity content is less, the calibration signal value and the discrimination degree are better, which is beneficial to improving the accuracy and precision of clinical detection.

[0036] In the above step S120, in some embodiments, the label is a long-chain molecule; preferably, the long-chain molecule is a hydrophilic long-chain molecule; more preferably, the hydrophilic long-chain molecule is selected from polyethylene glycol or dextran. Of course, the hydrophilic long-chain molecule can also be other molecules with hydrophilicity and within the specified molecular weight range. Only examples are given here and no limitation is made.

[0037] Correspondingly, the biotinylated label is a biotinylated long-chain molecule. Preferably, the biotinylated label is a biotinylated hydrophilic long-chain molecule; more preferably, the biotinylated label is biotinylated polyethylene glycol or dextran.

[0038] In some embodiments, the biotin-labeled small molecule reagent prepared according to the above preparation method may have the following simplified formula: small molecule-label-biotin. Preferably, when the small molecule is a sex hormone derivative, the simplified formula may be: sex hormone derivative-label-biotin.

[0039] Correspondingly, when the label is polyethylene glycol, the simplified formula of the biotin-labeled small molecule reagent is small molecule-PEG n-Biotin, where n represents the number of polyethylene glycol (PEG) monomers, and Biotin represents biotin. When the labeling agent is dextran, the simplified formula of the biotin-labeled small molecule reagent is small molecule - dextran - biotin, that is, small molecule - [C6H 10 O5] n -Biotin, where n represents the number of C6H 10 O5 monomers. Similarly, according to the variation of the types of small molecules, for example, it can also be expressed as sex hormone derivative - PEG n -Biotin, sex hormone derivative - [C6H 10 O5] n -Biotin.

[0040] In some embodiments, different labeling agents can be selected according to the types of small molecules. For example, when the small molecule is a testosterone derivative, the labeling agent can be polyethylene glycol PEG n . Correspondingly, the biotin-labeled sex hormone derivative can be expressed as: testosterone derivative - PEG n -Biotin. When the small molecule is an estradiol derivative, the labeling agent can be dextran [C6H 10 O5] n . Correspondingly, the biotin-labeled sex hormone derivative can be expressed as: estradiol derivative - [C6H 10 O5] n -Biotin. Of course, the combination of the types of small molecules and the types of labeling agents is only illustrated here and is not limited.

[0041] In some embodiments, when the labeling agent is selected from polyethylene glycol PEG n , polyethylene glycol PEG n is selected from at least one of PEG 25 to PEG 200 ; preferably, it is selected from at least one of PEG 30 to PEG 180 ; more preferably, it is selected from at least one of PEG 40 to PEG 150 ; further preferably, it is selected from at least one of PEG 70 to PEG 120 . That is to say, the labeling agent is selected from at least one or a mixture of multiple ones of PEG 25 to PEG 200 such that the molecular weight of the biotinylated labeling agent is between 1000 Da and 10000 Da.

[0042] In some embodiments, when the labeling agent is selected from dextran [C6H 10 O5] n when, dextran [C6H10 O5] n At least one selected from dextrans with n = 7 - 55; preferably, at least one selected from dextrans with n = 10 - 50; more preferably, at least one selected from dextrans with n = 16 - 40; still more preferably, at least one selected from dextrans with n = 20 - 30. That is to say, the label is selected from [C6H 10 O5]7 - [C6H 10 O5] 55 or a mixture of at least one or more thereof, such that the molecular weight of the biotinylated label is between 1000 Da and 10000 Da.

[0043] This application also provides a photochemiluminescence detection kit for detecting sex hormones, which includes reagent 1 and reagent 2. Reagent 1 is the biotin-labeled small molecule reagent of any of the above embodiments; Reagent 2 includes luminescent microparticles, and the surface of the luminescent microparticles is coated with an antibody against the sex hormone to be detected.

[0044] Furthermore, in some embodiments, the sex hormone to be detected is testosterone, progesterone or estradiol; the small molecule and the sex hormone to be detected can be recognized and bound by the same antibody coated on the surface of the luminescent microparticles. Among them, according to the type of the sex hormone to be detected, the antibody can be a testosterone antibody, a progesterone antibody or an estradiol antibody.

