A test strip for combined detection of heparin-binding protein (HBP) and procalcitonin (PCT) in peripheral blood

Abstract

The present invention discloses a test strip for the combined detection of heparin-binding protein (HBP) / procalcitonin (PCT) in peripheral blood, a preparation method thereof, and an application thereof. The test strip comprises a PVC base plate, a nitrocellulose membrane, a glass cellulose membrane conjugate pad, a sample pad, and a water-absorbing pad, wherein the PVC base plate is located at the bottom, and a sample pad, a glass cellulose membrane conjugate pad, a nitrocellulose membrane, and a water-absorbing pad are sequentially arranged above the PVC base plate; the nitrocellulose membrane has HBP, PCT detection lines, and a mouse anti-DNP monoclonal antibody quality control line; the glass nitrocellulose membrane conjugate pad has a fluorescent microsphere-labeled HBP antibody, PCT antibody, and a fluorescent microsphere-labeled DNP-BSA conjugate combination. The test strip utilizes the principle of a double antibody sandwich method combined with the characteristics of fluorescent microspheres to perform a fluorescent immunochromatography method, thereby achieving rapid, specific, simple, and sensitive combined detection of HBP and PCT.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a test strip for combined detection of heparin binding protein (HBP) and procalcitonin (PCT) in peripheral blood, a preparation method thereof and applications thereof. Background Art

[0002] Sepsis is a fatal organ dysfunction caused by a dysregulated host response to infection. It is a systemic inflammatory response syndrome (SIRS) resulting from an imbalance in the inflammatory and anti-inflammatory responses. Sepsis is categorized by severity into sepsis and septic shock. When an infection occurs, polymorphonuclear leukocytes migrate, accompanied by plasma extravasation. This worsens the infection, leading to SIRS and eventually to sepsis. Further development of sepsis, combined with severe circulatory, cellular, and metabolic disorders, can lead to septic shock and an increased risk of death.

[0003] Sepsis and septic shock remain major public health challenges in critical care medicine. Early diagnosis and treatment are key to improving the prognosis of patients with sepsis and septic shock. To more quickly and accurately diagnose and treat patients with sepsis or those at risk of developing sepsis, the American Society of Critical Care Medicine and the European Society of Intensive Care Medicine have released new diagnostic criteria for sepsis and septic shock. The Sequential Organ Failure Assessment (SOFA) and the Acute Physiology and Chronic Health Evaluation Scoring System (APACHE II) for sepsis diagnosis are the most commonly used organ failure scores in ICUs. However, existing microbiological culture and traditional infection indices cannot meet the clinical needs for timely, accurate, and rapid diagnosis due to time delays, low sensitivity, and lack of specificity. Early and accurate diagnosis of sepsis and septic shock remains challenging, and therefore, there is an urgent need for laboratory tests with high sensitivity and specificity to assist in diagnosis.

[0004] Chinese invention patent 201810318917.4 discloses a heparin-binding protein assay kit and method using an immunofluorescence dry-blot quantitative method. This invention uses an immunofluorescence dry-blot quantitative method to quantitatively determine heparin-binding protein in human plasma in vitro. This novel immunoassay kit overcomes the human error associated with EIA techniques (e.g., technique, temperature, time, and result interpretation) and objectively and quantitatively reflects the presence of HBP, providing a more robust experimental diagnostic basis for clinical diagnosis, efficacy observation, and prognosis assessment. It is widely recognized by physicians and patients. However, this invention uses an immunofluorescence dry-blot quantitative method to quantitatively determine heparin-binding protein in human plasma in vitro, objectively and quantitatively reflecting the presence of HBP; it cannot accurately measure the content of heparin-binding protein in whole blood or peripheral blood.

[0005] Chinese invention patent 201711415117.6 discloses a single-reagent heparin-binding protein detection kit and its preparation method. The invention discloses a single-reagent heparin-binding protein (HBP) detection kit and its preparation method. The kit is a single-reagent detection kit. The main components of the single-reagent detection kit are a reaction solution and a calibrator and quality control product containing HBP. The reaction solution includes latex particles labeled with HBP antibodies, as well as a buffer solution, a surfactant, salt, a stabilizer, a suspending agent, and a preservative. This invention detects the concentration of heparin-binding protein (HBP) in serum and plasma, and cannot accurately detect the content of HBP in peripheral blood.

[0006] The data in the aforementioned invention patents only describe the levels of inflammatory factors in serum and plasma. The relationship between inflammatory factors and disease, particularly whether a specific cutoff value is statistically significant for sepsis and septic shock at a certain level of significance, is not clarified. Furthermore, regarding the diagnostic value of combined inflammatory factor detection, the receiver operating characteristic (ROC) curve statistical method was not used to evaluate the diagnostic value of the HBP / PCT combined test for specific diseases. However, the clinical evaluation performance and diagnostic value of a product are the foundation of medical research.

[0007] Previous studies on HBP / PCT have shown that HBP testing alone cannot distinguish between localized and systemic infections. HBP is more sensitive and provides early warning, compensating for the PCT blank period (2 hours) and peaking within 12-24 hours. HBP can rise even before clinical symptoms appear, peaking within 1 hour, during the PCT blank period. Therefore, the necessity of combined testing of the two is extremely important. Furthermore, current tests for heparin-binding protein and procalcitonin mostly use serum and plasma samples, with few reports on measuring their levels in peripheral blood. As biomarkers for assessing sepsis and septic shock, there remains a pressing clinical need for diagnosing sepsis and septic shock early, with high specificity and sensitivity, to facilitate targeted clinical treatment.

