Test strip for heparin binding protein (HBP) / procalcitonin (PCT) joint detection of peripheral blood

Abstract

The invention discloses a test strip for heparin binding protein (HBP) / procalcitonin (PCT) joint detection of peripheral blood, and a preparation method and application thereof. The test strip comprises a PVC bottom plate, a nitrocellulose membrane, a glass cellulose membrane combination pad, a sample pad and a water absorption pad, the PVC bottom plate is located at the bottommost part, and the sample pad, the glass cellulose membrane combination pad, the nitrocellulose membrane and the water absorption pad are sequentially arranged above the PVC bottom plate; the nitrocellulose membrane is provided with an HBP and PCT detection line and a mouse anti-DNP monoclonal antibody quality control line; the glass nitrocellulose membrane combination pad is provided with a combination of a fluorescent microsphere-labeled HBP antibody, a fluorescent microsphere-labeled PCT antibody and a fluorescent microsphere-labeled DNP-BSA conjugate. According to the test strip, the principle of a double-antibody sandwich method is combined with the characteristics of fluorescent microspheres to carry out a fluorescence immunochromatography method, and rapid, specific, simple, convenient and sensitive joint detection on HBP and PCT is realized at the same time.

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) / procalcitonin (PCT) in peripheral blood, a preparation method thereof and application thereof. Background Art

[0002] Sepsis is a fatal organ dysfunction caused by an imbalance in the host's response to infection. It is a systemic inflammatory response syndrome (SIRS) caused by an imbalance in the balance of inflammation and anti-inflammatory response. Sepsis is divided into sepsis and septic shock according to its severity. When the body is infected, polymorphonuclear leukocytes migrate with plasma extravasation, and the infection worsens and SIRS occurs, which then develops into sepsis. Sepsis further develops and is combined with severe circulatory and cellular / metabolic disorders, leading to septic shock and an increased risk of death.

[0003] Sepsis and septic shock remain one of the major public health problems facing critical care medicine. Early diagnosis and treatment are the key to improving the prognosis of patients with sepsis and septic shock. In order to diagnose and treat patients with sepsis or those who may develop sepsis more quickly and accurately, 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 acute physiology and chronic health evaluation scoring system (APACHE II) for sepsis diagnosis are the most commonly used organ failure scores in ICUs. However, the existing microbial culture and traditional infection indicators cannot meet the clinical needs for timely, accurate and rapid diagnosis due to time delays, low sensitivity and lack of specificity. The early and accurate diagnosis of sepsis and septic shock remains challenging. Therefore, there is an urgent need for a laboratory test indicator with good sensitivity and specificity to assist in its diagnosis.

[0004] Chinese invention patent 201810318917.4 discloses a heparin binding protein assay kit and assay method using an immunofluorescence dry quantitative method. The invention uses an immunofluorescence dry quantitative method to quantitatively determine heparin binding protein in human plasma in vitro. It is a new type of immunoassay kit that overcomes the human errors of the EIA method in terms of operator skills (e.g., technique, temperature, time, and result judgment, etc.), and can objectively reflect the presence of HBP through quantitative analysis, providing a more powerful experimental diagnostic basis for clinical diagnosis, efficacy observation, and prognosis judgment, and is widely recognized by doctors and patients. However, the invention uses an immunofluorescence dry quantitative method to quantitatively determine heparin binding protein in human plasma in vitro, which objectively reflects the presence of HBP; it cannot accurately determine the content of heparin binding protein in whole blood and peripheral blood.

[0005] Chinese invention patent 201711415117.6 discloses a single-reagent heparin binding protein detection kit and a preparation method thereof. The invention discloses a single-reagent heparin binding protein (HBP-Heparin Binding Protein) detection kit and a 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 a quality control product containing HBP. The reaction solution includes latex particles labeled with HBP antibodies, as well as a buffer solution, a surfactant, a salt, a stabilizer, a suspending agent, and a preservative. The 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 above invention patents only illustrate the content of inflammatory factors in serum and plasma; as for the relationship between inflammatory factors and diseases, especially whether there is a certain cutoff value and whether there is statistical significance at a certain significant level for sepsis and septic shock, it has not been clarified. Secondly, in terms of the diagnostic value of the combined detection of inflammatory factors, the receiver operator characteristic curve (ROC curve) statistical method was not used to evaluate the diagnostic value of the HBP / PCT combined detection product in a certain disease. However, the clinical evaluation performance and diagnostic value of the product are the basis of medical research.