[0045] Preferably, in some embodiments, the binding force of the biotin-labeled small molecule and the corresponding sex hormone to be detected to the antibody is different. Specifically, in order to better enable the biotin-labeled small molecule and the sex hormone to be detected to be recognized and bound by the same antibody, according to the type of the sex hormone to be detected, the biotin-labeled small molecule is selected as the corresponding biotin-labeled sex hormone derivative. For example, the biotin-labeled small molecule can be a biotin-labeled testosterone derivative, a biotin-labeled estradiol derivative or a biotin-labeled progesterone derivative. Correspondingly, reagent 1 can be a reagent of biotin-labeled sex hormone derivative.

[0046] In some embodiments, the binding force of the biotin-labeled small molecule and the sex hormone to be detected to the antibody is different. More preferably, the binding force of the biotin-labeled small molecule to the antibody is lower than the binding force of the sex hormone to be detected to the antibody. By selecting the derivative of the sex hormone to be detected as the small molecule in reagent 1, it is possible to enable both reagent 1 and the sex hormone to be detected to competitively bind to the same antibody, but with different binding forces, and then an obvious competitive effect can be achieved, making the detection result have a clearer discrimination.

[0047] For more accurate detection results, in some embodiments, the concentration of reagent 1 can be 3 ng / mL to 10 ng / mL. Preferably, the concentration of reagent 1 can be 4 ng / mL to 8 ng / mL. More preferably, the concentration of reagent 1 can be, for example, 5 ng / mL, 6 ng / mL, 7 ng / mL, etc. Preferably, reagent 1 further includes a buffer. For example, the buffer can be a HEPES buffer, etc. Among them, reagent 1 can be diluted to the required concentration by the buffer.

[0048] In some embodiments, the concentration of reagent 2 can be 10 μg / mL to 30 μg / mL. Preferably, the concentration of reagent 2 can be 15 μg / mL to 25 μg / mL. More preferably, the concentration of reagent 2 can be, for example, 15 μg / mL, 18 μg / mL, 23 μg / mL, 25 μg / mL, 30 μg / mL, etc. Preferably, reagent 2 further includes a buffer. For example, the buffer can be a Tris buffer, etc. Among them, reagent 2 can be diluted to the required concentration by the buffer.

[0049] In some embodiments, the photochemiluminescence immunoassay kit further includes reagent 3, and reagent 3 includes a releasing agent, and the releasing agent is a strong acid, a strong base, and / or a displacing agent. Preferably, the releasing agent is a citrate buffer. Specifically, the releasing agent of the present application refers to a chemical reagent that can release the sex hormone to be detected from sex hormone-binding globulin or albumin. In some embodiments, the concentration of reagent 3 can be 0.1 mol / L to 0.5 mol / L. Preferably, for example, it can be 0.1 mol / L, 0.2 mol / L, 0.4 mol / L, 0.5 mol / L, etc. This is only for illustration and not for limitation.

[0050] In some embodiments, the photochemiluminescence immunoassay kit further includes reagent 4, and reagent 4 includes avidin-coated photosensitive microparticles. Preferably, the concentration of reagent 4 can be 40 μg / mL to 50 μg / mL. More preferably, the concentration of reagent 4 can be, for example, 50 μg / mL. Among them, reagent 4 can be used as a general reagent and is applicable to photochemiluminescence immunoassay kits for detecting different target substances.

[0051] In summary, according to the above photochemiluminescence immunoassay kit, by using the biotin-labeled small molecule reagent in reagent 1 to perform chemiluminescence detection on the sex hormone to be detected according to the competitive method, it has better detection signal values and discrimination.

[0052] The present application also provides an application of a photochemiluminescence immunoassay kit in detecting a sex hormone to be detected. For example, the method for detecting a sex hormone to be detected according to the above photochemiluminescence immunoassay kit. Among them, the molecular weight of the sex hormone to be detected is less than 1000 Da.

[0053] In some embodiments, the detection method includes: mixing a sample to be tested, reagent 1, reagent 2, reagent 3, and reagent 4 in a preset manner for reaction, then irradiating with excitation light, measuring the amount of emitted light photons to obtain a light signal value, and quantitatively or qualitatively obtaining the detection result of the sex hormone to be detected in the sample to be tested according to the light signal value.

