Five-item combined detection test strip for inflammation of peripheral blood

Abstract

The invention discloses a test strip for five-item combined detection of inflammation of peripheral blood as well as a preparation method and application of the test strip. 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, PCT, IL-6, CRP and SAA 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, a fluorescent microsphere-labeled IL-6 antibody, a fluorescent microsphere-labeled CRP antibody, a fluorescent microsphere-labeled SAA antibody and a fluorescent microsphere-labeled DNP-BSA. According to the test strip, fluorescence immunochromatography is carried out by utilizing the principle of a double-antibody sandwich method in combination with the characteristics of fluorescent microspheres, and rapid, specific, simple, convenient and sensitive joint detection on HBP, PCT, IL-6, CRP and SAA 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 five combined detection items of inflammation in peripheral blood, a preparation method thereof and an 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 202210055782.3 discloses a test strip for combined detection of inflammation, a preparation method and its application, that is, the glass nitrocellulose membrane contains a glass cellulose membrane labeled with a mixture of quantum dot-labeled CRP antibody, quantum dot-labeled PCT antibody, quantum dot-labeled SAA antibody, quantum dot-labeled IL-6 antibody, quantum dot-labeled HBP antibody and quantum dot-labeled chicken IgY; however, the final experimental data only clarifies the content of inflammatory factors; 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 is not clarified. Secondly, in terms of the diagnostic value of the combined detection of inflammatory factors, the receiver operator characteristic curve (ROC curve) statistical method is not used to evaluate the diagnostic value of the five combined detection products of inflammation in a certain disease. However, the clinical evaluation performance of the product and the diagnostic value of the product are the basis of medical research. In addition, from a process perspective, the spacing between the conventional NC membrane test line and the quality control line is 4±0.2mm, while the spacing between the NC membrane test line and the quality control line described in this invention is approximately 2±0.2mm, where the width of the test line itself is 1±0.2mm; at the same time, since six lines (five test lines and one quality control line) are coated on the NC membrane; the main structure of this test strip will lead to two possibilities: first, diffusion occurs between the detection antibodies and between the detection antibodies and the quality control antibodies; second, the value range of the detection system will be reduced exponentially; the above two situations may cause errors in the test signals of the detection lines and the quality control lines, and affect product performance.

[0005] Chinese invention patent 202311002647.3 discloses a joint test kit for detecting sepsis and its application, which discloses the application of CD14, CD95, HBP, HMGB-1, IFN-α, IFN-γ, IL-1β, IL-8, MIP-1β, MIF, PCT, TNF-α, sTREM-1, TLR-4, and TSP-1 in the detection of sepsis. The concentrations of the above 15 biomarkers in the sample were determined using flow fluorescence technology. After reading the concentrations, logistic regression analysis was performed using Medcalc software / R language / Python programming to obtain the correlation coefficient and equation, and the receiver operating curve (ROC) was drawn to obtain the area under the receiver operating curve (AUC), sensitivity, and specificity. For the combined diagnostic results of CD14, CD95, HBP, IL-1β, IL-8, MIP-1β, MIF, PCT, TNF-α, sTREM-1, TLR-4, and TSP-1, the AUC of the sepsis and healthy groups was 0.99, the specificity was 98%, and the sensitivity was 98%, which has a very high diagnostic value for sepsis. Flow cytometry is a new generation of high-throughput multi-index combined detection technology, and it is currently one of the hottest tracks in the field of in vitro diagnosis. Flow cytometry has the characteristics of high detection throughput and high sensitivity, but the relatively high cost, and up to 12 biomarkers need to be detected at the same time, which limits its clinical application. Lateral flow immunochromatography has the advantages of high sensitivity, high specificity, rapidity, and simplicity, which facilitates clinical promotion.

