Marked hormone derivative and application thereof

Abstract

The invention relates to a labeled sex hormone derivative for light-activated chemiluminescence detection, a sex hormone light-activated chemiluminescence detection kit and a detection method. The labeled sex hormone derivative is obtained by labeling a sex hormone derivative with a biotinylated marker, the simplified formula of the labeled sex hormone derivative is sex hormone derivative-marker-biotin, the molecular weight of the sex hormone derivative is less than 1000 Da, and the molecular weight of the biotinylated marker is 1000-10000 Da. According to the application, the biotinylated marker is used for marking the sex hormone derivative, so that the molecular weight after biotinylation can be improved, the subsequent purification is relatively easy, the impurity content after purification is relatively low, and the detection signal value and the discrimination degree are relatively good.

Description

Technical Field

[0001] The present application relates to the technical field of biological reagents, and particularly relates to a labeled sex hormone derivative for photochemiluminescence detection, a sex hormone photochemiluminescence detection kit, and a detection method. Background Art

[0002] In the related art, when sex hormones are detected on a photochemiluminescence platform, due to the relatively small molecular weight of the hormones themselves, after biotinylation with a common Biotin-LCLC-NHS reagent (N-succinimidyl 6-biotinamidohexanoic acid), it is very difficult to purify the reagent, resulting in a low purity and a large amount of impurities. Furthermore, when it is applied to chemiluminescence detection, the detection signal value and the discrimination degree are both poor, which is not conducive to improving the accuracy and precision of detection. Summary of the Invention

[0003] To solve or partially solve the problems existing in the related art, the present application provides a labeled sex hormone derivative for photochemiluminescence detection, a sex hormone photochemiluminescence detection kit, and a detection method. The labeled sex hormone derivative has a high purity, a small amount of impurities, good calibration signal values and discrimination degrees, which is conducive to improving the accuracy and precision of clinical detection.

[0004] The first aspect of the present application provides a labeled product obtained by labeling a sex hormone derivative with a biotinylated label. The simple formula of the labeled sex hormone derivative is sex hormone derivative-label-biotin, wherein the molecular weight of the sex hormone derivative is less than 1000 Da, and the molecular weight of the biotinylated label is 1000 Da to 10000 Da;

[0005] When performing photochemiluminescence detection, the labeled sex hormone derivative is used to react with a luminescent particle coated with an antibody against the sex hormone to be detected, a photosensitive particle coated with avidin, a releasing agent, and a sample to be detected. Under the irradiation of excitation light, the amount of luminescent photons is measured to obtain a light signal value.

[0006] In some embodiments, the label is a hydrophilic long-chain molecule. Preferably, the hydrophilic long-chain molecule is selected from polyethylene glycol or dextran.

[0007] In some embodiments, the polyethylene glycol (PEG) n is selected from at least one of PEG 25 ~PEG 200 ; preferably, it is selected from at least one of PEG 30 ~PEG 180 ; more preferably, it is selected from at least one of PEG 40 ~PEG 150 ; further preferably, it is selected from at least one of PEG 70 ~PEG 120at least one of; and / or

[0008] said dextran [C6H 10 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; further preferably, at least one selected from dextrans with n = 20 - 30.

[0009] In some embodiments, the sex hormone to be detected is selected from testosterone, progesterone or estradiol, and the sex hormone derivative and the corresponding sex hormone to be detected can be recognized and bound by the same antibody; preferably, the binding force between the sex hormone derivative and the corresponding sex hormone to be detected and the antibody is different; more preferably, the binding force between the sex hormone derivative and the antibody is lower than the binding force between the sex hormone to be detected and the antibody.

[0010] The second aspect of the present application provides a photochemiluminescence immunoassay kit for detecting sex hormones, which includes reagent 1 and reagent 2. Reagent 1 includes the labeled sex hormone derivative described in any of the above embodiments, and reagent 2 includes luminescent particles with the antibody against the sex hormone to be detected coated on the surface.

[0011] In some embodiments, the concentration of reagent 1 is 3 ng / mL - 10 ng / mL, preferably, the concentration of reagent 1 is 5 ng / mL; and / or

[0012] the concentration of reagent 2 is 10 μg / mL - 30 μg / mL; preferably, the concentration of reagent 2 is 20 μg / mL.

[0013] In some embodiments, the photochemiluminescence immunoassay kit for sex hormones 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 solution; and / or

[0014] It further includes reagent 4, and reagent 4 includes photosensitive particles coated with avidin; preferably, the concentration of reagent 4 is 40 ug / mL - 50 ug / mL.