[0008] Based on market and clinical needs, there is an urgent need for a detection reagent for heparin-binding protein and procalcitonin in peripheral blood, which can quickly and accurately predict whether a patient has sepsis and determine the severity of sepsis. Summary of the Invention

[0009] The object of the present invention is to provide a test strip suitable for HBP / PCT combined detection of peripheral blood, a preparation method and application thereof, thereby solving the above-mentioned problems existing in the prior art.

[0010] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0011] In one aspect, the present invention provides a test strip suitable for the combined detection of HBP / PCT in peripheral blood, comprising a PVC base plate, a nitrocellulose membrane (NC membrane), a glass cellulose membrane conjugate pad, a sample pad, and a water absorbent pad, wherein the PVC base plate is located at the bottom, and the sample pad, glass cellulose membrane conjugate pad, nitrocellulose membrane, and water absorbent pad are sequentially arranged above the PVC base plate; the nitrocellulose membrane has HBP and PCT detection lines and a mouse anti-DNP monoclonal antibody quality control line; the nitrocellulose membrane conjugate pad has an HBP antibody and a PCT antibody labeled with fluorescent microspheres, and a DNP-BSA (2,4-dinitrophenyl-albumin) conjugate combination labeled with fluorescent microspheres.

[0012] Preferably, the PVC bottom plate is a long strip structure.

[0013] In some embodiments, the top end of the sample pad presses and sticks to the bottom end of the glass cellulose membrane conjugate pad, the top end of the glass cellulose membrane conjugate pad presses and sticks to the bottom end of the nitrocellulose membrane, and the top end of the nitrocellulose membrane is pressed and stuck to the bottom end of the absorbent pad.

[0014] In one aspect, the present invention provides a method for preparing the above-mentioned test strip, comprising the following steps:

[0015] S1: Preparation of fluorescent microsphere-labeled HBP antibody

[0016] 1. Add 10 μL of 200 nm fluorescent microspheres to 90 μL of 0.05 M MES buffer at pH 7.5, then add 10 μL of 5 mM EDC solution and react on a silent mixer for 30 min.

[0017] 2. Centrifuge at 14,000 rcf for 20 min, discard the supernatant, and resuspend in 100 μL MES buffer.

[0018] 3. Add 100 μL of 0.5 mg / mL HBP monoclonal antibody that has been dialyzed against 10 mM PBS and place on a silent mixer for reaction for 1 hour.

[0019] 4. Centrifuge at 14,000 rcf for 20 min, discard the supernatant, resuspend with 200 μL 1% BSA, and block in a 4°C refrigerator overnight.

[0020] 5. Centrifuge at 14000 rcf for 20 min, discard the supernatant, resuspend with 100 μL fluorescent microsphere storage solution, and store in a 4°C refrigerator for later use.

[0021] S2: Prepare fluorescent microsphere-labeled PCT antibody and DNP-BSA conjugate respectively according to the same method as step S1;

[0022] S3: Preparation of conjugate pad containing antibody mixture

[0023] 1. Cut the glass cellulose membrane into 9mm width for later use.

[0024] 2. Prepare a conjugate pad blocking solution containing 20% ​​trehalose, 10% BSA, and TE buffer. Soak a 9 mm wide glass cellulose membrane in the conjugate pad blocking solution for 1 minute, and then dry it at 37°C for 12 hours to obtain a blocked conjugate pad.

[0025] 3. Mix the fluorescent microsphere-labeled HBP antibody and fluorescent microsphere-labeled PCT antibody with the fluorescent microsphere-labeled DNP-BSA conjugate and microsphere diluent at a mass ratio of 3:1:6, respectively. Spray them onto the sealed conjugate pad using a film sprayer at a spray volume of 4 μL / cm, a length of 300 mm, and a speed of 50 cm / min. Dry at 37°C for 12 h to obtain the conjugate pad containing the antibody mixture for use.

[0026] S4: Preparation of nitrocellulose membrane

[0027] 1. Stick the Millipore nitrocellulose chromatography membrane on the polyvinyl chloride polymer (PVC) bottom plate.

[0028] 2. Use stripping solution to dilute the capture antibody for the test line and the DNP mouse monoclonal antibody for the quality control line to 1.5 mg / mL and 0.8 mg / mL, respectively. Use a stripping sprayer to stripe the capture antibody and DNP mouse monoclonal antibody onto the NC membrane at a rate of 50 cm / min over a length of 300 mm and a spray rate of 1 μL / cm. Bake at 60°C for 12 hours before use.

[0029] S5: Following the same method as in step S4, the following capture antibodies, HBP antibody, PCT antibody and DNP mouse monoclonal antibody, are coated in sequence, and sprayed onto the corresponding test line and quality control line respectively. After drying, the coated nitrocellulose membrane substrate is obtained.

[0030] S6: Prepare the sample pad

[0031] 1. Cut the glass cellulose membrane into 15mm width for later use.