[0007] Previous studies on HBP / PCT have shown that: testing HBP alone cannot distinguish between local infection and systemic infection; HBP is more sensitive, provides early warning, and makes up for the PCT blank period (2 hours) of 12-24 hours. HBP can rise as early as before clinical symptoms appear. HBP can reach its peak in 1 hour, which is the blank period of PCT. Therefore, the necessity of combined detection of the two is extremely important. In addition, most of the sample types for detecting heparin-binding protein and procalcitonin are serum and plasma, and there are very few reports on the detection of heparin-binding protein and procalcitonin in peripheral blood. As markers for evaluating sepsis and septic shock, how to diagnose sepsis and septic shock with high specificity and sensitivity at an early stage to facilitate targeted clinical treatment is still a very urgent clinical need.

[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 meet the requirements of quickly and accurately predicting whether a patient has sepsis and judging 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, so as to solve 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 HBP / PCT combined detection of peripheral blood, comprising a PVC base plate, a nitrocellulose membrane (NC membrane), a glass cellulose membrane binding pad, a sample pad, and a water absorbent pad, wherein the PVC base plate is located at the bottom, and the sample pad, the glass cellulose membrane binding pad, the nitrocellulose membrane, and the water absorbent 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 binding pad has HBP antibodies, PCT antibodies, 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 binding pad, the top end of the glass cellulose membrane binding 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 pH 7.5 0.05 M MES buffer, then add 10 μL of 5 mM EDC solution and react on a silent mixer for 30 min.

[0017] 2. Centrifuge at 14000 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 with 10 mM PBS and place on a silent mixer to react for 1 hour.

[0019] 4. Centrifuge at 14000 rcf for 20 min, discard the supernatant, resuspend with 200 μL 1% BSA, and seal 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 PCT antibody and DNP-BSA conjugate labeled with fluorescent microspheres respectively according to the same method as in 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 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 in a mass ratio of 3:1:6, respectively, and spray them onto the sealed binding 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 binding pad containing the antibody mixture was obtained 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 the film-scraping solution to dilute the capture antibody of the test line and the DNP mouse monoclonal antibody of the quality control line to 1.5mg / mL and 0.8mg / mL respectively. Use the film-scraping gold spraying instrument to spray the capture antibody and DNP mouse monoclonal antibody onto the NC membrane at a spraying amount of 1μL / cm, a length of 300mm, and a speed of 50cm / min, and bake at 60℃ for 12h for use.

[0029] S5: According to the same method as step S4, the following capture antibodies, HBP antibody, PCT antibody and DNP mouse monoclonal antibody are coated in sequence respectively, and sprayed onto the corresponding detection line and quality control line respectively. After drying, the coated nitrocellulose membrane bottom plate 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, and dry it at 37°C for 12 hours to obtain a blocked sample pad for use.

[0033] S7: preparing a liquid absorption pad: drying 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 binding 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 face up and be 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 for 5 times at each concentration point of the calibrator, with the measured fluorescence signal value (T / C) as the Y-axis and the concentration value as the X-axis, and fit the scatter point by 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 7.50.05 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 selected from fluorescent microspheres labeled by a two-step method (i.e., adding EDC to activate the surface carboxyl groups, centrifuging to remove the supernatant, re-dissolving and then connecting the antibody).

[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 14000 rcf for 20 minutes, the supernatant is discarded, and the microspheres are resuspended with 200 μL 1% BSA and sealed in a 4°C refrigerator overnight; thereafter, the microspheres are centrifuged at 14000 rpm for 20 minutes, and the precipitated particles are redissolved in the dispersion to obtain antibodies labeled with fluorescent microspheres.

[0041] In some embodiments, the glass cellulose membrane used for the conjugate pad containing the antibody mixture in step S3 is 9 mm, and the conjugate pad blocking solution components are 20% trehalose, 10% BSA, and TE buffer. After the HBP antibody labeled with fluorescent microspheres, the PCT antibody labeled with fluorescent microspheres, the DNP-BSA conjugate labeled with fluorescent microspheres, and the microsphere diluent are mixed at a mass ratio of 3:1:6, they are sprayed onto the blocked conjugate pad using a film spraying instrument at a spraying amount of 4 μL / cm, a length of 300 mm, and a speed of 50 cm / min; the drying process is dried at 37°C for 12 hours.

[0042] In some embodiments, in step S4, HBP antibody, PCT antibody and DNP mouse monoclonal antibody are respectively taken and sprayed onto the corresponding test line and quality control line, respectively, which specifically includes: taking HBP antibody, PCT antibody and 0.8 mg / ml DNP mouse monoclonal antibody with 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 50 cm / min; the parameters are baking at 60° C. for 12 hours.

[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 carried out at 37° C. for 12 h.

