A homogenous recognition kit for detecting the venom of the bungarus multicinctus and its application

Abstract

The present application relates to the technical field of biological detection, and particularly relates to a homogenous luminescence identification kit for the venom of Daboia russelli and application thereof.The kit comprises: photosensitive microspheres, the photosensitive microspheres being connected with a first antibody capable of specifically binding with the venom of Daboia russelli; luminescent microspheres, the luminescent microspheres being connected with a second antibody capable of specifically binding with the venom of Daboia russelli; when the venom of Daboia russelli exists in a sample to be detected, the first antibody and the second antibody can simultaneously specifically bind with the venom of Daboia russelli, forming a complex in which the photosensitive microspheres and the luminescent microspheres are bridged by the venom of Daboia russelli, so that the distance between the photosensitive microspheres and the luminescent microspheres is shortened, and then a detectable luminescence signal is generated under excitation light irradiation.The kit and the detection method provided by the present application have the advantages of fewer operation steps, shorter detection time, higher specificity and higher sensitivity, and do not need washing and separation, and are suitable for rapid detection of the venom of Daboia russelli.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and in particular to a homogeneous luminescence identification kit for the venom of the five-step snake and its application. Background Technology

[0002] Snakebites are a common accidental injury in clinical emergency departments. After a bite from the five-step snake (also known as the sharp-nosed viper), the hemorrhagic and necrotic toxins in its venom can rapidly cause severe local pain, swelling, and tissue necrosis. In severe cases, it can lead to systemic bleeding, shock, and multiple organ dysfunction. For five-step snake bites, timely administration of the corresponding specific antivenom is crucial to improving the success rate of treatment.

[0003] Currently, the common method for clinically identifying the type of venomous snakebite relies primarily on the patient's description of the snake's appearance, or on live or dead snakes brought by the patient or others for identification. However, this traditional method has significant limitations. On the one hand, patients often struggle to remain calm after being bitten, or lack snake-specific knowledge, making it difficult to accurately describe the snake's characteristics. On the other hand, medical personnel who are not snake classification experts are prone to misdiagnosis based solely on verbal descriptions or cursory observations of the snake. This is especially true in areas where multiple venomous snake species coexist, as different species may appear similar, and local symptoms after bites often overlap, further complicating accurate identification. The uncertainty in diagnosis can lead to delayed or misused antivenom.

[0004] In addition, existing testing methods are cumbersome to operate, time-consuming, and rely on specialized equipment and operators for multi-step processing, making it difficult to meet the needs of rapid testing in emergency departments.

[0005] Therefore, it is necessary to provide a tool for rapid, accurate, and easy-to-use identification of the venom of the five-step snake to assist clinicians in timely selection of the correct antivenom serum. Summary of the Invention

[0006] The purpose of this invention is to address the problems existing in the prior art by providing a homogeneous luminescence identification kit for the venom of the five-step snake and its application, which achieves rapid, sensitive, and specific detection of the venom of the five-step snake through homogeneous luminescence technology.

[0007] To achieve the above objectives, the present invention provides a homogeneous luminescence identification kit for the venom of the five-step snake, comprising: Photosensitive microspheres, wherein a first antibody capable of specifically binding to the venom of the five-step snake is attached to the photosensitive microspheres; Luminescent microspheres, wherein a second antibody capable of specifically binding to the venom of the five-step snake is attached to the luminescent microspheres; When the sample to be tested contains the venom of the five-step snake, the first antibody and the second antibody can simultaneously bind specifically to the venom, forming a complex of photosensitive microspheres and luminescent microspheres bridged by the venom. This shortens the distance between the photosensitive microspheres and the luminescent microspheres, thereby generating a detectable luminescent signal under excitation light.

[0008] In an optional embodiment, the first antibody is a five-step snake thrombin antibody (labeled TLE-IgG) that specifically recognizes thrombin-like antigenic epitopes in the venom of the five-step snake.

[0009] In one optional embodiment, the second antibody is a five-step snake venom-specific whole venom antibody (labeled WB-IgG), which is a polyclonal antibody obtained by immunizing the host with five-step snake whole venom and capable of binding to multiple antigenic components in five-step snake venom.