[0015] The third aspect of the present application provides a photochemiluminescence immunoassay method for detecting sex hormones, which includes:

[0016] Using a competitive immunoassay method, mixing and reacting a sample to be detected, the above-mentioned labeled sex hormone derivative, luminescent particles with the antibody against the sex hormone to be detected coated thereon, a releasing agent and photosensitive particles coated with avidin, then irradiating with excitation light, measuring the amount of luminescent 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 detected according to the light signal value.

[0017] In some embodiments, after mixing the sample to be tested, the labeled sex hormone derivative, the releasing agent, and the luminescent particles coated with an antibody against the sex hormone to be tested and incubating for a preset period of time, the photosensitive particles coated with avidin are added and mixed for incubation reaction.

[0018] The fourth aspect of the present application provides an application of the above-mentioned labeled sex hormone derivative or the above-mentioned sex hormone photochemiluminescence detection kit in sex hormone detection.

[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 Embodiments

[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 described specific embodiments. It should also be understood that the terms used herein are only for describing specific embodiments and do not represent restrictive.

[0021] When 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 explicit exclusions in the specified range. When 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 the present invention belongs. Although any methods and materials similar or equivalent to those described herein can 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 herein, the term "photosensitive particle" refers to a particle containing a sensitizer that can generate active intermediates such as reactive oxygen species that can react with luminescent particles upon activation by energy or an active compound. In some specific embodiments of the present application, the photosensitive particle is a polymer particle filled with a photosensitizer, and the photosensitizer may 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 1-50 atomic substituents, which are used to make these compounds more lipophilic or more hydrophilic and / or as a linking group for attachment to a specific binding partner. Examples of other photosensitizers known to those skilled in the art may also be used in the present application.

[0025] As used herein, 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 particle, thereby transferring energy to activate the luminescent particle. In some specific embodiments of the present 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] The present application provides a labeled sex hormone derivative for photochemiluminescence detection, which is obtained by labeling a sex hormone derivative with a biotinylated label. The simple formula of the labeled sex hormone derivative is sex hormone derivative - label - biotin, wherein the molecular weight of the sex hormone derivative is less than 1000 Da, and the molecular weight of the biotinylated label is 1000 Da - 10000 Da. By using a biotinylated label within a specified molecular weight range to label the sex hormone derivative, the present application can increase the molecular weight of the sex hormone derivative after biotinylation, making subsequent purification easier, with higher purity, less impurity content, better calibration signal value and discrimination, which is beneficial to improving the accuracy and precision of clinical sex hormone detection.

[0027] In some embodiments, when performing photochemiluminescence detection, the labeled sex hormone derivative of the present application is used to react with a luminescent particle coated with an antibody against the sex hormone to be detected, a releasing agent, a photosensitive particle coated with avidin, and a sample to be tested. Under the irradiation of excitation light, the amount of luminescent photons is measured to obtain a light signal value. According to the obtained light signal value, the content of the sex hormone to be detected can be quantitatively known, or it can be qualitatively known whether the sample to be tested contains the sex hormone to be detected.

[0028] It should be noted that when using the common Biotin-LCLC-NHS reagent, that is, N-succinimidyl 6-biotinamidohexanoic acid to biotinylate sex hormone derivatives, the molecular weight is still relatively small, usually less than 1000 Da. As a result, it is very difficult to purify the sex hormones biotinylated with the Biotin-LCLC-NHS reagent, leading to a relatively high impurity content in the detection, poor detection signal values and discrimination, which is not conducive to improving the accuracy and precision of the detection. In this application, however, by using a biotinylated marker to label sex hormones, the molecular weight after labeling is relatively high, making purification easier, with a higher purity and less impurity content, better calibration signal values and discrimination, which is conducive to improving the accuracy and precision of clinical detection.

[0029] It can be understood that the labeled sex hormone derivatives in this application refer to sex hormone derivatives with markers. When used for specifically testing a certain sex hormone to be detected, select the sex hormone derivative corresponding to the sex hormone to be detected, so as to perform photoinduced chemiluminescence detection using the competitive method. In some embodiments, the sex hormone to be detected can be testosterone, estradiol or progesterone. Correspondingly, the sex hormone derivatives in this application can be testosterone derivatives, progesterone derivatives or estradiol derivatives.