[0054] In a specific embodiment, the sample to be tested, reagent 1, reagent 2, and reagent 3 are mixed and incubated, and then reagent 4 is added and mixed and incubated. For example, the sample to be tested, reagent 1, reagent 2, and reagent 3 are mixed and incubated at 37 °C for 15 min, then an appropriate amount of reagent 4 is added, and after incubating at 37 °C for 10 min, the light signal value is measured by irradiating with the excitation light of a photochemiluminescence detection system.

[0055] The method of the present application is detected according to the test principle of the competition method, and is used for quantitatively or qualitatively detecting the sex hormone to be detected in the sample to be tested. The operation is simple and convenient, and the small molecule reagent labeled with biotin has good signal value and discrimination, and more accurate detection results can be obtained.

[0056] To make the present invention easier to understand, the following will further illustrate the present invention in detail with reference to embodiments. These embodiments are only illustrative and are not limited to the application scope of the present invention. The raw materials or components used in the present invention can be obtained through commercial channels or conventional methods without special instructions.

[0057] The following takes the sex hormone to be detected as testosterone, and the small molecule as a derivative of testosterone T, hereinafter referred to as T derivative as an example. Biotin-PEG n -NH2 reagents with molecular weights of 588 Da, 1000 Da, 5000 Da, 10 kDa, and 20 kDa are used for labeling to obtain T derivative-PEG n -Biotin. Then it is purified with a desalting column, and finally the signal value and discrimination are detected on a photochemiluminescence platform. The Biotin-PEG n -NH2 reagent is biotin polyethylene glycol active ester reagent, hereinafter referred to as Biotin-PEG n -NH2.

[0058] Example 1: Preparation of T derivative-PEG n -Biotin

[0059] 1.1 The main experimental raw materials and equipment are shown in Table 1:

[0060] Table 1

[0061]

[0062] 1.2 Take a centrifuge tube and weigh 0.2 mg of T derivative and dissolve it in 200 μL of DMSO solution to prepare a T derivative reagent. Weigh 5 mg of EDC and dissolve it in 500 μL of pure water to prepare an EDC reagent. Take 5 μL of the above EDC reagent and add it to the prepared T derivative reagent, mix well to obtain an activated T derivative reagent.

[0063] 1.3 Respectively dissolve 0.26 μmol of Biotin-PEG n -NH2 (588 Da), Biotin-PEG n -NH2 (1 K Da), Biotin-PEG n -NH2 (5 K Da), Biotin-PEG n -NH2 (10 K Da) and Biotin-PEG n -NH2 (20 K Da) in DMSO solution respectively to prepare 5 kinds of Biotin-PEG n -NH2 reagents with different molecular weights. Mix each Biotin-PEG n -NH2 reagent with the activated T derivative reagent respectively, and let it stand and react at 2 °C - 8 °C for 18 h to obtain the corresponding reaction solution.

[0064] 1.4 Desalting and purification process:

[0065] Desalt and purify the 5 kinds of reaction solutions prepared in the above step 1.3 according to the following steps.

[0066] First centrifugation: Take a Zeba desalting column, remove the tail of the desalting column, and loosen the lid of the desalting column. Place it in a suitable centrifuge tube and weigh it on an electronic balance for balance. Centrifuge at 2 °C - 8 °C (set at 4 °C), 1500 g, for 2 min.

[0067] Second centrifugation: Replace with a new centrifuge tube, add 0.1 M NaHCO3 (pH 8.5) buffer solution to the desalting column, and weigh it on an electronic balance for balance. Centrifuge at 2 °C - 8 °C (set at 4 °C), 1500 g, for 2 min.

[0068] Third centrifugation: Replace with a new centrifuge tube, add 0.1 M NaHCO3 (pH 8.5) buffer solution to the desalting column, and weigh it on an electronic balance for balance. Centrifuge at 2 °C - 8 °C (set at 4 °C), 1500 g, for 2 min. Recover the liquid in the centrifuge tube.

[0069] Fourth centrifugation: Replace with a new centrifuge tube, add T derivative-PEG n-Biotin, weigh and balance with an electronic balance. Centrifuge at 2°C to 8°C (set at 4°C), 1500 g, for 2 min. The liquid in the centrifuge tube is the purified T derivative-PEG n -Biotin reagent is biotin polyethylene glycol active ester labeled T derivative.