[0010] In one optional embodiment, the photosensitive microspheres are polystyrene microspheres containing a photosensitizer, and the luminescent microspheres are polystyrene microspheres containing a luminescent material.

[0011] In an optional embodiment, the luminescent material is Eu(TFDH)3phen.

[0012] In an optional embodiment, the Eu(TFDH)3phen is a complex formed by europium with 1,1,1-trifluoro-5,5-dimethyl-2,4-hexanedione and 1,10-phenanthroline.

[0013] In one optional embodiment, the photosensitive microspheres are coupled with the first antibody to obtain a photosensitive microsphere-IgG-TLE complex; the luminescent microspheres are coupled with the second antibody to obtain a luminescent microsphere-IgG-WB complex.

[0014] In an optional implementation, when the sample to be tested contains the venom of the five-step snake, the first antibody and the second antibody specifically bind to the venom, forming a photosensitive microsphere-IgG-TLE-five-step snake venom-WB-IgG-luminescent microsphere complex, thereby shortening the distance between the photosensitive microspheres and the luminescent microspheres to below 200 nm. When the photosensitive microspheres are excited by 680 nm excitation light, excited-state oxygen is generated, transferring energy to the luminescent microspheres, which then generate a luminescence signal of approximately 610 nm.

[0015] In an optional embodiment, the five-step snake venom homogeneous luminescence identification kit further includes at least one of a diluent, a blocking solution, and an activation buffer for homogeneous luminescence detection.

[0016] The present invention also provides the application of the homogeneous luminescence identification kit for the venom of the five-step snake in the preparation of products for diagnosing bites from the five-step snake.

[0017] This invention also provides a method for detecting the venom of the five-step snake using the aforementioned homogeneous luminescence identification kit for the venom of the five-step snake. The method is for non-diagnostic purposes and includes the following steps: The sample to be tested was mixed with luminescent microspheres connected to the second antibody and incubated for the first time; then photosensitive microspheres connected to the first antibody were added and incubated for the second time. The luminescence signal value of the incubated system was detected using a photo-induced chemiluminescence detector.

[0018] In an optional implementation, the method is used for non-diagnostic purposes, such as, but not limited to: laboratory scientific research, snake venom toxicity determination, antivenom titer detection, monitoring of snake venom contamination in environmental samples, and quality control of snake venom-related biological products.

[0019] Compared with the prior art, the present invention has the following beneficial effects: (1) High sensitivity and wide linear range. The kit provided by this invention is based on photo-induced chemiluminescence technology, which can achieve sensitive detection of snake venom from the five-step snake. It has a wide linear range and a low detection limit, and can meet the detection needs of samples from low to high concentrations.

[0020] (2) Good specificity. The present invention uses a first antibody that can specifically bind to a specific antigenic component in the venom of the five-step snake and a second antibody that can capture all antigenic components in the venom of the five-step snake. The two antibodies recognize different antigenic epitopes, which can achieve specific recognition of the venom of the five-step snake and have basically no cross-reaction with the venom of other common venomous snakes.

[0021] (3) Good repeatability. The detection method of the kit of the present invention has good repeatability, low intra-batch coefficient of variation, and relatively stable detection results.

[0022] (4) Strong anti-interference ability. The kit of the present invention can detect the venom of the five-step snake in the presence of a variety of common serum matrix components, and the spiked recovery rate is within a certain range, making it suitable for the detection of complex biological samples.

[0023] (5) The detection time is short and the operation steps are few. Based on homogeneous luminescence technology, this invention does not require washing and separation steps, the operation is relatively simple, the detection time is short, and it can meet the time requirements of emergency detection.

