Trypsinogen-2 detection method and kit based on homogeneous light-activated chemiluminescence immunoassay technology

Abstract

The invention discloses a trypsinogen-2 detection method and kit based on a homogeneous light-excited chemiluminescence immunoassay technology, and belongs to the technical field of immunodetection analysis. The method provided by the invention comprises the following steps: preparing donor microspheres and receptor microspheres coupled with a trypsinogen-2 capture antibody, adding the coupled donor microspheres and receptor microspheres into a sample to be detected, uniformly mixing and incubating without cleaning, and finally detecting by using a fluorescence detector. The kit comprises donor microspheres coupled with a trypsinogen-2 capture antibody, receptor microspheres coupled with a trypsinogen-2 detection antibody, a trypsinogen-2 standard substance solution and an analysis buffer solution. The trypsinogen-2 content in a sample to be detected can be rapidly detected by using the characteristics of a double-antibody sandwich immunoassay and light-activated chemiluminescence, and the trypsinogen-2 detection kit has the characteristics of wide detection range, high sensitivity, high accuracy, rapid and simple detection and the like, can be used for early auxiliary diagnosis of acute pancreatitis clinically, and has a relatively good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a trypsinogen-2 detection method and a kit based on homogeneous photoinduced chemiluminescence immunoassay technology. Background Art

[0002] Trypsinogen is a protease secreted by pancreatic exocrine cells. There are two main types of trypsinogen in human pancreatic juice, namely trypsinogen-1 and trypsinogen-2. Although the concentration of trypsinogen-1 in the serum of healthy people is higher than that of trypsinogen-2, the concentration of trypsinogen-2 is much higher than that of trypsinogen-1 in patients with pancreatitis, and a large amount of literature shows that trypsinogen-2 is a biomarker for diagnosing acute pancreatitis, which is more reliable than amylase and lipase commonly used in clinical practice.

[0003] Acute pancreatitis (AP) is a common inflammatory disease of the exocrine pancreas, with abnormal activation of trypsinogen and destruction of secretory cells as the main pathogenesis. It can lead to local and systemic inflammatory response syndrome (SIRS), with a high incidence rate, and is the main reason for hospitalization for gastrointestinal diseases. Most patients have mild pancreatitis, which is not life-threatening, but about 20% of patients will develop moderate or severe acute pancreatitis, accompanied by pancreatic or peripancreatic tissue necrosis and organ failure, with a high mortality rate. Early diagnosis and intervention can not only provide better treatment options for AP patients, but also improve prognosis and reduce mortality.

[0004] Accurate diagnosis of patients with suspected acute pancreatitis can avoid unnecessary surgery, but if the detection technology is to be applied to emergency departments and outpatient clinics, it must be tested promptly, accurately, and quickly. Although the use of serum amylase and lipase to diagnose AP is widely supported in clinical practice, their sensitivity and specificity are not satisfactory, and imaging examinations such as CE-CT are not readily available due to cost and harm. Existing detection methods, such as enzyme-linked immunosorbent assay (ELISA) and chemiluminescent immunoassay (CLIA), are also unable to provide clinicians with timely and reliable results due to long detection time, complex operation, and poor stability. Therefore, the development of a rapid detection method for trypsinogen-2 is of great significance for the early diagnosis of acute pancreatitis.

[0005] Homogeneous photoinduced chemiluminescence immunoassay (AlphaLISA) is an innovative immunoassay technology that uses the specific interaction between biological molecules to achieve signal amplification through the principle of fluorescence resonance energy transfer (FRET). AlphaLISA technology uses silica microspheres as carriers and adopts time-resolved fluorescence detection mode. Its detection principle is to bring the donor and acceptor microspheres into the diffusion range of singlet oxygen through the specific interaction between antigen and antibody, thereby stimulating the chemiluminescence reaction. The luminescence principle of this technology is based on photoinduced chemiluminescence, in which the photosensitizer benzodimethyl blue coated on the surface of the donor microspheres decomposes oxygen in the environment to generate monomeric oxygen molecules under 680nm laser irradiation. These molecules then diffuse to the acceptor microspheres and transfer energy to the rare earth element europium, generating 615nm excitation light.