[0032] 2. Prepare a sample pad blocking solution containing 20% ​​trehalose, 10% BSA, and TE buffer. Soak a 15 mm wide glass cellulose membrane in the sample pad blocking solution for 1 minute. Dry it at 37°C for 12 hours to obtain a blocked sample pad for use.

[0033] S7: Prepare a liquid absorption pad: Dry a 20 mm absorbent paper at 60° C. for 2 hours to obtain a liquid absorption pad for use.

[0034] S8: Test Card Lamination

[0035] 1. Separately laminate the coated nitrocellulose membrane base, the dried liquid absorption pad, the sealed sample pad, and the conjugate pad containing the antibody mixture.

[0036] 2. At the lamination position, the liquid absorption pad should be 1mm away from the edge of the coated nitrocellulose membrane base isolation paper, and the overlapping distance between the liquid absorption pad and the nitrocellulose membrane base should not exceed 2±0.5mm; the binding pad should be facing up and close to the upper edge of the sample pad isolation paper, and the overlapping distance between the binding pad and the nitrocellulose membrane should not exceed 2±0.5mm; the sample pad should be close to the lower edge of the sample pad isolation paper.

[0037] S9: Assemble the test strips using the same cartridge: overlap the components of the test strips in the chromatographic direction, cut them into 4mm wide strips, install the two test strips into the corresponding positions of the customized cartridge, and close the upper cover to obtain the duplex test strips.

[0038] S10: Repeat the measurement five times for each concentration point of the calibrator. Use the measured fluorescence signal value (T / C) as the Y-axis and the concentration value as the X-axis. Fit the scatter point of the logistic curve (four parameters) to develop a standard curve for the determination of sample concentration.

[0039] In some embodiments, in step S1, 200 nm fluorescent microspheres are used as labeling substances, pH 7.5 0.05 M MES solution is used as fluorescent microsphere labeling buffer, and the volume ratio of microspheres to MES buffer is 1:10; 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) is used as the activator; the activation time is 30 min, and the order of adding EDC and the antibody is a two-step labeling method (i.e., adding EDC to activate the surface carboxyl groups, centrifuging to remove the supernatant, re-dissolving and then connecting the antibody) of the fluorescent microspheres.

[0040] In some embodiments, in step S1, the reaction time of the activated microspheres and the dialyzed monoclonal antibody is 1 hour. After the reaction, the microspheres are centrifuged at 14,000 rcf for 20 minutes, the supernatant is discarded, and the microspheres are resuspended in 200 μL of 1% BSA and sealed in a refrigerator at 4°C overnight. The microspheres are then centrifuged at 14,000 rpm for 20 minutes, and the precipitated particles are redissolved in the dispersion to obtain fluorescent microsphere-labeled antibodies.

[0041] In some embodiments, the conjugate pad containing the antibody mixture in step S3 is made of a 9 mm glass cellulose membrane, and the conjugate pad blocking solution comprises 20% trehalose, 10% BSA, and TE buffer. Fluorescent microsphere-labeled HBP antibody, fluorescent microsphere-labeled PCT antibody, fluorescent microsphere-labeled DNP-BSA conjugate, and microsphere diluent are mixed at a mass ratio of 3:1:6 and then sprayed onto the blocked conjugate pad using a streaking gold sprayer at a spray rate of 4 μL / cm, a length of 300 mm, and a speed of 50 cm / min. Drying is performed at 37°C for 12 h.

[0042] In some embodiments, step S4 comprises spraying HBP antibody, PCT antibody, and DNP mouse monoclonal antibody onto the corresponding test line and quality control line, respectively, which specifically comprises: taking HBP antibody, PCT antibody, and DNP mouse monoclonal antibody at a concentration of 1.5 mg / ml, respectively, and drawing them onto the test line and quality control line of the nitrocellulose membrane at a speed of 1 μL / cm, a length of 300 mm, and a speed of 50 cm / min; and the parameters are baking at 60° C. for 12 h.

[0043] In some embodiments, the glass cellulose membrane used for the sample pad in step S6 is 15 mm, and the sample pad blocking solution comprises 20% trehalose, 10% BSA, and TE buffer. The drying process is performed at 37° C. for 12 hours.

[0044] It should be noted that, in the above-mentioned test strips, the same sample and the same test strip can be used to simultaneously detect both HBP and PCT.

[0045] Preferably, during testing, the test strip only needs to add 15 μL of peripheral blood and 100 μL of diluent to the sample injection port at one time. After the test strip is chromatographed for 15 minutes, it can be matched with a specific immunoassay analyzer and the results can be read.

[0046] In one aspect, the present invention provides use of a test strip in preparing a detection kit for detecting heparin binding protein (HBP) and procalcitonin (PCT) in a patient's peripheral blood.

[0047] In some embodiments, the detection kit includes the test strip, buffer, desiccant, instructions for use, etc.

[0048] In some embodiments, the buffer is phosphate buffer, such as 10 mM phosphate buffer.

[0049] In some embodiments, the desiccant is silica gel.

[0050] In some embodiments, the patient has sepsis or septic shock.