[0024] (6) Wide range of applications. This invention can be used not only for the auxiliary diagnosis of patients with snake bites in clinical practice, but also for laboratory scientific research, snake venom toxicity determination, antivenom serum titer detection, monitoring of snake venom contamination in environmental samples, and quality control of snake venom-related biological products. Attached Figure Description

[0025] Figure 1 This is the infrared spectrum of Eu(TFDH)3phen of the present invention; Figure 2 This is the UV-Vis absorption spectrum of Eu(TFDH)3phen of this invention; Figure 3 This is the fluorescence spectrum of Eu(TFDH)3phen of the present invention; Figure 4 This is the energy distribution diagram of Eu(TFDH)3phen of the present invention; Figure 5 This is the standard curve diagram for the detection of five-step snake venom in this invention; Figure 6 This is a diagram showing the repeatability test results of the five-step snake venom detection method of this invention; Figure 7 This is a diagram showing the specific experimental results of the five-step snake venom detection method of this invention; Figure 8 This is a spiked recovery rate graph for the detection of snake venom from the five-step snake under the presence of different interfering substances according to the present invention. Detailed Implementation

[0026] The following embodiments are provided to better understand the present invention and are not limited to the described embodiments. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0027] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0028] In the following embodiments and comparative examples of the present invention, both TLE-IgG and WB-IgG were provided by the Disease Prevention and Control Center of the Southern Theater Command of the Chinese People's Liberation Army.

[0029] Example 1 This embodiment provides a homogeneous luminescence identification kit for the venom of the five-step snake.

[0030] (1) Preparation of luminescent material Eu(TFDH)3phen First, take 120 µL of 1,1,1-trifluoro-5,5-dimethyl-2,4-hexanedione (TFDH), add it to 5 mL of anhydrous ethanol, and sonicate until completely dissolved. Place the solution at room temperature, protected from light, and stir with a stir bar for 10 min. Then, add 5 mL of 25% ammonia solution and continue stirring until the ammonia odor dissipates. Adjust the pH of the solution to neutral to obtain solution a, which is ready for use. Separately, take 0.08 g of europium chloride hexahydrate and 0.04 g of 1,10-phenanthroline, place them in a beaker, add 5 mL of anhydrous ethanol, and sonicate until completely dissolved to obtain solution b, which is also ready for use.

[0031] Subsequently, solution b was mixed with solution a under magnetic stirring and stirred overnight at 600 rpm at room temperature. After the reaction was complete, the mixture was filtered, and the precipitate was washed with anhydrous ethanol. The precipitate was placed in a drying oven and dried at 40°C for 2 hours. The resulting powder was collected and stored in a desiccator in the dark to obtain Eu(TFDH)3phen.

[0032] (2) Preparation of luminescent microspheres Take 400 µL of a 10 mg / mL Eu(TFDH)3phen solution, 60 µL of a 10 mg / mL thiophene solution, and 38 µL of a 2 mg / mL 9,10-bisphenylethynylanthracene solution, mix thoroughly, and then add 1162 µL of ethylene glycol and 140 µL of benzyl alcohol. Add the mixture dropwise to a vial containing 200 µL of carboxylated polystyrene microspheres (from Du Biotechnology Co., Ltd., 10% solids content) while stirring. Then, place the vial in an oil bath at 110 °C and react for 10 min with magnetic stirring. After the reaction, centrifuge the solution for 20 min, discard the supernatant, and wash the precipitate repeatedly with anhydrous ethanol until the supernatant shows no fluorescence under UV light. Finally, resuspend the precipitate in 1 mL of ultrapure water and store in the dark at 4 °C to obtain luminescent microspheres.

[0033] (3) Preparation of photosensitive microspheres Weigh 0.4 g of dihydroxysilyl phthalocyanine and add 7 mL of N,N-dimethylformamide under nitrogen protection, stirring for 10 min. Then add 0.08 mL of a mixture of 3-(trimethoxysilyl)propyl methacrylate and 1 mL of N,N-dimethylformamide, and heat to 40 °C with stirring for 6 h. After the reaction is complete, pour the resulting reaction mixture into ice water and place it in a 4 °C refrigerator overnight. Filter the precipitated solid, collect the precipitate, and wash repeatedly with deionized water. Finally, dry under vacuum to obtain SiPC(Osi(C9H) 17 O4)2), store in a desiccator for later use.