[0006] Currently, there are products on the market that can diagnose sepsis based on single or combined detection of heparin binding protein (HBP), procalcitonin (PCT), interleukin-6 (IL-6), C-reactive protein (CRP), and serum amyloid A (SAA) markers; however, the sensitivity and specificity of the above products as early clinical diagnosis of sepsis need further study.

[0007] In particular, for patients who monitor their inflammation levels, venous blood sampling is not suitable for repeated use. Peripheral blood sampling is convenient and causes less wounds to people, making it a better source of blood samples. However, there are significant differences in the composition of peripheral blood and venous blood. For example, in general peripheral blood, red blood cells, hemoglobin, hematocrit, mean corpuscular hemoglobin content, mean corpuscular hemoglobin concentration, reticulocyte count and neutrophil content are higher than those in venous blood. Platelets, mean corpuscular volume, lymphocyte content, eosinophil content and basophil content are lower than those in venous blood. Therefore, although current testing products can measure the content of inflammatory factors in the serum or plasma of venous blood, these products cannot accurately detect the content of inflammatory factors in peripheral blood.

[0008] Based on the above background, how to diagnose sepsis at an early stage with high specificity and sensitivity to facilitate targeted clinical treatment is still a very urgent clinical need.

[0009] Therefore, it is particularly important to develop a product that can quickly and accurately detect peripheral blood; the combined detection of five inflammation items can more accurately predict whether a patient has sepsis and determine the severity of sepsis. Summary of the invention

[0010] The object of the present invention is to provide a method for preparing a test strip for five combined detection items of inflammation in peripheral blood and its application, so as to solve the above-mentioned problems existing in the prior art.

[0011] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0012] In one aspect, the present invention provides a test strip suitable for the combined detection of five inflammation items in 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, IL-6, CRP, SAA detection lines and a mouse anti-DNP monoclonal antibody quality control line; the glass nitrocellulose membrane binding pad has HBP antibodies labeled with fluorescent microspheres, PCT antibodies labeled with fluorescent microspheres, IL-6 antibodies labeled with fluorescent microspheres, CRP antibodies labeled with fluorescent microspheres, SAA antibodies labeled with fluorescent microspheres and a DNP-BSA combination labeled with fluorescent microspheres.

[0013] Preferably, the PVC bottom plate is a long strip structure.

[0014] 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.

[0015] In one aspect, the present invention provides a method for preparing the above-mentioned test strip, comprising the following steps:

[0016] S1: Preparation of fluorescent microsphere-labeled HBP antibody

[0017] 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.

[0018] 2. Centrifuge at 14000 rcf for 20 min, discard the supernatant, and resuspend in 100 μL MES buffer.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] S2: Preparation of microsphere-labeled PCT antibody, IL-6 antibody, CRP antibody, SAA antibody and DNP-BSA: According to the same method as step S1, microsphere-labeled PCT antibody, IL-6 antibody, CRP antibody, SAA antibody and DNP-BSA are prepared respectively;

[0023] S3: Preparation of conjugate pad containing antibody mixture

[0024] 1. Cut the glass cellulose membrane into 9mm width for later use.

[0025] 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.

[0026] 3. The microsphere-labeled HBP antibody, microsphere-labeled PCT antibody, microsphere-labeled IL-6 antibody, microsphere-labeled CRP antibody, and microsphere-labeled SAA antibody, the above microsphere coupling complexes were mixed with microsphere-labeled DNP-BSA and microsphere diluent at a mass ratio of 3:1:6, and then 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.

[0027] S4: Preparation of nitrocellulose membrane

[0028] 1. Stick the nitrocellulose chromatography membrane on the polyvinyl chloride polymer (PVC) support plate.

[0029] 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.

[0030] S5: According to the same method as step S4, the following capture antibodies, HBP antibody, PCT antibody, IL-6 antibody, CRP antibody, SAA antibody and DNP mouse monoclonal antibody, are coated in sequence respectively, and sprayed onto the corresponding test line and quality control line respectively. After drying, the coated nitrocellulose membrane bottom plate is obtained.