[0070] Example 2: Preparation of a photochemiluminescence immunoassay kit for detecting testosterone (T)

[0071] 1. Dilute the T derivative-PEG n -Biotin reagents with different molecular weights prepared in Example 1 above to 5 ng / mL with HEPES buffer to obtain 5 corresponding reagents 1.

[0072] 2. Dilute the luminescent microparticles coated with testosterone antibody to 20 μg / mL with Tris buffer to obtain reagent 2.

[0073] 3. Prepare 0.2 mol / L citric acid buffer and dispense it to obtain reagent 3.

[0074] 4. Assemble reagent 1, reagent 2 and reagent 3 into a corresponding set of complete reagents respectively to obtain 5 sets of complete reagents.

[0075] 5. Prepare a photosensitive microparticle solution with a concentration of 50 μg / mL in advance as a universal reagent, i.e., reagent 4.

[0076] Example 3: Detection of testosterone (T)

[0077] Prepare 6 different concentrations of testosterone reagents as calibration standards, and detect the 5 sets of complete reagents prepared in Example 2 above and the test sample on a 500 photochemiluminescence immunoassay system.

[0078] 1. Mix 20 μL of the test sample, 25 μL of reagent 1, 25 μL of reagent 2 and 20 μL of reagent 3 evenly and incubate at 37°C for 15 min.

[0079] 2. Add 175 μL of the universal reagent, i.e., reagent 4, and incubate at 37°C for 10 min.

[0080] 3. After the photoexcitation reaction, read the light signal values of each group of tests respectively. The experimental data are shown in Table 2 below.

[0081] Table 2

[0082]

[0083] It can be seen from the data in Table 2 that when Biotin-PEG nWhen the molecular weight of the -NH2 reagent is 588Da, the corresponding calibration signal value is relatively low overall, and the overall discrimination of the reagent is 10.2; when Biotin-PEG n When the molecular weight of the -NH2 reagent increased from 588Da to 1KDa, based on the data corresponding to 588Da, the signal values ​​corresponding to different calibrant concentrations increased by an average of 7.8 times, and the highest concentration inhibition rate increased by 1.8 times; the overall discrimination of the reagent was 18.2. n When the molecular weight of the -NH2 reagent continued to increase from 1K Da to 5K Da, based on the 588Da data, the signal value increased by an average of 10 times, the highest concentration inhibition rate increased by 5.2 times, and the overall discrimination of the reagent was 53.1. n When the molecular weight of the -NH2 reagent continued to increase from 5K Da to 10K Da, based on the 588Da data, the signal value increased by an average of 7.8 times, the highest concentration inhibition rate increased by 1.8 times, and the overall discrimination of the reagent was 18.8. n When the molecular weight of the -NH2 reagent continued to increase from 10K Da to 20K Da, based on the 588Da data, the signal value increased by an average of 5.9 times, the highest concentration inhibition rate increased by 1.3 times, and the overall discrimination of the reagent was 13.3.

[0084] It should be noted that the five Biotin-PEG n The theoretical molecular weights of -NH2 are 948Da, 1360Da, 5360Da, 10360Da and 20360Da respectively, and the molecular weight of the T derivative is 360. The retention efficiency of the desalting column for salts and small molecules (molecular weight <1000Da) is ≥95%. n -When the molecular weight of Biotin is around 1000Da, it will also be retained in the desalting column with a high probability, resulting in the T derivative-PEG in the obtained reagent n -Biotin content decreases, and the signal value decreases. n When the molecular weight of -NH2 reagent reaches 5K Da, T derivative and T derivative-PEG can be fully separated n -Biotin, the effective reagent content is high, and the signal value is correspondingly improved. However, the separation effect will not increase significantly if the molecular weight continues to increase. On the contrary, after exceeding 10K Da, the reaction efficiency is affected due to the large molecular chain length, resulting in a decrease in signal value and poor discrimination.

[0085] In summary, Biotin-PEG n -NH2 has a better effect when its molecular weight is between 1KDa and 10KDa, and it has the best effect on labeling small molecule hormones when its molecular weight is 5K Da.