[0034] Take 10 mg of SiPC (Osi(C9H) 17O4)2) Place the solution in a brown EP tube, add 1 mL of benzyl alcohol, and sonicate to dissolve. Take 0.8 mL of the above solution and mix it with 1 mL of ethylene glycol. Add this mixture dropwise to a vial containing 200 µL of carboxylated polystyrene microspheres (from Du Biotechnology Co., Ltd., 10% solid content) while stirring at 60 °C. Then place the vial in a 110 °C oil bath and react for 10 min with magnetic stirring. After the reaction is complete, centrifuge the solution for 20 min, wash the precipitate several times with anhydrous ethanol until the supernatant is colorless, and finally wash once with ultrapure water. Resuspend the precipitate in 1 mL of ultrapure water and store it in the dark at 4 °C to obtain photosensitive microspheres.

[0035] (4) Photosensitive microspheres conjugated with antibodies Take 20 μL of the photosensitive microspheres prepared in (3) (concentration of 10 mg / mL) and add them to a transparent centrifuge tube containing 1 mL of activation buffer (pH 6.2, 0.05 mmol / L 2-morpholinoethanesulfonic acid). Mix well by sonication. Then centrifuge at 11000 r / min and 4℃ for 35 min and discard the supernatant.

[0036] Add 1 mL of the aforementioned activation buffer to the precipitate and sonicate to mix. Immediately add 6 μL of 10 μg / μL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution, vortex to mix, then add 60 μL of 10 μg / μL hydroxysuccinimide solution, and vortex again to mix. Seal with sealing film and place in a constant temperature shaking instrument at 70 rpm and 25 °C for 20 min. After activation, immediately place the centrifuge tube in a centrifuge and centrifuge at 11000 rpm and 4 °C for 35 min, discarding the supernatant.

[0037] Add 1 mL of the aforementioned activation buffer to the precipitate, sonicate to mix, and then centrifuge again at 11000 rpm and 4°C for 35 min, discarding the supernatant. Add 1 mL of ultrapure water to the precipitate, sonicate to wash, and then centrifuge again at 11000 rpm and 4°C for 35 min, discarding the supernatant.

[0038] Add 1 mL of phosphate buffer (pH 7.4) to the washed microspheres and disperse by sonication. Then add 4 μL of TLE-IgG solution (concentration 1 mg / mL) to form a photosensitive microsphere-IgG-TLE complex. After careful mixing, seal with sealing film and incubate in a constant temperature shaker at 70 rpm and 25 °C for 120 min in the dark. After incubation, add 100 μL of Quick Block to the microspheres. TMThe Western blot blocking solution was mixed by shaking and incubated for another 120 min. After incubation, the centrifuge tubes were placed in a centrifuge and centrifuged at 13500 r / min and 4 °C for 35 min, and the supernatant was discarded.

[0039] Add 1 mL of 0.15% Tween-20 solution to the precipitate, sonicate to mix, and centrifuge again at 13500 r / min and 4℃ for 35 min. Discard the supernatant. Finally, add 1 mL of homogeneous luminescent diluent (from Du Biotechnology Co., Ltd.), resuspend the precipitate, and store at 4℃ in the dark to obtain antibody-labeled photosensitive microspheres (photosensitive microsphere-IgG-TLE complex).

[0040] (5) Luminescent microspheres conjugated with antibodies The process is basically the same as (4), except that the photosensitive microspheres (concentration of 10 mg / mL) are replaced with the luminescent microspheres (concentration of 10 mg / mL) prepared in (2); and the TLE-IgG solution is replaced with the WB-IgG solution (concentration of 13 mg / mL). Antibody-labeled luminescent microspheres (luminescent microsphere-IgG-WB complex) are prepared.

[0041] Example 2 This embodiment is basically the same as that of embodiment 1, except that (4) the amount of TLE-IgG solution added to the photosensitive microsphere conjugate antibody is 2 μL; (5) the amount of WB-IgG solution added to the luminescent microsphere conjugate antibody is 2 μL.

[0042] Example 3 This embodiment is basically the same as that of embodiment 1, except that (4) the amount of TLE-IgG solution added to the photosensitive microsphere conjugate antibody is 6 μL; (5) the amount of WB-IgG solution added to the luminescent microsphere conjugate antibody is 6 μL.