[0031] S6: Prepare the sample pad

[0032] 1. Cut the glass cellulose membrane into 15mm width for later use.

[0033] 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.

[0034] 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.

[0035] S8: Test Card Lamination

[0036] 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.

[0037] 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.

[0038] 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 five test strips into the corresponding positions of the customized cartridge, and close the top cover to obtain the five-combination test strips.

[0039] 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.

[0040] 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).

[0041] 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 the microsphere-labeled antibody.

[0042] 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. The HBP antibody labeled with fluorescent microspheres, the PCT antibody labeled with fluorescent microspheres, the CRP antibody labeled with fluorescent microspheres, the SAA antibody labeled with fluorescent microspheres, and the IL-6 antibody labeled with fluorescent microspheres, the above microsphere coupling complexes are respectively mixed with the microsphere-labeled DNP-BSA and the microsphere diluent at a mass ratio of 3:1:6, and then sprayed onto the sealed 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 all dried at 37°C for 12 hours.

[0043] In some embodiments, in step S4, HBP antibody, PCT antibody, IL-6 antibody, CRP antibody, SAA antibody and DNP mouse monoclonal antibody are taken respectively and sprayed onto the corresponding detection line and quality control line respectively, specifically comprising: taking HBP antibody, PCT antibody, IL-6 antibody, CRP antibody, SAA 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.

[0044] 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.

[0045] It should be noted that, in the above-mentioned test strips, five different items, namely HBP, PCT, IL-6, CRP, and SAA, can be detected simultaneously using the same sample and the same reagent.

[0046] Preferably, during testing, the test strip only needs to add 15 μL of peripheral blood + 100 μL of diluent sample to the sample addition port at one time. After the test strip is chromatographed for 15 minutes, it can be matched with a specific immunoassay analyzer and the result can be read.

[0047] In one aspect, the present invention provides use of a test strip in preparing a detection kit for detecting HBP, PCT, IL-6, CRP and SAA in peripheral blood of a patient.

[0048] In some embodiments, the detection kit includes the test strip, buffer, desiccant, instructions for use, etc.

[0049] In some embodiments, the buffer is a phosphate buffer, such as a 10 mM phosphate buffer.

[0050] In some embodiments, the desiccant is silica gel.

[0051] In some embodiments, the patient has sepsis or septic shock.

[0052] The beneficial effects of the present invention include at least the following:

[0053] 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 magnetic particle chemiluminescence, electrochemiluminescence, immunoturbidimetry 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

[0054] 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:

[0055] Figure 1 It is a side structural schematic diagram of the test strip of the present invention.

[0056] Figure 2 It is a schematic diagram of the internal structure of the five-component test strip after assembly of the present invention.

[0057] Figure 3 It is a schematic diagram of the external structure of the five-component test strip after assembly of the present invention.

[0058] Figure 4 It is a diagram of the preparation process of the product of the present invention.

[0059] Figure 5 It is a ROC curve used to evaluate the ability of combined detection of peripheral blood HBP and PCT in diagnosing sepsis.

[0060] 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

[0061] 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 ...").

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] As used herein, "interleukin-6" (IL-6) is an important member of the cytokine network and is widely used in many clinical fields, such as regulating the growth and differentiation of various cells, immune response, etc., and participating in the pathological process of various clinical diseases, including bacterial infection, neonatal sepsis, respiratory failure, systemic lupus erythematosus, enteritis, cardiovascular disease, rheumatoid arthritis and other acute and chronic inflammatory diseases. If the human body is stimulated by inflammation, IL-6 will be secreted by T cells, B cells, mononuclear giant cells, etc., which will then lead to the acute phase reaction of the liver and promote the production of acute phase proteins such as C-reactive protein (CRP) and serum amyloid A (SAA). Therefore, IL-6 is the earliest marker to increase when inflammation occurs, starting to increase in 1 hour and reaching a peak in 2 hours.