[0006] The advantages of AlphaLISA technology are its high sensitivity, rapid detection, simple operation and no need for complicated cleaning steps, making it very suitable for the rapid diagnosis needs of emergency departments and outpatient clinics. At present, there are related patents involving the application of AlphaLISA technology in biomedical testing. Existing patents such as CN110907636A propose a photoinduced chemiluminescence homogeneous immunoassay method for detecting target molecules in biological samples. This method achieves the transfer of high-energy reactive oxygen through antigen-antibody binding between two microspheres, inducing a photoinduced chemiluminescence process, and has the characteristics of high sensitivity, rapidity, and easy operation. However, the patent did not study the detection method for the specific biomarker trypsinogen-2.

[0007] Based on the existing technology, the present invention proposes a trypsinogen-2 concentration detection method based on AlphaLISA technology, aiming to solve the shortcomings of the existing diagnostic methods in terms of sensitivity and specificity, as well as the long detection time and complicated operation. Through the implementation of the present invention, it can not only provide clinicians with a fast and reliable diagnostic tool, but also help improve the treatment options and prognosis of patients with acute pancreatitis, thereby reducing the mortality rate, which is of great significance for improving the early diagnosis level of AP and improving the prognosis of patients. Summary of the invention

[0008] In view of the problems in the related art, the present invention discloses a method and a kit for detecting trypsinogen-2 based on homogeneous photochemiluminescence immunoassay technology. The method achieves high-sensitivity detection of trypsinogen-2 through specific antibody coupling technology, formation of fluorescent immune complexes and time-resolved fluorescence measurement. At the same time, the present invention also provides a kit containing all necessary reagents, which simplifies the detection process.

[0009] To achieve the above object, the present invention provides the following technical solutions:

[0010] A method for detecting trypsinogen-2 based on homogeneous photoinduced chemiluminescence immunoassay technology comprises: setting a solid phase carrier, wherein the solid phase carrier is a donor microsphere coupled with a trypsinogen-2 capture antibody; covalently coupling the trypsinogen-2 antigen to the surface of the donor microsphere; and coupling the trypsinogen-2 detection antibody to an acceptor microsphere at the same time; combining the donor microsphere coupled with the trypsinogen-2 capture antibody and antigen and the acceptor microsphere coupled with the trypsinogen-2 detection antibody by a double antibody sandwich method to form a fluorescent immune complex of the donor microsphere coupled with the capture antibody-antigen-acceptor microsphere coupled with the detection antibody; irradiating a photosensitizer on the donor microsphere with excitation light to generate singlet oxygen; the luminescent agent on the acceptor microsphere receives the energy transfer of the singlet oxygen generated by the donor microsphere irradiated with the excitation light to generate fluorescence; and finally, performing detection and analysis according to the fluorescence intensity.

[0011] The fluorescence detection analysis refers to the use of the long-life fluorescence characteristic of lanthanide rare earth chelates. After the sample is excited and before the fluorescence signal is collected, the fluorescence decay time of different fluorescent substances contained in the sample is different. After the short-life background fluorescence in the sample to be tested is completely quenched during this time period, the long-life rare earth chelate specific fluorescence signal is collected and detected. The time-resolved delay can effectively eliminate the interference from other non-specific fluorescence from samples, reagents, instruments, etc., thereby greatly improving the sensitivity of the detection.

[0012] By adopting the above technical scheme, based on the principle of interaction between biological molecules, fluorescence resonance energy transfer luminescence, and silica microspheres as carriers, by identifying and utilizing time-resolved fluorescence measurement and spectral separation technology to eliminate nonspecific interference in the sample, and measuring the fluorescence intensity in the final product, the concentration of trypsinogen-2 in the sample to be tested can be more conveniently and accurately detected, thereby effectively improving the sensitivity and efficiency of detection, and providing an important basis for the diagnosis and treatment of pancreatic diseases.

[0013] As a further solution of the present invention: the detection method also includes the following steps: purifying the trypsinogen-2 capture antibody and the trypsinogen-2 detection antibody by centrifugal filtration, wherein the centrifugal filtration method is to centrifuge and wash the capture antibody and the detection antibody multiple times using a centrifuge tube with a filter membrane.

[0014] As a further solution of the present invention: the detection method also includes the following steps: activating the donor microspheres and the acceptor microspheres using a cross-linking agent, wherein the cross-linking agent is N-hydroxysuccinimide (NHS) or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC).

[0015] As a further solution of the present invention: the detection method further comprises the following steps: the double antibody sandwich method is to add optimized doses of donor microspheres coupled with capture antibodies, acceptor microspheres coupled with detection antibodies and the antigen to be detected into a polypropylene well plate for reaction to form a fluorescent immune complex.