[0043] Experimental Example 1 In this experiment, infrared spectroscopy, ultraviolet-visible absorption spectroscopy, fluorescence spectroscopy analysis, and theoretical calculations were performed on the luminescent material Eu(TFDH)3phen prepared in Example 1 to confirm its structure and luminescent properties.

[0044] (1) Infrared spectrum of Eu(TFDH)3phen, as shown Figure 1 As shown. From Figure 1 As can be seen from this, it is located at 1600-1650cm -1 The absorption peaks within the range are attributed to the stretching vibrations of the C=O bonds in the coordinated TFDH. Compared to the free ligands, this peak shifts significantly to lower wavenumbers, indicating that Eu... 3+ It coordinated with the carboxyl oxygen of the ligand. (At 1350-1100 cm⁻¹) -1The strong, broad absorption peaks appearing within this range correspond to the vibrations of the CF bond. At 1631 cm⁻¹... -1 1516cm -1 and 1424cm -1 The sharp absorption peaks appearing nearby are attributed to the C=C and C=N stretching vibrations of the Phen aromatic ring skeleton. (900-650 cm⁻¹) -1 The absorption peaks within this range correspond to the out-of-plane bending vibration of the CH group of the Phen aromatic ring. Below 600 cm⁻¹ -1 The vibrational peaks of the metal-ligand (Eu-O, Eu-N) compounds were not directly observed. Comprehensive analysis indicates that the aforementioned infrared spectral characteristics are consistent with the molecular structure of the target complex, confirming the successful binding of the ligands to Eu... 3+ Coordination.

[0045] (2) The UV-Vis absorption spectrum of Eu(TFDH)3phen, as shown in the figure. Figure 2 As shown. From Figure 2 As can be seen, the complex exhibits strong absorption in the ultraviolet region (200-400 nm), which is attributed to the π→π* electronic transition within the ligand (TFDH) molecule. Compared to the free ligand, the absorption band of the complex shows a significant shift, indicating that the ligand interacts with Eu... 3+ Successful coordination between the molecules influenced the electronic structure. The broad and strong ultraviolet absorption band allowed the complex to effectively capture ultraviolet light energy and excite the Eu center through intramolecular energy transfer (antenna effect). 3+ Ions provide the basis for subsequent characteristic luminescence.

[0046] (3) The fluorescence spectrum of Eu(TFDH)3phen, as shown in the figure. Figure 3 As shown. From Figure 3 As can be seen, the emission spectrum of the complex exhibits a series of sharp emission peaks near 592 nm, 611 nm, 650 nm, and 700 nm, which are attributed to Eu. 3+ Ionic 5 D0→ 7 F1 (magnetic dipole transition) 5 D0→ 7 F2 (electric dipole transition, main emitter) 5 D0→ 7 F3 and 5 D0→ 7 The F4 energy level transition occurs. Among them, the electric dipole transition peak near 611 nm has the highest intensity, which is the main reason why the material exhibits high-purity red luminescence.

[0047] (4) The energy transfer, storage and release capabilities of the material in the excited state were evaluated using the TD-DFT theoretical calculation method, and the energy distribution map of Eu(TFDH)3phen was obtained, as shown in the figure. Figure 4 As shown. Figure 4 In this context, "Absorbing Energy" refers to the absorption of energy, "Phosphorescence" refers to phosphorescent radiation, "Energy Transfer" refers to energy transfer, and "luminescence" refers to the emission of light. Figure 4 As can be seen, after electrons are excited to singlet states higher than S1 (S2, S3, S4), they return to the S1 state through an extremely fast internal conversion process, and then transfer energy to the S1 state. 5 D1 state, then rapid internal relaxation or direct transmission to 5 D0 state, finally from 5 D0 jump to 7 F 0-4 The ground state energy level emits a sharp line spectrum.

[0048] Experimental Example 2 This experimental example verifies the linear range, repeatability, and specificity of the homogeneous luminescence identification kit for five-step snake venom prepared in Example 1.

[0049] (1) Linear range and detection limit A series of standard samples of five-step snake venom at different concentrations were prepared, namely: 0.19 ng / mL, 0.76 ng / mL, 97.6 ng / mL, 195.0 ng / mL, 780.0 ng / mL, 1560.0 ng / mL, and 3125.0 ng / mL.