[0070] As used herein, "C-reactive protein", also known as CRP, is an acute phase reaction protein that can be synthesized by liver cells. When the pathogen is mildly stimulated or the tissue is damaged, the synthesis of liver cells starts rapidly, and the concentration of C-reactive protein reaches the pathological level (>8mg / L) within 6 hours, and reaches the peak within 48-72 hours, which directly reflects the early tissue inflammatory necrosis. As an extremely sensitive indicator of acute phase reaction, CRP has a variety of biological activities, and its concentration and secretion level are not changed by eating and anti-inflammatory drugs. Therefore, the high and low changes of C-reactive protein are closely related to the inflammatory response and severity of the disease. C-reactive protein is the most effective and most common inflammatory response marker. The detection of C-reactive protein is of certain significance in many diseases such as bacterial and viral infections, tissue trauma, cerebral infarction, myocardial infarction, cardiovascular disease, etc.

[0071] As used herein, "serum amyloid A" (SAA) is a sensitive acute inflammatory marker. When the body is stimulated by infection, inflammation, trauma, tumor, etc., liver cells secrete a large amount of SAA. The SAA level in the serum can rapidly increase by about 1,000 times within 5-6 hours as the body's inflammatory response develops. In addition, the half-life of SAA is only 50 minutes. When the body's inflammatory response is controlled, SAA quickly drops to normal levels. This feature makes SAA a sensitive indicator reflecting the inflammatory state of the body such as infection or trauma, and can also be used as an evaluation indicator for inflammation recovery.

[0072] 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.

[0073] 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.

[0074] Example 1

[0075] Preparation of Heparin Binding Protein (HBP) / Procalcitonin (PCT) / Interleukin-6 (IL-6) / C-Reactive Protein (CRP) / Serum Amyloid A (SAA) Combined Assay Kit (Fluorescence Immunochromatography)

[0076] S1: Preparation of fluorescent microsphere-labeled HBP antibody

[0077] 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 0.05 M MES buffer, then add 5 mM 10 μL EDC solution and react on a silent mixer for 30 minutes.

[0078] 2. Centrifuge at 14000 rcf for 20 min, discard the supernatant, and resuspend in 100 μL MES buffer.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] S2: Preparation of microsphere-labeled PCT antibody, IL-6 antibody, CRP antibody, SAA antibody and DNP-BSA: According to the same method as step S1, microsphere-labeled PCT antibody, IL-6 antibody, CRP antibody, SAA antibody and DNP-BSA are prepared respectively;

[0083] S3: Preparation of conjugate pad containing antibody mixture

[0084] 1. Cut the glass cellulose membrane into 9mm width for later use.

[0085] 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.

[0086] 3. The microsphere-labeled HBP antibody, microsphere-labeled PCT antibody, microsphere-labeled IL-6 antibody, microsphere-labeled CRP antibody, and microsphere-labeled SAA antibody, the above microsphere coupling complexes were mixed with microsphere-labeled DNP-BSA and microsphere diluent at a mass ratio of 3:1:6, and then 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.

[0087] S4: Preparation of nitrocellulose membrane

[0088] 1. Stick the nitrocellulose chromatography membrane on the polyvinyl chloride polymer (PVC) support plate.

[0089] 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.

[0090] S5: According to the same method as step S4, the following capture antibodies, HBP antibody, PCT antibody, IL-6 antibody, CRP antibody, SAA antibody and DNP mouse monoclonal antibody, are coated in sequence respectively, and sprayed onto the corresponding test line and quality control line respectively. After drying, the coated nitrocellulose membrane bottom plate is obtained.

[0091] S6: Prepare the sample pad

[0092] 1. Cut the glass cellulose membrane into 15mm width for later use.

[0093] 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.

[0094] 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.

[0095] S8: Test Card Lamination

[0096] 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.

[0097] 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.