[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 inlet 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 peripheral blood of a patient.

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

[0048] In some embodiments, the buffer is a phosphate buffer, such as a 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 strip adopts fluorescent immunochromatography technology, combines immunofluorescence with immunochromatography technology, and is established by using antigen-antibody specific reaction. 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 given. The following is a brief introduction to the drawings:

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

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

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

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

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

[0059] Figure 6 It is a 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 intended only to describe specific embodiments 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 also intended to include plural forms. In addition, the open-ended expressions "including" and "comprising" are interpreted as also containing unmentioned structural components or method steps, but it should be noted that the open-ended expressions also cover situations consisting only of the described components and method steps (i.e., covering the situation of the closed expression "consisting of ...").

[0061] As used throughout, range is used as a shorthand form for describing each and all numerical values ​​within the range. Any numerical value within the range, such as an integer value, a value that increases by one tenth (when the end value of the range is one decimal place), or a value that increases by one hundredth (when the end value of the range is two decimal places), can be selected as the end point of the range. For example, the range 0.1-10 is used to describe 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 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 generally understood by those skilled in the art. In case of 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 a certain wavelength of light and emits another wavelength of light different from the absorbed wavelength. When a substance is irradiated by a certain wavelength of incident light, the substance will emit visible light of different wavelengths and intensities. When the irradiation of the excitation light stops, the luminescence phenomenon will quickly disappear. This emitted light is fluorescence. Fluorescence is a widely used signal tracing reagent. Most of them are compounds containing conjugated double bonds of benzene rings or heterocyclic rings. They can be used alone or combined into composite fluorescent dyes.

[0064] As used herein, "fluorescent microsphere (FM)" refers to microspheres with fluorescent substances on the surface of the microspheres (including surface coating) or with fluorescent substances in the internal structure of the microspheres (embedded or polymerized), which can excite fluorescence when excited by a certain amount of energy. It is a functional microsphere carrying fluorescent molecules, made of silica or polyethylene, etc. Its shape is generally spherical with a diameter of nanometer to micron (0.01-10μm), but it can also be any shape. As a special type of functional microsphere, it has important applications in many fields, especially in the biomedical field, with its stable morphological structure, narrow particle size distribution, good monodispersity and high luminous efficiency. It is now widely used in immunochromatography technology.

[0065] As used herein, "fluorescent microspheres labeled with antibodies" refers to a biomarker technology, in which the target antibody is specifically bound 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 realizing the coupling of the microspheres and antibodies, which is used for immunochromatography application research. It refers to polymer microspheres that can emit fluorescence by introducing the target fluorescent material into an organic or inorganic matrix by 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 antibodies and stir at room temperature for a specific time to couple the microspheres with antibodies; further add a specific mass of BSA blocking solution and continue stirring to achieve the blocking of the microsphere coupling complex; at low temperature, centrifuge at a high speed 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 antibodies.

[0066] As used herein, "heparin-binding protein" (HBP), also known as azurocidin or CAP37, is derived from neutrophils and is mainly stored in azurocidin granules, with a small portion stored in secretory vesicles. HBP is a member of the serine protease family and has no protease activity. It has a high affinity for lipopolysaccharide lipid A. Neutrophils degranulate after activation, releasing HBP. When acute bacterial infection occurs, the concentration of HBP in the blood can increase significantly within 1-2 hours, while HBP does not increase or increases slightly during viral infection. HBP also acts as a pathogenic factor, and the HBP concentration decreases rapidly after the patient receives effective treatment. Therefore, detecting the HBP content in the patient's blood can assist in the clinical diagnosis and prediction of acute bacterial infection, the judgment of the severity of infection, and the monitoring of the efficacy of antibiotics.

[0067] As used in this article, "procalcitonin" (PCT) is a precursor of calcitonin (CT) without hormonal activity. It is a glycoprotein composed of 116 amino acids and has a molecular weight of 13,000. The half-life of PCT is 25 to 30 hours, and it has good stability in vivo and in vitro. The increase of PCT is closely related to bacterial infection. In severe systemic infection, PCT can be increased early. After the infection is controlled by antibiotic treatment, PCT in the blood will decrease. In patients with viral infection and local bacterial infection without systemic manifestations, PCT is only slightly increased. Therefore, PCT has been used as an important new observation indicator for severe systemic infection or sepsis.

[0068] 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. Many changes to 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 pH 7.5 50 mM MES buffer, then add 5 mM 10 μL EDC solution and react on a silent mixer for 30 minutes.

[0074] 2. Centrifuge at 14000 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 with 10 mM PBS and place on a silent mixer to react for 1 hour.