[0051] The beneficial effects of the present invention include at least the following:

[0052] The test strips use fluorescent immunochromatography technology, combining immunofluorescence with immunochromatography technology, and are established by utilizing antigen-antibody specific reactions. Compared with colloidal gold test paper, fluorescent test paper has stronger anti-interference performance for samples, higher detection sensitivity, and better result repeatability. On the other hand, compared with other detection technologies such as gas chromatography, liquid chromatography, gas chromatography-mass spectrometry and other methods, the combined immunofluorescence chromatography test paper not only solves the problem that large instruments cannot be used for on-site and immediate detection, but also saves detection time and funding costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] In order to more clearly illustrate the test strip of the present invention, its preparation and use, the corresponding drawings are provided. The following is a brief introduction to the drawings:

[0054] Figure 1 It is a side structural schematic diagram of the fluorescent immunoassay paper of the present invention.

[0055] Figure 2 It is a schematic diagram of the internal structure of the assembled fluorescence detection card of the present invention.

[0056] Figure 3 It is a schematic diagram of the external structure of the assembled fluorescent dual card of the present invention.

[0057] Figure 4It is a diagram of the preparation process of the product of the present invention.

[0058] Figure 5 This is a ROC curve used to evaluate the ability of combined detection of peripheral blood HBP and PCT to diagnose sepsis.

[0059] Figure 6 This is the ROC curve used to evaluate the diagnostic value of combined detection of peripheral blood HBP and PCT for sepsis. DETAILED DESCRIPTION

[0060] The terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the present invention. Unless the context clearly indicates otherwise, the singular forms "a," "an," and "the" used herein are intended to include the plural forms. In addition, the open-ended expressions "comprise" and "include" are interpreted as also including unmentioned structural components or method steps, but it should be noted that such open-ended expressions also cover situations consisting only of the described components and method steps (i.e., covering situations where the closed-ended expression "consisting of..." is used).

[0061] As used in full, range is used as a shorthand form for describing each numerical value and all numerical values ​​within the range. Any numerical value within the range, such as an integer value, a value that increases progressively by one tenth (when the end value of the range is one decimal place), or a value that increases progressively by one hundredth (when the end value of the range is two decimal places) can be selected as the endpoint of the range. For example, the range 0.1-10 is used as describing all numerical values ​​within the range, such as 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 ... 9.5, 9.6, 9.7, 9.8, 9.9 and 10 (values ​​that increase progressively by one tenth), and includes all subranges, such as 0.1-1.0, 2.0-3.0, 4.0-5.0, 6.0-7.0, 8.0-9.0, etc.

[0062] All scientific and technical terms mentioned in this specification have the same meanings as those commonly understood by those skilled in the art. In the event of a conflict, the definitions in this specification shall prevail. In order to make the description of the present invention easier to understand, some terms are explained below.

[0063] As used herein, "fluorescence" refers to a substance that absorbs light of a certain wavelength and simultaneously emits light of a different wavelength. When a substance is exposed to incident light of a certain wavelength, it emits visible light of varying wavelengths and intensities. When the excitation light ceases, the luminescence quickly disappears. This emitted light is known as fluorescence. Fluorescence is a widely used signal-tracing reagent. Most of these compounds are benzene or heterocyclic compounds containing conjugated double bonds. These compounds can be used alone or in combination to form complex fluorescent dyes.

[0064] As used herein, "fluorescent microspheres (FM)" refer to microspheres with fluorescent substances on their surface (including surface coatings) or in their internal structure (embedded or aggregated), which can emit fluorescence when excited by a certain amount of energy. FMs are functional microspheres loaded with fluorescent molecules, made of materials such as silica or polyethylene. They are generally spherical with diameters ranging from nanometers to micrometers (0.01 to 10 μm), but can also be any shape. As a special type of functional microsphere, FMs have important applications in many fields, especially biomedicine, due to their stable morphology, narrow particle size distribution, good monodispersity, and high luminescence efficiency. They are now widely used in immunochromatography technology.

[0065] As used herein, "fluorescent microspheres labeled with antibodies" refers to a biomarker technology that specifically binds the target antibody to the fluorescent microspheres by washing the microspheres, activating the microspheres, and other steps. The antibody can be covalently bound to the active groups on the surface of the microspheres, thereby achieving the coupling of the microspheres and the antibody for use in immunochromatography application research. It refers to polymer microspheres that can emit fluorescence by introducing the target fluorescent material into an organic or inorganic matrix through a certain chemical or physical method. Add a specific mass of EDC and NHS to the fluorescent microspheres and stir at room temperature for a specific time to activate the microspheres; then add a specific amount of monoclonal antibody and stir at room temperature for a specific time to couple the microspheres with the antibody; further add a specific mass of BSA blocking solution and continue stirring to achieve the blocking of the microsphere coupling complex; centrifuge at a high speed at low temperature and remove the supernatant; finally, use a specific buffer solution to redissolve the solid precipitate to a specific volume to achieve the labeling of the fluorescent microspheres on the antibody.

[0066] As used herein, "heparin-binding protein" (HBP), also known as azurocidin or CAP37, originates from neutrophils and is primarily stored in azurocidin granules, with a smaller portion stored in secretory vesicles. HBP is a member of the serine protease family and lacks protease activity. It has a high affinity for lipopolysaccharide lipid A. Upon activation, neutrophils degranulate, releasing HBP. In acute bacterial infections, HBP concentrations in the blood can rise significantly within 1-2 hours, whereas in viral infections, HBP levels remain unchanged or increase only slightly. HBP also acts as a pathogenic factor, and HBP concentrations rapidly decrease after patients receive effective treatment. Therefore, measuring HBP levels in a patient's blood can assist in the diagnosis and prediction of acute bacterial infections, assessment of infection severity, and monitoring of antibiotic efficacy.