[0050] 20 µL of each concentration of standard was added to different wells of a 96-well plate. Then, 25 µL of the luminescent microsphere-IgG-WB complex prepared in Example 1 was added to each well, mixed thoroughly, and incubated at 37 °C for 5 min. Subsequently, 25 µL of the photosensitive microsphere-IgG-TLE complex prepared in Example 1 was added to each well, mixed thoroughly, and incubated for another 5 min. After incubation, the luminescence signal value of each well was immediately measured using a photochemiluminescence detector.

[0051] A standard curve was plotted with the concentration of the five-step snake venom on the x-axis and the luminous signal value on the y-axis, resulting in a standard curve graph for the detection of five-step snake venom. Figure 5 As shown. The obtained linear equation is I = 110.8c + 136.47, and the correlation coefficient R0 is... 2 =0.9923, the linear range was 0.19 ng / mL to 3125 ng / mL, and the limit of detection was 0.18 ng / mL.

[0052] (2) Repeatability Five-step snake venom samples prepared in the same batch (concentration of 0.09 μg / mL) were aliquoted into 14 different wells of a 96-well plate, 20 µL per well. The luminescence signal value of each well was measured according to the method in (1) of this experimental example.

[0053] Calculate the relative standard deviation (RSD) of the 14 measurements. Obtain the repeatability test results for the detection of *Phaseosaurus* venom, as shown in the figure. Figure 6 As shown in the figure, the RSD is 1.4%, indicating that this kit has good reproducibility.

[0054] (3) Specificity The following other snake venoms that may interfere with the detection of the five-step snake venom were selected for specific assay: viper venom (FS), viper venom (KS), king cobra venom (YW), pit viper venom (YM), bamboo pit viper venom (ZYQ), banded krait venom (YH), ​​golden krait venom (JH), and cobra venom (YJ). Antigen blank buffer (CG) without snake venom was used as a negative control, and five-step snake venom (WB) was used as a positive control.

[0055] 20 µL of each snake venom sample was added to different wells of a 96-well plate, and the luminescence signal value was measured according to method (1) in this experimental example. The specificity experimental results for the detection of Five-Step Snake venom are shown in the figure below. Figure 7 As shown. From Figure 7 The results showed that, compared with the positive control, the luminescent signal values ​​of other snake venoms were basically the same as those of the blank control, and could be almost ignored. This indicates that the kit has high specificity for the venom of the five-step snake and no cross-reaction with the other snake venoms mentioned above.

[0056] Experimental Example 3 This experiment evaluates the anti-interference ability of the homogeneous luminescence recognition kit for five-step snake venom prepared in Example 1 under physiologically relevant conditions.

[0057] Eight common serum matrix components were added to a standard sample of *Spathea pilosa* venom (concentration 0.1 µg / mL): ethylenediaminetetraacetic acid (EDTA), heparin, fibrinogen, trilaurin, cholesterol, hemoglobin, immunoglobulin, and bilirubin. The concentrations of each interfering substance were added at 10, 100, and 1000 times the concentration of *Spathea pilosa* venom. A blank control (CG) was used without the addition of any interfering substances.

[0058] 20 µL of the sample with added interfering substances and the blank control sample were added to a 96-well plate, and the luminescence signal value was measured according to the method in Experimental Example 2 (1), and the spiked recovery rate was calculated. The spiked recovery rate graphs for the detection of snake venom in the presence of different interfering substances were obtained, as shown below. Figure 8 As shown. From Figure 8The results showed that the recoveries of spiked samples in the presence of various interfering substances ranged from 85.6% to 107.5%, all within acceptable ranges. This indicates that the kit has good anti-interference performance and can accurately detect the venom of the five-step snake in complex samples.

[0059] Experiment Example 4 This experimental example verifies the detection accuracy of the homogeneous luminescence recognition kit for snake venom prepared in Example 1 in actual biological samples.