[0030] In order to detect the sex hormone to be detected using the competitive method, in some embodiments, the sex hormone derivatives in this application and the corresponding sex hormones to be detected can be recognized and bound by the same antibody. Preferably, the antibody coated on the surface of the luminescent particles is the antibody of the sex hormone to be detected. Specifically, 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.

[0031] In some embodiments, the binding forces of the sex hormone derivatives and the corresponding sex hormones to be detected with the antibody are different. More preferably, the binding force of the sex hormone derivative with the antibody is lower than the binding force of the sex hormone to be detected with the antibody. By using substances with different binding forces for competitive detection, an obvious competitive effect can be achieved, making the detection results have a clearer discrimination.

[0032] In order to reliably increase the molecular weight of the sex hormone derivatives and reduce the interference with the test results, in some embodiments, the marker is a hydrophilic long-chain molecule. Preferably, the hydrophilic long-chain molecule is selected from polyethylene glycol or dextran. That is to say, the biotinylated marker can be a biotinylated hydrophilic long-chain molecule. More preferably, the biotinylated marker can be biotinylated polyethylene glycol or dextran. Of course, the hydrophilic long-chain molecule can also be other hydrophilic molecules with a molecular weight within the specified range. Only examples are given here and are not limited.

[0033] Correspondingly, the molecular formula of the sex hormone derivative after being labeled with the corresponding biotinylated marker can be sex hormone derivative - polyethylene glycol - biotin, that is, sex hormone derivative - PEGn -Biotin, where n represents the number of PEG monomers; or a sex hormone derivative - dextran - biotin, that is, a sex hormone derivative - [C6H 10 O5] n *Biotin, where n represents the number of C6H 10 O5 monomers.

[0034] In some embodiments, when the label is selected from polyethylene glycol PEG n , polyethylene glycol PEG n is selected from at least one of PEG 25 to PEG 200 ; preferably, selected from at least one of PEG 30 to PEG 180 ; more preferably, selected from at least one of PEG 40 to PEG 150 ; further preferably, selected from at least one of PEG 70 to PEG 120 . That is to say, the label is selected from at least one or a mixture of polyethylene glycol PEG 25 to PEG 200 such that the molecular weight of the biotinylated label is in the range of 1000 Da to 10000 Da.

[0035] In some embodiments, when the label is selected from dextran [C6H 10 O5] n , dextran [C6H 10 O5] n is selected from at least one of dextrans with n = 7 to 55; preferably, selected from at least one of dextrans with n = 10 to 50; more preferably, selected from at least one of dextrans with n = 16 to 40; further preferably, selected from at least one of dextrans with n = 20 to 30. That is to say, the label is selected from at least one or a mixture of dextran [C6H 10 O5]7 to [C6H 10 O5] 55 such that the molecular weight of the biotinylated label is in the range of 1000 Da to 10000 Da.

[0036] In some embodiments, different labels can be selected according to the type of sex hormone derivative. Correspondingly, when the sex hormone derivative can be a derivative of testosterone, the label can be polyethylene glycol PEG n . When the sex hormone derivative can be a derivative of estradiol or progesterone, the label can be dextran [C6H 10 O5] nOf course, for the same sex hormone derivative, other types of labels can also be selected. Here, only examples are given and no limitations are imposed.

[0037] In order to prepare the labeled sex hormone derivative, in some embodiments, the sex hormone derivative is pre-mixed with an activator in a certain proportion; after obtaining the activated sex hormone derivative, it is then reacted with a labeling reagent, and after desalting and purification, the labeled sex hormone derivative is obtained. Among them, the labeling reagent contains the biotinylated label in any of the above embodiments.

[0038] In some embodiments, before mixing the sex hormone derivative with the activator, it further includes dissolving the sex hormone derivative in a solvent to a preset concentration, preparing the activator in water to a preset concentration, and dissolving the labeling reagent in a solvent to a preset concentration.

[0039] Preferably, the activator can be EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide). Preferably, the liquid after the reaction of the activated sex hormone derivative with the labeling reagent can be desalted and purified according to a desalting column. It can be understood that the molecular weight of the biotinylated label is 1000 Da to 10000 D, and 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 sex hormone derivative, the molecular weight of the labeled sex hormone derivative is greater than 1000 Da, and then the molecular weight after biotinylation can be increased. After desalting and purification, it is easier to obtain a labeled sex hormone derivative reagent with higher purity and less impurity content, which is beneficial to improving the accuracy and precision of clinical detection.

[0040] The present application also provides a sex hormone photoinduced chemiluminescence detection kit, which includes reagent 1 and reagent 2. Reagent 1 includes the above-mentioned labeled sex hormone derivative, and reagent 2 includes luminescent microparticles with an antibody against the sex hormone to be detected coated on the surface.