[0016] As a further solution of the present invention: the reaction conditions of the double antibody sandwich method in the detection method are shaking at 37° C. in the dark for 15-20 minutes.

[0017] As a further solution of the present invention: the coating ratio of the donor microspheres and the acceptor microspheres to form conjugates with the trypsinogen-2 capture antibody and the trypsinogen-2 detection antibody is 1 mg microsphere labeled with 0.1 mg antibody.

[0018] As a further solution of the present invention: the coupling conditions of the trypsinogen-2 capture antibody and the detection antibody with the donor microspheres and the acceptor microspheres respectively are set to incubate at room temperature in the dark for 2 hours with shaking.

[0019] As a further solution of the present invention: a trypsinogen-2 detection kit based on homogeneous photochemiluminescence immunoassay technology, including a microplate, a kit body and a fluorescence detector, characterized in that: the kit body includes a trypsinogen-2 standard solution, donor microspheres coupled with trypsinogen-2 capture antibodies, and acceptor microspheres coupled with trypsinogen-2 detection antibodies. The kit body is also provided with an expandable polystyrene foam layer to protect the reagents from damage during transportation and storage.

[0020] By adopting the above technical solution and using homogeneous photochemiluminescence immunoassay technology, the sensitivity and specificity of trypsinogen-2 detection are improved, the operation process is simplified, and the detection time is shortened.

[0021] As a further solution of the present invention: the kit body also includes auxiliary detection reagents, and the auxiliary detection reagents include one or more of a labeling buffer, an analysis buffer, a blocking solution, a stop solution, a cross-linking agent and a concentrated washing solution.

[0022] As a further solution of the present invention: the analysis buffer is an analysis buffer with a pH of 7.8, and the concentrated washing solution is a PBS buffer or a Tris-HCl washing solution.

[0023] By adopting the above technical solution, the content of trypsinogen-2 in the sample to be tested can be quickly detected. It has the characteristics of strong specificity, high sensitivity, good stability, and rapid detection. It can be used for clinical auxiliary diagnosis of AP and has good application prospects. The kit has a simple structure and strong practicality.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. High sensitivity: The homogeneous photochemiluminescence immunoassay technology can accurately and quickly detect the concentration of trypsinogen-2 in the sample to be tested, providing an important basis for the early diagnosis and treatment of pancreatic diseases such as pancreatitis.

[0026] 2. Easy operation: The kit has a simple composition, few detection steps, easy operation, and no need for cumbersome sample processing, which improves the detection sensitivity and specificity.

[0027] 3. Reliable detection: By using specific cross-linking agents, labeling buffers and concentrated washing solutions, the detection process is further optimized, the stability and reliability of the detection are improved, and the clinical demand for rapid detection is met. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention.

[0029] In the attached picture:

[0030] Figure 1 The standard curve for trypsinogen-2 detection.

[0031] Figure 2 Schematic diagram of the fluorescent immune complex of donor microspheres coupled to capture antibodies - antigens - acceptor microspheres coupled to detection antibodies, where 1-1 is the capture antibody, 1-2 is trypsinogen-2, and 2-1 is the detection antibody.

[0032] Figure 3 It is a top view of the main body of the trypsinogen-2 detection kit, wherein 11 is the kit body, 22 is the auxiliary reagent, 1 is the trypsinogen-2 standard solution, 2 is the donor microspheres coupled to the trypsinogen-2 capture antibody, 3 is the acceptor microspheres coupled to the trypsinogen-2 detection antibody, 4 is the labeling buffer, 5 is the analysis buffer, 6 is the blocking solution, 7 is the stop solution, 8 is the cross-linking agent, and 9 is the concentrated washing solution. DETAILED DESCRIPTION

[0033] Exemplary embodiments will be described in detail herein, examples of which are shown in the accompanying drawings. When the following description refers to the drawings, the same numbers in different drawings represent the same or similar elements unless otherwise indicated. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present disclosure. Instead, they are merely examples of devices and methods consistent with some aspects of the present embodiment disclosure as detailed in the appended claims.

[0034] It should be noted that all directional indications in the embodiments (such as up, down, left, right, front, back, etc.) are only used to explain the relative position relationship, movement status, etc. between the components under a certain specific posture (as shown in the accompanying drawings). If the specific posture changes, the directional indication will also change accordingly.