[0067] As used herein, "procalcitonin" (PCT) is the inactive precursor of calcitonin (CT). It is a glycoprotein composed of 116 amino acids with a molecular weight of 13,000. PCT has a half-life of 25 to 30 hours and is very stable in vivo and in vitro. Elevated PCT levels are closely associated with bacterial infection. PCT levels can be elevated early in severe systemic infections and decrease after infection control with antibiotic treatment. In patients with viral infections and localized bacterial infections without systemic manifestations, PCT levels are only mildly elevated. Therefore, PCT has become an important new indicator for observation in severe systemic infections or sepsis.

[0068] Although various embodiments of the present invention have been described above, it should be understood that these are provided by way of example only and not limitation. Many variations of the disclosed embodiments may be made in accordance with the disclosure herein without departing from the spirit or scope of the present invention. Therefore, the breadth and scope of the present invention should not be limited by any of the above-described embodiments.

[0069] All documents mentioned herein are incorporated herein by reference.All publications and patent documents cited in this application are incorporated by reference for all purposes to the same extent as if each individual publication or patent document were individually denoted.

[0070] Example 1

[0071] Preparation of Heparin Binding Protein (HBP) / Procalcitonin (PCT) Detection Kit (Immunofluorescence Chromatography)

[0072] S1: Preparation of fluorescent microsphere-labeled HBP antibody

[0073] 1. Add 10 μL of 200 nm fluorescent microspheres (source: Merck Chemicals, Germany, the specific name of the microspheres is Estapor Fluorescent Carboxylated microspheres) to 90 μL of 50 mM MES buffer at pH 7.5, then add 10 μL of 5 mM EDC solution and react on a silent mixer for 30 min.

[0074] 2. Centrifuge at 14,000 rcf for 20 min, discard the supernatant, and resuspend in 100 μL MES buffer.

[0075] 3. Add 100 μL of 0.5 mg / mL HBP monoclonal antibody that has been dialyzed against 10 mM PBS and place on a silent mixer for reaction for 1 hour.

[0076] 4. Centrifuge at 14,000 rcf for 20 min, discard the supernatant, resuspend in 200 μL 1% BSA, and block in a 4°C refrigerator overnight.

[0077] 5. Centrifuge at 14000 rcf for 20 min, discard the supernatant, resuspend with 100 μL fluorescent microsphere storage solution, and store in a 4°C refrigerator for later use.

[0078] S2: Prepare fluorescent microsphere-labeled PCT antibody and DNP-BSA conjugate respectively according to the same method as step S1;

[0079] S3: Preparation of conjugate pad containing antibody mixture

[0080] 1. Cut the glass cellulose membrane into 9mm width for later use.

[0081] 2. Prepare a conjugate pad blocking solution containing 20% ​​trehalose, 10% BSA, and TE buffer. Soak a 9 mm wide glass cellulose membrane in the conjugate pad blocking solution for 1 minute, and then dry it at 37°C for 12 hours to obtain a blocked conjugate pad.

[0082] 3. Mix the fluorescent microsphere-labeled HBP antibody and fluorescent microsphere-labeled PCT antibody with the fluorescent microsphere-labeled DNP-BSA conjugate and microsphere diluent at a mass ratio of 3:1:6, respectively. Spray them onto the sealed conjugate pad using a film sprayer at a spray volume of 4 μL / cm, a length of 300 mm, and a speed of 50 cm / min. Dry at 37°C for 12 h to obtain the conjugate pad containing the antibody mixture for use.

[0083] S4: Preparation of nitrocellulose membrane

[0084] 1. Stick the Millipore nitrocellulose chromatography membrane on the polyvinyl chloride polymer (PVC) bottom plate.

[0085] 2. Use stripping solution to dilute the capture antibody for the test line and the DNP mouse monoclonal antibody for the quality control line to 1.5 mg / mL and 0.8 mg / mL, respectively. Use a stripping sprayer to stripe the capture antibody and DNP mouse monoclonal antibody onto the NC membrane at a rate of 50 cm / min over a length of 300 mm and a spray rate of 1 μL / cm. Bake at 60°C for 12 hours before use.

[0086] S5: Following the same method as in step S4, the following capture antibodies, HBP antibody, PCT antibody and DNP mouse monoclonal antibody, are coated in sequence, and sprayed onto the corresponding test line and quality control line respectively. After drying, the coated nitrocellulose membrane substrate is obtained.

[0087] S6: Prepare the sample pad

[0088] 1. Cut the glass cellulose membrane into 15mm width for later use.

[0089] 2. Prepare a sample pad blocking solution containing 20% ​​trehalose, 10% BSA, and TE buffer. Soak a 15 mm wide glass cellulose membrane in the sample pad blocking solution for 1 minute. Dry it at 37°C for 12 hours to obtain a blocked sample pad for use.

[0090] S7: Prepare a liquid absorption pad: Dry a 20 mm absorbent paper at 60° C. for 2 hours to obtain a liquid absorption pad for use.