[0076] 4. Centrifuge at 14000 rcf for 20 min, discard the supernatant, resuspend with 200 μL 1% BSA, and seal 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 PCT antibody and DNP-BSA conjugate labeled with fluorescent microspheres respectively according to the same method as in 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 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 in a mass ratio of 3:1:6, respectively, and spray them onto the sealed binding 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 binding pad containing the antibody mixture was obtained 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 the film-scraping solution to dilute the capture antibody of the test line and the DNP mouse monoclonal antibody of the quality control line to 1.5mg / mL and 0.8mg / mL respectively. Use the film-scraping gold spraying instrument to spray the capture antibody and DNP mouse monoclonal antibody onto the NC membrane at a spraying amount of 1μL / cm, a length of 300mm, and a speed of 50cm / min, and bake at 60℃ for 12h for use.

[0086] S5: According to the same method as step S4, the following capture antibodies, HBP antibody, PCT antibody and DNP mouse monoclonal antibody are coated in sequence respectively, and sprayed onto the corresponding detection line and quality control line respectively. After drying, the coated nitrocellulose membrane bottom plate 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, and dry it at 37°C for 12 hours to obtain a blocked sample pad for use.

[0090] S7: preparing a liquid absorption pad: drying 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 binding 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 face up and be 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 cartridges, and close the top 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) prepared according to Example 1.

[0100] 2. Selection of trial participants

[0101] From January 1, 2023 to September 30, 2023, 200 patients aged 20 to 83 years were admitted to the ICU of Linfen People's Hospital aged 18 years and older. According to the sepsis diagnostic criteria, the patients were divided into two groups according to whether they had sepsis and shock: sepsis without shock group (n = 100 cases) and septic shock group (n = 100 cases); 100 healthy people were selected as the healthy control group.

[0102] 3. Sample Collection

[0103] Peripheral blood was collected, and 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 comparison between groups, and the chi-square test was used for count data. 2 Test; binary logistic regression was used to calculate the probability of diagnosing sepsis by combined biomarker detection. Receiver operating characteristic (ROC) curve was constructed and the area under the curve (AUC) was calculated to evaluate the diagnostic value of each indicator and combined detection. The chi-square test was used to compare the percentages, and P < 0.05 was considered statistically significant.

[0106] V. 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: Test results for PCT (unit: ng / mL)

[0114]

[0115]

[0116]

[0117] 2. There was no statistically significant difference in the subjects' 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

[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 in the sepsis group and septic shock group were significantly increased, and the difference was 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] In order to evaluate the diagnostic value of peripheral blood HBP and PCT detection indicators for sepsis, we performed ROC curve analysis and evaluated the diagnostic ability based on the area under the curve (AUC). The detection 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 of HBP and PCT are both greater than 0.7, indicating that these indicators have relatively good diagnostic efficacy for diagnosing sepsis. The PCT with the highest sensitivity 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 sensitivity and specificity are simultaneously made to reach the maximum critical point, that is, the Youden index (sensitivity + specificity - 1) reaches the maximum or the value corresponding to the maximum positive likelihood ratio is the optimal diagnostic threshold. At present, the generally recognized ideal diagnostic test AUC is 1, and the diagnostic value is higher when it is above 0.9. Since the sensitivity of single-test products is low, which limits the positive detection rate in diagnosis, we use the combined detection method to improve the detection rate of products in clinical practice. We perform ROC curve analysis and evaluate the diagnostic ability of different combined tests based on the area under the curve (AUC). The detection performance parameters of each indicator are shown in Table 6 below. After the combined detection, the AUC increased significantly, the sensitivity of HBP / PCT (combination 1 and combination 2) was 83%, the specificity was 98%, and the Youden index of the two-combination test was 0.81, which was higher than that of HBP single test or PCT single test. By testing the HBP / PCT test with the sample type of peripheral blood, the combined detection of the two can improve the diagnostic value of sepsis.

[0132] Table 6: Detection performance of each indicator of combined detection of HBP / PCT

[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 by way of limitation. The present invention may be varied and improved without departing from the spirit and scope of the present invention, and these variations and improvements 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) / procalcitonin (PCT) in peripheral blood, comprising a PVC base plate, a nitrocellulose membrane, a glass cellulose membrane binding pad, a sample pad, and a water absorbent pad, wherein the PVC base plate is located at the bottom, and the sample pad, the glass cellulose membrane binding pad, the nitrocellulose membrane, and the 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 glass nitrocellulose membrane binding pad has HBP antibodies, PCT antibodies, and a DNP-BSA conjugate combination labeled with fluorescent microspheres.