[0035] In addition, in the embodiments, the descriptions involving "first", "second", etc. are only for descriptive purposes, and do not specifically refer to the order or sequence, nor are they used to limit the present invention. They are only used to distinguish components or operations described with the same technical terms, and cannot be understood as indicating or implying their relative importance or implicitly indicating the number of technical features indicated. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In addition, the technical solutions between the various embodiments can be combined with each other, but they must be based on the ability of ordinary technicians in the field to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0036] In order to further understand the content, features and effects of the present invention, the following embodiments are given as examples and described in detail with reference to the accompanying drawings:

[0037] Example 1: Donor (acceptor) microsphere-trypsinogen-2 capture (detection) antibody coupling

[0038] The donor (acceptor) microspheres were activated and washed with labeling buffer (MES), NHS and EDC, and then incubated with the purified capture (detection) antibodies at room temperature for 2 hours in the dark. Then 100 μL of BSA blocking solution was added and incubated at room temperature for 1 hour in the dark. Finally, Tris-HCl washing solution was used for washing and separation to remove unbound antibodies, and ELISA or flow cytometry was used to verify the binding amount of antibodies on the coupled microspheres to ensure the optimal coupling efficiency. The prepared microspheres were stored in a 4°C refrigerator.

[0039] Example 2: Antibody Purification

[0040] Add the antibody material to be coupled to a 50KD centrifuge tube with a filter membrane, add 300μL of labeling buffer to the concentrated antibody material, centrifuge at 10000rpm for 5min, discard the filtrate, add 300μL of elution buffer, and repeat the above centrifugation steps 8 times. Take out the centrifuge tube after several centrifugations, discard the filtrate, reverse the filter membrane of the centrifuge tube, centrifuge at 3000rpm for 1min, and collect the filtrate. Then reverse the filter membrane again, add 50μL of labeling buffer, let it stand for a while, let the filter membrane absorb the labeling buffer, and dissolve the residual antibody material left on the filter membrane, and then reverse the filter membrane again and centrifuge at 3000rpm for 1min, collect the liquid on the filter membrane, so that as much antibody material on the filter membrane is recovered as much as possible, that is, the concentrated and purified antibody material required. The optimal volume for collecting antibodies during the whole process is about 100μL.

[0041] Example 3: Microsphere Activation

[0042] The microsphere raw material to be coupled was soaked in acetic acid buffer at pH 4.5 for 5 minutes to expose more binding sites. The soaked microsphere raw material was added to a 2mL centrifuge tube, 900μL labeling buffer was added, centrifuged at 17000rpm for 20min, the supernatant was discarded, 1mL labeling buffer was added for 15min, and the mixture was shaken and dispersed. Then, 10μL of 20mg / mL NHS solution and 5μL of 20mg / mL EDC solution were added, and the mixture was shaken at room temperature and away from light for 20min. Subsequently, the activated microspheres were centrifuged at 17000rpm for 20min, the supernatant was discarded, 1000μL of labeling buffer was added for ultrasonic mixing, and repeated twice.

[0043] Example 4: Formation of fluorescent immune complex of trypsinogen-2 capture antibody coupled to donor microspheres-trypsinogen-2 antigen-trypsinogen-2 detection antibody coupled to acceptor microspheres

[0044] Donor microspheres coupled with capture antibodies, acceptor microspheres coupled with detection antibodies and the sample to be tested are added to a polypropylene well plate together, and the incubation condition is 37°C with shaking in the dark for 15 minutes to obtain a fluorescent immune complex of trypsinogen-2 capture antibody coupled to donor microspheres-trypsinogen-2 antigen-trypsinogen-2 detection antibody coupled to acceptor microspheres. The fluorescent immune complex is irradiated with excitation light to cause the photosensitizer on the donor microspheres to produce singlet oxygen, and the luminescent agent on the acceptor microspheres receives the energy transfer from singlet oxygen to produce fluorescence. Finally, the fluorescence intensity is detected on a machine to determine the content of the detection index.

[0045] Example 5: Detection of the effective binding amount of trypsinogen-2 detection antibody coupled to trypsinogen-2 capture antibody-trypsinogen-2 antigen-coupled receptor microspheres

[0046] Enzyme-linked immunosorbent assay (ELISA): Add the donor microspheres coupled with the capture antibody, the acceptor microspheres coupled with the detection antibody and the sample to be tested to the ELISA plate in sequence, incubate at 37°C in the dark for 15 minutes, then add the enzyme-labeled secondary antibody and continue incubating for 30 minutes. After 5 washes, add the substrate colorimetric solution and react at room temperature in the dark for 15 minutes. Finally, add the stop solution, detect the absorbance on the machine, and determine the content of the detection index.