[0091] S8: Test Card Lamination

[0092] 1. Separately laminate the coated nitrocellulose membrane base, the dried liquid absorption pad, the sealed sample pad, and the conjugate pad containing the antibody mixture.

[0093] 2. At the lamination position, the liquid absorption pad should be 1mm away from the edge of the coated nitrocellulose membrane base isolation paper, and the overlapping distance between the liquid absorption pad and the nitrocellulose membrane base should not exceed 2±0.5mm; the binding pad should be facing up and close to the upper edge of the sample pad isolation paper, and the overlapping distance between the binding pad and the nitrocellulose membrane should not exceed 2±0.5mm; the sample pad should be close to the lower edge of the sample pad isolation paper.

[0094] S9: Assemble the test strips using the same cartridge: overlap the components of the test strips in the chromatographic direction, cut them into 4mm wide strips, install them into the corresponding positions of the customized cartridge, and close the upper cover to obtain the duplex test strips.

[0095] Example 2

[0096] Clinical value of the product

[0097] For specific clinical trial methods not listed, please refer to relevant national standards and common industry practices and will not be elaborated here.

[0098] 1. Test products

[0099] The heparin binding protein (HBP) / procalcitonin (PCT) detection kit (immunofluorescence chromatography) was prepared according to Example 1.

[0100] 2. Selection of trial participants

[0101] From January 1, 2023 to September 30, 2023, 200 patients aged 18 years and above were admitted to the ICU of Linfen People's Hospital, including 20 ~Patients aged 83 years were divided into two groups based on whether they developed sepsis and shock according to the sepsis diagnostic criteria: a sepsis-without-shock group (n=100) and a septic shock group (n=100). Another 100 healthy subjects were selected as the control group.

[0102] 3. Sample Collection

[0103] Peripheral blood was collected. After the specimen collection was completed, the HBP / PCT levels were detected using a heparin-binding protein (HBP) / procalcitonin (PCT) detection kit (immunofluorescence chromatography) and the test results were recorded.

[0104] 4. Statistical Processing

[0105] SPSS 25.0 statistical software was used for analysis. Continuous variable data were expressed as mean ± standard deviation or median (interquartile range), and categorical variable data were expressed as frequency. The t-test was used for group design data, and the chi-square test was used for count data. 2 Binary logistic regression was used to calculate the probability of diagnosing sepsis using combined biomarker testing. Receiver operating characteristic (ROC) curves were constructed, and the area under the curve (AUC) was calculated to evaluate the diagnostic value of each marker and combined testing. The chi-square test was used to compare percentages; P < 0.05 was considered statistically significant.

[0106] 5. Test Results

[0107] 1. General Information

[0108] The study enrolled 300 subjects, including 100 healthy subjects, 100 sepsis subjects, and 100 septic shock subjects. The test results are shown in Tables 1 and 2 below.

[0109] Table 1: Test results for HBP (unit: ng / mL)

[0110]

[0111]

[0112]

[0113] Table 2: PCT test results (unit: ng / mL)

[0114]

[0115]

[0116]

[0117] 2. There was no statistically significant difference in the demographic characteristics of the subjects in terms of gender, age, and body mass index (P>0.05), see Table 3.

[0118] Table 3: Gender, age and body mass index distribution characteristics of subjects in each group ( ±s)

[0119]

[0120] 3. Comparison of peripheral blood levels between the healthy control group, sepsis group, and septic shock group

[0121] Compared with the healthy control group, the peripheral blood levels of the sepsis group and the septic shock group were significantly increased, and the differences were statistically significant (P<0.05).

[0122] Table 4: Comparison of peripheral blood levels between the healthy control group, sepsis group, and septic shock group

[0123]

[0124]

[0125] Note: Compared with the sepsis group, a P<0.05; compared with the septic shock group, b P<0.05.

[0126] 4. ROC curve analysis results of various indicators

[0127] To evaluate the diagnostic value of peripheral blood HBP and PCT in the diagnosis of sepsis, we performed ROC curve analysis and evaluated the diagnostic ability based on the area under the curve (AUC). The performance parameters of each indicator are detailed in Table 5 and Figure 5 As shown in the table below, we can see that the AUCs for HBP and PCT are both greater than 0.7, indicating that these indicators have relatively good diagnostic efficacy for diagnosing sepsis. However, the highest sensitivity of PCT is only 78%, which seriously limits its application in the diagnosis of sepsis and septic shock.

[0128] Table 5: ROC curve analysis results of various indicators

[0129]

[0130] 5. The diagnostic value of combined detection of HBP and PCT in sepsis

[0131] In a diagnostic system, the optimal diagnostic cutoff is achieved by simultaneously maximizing sensitivity and specificity, i.e., by maximizing the Youden index (sensitivity + specificity - 1) or the positive likelihood ratio. Currently, the generally accepted ideal diagnostic test area under the curve (AUC) is 1, with diagnostic value higher when it is above 0.9. Because the low sensitivity of single-test products limits the positive detection rate in diagnosis, we are using combined testing to improve the clinical detection rate. We performed receiver operating characteristic (ROC) curve analysis to evaluate the diagnostic performance of different combined tests based on the area under the curve (AUC). The performance parameters for each indicator are shown in Table 6 below. The AUC significantly increased with combined testing. The sensitivity of HBP / PCT (combinations 1 and 2) was 83%, the specificity was 98%, and the Youden index of the combined test was 0.81, which is higher than that of HBP or PCT alone. By testing HBP / PCT in peripheral blood, combined testing can improve the diagnostic value of sepsis.