2. The test strip according to claim 1, wherein the top end of the sample pad presses and sticks to the bottom end of the glass cellulose membrane binding pad, the top end of the glass cellulose membrane binding 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.

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

4. A method for preparing a test strip according to any one of claims 1 to 3, comprising the following steps: Step S1: Preparation of fluorescent microsphere-labeled HBP antibody (1): Add 10 μL of 200 nm fluorescent microspheres to 90 μL of MES buffer (pH 7.5, 0.05 M), then add 5 mM 10 μL LEDC solution and react on a silent mixer for 30 min; (2): Centrifuge at 14000 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 with 10 mM PBS and place on a silent mixer to react for 1 h; (4): Centrifuge at 14000 rcf for 20 min, discard the supernatant, resuspend with 200 μL 1% BSA, and seal 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 PCT antibody and DNP-BSA conjugate labeled with fluorescent microspheres respectively according to the same method as in step S1. S3: Preparation of glass cellulose membrane binding pad containing antibody mixture (1): Cut the glass cellulose membrane into 9 mm wide pieces for later use; (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 dry it at 37°C for 12 hours to obtain a blocked conjugate pad; (3): After the fluorescent microsphere-labeled HBP antibody and the fluorescent microsphere-labeled PCT antibody were mixed with the fluorescent microsphere-labeled DNP-BSA conjugate and the 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): Stick the Millipore nitrocellulose chromatography membrane on the PVC base plate; (2): Use the film-scraping solution to dilute the capture antibody of the test line and the DNP mouse monoclonal antibody of the quality control line to 1.5 mg / mL and 0.8 mg / mL respectively; use the film-scraping gold spraying instrument to spray the capture antibody and the 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°C for 12 h for use. S5: Following the same method as in step S4, 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 bottom plate is obtained. S6: Prepare the sample pad (1): Cut the glass cellulose membrane into 15 mm wide pieces for later use; (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, and dry it at 37°C for 12 hours to obtain a blocked sample pad for use. 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. S8: Test Card Lamination (1): laminating the coated nitrocellulose membrane base, the dried liquid absorption pad, the sealed sample pad, and the binding pad containing the antibody mixture respectively; (2): At the lamination position, the liquid absorption pad should be 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 should not exceed 2±0.5mm; the binding pad should face 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. 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, 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 an activator; the activation time is 30 min, and the order of adding EDC and antibodies is selected from fluorescent microspheres labeled by a two-step method (i.e., adding EDC to activate surface carboxyl groups, centrifuging to remove the supernatant, re-dissolving and then connecting the antibody), and / or 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 14000 rcf for 20 minutes, the supernatant is discarded, and the microspheres are resuspended with 200 μL 1% BSA and sealed in a 4°C refrigerator overnight; then, the microspheres are centrifuged at 14000 rpm for 20 minutes, and the precipitated particles are redissolved in the dispersion to obtain the fluorescent microsphere-labeled antibody.

6. The method according to claim 4, characterized in that In step S3, the glass cellulose membrane used for the binding pad containing the antibody mixture is 9 mm, and the components of the binding pad blocking solution are 20% trehalose, 10% BSA, and TE buffer; the HBP antibody labeled with fluorescent microspheres, the PCT antibody labeled with fluorescent microspheres, the DNP-BSA conjugate labeled with fluorescent microspheres, and the microsphere diluent are mixed in a mass ratio of 3:1:6, and then sprayed onto the blocked binding pad using a film spraying instrument at a spraying amount of 4 μL / cm, a length of 300 mm, and a speed of 50 cm / min; the drying process is carried out at 37°C for 12 hours.

7. The method according to claim 4, characterized in that In step S4, HBP antibody, PCT antibody and DNP mouse monoclonal antibody are respectively taken and sprayed onto the corresponding detection line and quality control line, respectively, which specifically includes: taking HBP antibody, PCT antibody and 0.8 mg / ml DNP mouse monoclonal antibody with a concentration of 1.5 mg / ml, respectively, and drawing them onto the detection line and quality control line of the nitrocellulose membrane at a speed of 1 μL / cm, a length of 300 mm, and 50 cm / min; the parameters are baking 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 is 15 mm, and the sample pad sealing solution components are 20% trehalose, 10% BSA, and TE buffer; the drying process is carried out at 37° C. for 12 h.

9. Use of the test strip according to any one of claims 1 to 3 in preparing a test kit for detecting heparin binding protein (HBP) and procalcitonin (PCT) in peripheral blood of a patient.

10. The use according to claim 9, wherein the patient suffers from sepsis or septic shock.

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