[0047] Trypsinogen-2 capture antibody-trypsinogen-2 antigen-trypsinogen-2 detection antibody time-resolved fluorescence immunoassay of receptor microspheres: Add the donor microspheres coupled with capture antibody, the receptor microspheres coupled with detection antibody and the sample to be tested into the reaction tube, incubate at 37℃ in the dark for 15 minutes. Then add Eu 3 + labeled secondary antibody, continue incubation for 30 minutes. After 5 washes, add enhancement solution and react at room temperature in the dark for 5 minutes. Finally, the time-resolved fluorescence intensity is measured on the machine to determine the content of the detection index.

[0048] Trypsinogen-2 capture antibody-trypsinogen-2 antigen-trypsinogen-2 detection antibody chemiluminescent immunoassay of coupled receptor microspheres: Add the donor microspheres coupled with the capture antibody, the acceptor microspheres coupled with the detection antibody and the sample to be tested into the reaction tube, and incubate at 37°C in the dark for 15 minutes. Then add the HRP-labeled secondary antibody and continue incubation for 30 minutes. After 5 washes, add the chemiluminescent substrate and react at room temperature in the dark for 5 minutes. Finally, the luminescence intensity is detected on the machine to determine the content of the detection index.

[0049] Trypsinogen-2 capture antibody-trypsinogen-2 antigen-coupled receptor microspheres for trypsinogen-2 detection antibody electrochemiluminescence immunoassay: Add the donor microspheres coupled with the capture antibody, the receptor microspheres coupled with the detection antibody, and the sample to be tested to the electrochemical sensor, and incubate at 37°C in the dark for 15 minutes. Subsequently, add the secondary antibody labeled with electrochemically active substances and continue incubation for 30 minutes. After washing, use an electrochemical workstation to detect the current signal, and determine the content of the detection index by the change in the current signal.

[0050] Trypsinogen-2 capture antibody-trypsinogen-2 antigen-trypsinogen-2 detection antibody surface enhanced Raman scattering immunoassay of receptor microspheres: Add the donor microspheres coupled with the capture antibody, the receptor microspheres coupled with the detection antibody and the sample to be tested to the surface enhanced Raman scattering (SERS) active substrate and incubate at 37°C in the dark for 15 minutes. Subsequently, add the Raman-labeled secondary antibody and continue incubation for 30 minutes. After washing, use a Raman spectrometer to detect the SERS signal, and determine the content of the detection index by the signal intensity.

[0051] Trypsinogen-2 capture antibody-trypsinogen-2 antigen-coupled receptor microspheres trypsinogen-2 detection antibody biosensor detection: The donor microspheres coupled with the capture antibody, the receptor microspheres coupled with the detection antibody and the sample to be tested are fixed on the surface of the biosensor and incubated at 37°C in the dark for 15 minutes. Subsequently, the biotin-labeled secondary antibody and the streptavidin-labeled enzyme are added and incubated for another 30 minutes. After washing, the enzyme substrate is added, and the reaction product of the substrate is detected by the biosensor to determine the content of the detection index.

[0052] Trypsinogen-2 capture antibody-trypsinogen-2 antigen-trypsinogen-2 detection antibody microfluidic chip detection of coupled receptor microspheres: In the microfluidic chip, the donor microspheres coupled with the capture antibody, the receptor microspheres coupled with the detection antibody and the sample to be tested are mixed according to the preset process and incubated at 37°C in the dark for 15 minutes. Subsequently, the HRP-labeled secondary antibody is introduced through the microfluidic channel and incubated for another 30 minutes. After washing, the substrate solution is added, and the detection index content is determined by detecting the fluorescence signal or chemiluminescence signal in the microfluidic chip.

[0053] Trypsinogen-2 capture antibody-trypsinogen-2 antigen-trypsinogen-2 detection antibody quantum dot immunoassay of coupled receptor microspheres: Add the donor microspheres coupled with the capture antibody, the receptor microspheres coupled with the detection antibody and the sample to be tested to the reaction system containing quantum dots, and incubate at 37°C in the dark for 15 minutes. Subsequently, add the secondary antibody that specifically recognizes the quantum dots and continue incubation for 30 minutes. After washing, the fluorescence signal of the quantum dots is detected by a fluorescence spectrometer to determine the content of the detection index.