[0098] 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 five test strips into the corresponding positions of the customized cartridge, connect them together using the sample connection pads, and cover them with the upper cover to obtain the five-combination test strips.

[0099] Example 2

[0100] Clinical effect of the product

[0101] For specific clinical trial methods not listed, please refer to relevant national standards and common industry practices and will not be elaborated here.

[0102] 1. Test products

[0103] A heparin binding protein (HBP) / procalcitonin (PCT) / interleukin-6 (IL-6) / C-reactive protein (CRP) / serum amyloid A (SAA) combined assay kit (fluorescence immunochromatography) prepared according to Example 1.

[0104] 2. Selection of trial participants

[0105] From May 1, 2023 to December 1, 2024, 100 patients aged 18 years and over admitted to the ICU of Linfen People's Hospital, aged 26 years and over ～ 80 years old. 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 = 50 cases) and septic shock group (n = 50 cases). Another 50 healthy people were selected as the healthy control group.

[0106] 3. Sample Collection

[0107] Peripheral blood samples were collected, and after the specimen collection was completed, the levels of five biomarkers were tested using the HBP / PCT / IL-6 / CRP / SAA assay kit (fluorescence immunochromatography) and the test results were recorded.

[0108] 4. Statistical Processing

[0109] 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.

[0110] V. Test Results

[0111] 1. General Information

[0112] A total of 150 subjects were included. The test results are shown in Tables 1-5.

[0113] Table 1: Test results for HBP (unit: ng / mL)

[0114]

[0115]

[0116]

[0117] Table 2: Test results for PCT Unit: ng / mL

[0118]

[0119]

[0120]

[0121] Table 3: Test results for IL-6 Unit: pg / mL

[0122]

[0123]

[0124]

[0125] Table 4: Test results for CRP Unit: μg / mL

[0126]

[0127]

[0128]

[0129] Table 5: Test results for SAA μg / mL

[0130]

[0131]

[0132]

[0133] 2. There was no statistically significant difference in the subjects' gender, age and body mass index (P>0.05), see Table 6.

[0134] Table 6: Gender, age and body mass index distribution characteristics of subjects in each group

[0135]

[0136] 3. Comparison of peripheral blood levels between the healthy control group, sepsis group, and septic shock group

[0137] Compared with the healthy control group, the sample levels in the sepsis group and septic shock group were significantly increased, and the difference was statistically significant (P<0.05).

[0138] Table 7: Comparison of peripheral blood levels between the healthy control group, sepsis group, and septic shock group

[0139]

[0140]

[0141] Note: Compared with the sepsis group, a P<0.05; compared with the septic shock group, b P<0.05.

[0142] 4. ROC curve analysis results of various indicators

[0143] In order to evaluate the diagnosis of sepsis by five test indicators of peripheral blood HBP, PCT, IL-6, CRP, and SAA, we performed ROC curve analysis and evaluated the diagnostic ability based on the area under the curve (AUC). The test performance parameters of each indicator are detailed in Table 8 and Figure 5 As shown in the table below, we can see that the AUCs of the five indicators, including HBP, PCT, and IL-6, are all greater than 0.7, indicating that these indicators have relatively good diagnostic efficacy for diagnosing sepsis. The AUCs of CRP and SAA are relatively poor, but even HBP, which has the largest AUC, is only 0.854, and its diagnostic efficacy is still very limited. The main reason is their low sensitivity. The PCT with the highest sensitivity is only 76%, which seriously limits its application in the diagnosis of sepsis.

[0144] Table 8: ROC curve analysis results of various indicators

[0145]

[0146] 5. The diagnostic value of combined detection of HBP, PCT, IL-6, CRP, and SAA for sepsis

[0147] 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 a combined test 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 test performance parameters of each indicator are detailed in Table 9 and Figure 6 After adopting the combined test, our AUC increased significantly, reaching 0.935 when using the five-joint test, and the Yuden index was 0.82, which was higher than any single test as well as the two-joint test, the three-joint test and the four-joint test.