[0041] In order to obtain 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 solution. For example, the buffer solution can be a HEPES buffer solution, etc. Among them, reagent 1 can be diluted to the required concentration through the buffer solution.

[0042] 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 solution. For example, the buffer solution can be Tris buffer solution, etc. Among them, reagent 2 can be diluted to the required concentration by the buffer solution.

[0043] In some embodiments, the photochemiluminescence detection kit further includes reagent 3. 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 solution. Specifically, taking the small molecule of the target to be detected as sex hormone as an example, the releasing agent in the present application refers to a chemical reagent that can release the small molecule of the target 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 here and is not limiting.

[0044] In some embodiments, the photochemiluminescence detection kit further includes reagent 4. 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 the photochemiluminescence detection kit for detecting different sex hormones to be detected.

[0045] The present application also provides a photochemiluminescence detection method for detecting sex hormones. This detection method can quantitatively or qualitatively detect the sex hormones to be detected according to the above-mentioned photochemiluminescence detection kit.

[0046] In some embodiments, the competitive detection method is adopted. The sample to be detected, reagent 1 containing the above-mentioned labeled sex hormone derivative, reagent 2 containing the luminescent microparticles coated with the antibody of the sex hormone to be detected, reagent 3 containing the releasing agent, and reagent 4 which is the avidin-coated photosensitive microparticles are mixed and reacted, and then irradiated with excitation light, and the amount of luminescent photons is measured to obtain the optical signal value, and the detection result of the sex hormone to be detected in the sample to be detected is quantitatively or qualitatively obtained according to the optical signal value.

[0047] In some specific embodiments, the sample to be detected, the labeled sex hormone derivative, and the luminescent microparticles coated with the antibody of the sex hormone to be detected are mixed and incubated for a preset time, and then the avidin-coated photosensitive microparticles are added and incubated for reaction.

[0048] Preferably, after mixing and incubating the sample to be tested, Reagent 1, Reagent 2, and Reagent 3, add Reagent 4 and mix and incubate. For example, mix the sample to be tested, Reagent 1, Reagent 2, and Reagent 3 and incubate at 37°C for 15 min, then add an appropriate amount of Reagent 4 and incubate at 37°C for 10 min, and then measure the light signal value through the excitation light irradiation of the photochemiluminescence detection system.

[0049] The detection method of this application is detected according to the test principle of the competitive method, and is used for quantitative or qualitative detection of sex hormones in the sample to be tested. The operation is simple and convenient, and the sex hormone derivative - label - biotin has good signal values and discrimination, and more accurate detection results can be obtained.

[0050] 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.

[0051] Taking the sex hormone to be tested as testosterone and the corresponding sex hormone derivative as testosterone derivative as an example, the testosterone derivative is labeled with labeling reagent Biotin - PEG n -NH2 reagents with molecular weights of 588 Da, 1000 Da, 5000 Da, 10 kDa, and 20 kDa respectively to obtain labeled sex hormone derivative, namely testosterone derivative - PEG n -Biotin. Then it is purified with a desalting column, and finally the signal value and discrimination are detected on the photochemiluminescence platform. Biotin - PEG n -NH2 reagent is biotin polyethylene glycol active ester reagent, hereinafter referred to as Biotin - PEG n -NH2.

[0052] Example 1: Preparation of testosterone derivative - PEG n -Biotin

[0053] 1.1 The main experimental raw materials and equipment are shown in Table 1:

[0054] Table 1

[0055]

[0056] 1.2 Take a centrifuge tube, weigh 0.2 mg of testosterone derivative and dissolve it in 200 μL of DMSO solution to prepare a testosterone 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 - prepared EDC reagent and add it to the prepared testosterone derivative reagent, mix well to obtain an activated testosterone derivative reagent.

[0057] 1.3 Respectively dissolve 0.26 μmol of Biotin-PEG n -NH2 (588 Da), Biotin-PEG n -NH2 (1 kDa), Biotin-PEG n -NH2 (5 kDa), Biotin-PEG n -NH2 (10 kDa) and Biotin-PEG n -NH2 (20 kDa) 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 testosterone derivative reagent respectively, and let it stand and react at 2°C to 8°C for 18 h to obtain the corresponding reaction solution.

[0058] 1.4 Desalting and purification process:

[0059] Desalt and purify the above 5 kinds of reaction solutions according to the following steps.