[0054] Trypsinogen-2 capture antibody-trypsinogen-2 antigen-trypsinogen-2 detection antibody magnetic separation immunoassay of coupled receptor microspheres: Add the donor microspheres coupled with the capture antibody, the receptor microspheres coupled with the detection antibody and the sample to be tested to the reaction system containing magnetic particles, and incubate at 37°C in the dark for 15 minutes. Subsequently, add the magnetically labeled secondary antibody and continue incubation for 30 minutes. Wash using magnetic separation technology, then add the substrate solution, and determine the content of the detection index by detecting the fluorescence or color change of the magnetic particles.

[0055] Trypsinogen-2 capture antibody-trypsinogen-2 antigen-coupled receptor microspheres for trypsinogen-2 detection antibody microsphere array immunoassay: On the microsphere array platform, incubate the donor microspheres coupled with the capture antibody, the receptor microspheres coupled with the detection antibody, and the sample to be tested at 37°C in the dark for 15 minutes. Subsequently, add the fluorescently labeled secondary antibody and continue incubation for 30 minutes. After washing, use a microsphere array reader to detect the fluorescent signal on each microsphere, and determine the content of the detection index by the signal intensity.

[0056] Trypsinogen-2 capture antibody-trypsinogen-2 antigen-coupled receptor microspheres for trypsinogen-2 detection antibody immunochromatographic test: Apply the donor microspheres coupled with the capture antibody, the receptor microspheres coupled with the detection antibody, and the sample to be tested to the immunochromatographic test strips, and incubate at 37°C in the dark for 15 minutes. Subsequently, the sample moves along the test strip and binds to the secondary antibody fixed on the test strip. By observing the changes in the color bands on the test strips, the content of the test index can be determined qualitatively or semi-quantitatively.

[0057] Example 6: Linear range, sensitivity, specificity, precision and recovery of trypsinogen-2 detection.

[0058] Prepare trypsinogen-2 antigen standard, use this kit for detection, draw standard curve, use fluorescence value and concentration value to draw standard curve, the linear equation of trypsinogen-2 antigen detection is: Log10Y=1.2057×Log10X+2.4055, R 2 =0.99296, the linear range is 0.99ng / mL~300ng / mL, the results Figure 1 shown.

[0059] The fluorescence value of the 0 ng / mL reference standard was measured 10 times in parallel, and the mean (mean) and standard deviation (SD) were calculated. Mean+2SD was substituted into the standard curve equation, and the detection sensitivity of this kit for trypsinogen-2 was calculated to be 0.99 ng / mL.

[0060] The antigens of Tim-3 and CA242 were measured three times, and their concentrations were calculated by the standard curve. Cross-reaction rate (%) = detection concentration / actual concentration. The results are shown in Table 1. It was calculated that the kit had almost no cross-reaction rate for Tim-3 and CA242, and had good specificity.

[0061] Table 1 Specificity of trypsinogen-2 detection

[0062]

[0063] The low-value samples, medium-value samples and high-value samples of trypsinogen-2 were tested 10 times respectively. The results are shown in Table 2. The intra-batch coefficient of variation of trypsinogen-2 detection was 1.54%-2.20% (<10%), and the inter-batch coefficient of variation was 3.17%-6.94% (<15%).

[0064] Table 2 Trypsinogen-2 analytical precision

[0065]

[0066] Three trypsinogen-2 antigen standards (45, 150, and 300 ng / mL) were mixed with a serum sample of known concentration of 39.81 ng / mL in a ratio of 1:9. The AlphaLISA signal of the mixed solution was calculated using the standard curve to determine the actual concentration of the mixed solution. The serum and antigen standard solutions were mixed in proportion to prepare mixed solutions with theoretical concentrations of 40.33, 50.83, and 65.83 ng / mL. The recovery rate (%) was calculated as follows: Recovery rate (%) = actual concentration / theoretical concentration. The recovery rate was 96.23%-103.45%.

[0067] In summary, the present invention can detect the trypsinogen-2 antigen content in the sample to be tested, has high efficiency, strong specificity, high sensitivity, good stability, and rapid detection, can be used for AP diagnosis and screening, and has good application prospects.

[0068] Finally, it should be noted that the above disclosure is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments or to replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention. The scope of this application is limited only by the attached claims.