[0148] Table 9: Detection performance of each index of combined detection of HBP, PCT, IL-6, CRP, and SAA

[0149]

[0150]

[0151] 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 five items of peripheral blood inflammation, 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, PCT, IL-6, CRP, SAA detection lines and a mouse anti-DNP monoclonal antibody quality control line; the glass nitrocellulose membrane binding pad has HBP antibody labeled with fluorescent microspheres, PCT antibody labeled with fluorescent microspheres, IL-6 antibody labeled with fluorescent microspheres, CRP antibody labeled with fluorescent microspheres, SAA antibody labeled with fluorescent microspheres and a combination of DNP-BSA 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 five items: HBP, PCT, IL-6, CRP and SAA.

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 hour; 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; Step S2: preparing microsphere-labeled PCT antibody, IL-6 antibody, CRP antibody, SAA antibody and DNP-BSA: preparing microsphere-labeled PCT antibody, IL-6 antibody, CRP antibody, SAA antibody and DNP-BSA respectively according to the same method as in step S1; Step 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) The microsphere-labeled HBP antibody, microsphere-labeled PCT antibody, microsphere-labeled IL-6 antibody, microsphere-labeled CRP antibody, and microsphere-labeled SAA antibody, the above microsphere coupling complexes were mixed with microsphere-labeled DNP-BSA and microsphere diluent at a mass ratio of 3:1:6, and then 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. Step S4: Preparation of nitrocellulose membrane 1) Paste the Millipore nitrocellulose chromatography membrane onto 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 DNP mouse monoclonal antibody onto the NC membrane 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 hours for use. Step S5: According to the same method as in step S4, the following capture antibodies, HBP antibody, PCT antibody, IL-6 antibody, CRP antibody, SAA antibody and DNP mouse monoclonal antibody, are coated in sequence, respectively, and sprayed onto the corresponding detection line and quality control line, and the coated nitrocellulose membrane bottom plate is obtained after drying. Step S6: Prepare 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. Step 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. Step 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) The liquid absorption pad at the lamination position is 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 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, Step 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 test strips, install the five test strips into the corresponding positions of the customized cartridge, and cover them with the upper cover to obtain the five-combination 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 the antibody is selected to be 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 h, after the reaction, centrifuge at 14000 rcf for 20 min, discard the supernatant, resuspend with 200 μL 1% BSA, and seal in a 4°C refrigerator overnight; then centrifuge at 14000 rpm for 20 min, and re-dissolve the precipitated particles in the dispersion to obtain the microsphere-labeled antibody.

6. The method according to claim 4, characterized in that Step S3: The glass cellulose membrane used for the binding pad containing the antibody mixture is 9 mm, and the binding pad blocking solution components are 20% trehalose, 10% BSA, and TE buffer. The HBP antibody labeled with fluorescent microspheres, the PCT antibody labeled with fluorescent microspheres, the CRP antibody labeled with fluorescent microspheres, the SAA antibody labeled with fluorescent microspheres, and the IL-6 antibody labeled with fluorescent microspheres, the above microsphere coupling complexes are mixed with the microsphere-labeled DNP-BSA and the microsphere diluent at a mass ratio of 3:1:6, and then sprayed onto the sealed 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 dried at 37°C for 12 hours.

7. The method according to claim 4, characterized in that In step S4, HBP antibody, PCT antibody, IL-6 antibody, CRP antibody, SAA 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, IL-6 antibody, CRP antibody, SAA 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 In step S6, the glass cellulose membrane used for the sample pad is 15 mm, and the sample pad blocking solution is composed of 20% trehalose, 10% BSA, and TE buffer. The drying process is carried out at 37° C. for 12 hours.

9. Use of the test strip according to any one of claims 1 to 3 in preparing a test kit for detecting HBP, PCT, IL-6, CRP and SAA in peripheral blood of patients.

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

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