[0060] 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 to 8°C (set at 4°C), 1500 g, for 2 min.

[0061] 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 to 8°C (set at 4°C), 1500 g, for 2 min.

[0062] 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 to 8°C (set at 4°C), 1500 g, for 2 min. Recover the liquid in the centrifuge tube.

[0063] Fourth centrifugation: Replace with a new centrifuge tube, add testosterone derivative-PEG n -Biotin, weigh it on an electronic balance for balance. Centrifuge at 2°C to 8°C (set at 4°C), 1500 g, for 2 min. Collect the liquid in the centrifuge tube, which is the purified testosterone derivative-PEG n -Biotin reagent, which is the biotin polyethylene glycol active ester-labeled testosterone derivative.

[0064] Example 2: Preparation of a photochemiluminescence immunoassay kit for detecting testosterone

[0065] 1. Dilute the testosterone derivatives - PEG with different molecular weights prepared in Example 1 above with HEPES buffer to 5 ng / mL respectively to obtain 5 corresponding reagents 1. n -Biotin reagents are diluted to 5 ng / mL respectively to obtain 5 corresponding reagents 1.

[0066] 2. Dilute the luminescent microparticles coated with testosterone antibody with Tris buffer to 20 μg / mL to obtain reagent 2.

[0067] 3. Prepare 0.2 mol / L citric acid buffer solution, dispense it to obtain reagent 3.

[0068] 4. Assemble reagent 1, reagent 2 and reagent 3 into corresponding complete sets of reagents respectively to obtain 5 complete sets of reagents.

[0069] 5. Prepare a photosensitive microparticle solution with a concentration of 50 μg / mL in advance as a general reagent, namely reagent 4.

[0070] Example 3: Detection of Testosterone

[0071] Prepare 6 different concentrations of testosterone reagents as calibration products, and detect the 5 complete sets of reagents prepared in Example 2 above and the sample to be tested on a 500 photochemiluminescence detection system.

[0072] 1. Mix 20 μL of the sample to be tested, 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.

[0073] 2. Add 175 μL of the general reagent of photosensitive microparticles, namely reagent 4, and incubate at 37 °C for 10 min.

[0074] 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.

[0075] Table 2

[0076]

[0077] It can be seen from the data in Table 2 that when the molecular weight of the Biotin - PEG n -NH2 reagent is 588 Da, the signal values of the corresponding calibration products are generally relatively low, and the overall discrimination degree of the reagent is 10.2; when the molecular weight of the Biotin - PEG n -NH2 reagent increases from 588 Da to 1 kDa, based on the data corresponding to 588 Da, the signal values corresponding to different calibration product concentrations increase by an average of 7.8 times, and the inhibition rate of the highest concentration increases by 1.8 times; the overall discrimination degree of the reagent is 18.2. When the Biotin - PEG nWhen 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.

[0078] It should be noted that the five Biotin-PEG n The theoretical values ​​of the molecular weight of -NH2 are 948Da, 1360Da, 5360Da, 10360Da and 20360Da respectively, and the molecular weight of the testosterone derivative is 360. The retention efficiency of the desalting column for salt 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 testosterone derivative-PEG in the obtained reagent n -Biotin content decreases, and the signal value decreases. When the molecular weight of Biotin-PEG-NH2 reagent reaches 5K Da, testosterone derivatives and testosterone derivatives-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.

[0079] 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.

[0080] Example 4: Detection of the sex hormone estradiol

[0081] Referring to the method of Example 1, estradiol derivatives were labeled with 3K Da and 10K Da biotin-dextran reagents to obtain desalted and purified estradiol derivative-dextran Biotin reagents.

[0082] Referring to the method of Example 2, a photochemiluminescence detection kit for detecting the sex hormone estradiol was prepared.

[0083] In this embodiment, 6 portions of estradiol reagents with different concentrations are prepared as calibration products. The 2 sets of complete reagents prepared in the above Embodiment 2 and the sample to be tested are tested on a 500 photochemiluminescence immunoassay system.

[0084] The detection process of this embodiment refers to Embodiment 3, and the experimental data are shown in Table 3 below.

[0085] Table 3

[0086]

[0087]

[0088] In this embodiment, it is illustrated that the molecular weight of the marker is not the larger the better. When the molecular weight is 3000 Da, the labeling effect of the biotin-dextran reagent is the best.