Claims

1. A method for detecting trypsinogen-2 based on homogeneous photochemiluminescence immunoassay technology, characterized in that: A solid phase carrier is provided, wherein the solid phase carrier is a donor microsphere coupled with a trypsinogen-2 capture antibody, the trypsinogen-2 antigen is covalently coupled to the surface of the donor microsphere in a labeling buffer, and the trypsinogen-2 detection antibody is coupled to the acceptor microsphere at the same time, and the donor microsphere coupled with the trypsinogen-2 capture antibody and antigen and the acceptor microsphere coupled with the trypsinogen-2 detection antibody are combined by a double antibody sandwich method to form a fluorescent immune complex of the donor microsphere coupled with the capture antibody-antigen-acceptor microsphere coupled with the detection antibody, wherein the antigen is the trypsinogen-2 in the sample to be tested, and singlet oxygen is generated by irradiating the photosensitizer on the donor microsphere with excitation light, and the luminescent agent on the acceptor microsphere receives the energy transfer of the singlet oxygen generated by the donor microsphere irradiated with the excitation light to generate fluorescence, and finally detection and analysis are performed according to the fluorescence intensity.

2. The method for detecting trypsinogen-2 based on homogeneous photochemiluminescence immunoassay according to claim 1, characterized in that: The detection method also includes the following steps: purifying the trypsinogen-2 capture antibody and the trypsinogen-2 detection antibody by a centrifugal filtration method, wherein the centrifugal filtration method is to use a centrifuge tube with a filter membrane to centrifuge and wash the capture antibody and the detection antibody multiple times, and use Tris-HCl washing solution to wash and separate to remove unbound antibodies.

3. The method for detecting trypsinogen-2 based on homogeneous photochemiluminescence immunoassay according to claim 1, characterized in that: The detection method further comprises the following steps: activating the donor microspheres and the acceptor microspheres using a cross-linking agent, wherein the cross-linking agent is N-hydroxysuccinimide (NHS) or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC).

4. The method for detecting trypsinogen-2 based on homogeneous photochemiluminescence immunoassay according to claim 1, characterized in that: The detection method further comprises the following steps: the double antibody sandwich method is to add optimized doses of donor microspheres coupled with capture antibodies, acceptor microspheres coupled with detection antibodies and the antigen to be detected into a polypropylene well plate for reaction to form a fluorescent immune complex.

5. The method for detecting trypsinogen-2 based on homogeneous photochemiluminescence immunoassay according to claim 1 or 4, characterized in that: The reaction conditions of the double antibody sandwich method in the detection method are shaking at 37° C. in the dark for 15-20 minutes.

6. A trypsinogen-2 detection method based on homogeneous photochemiluminescence immunoassay technology according to claims 1 to 5, characterized in that: The donor microspheres and the acceptor microspheres are respectively coated with the trypsinogen-2 capture antibody and the trypsinogen-2 detection antibody to form a conjugate, and the coating ratio is 1 mg microspheres labeled with 0.1 mg antibody.

7. The method for detecting trypsinogen-2 based on homogeneous photochemiluminescence immunoassay according to claim 1, characterized in that: The coupling conditions of the trypsinogen-2 capture antibody and the detection antibody to the donor microspheres and the acceptor microspheres were set to incubate at room temperature in the dark for 2 hours with shaking.

8. A trypsinogen-2 detection kit based on homogeneous photochemiluminescence immunoassay technology, comprising a microplate, a kit body and a fluorescence detector, characterized in that: The kit body comprises a trypsinogen-2 standard solution, donor microspheres coupled with a trypsinogen-2 capture antibody, and acceptor microspheres coupled with a trypsinogen-2 detection antibody. The kit body is also provided with an expandable polystyrene foam layer. The donor microspheres and the acceptor microspheres are silica microspheres.

9. The trypsinogen-2 detection kit based on homogeneous photochemiluminescence immunoassay technology according to claim 8, characterized in that: The kit body also includes auxiliary detection reagents, which include one or more of a labeling buffer, an analysis buffer, a blocking solution, a stop solution, a cross-linking agent, and a concentrated washing solution.

10. The trypsinogen-2 detection kit based on homogeneous photochemiluminescence immunoassay technology according to claim 9, characterized in that: The analysis buffer is an analysis buffer with a pH of 7.8, and the concentrated washing solution is a PBS buffer or a Tris-HCl washing solution.

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