[0132] Table 6: Detection performance of various indicators for combined HBP / PCT detection

[0133]

[0134] Although various embodiments of the present invention have been described above, it should be understood that they are provided by way of example only and not limitation. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention, and such changes and modifications will fall within the scope of the present invention as claimed. The scope of the present invention as claimed is defined by the appended claims and their equivalents.

Claims

1. A test strip for the combined detection of heparin-binding protein (HBP) and procalcitonin (PCT) in peripheral blood, comprising a PVC base, a nitrocellulose membrane, a glass cellulose membrane conjugate pad, a sample pad, and an absorbent pad, wherein the PVC base is located at the bottom, and the sample pad, glass cellulose membrane conjugate pad, nitrocellulose membrane, and absorbent pad are sequentially disposed above the PVC base; the nitrocellulose membrane has HBP and PCT detection lines and a mouse anti-DNP monoclonal antibody quality control line; the nitrocellulose membrane conjugate pad has fluorescent microsphere-labeled HBP antibodies, PCT antibodies, and a fluorescent microsphere-labeled DNP-BSA conjugate combination; and the fluorescent microspheres are Estapor Fluorescent Carboxylated microspheres. The test strip is prepared by a method comprising the following steps: Step S1: Preparation of fluorescent microsphere-labeled HBP antibody (1) After adding 10 μL of 200 nm fluorescent microspheres to 90 μL of MES buffer (pH 7.5, 0.05 M), add 10 μL of 5 mM EDC solution and react on a silent mixer for 30 min; (2) Centrifuge at 14,000 rcf for 20 min, discard the supernatant, and resuspend in 100 μL MES buffer; (3) Add 100 μL of 0.5 mg / mL HBP monoclonal antibody that has been dialyzed against 10 mM PBS and place on a silent mixer for 1 h; (4): Centrifuge at 14000 rcf for 20 min, discard the supernatant, resuspend in 200 μL 1% BSA, and block in a 4°C refrigerator overnight; (5): Centrifuge at 14000 rcf for 20 min, discard the supernatant, resuspend with 100 μL fluorescent microsphere storage solution and store in a 4°C refrigerator for later use. S2: Prepare fluorescent microsphere-labeled PCT antibody and DNP-BSA conjugate respectively according to the same method as step S1, and S3: Preparation of glass cellulose membrane binding pad containing antibody mixture (1): Cut the glass cellulose membrane into 9 mm width for later use; (2) Prepare a conjugate pad blocking solution containing 20% ​​trehalose, 10% BSA, and TE buffer. Soak a 9 mm wide glass fiber membrane in the conjugate pad blocking solution for 1 minute, and then dry it at 37°C for 12 hours to obtain a blocked conjugate pad. (3): After the fluorescent microsphere-labeled HBP antibody and fluorescent microsphere-labeled PCT antibody were mixed with the fluorescent microsphere-labeled DNP-BSA conjugate and microsphere diluent at a mass ratio of 3:1:6, they were sprayed onto the sealed conjugate pad using a film sprayer at a spray volume of 4 μL / cm, a length of 300 mm, and a speed of 50 cm / min. After drying at 37°C for 12 h, the conjugate pad containing the antibody mixture was obtained for use.

2. The test strip according to claim 1, wherein the top of the sample pad presses and sticks to the bottom of the glass cellulose membrane conjugate pad, the top of the glass cellulose membrane conjugate pad presses and sticks to the bottom of the nitrocellulose membrane, and the top of the nitrocellulose membrane is pressed and stuck to the bottom of the absorbent pad.

3. The test strip according to claim 1, wherein the same sample and the same test strip are used to detect both HBP and PCT.