[0089] When the labeled sex hormone derivative provided by the present application is applied in a photochemiluminescence immunoassay kit, due to its good purity, the detection signal value and the discrimination degree are better. On the photochemiluminescence platform, the detection signal value and the discrimination degree are superior to other reagents, such as the small molecule biotinylated with Biotin-PEG-NH2 reagent.

[0090] The embodiments of the present application have been described above. The above description is exemplary and 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 choice of 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 to understand the embodiments disclosed herein.

Claims

1. A labeled sex hormone derivative for photochemiluminescence detection, characterized in that, It is obtained by labeling with a biotinylated label of a sex hormone derivative. The simple formula of the labeled sex hormone derivative is sex hormone derivative - label - biotin, wherein the molecular weight of the sex hormone derivative is less than 1000 Da, and the molecular weight of the biotinylated label is 1000 Da to 10000 Da; When performing photochemiluminescence detection, the labeled sex hormone derivative is used to react with the luminescent particles coated with the antibody of the sex hormone to be detected, the releasing agent, the photosensitive particles coated with avidin, and the sample to be detected. Under the irradiation of the excitation light, the amount of luminescent photons is measured to obtain the optical signal value.

2. The labeled sex hormone derivative according to claim 1, characterized in that, The label is a hydrophilic long-chain molecule. Preferably, the hydrophilic long-chain molecule is selected from polyethylene glycol or dextran.

3. The labeled sex hormone derivative according to claim 2, wherein The polyethylene glycol PEG n is selected from PEG 25 to PEG 200 and is at least one of them; preferably, it is selected from PEG 30 to PEG 180 and is at least one of them; more preferably, it is selected from PEG 40 to PEG 150 and is at least one of them; further preferably, it is selected from PEG 70 to PEG 120 and is at least one of them; and / or The dextran [C6H 10 O5] n is at least one selected from dextrans with n = 7 to 55; preferably, at least one selected from dextrans with n = 10 to 50; more preferably, at least one selected from dextrans with n = 16 to 40; still more preferably, at least one selected from dextrans with n = 20 to 30.

4. The labeled sex hormone derivative according to any one of claims 1 to 3, characterized in that, The sex hormone to be detected is selected from testosterone, progesterone or estradiol, and the sex hormone derivative and the corresponding sex hormone to be detected can be recognized and bound by the same antibody; preferably, the binding force between the sex hormone derivative and the corresponding sex hormone to be detected and the antibody is different; more preferably, the binding force between the sex hormone derivative and the antibody is lower than the binding force between the sex hormone to be detected and the antibody.

5. A chemiluminescence immunoassay kit for sex hormone detection by photonic excitation, characterized in that, It includes reagent 1 and reagent 2. Reagent 1 includes the labeled sex hormone derivative described in any one of claims 1 to 4, and reagent 2 includes the luminescent particles coated with the antibody of the sex hormone to be detected.

6. The sex hormone photochemiluminescence detection kit according to claim 5, wherein: The concentration of reagent 1 is 3 ng / mL to 10 ng / mL; preferably, the concentration of reagent 1 is 5 ng / mL; and / or The concentration of reagent 2 is 10 μg / mL to 30 μg / mL; preferably, the concentration of reagent 2 is 20 μg / mL.

7. The sex hormone photochemiluminescence detection kit according to claim 5 or 6, characterized in that, It further includes reagent 3, and reagent 3 includes a releasing agent. 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 includes reagent 4, and reagent 4 includes photosensitive particles coated with avidin; preferably, the concentration of reagent 4 is 40 μg / mL to 50 μg / mL.

8. A photochemiluminescence detection method for detecting sex hormones, characterized in that, It includes: Using the competitive detection method, the sample to be detected, the labeled sex hormone derivative described in any one of claims 1 to 4, the releasing agent, the luminescent particles coated with the antibody of the sex hormone to be detected, and the photosensitive particles coated with avidin are mixed and reacted, and then irradiated with the excitation light, and the amount of luminescent photons is measured to obtain the optical signal value. According to the optical signal value, the detection result of the sex hormone to be detected in the sample to be detected is quantitatively or qualitatively obtained.

9. The detection method of the sex hormone according to claim 8, wherein, The sample to be detected, the labeled sex hormone derivative, the luminescent particles coated with the antibody of the sex hormone to be detected, and the releasing agent are mixed and incubated for a preset time, and then the photosensitive particles coated with avidin are added and mixed and incubated for reaction. The application of the labeled sex hormone derivative described in any one of claims 1 to 4 or the sex hormone photochemiluminescence detection kit described in any one of claims 5 to 7 in the detection of sex hormones.

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