[0060] Due to the difficulty in obtaining biological samples from actual snakebite patients, this experiment used healthy human serum as a matrix, adding different concentrations of *Vibrio vulgaris* venom standards to it for spiked recovery experiments. The healthy human serum samples used were obtained from the Department of Laboratory Medicine, Guangdong Provincial Second People's Hospital, and all procedures followed the hospital's ethical requirements.

[0061] Serum samples from healthy individuals were tested and confirmed to be free of *Phlebotomyces fasciatus* venom (initial concentration < 0.00001 μg / mL). *Phlebotomyces fasciatus* venom standards at low, medium, and high concentrations were added to the serum samples: 0.001 μg / mL, 0.1 μg / mL, and 2 μg / mL, respectively. Three parallel samples were prepared for each concentration. The luminescence signal values ​​of each sample were measured according to the method in Experimental Example 2 (1), and the measured concentrations were calculated by substituting them into the linear equation in Experimental Example 2 (1). The recovery rate and relative standard deviation (RSD) were then calculated.

[0062] The test results are shown in Table 1. The spiked recoveries ranged from 95.15% to 102.06%, and the RSDs were all less than 5%. This indicates that the kit has high accuracy and precision in complex biological samples and can be used for the detection of snake venom in actual serum samples.

[0063] Table 1 Test Results

[0064] In summary, the reagent kit and detection method provided by this invention have fewer operation steps, shorter detection time, higher specificity, higher sensitivity, and do not require washing and separation, making them suitable for rapid detection of the venom of the five-step snake.

[0065] Finally, it should be noted that the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A homogeneous luminescent identification kit for the venom of the five-step snake, characterized in that, include: Photosensitive microspheres, wherein a first antibody capable of specifically binding to the venom of the five-step snake is attached to the photosensitive microspheres; Luminescent microspheres, wherein a second antibody capable of specifically binding to the venom of the five-step snake is attached to the luminescent microspheres; When the sample to be tested contains the venom of the five-step snake, the first antibody and the second antibody can simultaneously bind specifically to the venom, forming a complex of photosensitive microspheres and luminescent microspheres bridged by the venom. This shortens the distance between the photosensitive microspheres and the luminescent microspheres, thereby generating a detectable luminescent signal under excitation light.

2. The homogeneous luminescence identification kit for five-step snake venom according to claim 1, characterized in that, The first antibody is a five-step snake thrombin antibody.

3. The homogeneous luminescence identification kit for five-step snake venom according to claim 1, characterized in that, The second antibody is a specific whole-venom antibody against the venom of the five-step snake.

4. The homogeneous luminescence identification kit for five-step snake venom according to claim 1, characterized in that, The photosensitive microspheres are polystyrene microspheres containing photosensitizers, and the luminescent microspheres are polystyrene microspheres containing luminescent materials.

5. The homogeneous luminescence identification kit for five-step snake venom according to claim 4, characterized in that, The luminescent material is Eu(TFDH)3phen.

6. The homogeneous luminescence identification kit for five-step snake venom according to claim 5, characterized in that, The Eu(TFDH)3phen is a complex formed by europium with 1,1,1-trifluoro-5,5-dimethyl-2,4-hexanedione and 1,10-phenanthroline.

7. The homogeneous luminescence identification kit for five-step snake venom according to claim 1, characterized in that, The photosensitive microspheres are coupled to the first antibody; the luminescent microspheres are coupled to the second antibody.

8. The homogeneous luminescence identification kit for five-step snake venom according to claim 1, characterized in that, It also includes at least one of a diluent, a blocking solution, and an activation buffer for homogeneous luminescence detection.

9. The use of the homogeneous luminescence identification kit for five-step snake venom according to any one of claims 1-8 in the preparation of products for diagnosing five-step snake bites.

10. A method for detecting *Spathea pilosa* venom using the homogeneous luminescence identification kit for *Spathea pilosa* venom according to any one of claims 1-8, wherein the method is for non-diagnostic purposes, characterized in that... Includes the following steps: The sample to be tested was mixed with luminescent microspheres connected to the second antibody and incubated for the first time; then photosensitive microspheres connected to the first antibody were added and incubated for the second time. The luminescence signal value of the incubated system was detected using a photo-induced chemiluminescence detector.

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