4. A method for preparing the test strip according to claim 1, comprising the following steps: Step S1: Preparation of fluorescent microsphere-labeled HBP antibody (1) After adding 10 μL of 200 nm fluorescent microspheres to 90 μL of MES buffer (pH 7.5, 0.05 M), add 10 μL of 5 mM EDC solution and react on a silent mixer for 30 min; (2) Centrifuge at 14,000 rcf for 20 min, discard the supernatant, and resuspend in 100 μL MES buffer; (3) Add 100 μL of 0.5 mg / mL HBP monoclonal antibody that has been dialyzed against 10 mM PBS and place on a silent mixer for 1 h; (4): Centrifuge at 14000 rcf for 20 min, discard the supernatant, resuspend in 200 μL 1% BSA, and block in a 4°C refrigerator overnight; (5): Centrifuge at 14000 rcf for 20 min, discard the supernatant, resuspend with 100 μL fluorescent microsphere storage solution and store in a 4°C refrigerator for later use. S2: Prepare fluorescent microsphere-labeled PCT antibody and DNP-BSA conjugate respectively according to the same method as step S1. S3: Preparation of glass cellulose membrane binding pad containing antibody mixture (1): Cut the glass cellulose membrane into 9 mm width for later use; (2) Prepare a conjugate pad blocking solution containing 20% ​​trehalose, 10% BSA, and TE buffer. Soak a 9 mm wide glass fiber membrane in the conjugate pad blocking solution for 1 minute, and then dry it at 37°C for 12 hours to obtain a blocked conjugate pad. (3): After the fluorescent microsphere-labeled HBP antibody and fluorescent microsphere-labeled PCT antibody were mixed with the fluorescent microsphere-labeled DNP-BSA conjugate and microsphere diluent at a mass ratio of 3:1:6, they were sprayed onto the sealed conjugate pad using a film sprayer at a spray volume of 4 μL / cm, a length of 300 mm, and a speed of 50 cm / min. After drying at 37°C for 12 h, the conjugate pad containing the antibody mixture was obtained for use. S4: Preparation of nitrocellulose membrane (1): Attach the Millipore nitrocellulose chromatography membrane to the PVC base plate; (2): Use film stripping solution to dilute the detection line capture antibody and the quality control line DNP mouse monoclonal antibody to 1.5 mg / mL and 0.8 mg / mL respectively; use a film stripping gold sprayer to spray the capture antibody and DNP mouse monoclonal antibody onto the NC film at a spraying amount of 1 μL / cm, a length of 300 mm, and a speed of 50 cm / min, and bake at 60℃ for 12 hours. S5: Following the same method as in step S4, HBP antibody, PCT antibody and DNP mouse monoclonal antibody were coated in sequence and sprayed onto the corresponding test line and quality control line respectively. After drying, the coated nitrocellulose membrane substrate was obtained. S6: Prepare the sample pad (1): Cut the glass cellulose membrane into 15 mm width for later use; (2): Prepare a sample pad blocking solution containing 20% ​​trehalose, 10% BSA, and TE buffer. Soak a 15 mm wide glass fiber membrane in the sample pad blocking solution for 1 minute, and then dry it at 37°C for 12 hours to obtain a blocked sample pad for use. S7: Prepare liquid absorption pad: Dry 20mm absorbent paper at 60℃ for 2 hours to obtain liquid absorption pad for use. S8: Test Card Lamination (1) Separately laminating the coated nitrocellulose membrane base, the dried liquid absorption pad, the sealed sample pad, and the conjugate pad containing the antibody mixture; (2): The liquid absorption pad at the lamination position is 1mm away from the edge of the coated nitrocellulose membrane base paper, and the overlapping distance between the liquid absorption pad and the nitrocellulose membrane base does not exceed 2±0.5mm; the binding pad is facing up and close to the upper edge of the sample pad isolation paper, and the overlapping distance between the binding pad and the nitrocellulose membrane should not exceed 2±0.5mm; the sample pad is close to the lower edge of the sample pad isolation paper, and S9: Assemble the test strips using the same cartridge: overlap the components of the test strips in the chromatographic direction, cut them into 4mm wide strips, install the two test strips into the corresponding positions of the customized cartridge, and close the upper cover to obtain the duplex test strips.

5. The method according to claim 4, characterized in that In step S1, 200 nm fluorescent microspheres are used as labeling substances, and MES solution at pH 7.5 and 0.05 M is used as fluorescent microsphere labeling buffer. The volume ratio of microspheres to MES buffer is 1:

10. The activator is 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC). The activation time is 30 min. The order of adding EDC and antibody is selected from the two-step labeling method for fluorescent microspheres, and / or In step S1, the activated microspheres and the dialyzed monoclonal antibody react for 1 hour. After the reaction, the microspheres are centrifuged at 14,000 rcf for 20 minutes, the supernatant is discarded, and the microspheres are resuspended in 200 μL of 1% BSA and sealed in a refrigerator at 4°C overnight. The microspheres are then centrifuged at 14,000 rpm for 20 minutes, and the precipitated particles are redissolved in the dispersion to obtain fluorescent microsphere-labeled antibodies.

6. The method according to claim 4, characterized in that In step S3, the conjugate pad containing the antibody mixture used a 9 mm glass cellulose membrane, and the conjugate pad blocking solution consisted of 20% trehalose, 10% BSA, and TE buffer. Fluorescent microsphere-labeled HBP antibody, fluorescent microsphere-labeled PCT antibody, fluorescent microsphere-labeled DNP-BSA conjugate, and microsphere diluent were mixed in a mass ratio of 3:1:6 and sprayed onto the blocked conjugate pad using a streaking gold sprayer at a spray volume of 4 μL / cm, a length of 300 mm, and a speed of 50 cm / min. The mixture was dried at 37°C for 12 h.

7. The method according to claim 4, characterized in that In step S4, HBP antibody, PCT antibody, and DNP mouse monoclonal antibody are respectively sprayed onto the corresponding test line and quality control line, specifically comprising: taking HBP antibody, PCT antibody, and DNP mouse monoclonal antibody at a concentration of 1.5 mg / ml, respectively, and drawing them onto the test line and quality control line of the nitrocellulose membrane at a speed of 1 μL / cm, a length of 300 mm, and a speed of 50 cm / min; the parameters are drying at 60°C for 12 hours.

8. The method according to claim 4, characterized in that The glass cellulose membrane used for the sample pad in step S6 was 15 mm, and the sample pad blocking solution consisted of 20% trehalose, 10% BSA, and TE buffer. The drying process was performed at 37° C. for 12 h.

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