[0086] Example 4: Detection of Estradiol (E2)

[0087] The detection target in this example is estradiol. The biotinylated labeling reagent is biotin-dextran reagent, and the small molecule is a derivative of estradiol E2, abbreviated as E2 derivative.

[0088] Referring to the method of Example 1, the E2 derivative was labeled with biotin-dextran reagents of 3K Da and 10KDa to obtain the desalted and purified E2 derivative -[C6H 10 O5] n -Biotin reagent.

[0089] Referring to the method of Example 2, a photochemiluminescence immunoassay kit for detecting sex hormone E2 was prepared.

[0090] In this example, 6 E2 reagents with different concentrations were prepared as calibration products. The two sets of complete reagents prepared in Example 2 above and the sample to be tested were detected on a 500 photochemiluminescence detection system.

[0091] The detection process of this example refers to Example 3, and the experimental data are shown in Table 3 below.

[0092] Table 3

[0093]

[0094]

[0095] In this example, it is illustrated that the molecular weight of the biotinylated labeling agent is not the larger the better, and the biotin-dextran reagent with a molecular weight of 3000Da has the best labeling effect.

[0096] When the biotin-labeled small molecule provided in this application is applied in a photochemiluminescence immunoassay kit, due to its good purity, the detection signal value and discrimination are better. On the photochemiluminescence platform, the detection signal value and discrimination are superior to other reagents, such as the small molecule biotinylated by Biotin-PEG-NH2 reagent.

[0097] The above has described the embodiments of the present application. The above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and variations are obvious to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The selection of the terms used herein is intended to best explain the principles of the embodiments, practical applications, or improvements to the technology in the market, or to enable other ordinary skill in the art in this technical field to understand the disclosed embodiments.

Claims

1. A preparation method of a biotin-labeled small molecule, characterized in that, Comprising: Mixing and reacting a small molecule with an activator to obtain an activated small molecule reagent; wherein, the molecular weight of the small molecule is less than 1000 Da; Mixing and reacting the activated small molecule reagent with a biotinylated labeling reagent to prepare a biotin-labeled small molecule reagent; wherein, the molecular weight of the biotinylated labeling agent is 1000 Da to 10000 Da.

2. The preparation method according to claim 1, characterized in that, After mixing and reacting the activated small molecule with the biotinylated labeling reagent, it further includes: Desalting and purifying the biotin-labeled small molecule reagent; preferably, the molar ratio of the activated small molecule to the biotinylated labeling agent is greater than 1.

3. The preparation method according to claim 2, wherein: Desalting and purifying the biotin-labeled small molecule reagent through a desalting column to remove the free activated small molecule.

4. The preparation method according to claim 1, wherein: The labeling agent is a long-chain molecule; preferably, the long-chain molecule is a hydrophilic long-chain molecule; more preferably, the hydrophilic long-chain molecule is selected from polyethylene glycol or dextran.

5. The preparation method according to claim 1, wherein: The small molecule is a derivative or structural analogue of a sex hormone.

6. A biotin-labeled small molecule reagent, characterized in that, Prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the biotin-labeled small molecule reagent according to claim 6 in the detection of sex hormones, or use in the preparation of a sex hormone detection kit.

8. A photochemiluminescence detection kit for detecting sex hormones, characterized in that, Comprising reagent 1 and reagent 2, wherein reagent 1 is the biotin-labeled small molecule reagent according to claim 6; reagent 2 comprises luminescent microparticles, and the surface of the luminescent microparticles is coated with an antibody against the sex hormone to be detected.

9. The detection kit according to claim 8, wherein: The sex hormone to be detected is testosterone, progesterone or estradiol; the small molecule and the sex hormone to be detected can be recognized and bound by the same antibody coated on the surface of the luminescent microparticles; preferably, the binding force of the small molecule and the corresponding sex hormone to be detected with the antibody is different.

10. The detection kit according to claim 8 or 9, wherein: It further comprises reagent 3, and reagent 3 comprises a releasing agent, and the releasing agent is a strong acid, a strong base and / or a displacing agent; preferably, the releasing agent is a citrate buffer solution; and / or It further comprises reagent 4, and reagent 4 comprises avidin-coated photosensitive microparticles; preferably, the concentration of reagent 4 is 40 μg / mL to 50 μg / mL.

Images