Time-resolved fluorescent microsphere labeled lateral flow immunochromatography test strip for peripheral blood procalcitonin / interleukin-6, and preparation method and application thereof

Abstract

The invention discloses a time-resolved fluorescence immunoassay test strip for joint detection of procalcitonin (PCT) / interleukin-6 (IL-6) in peripheral blood as well as a preparation method and application of the time-resolved fluorescence immunoassay test strip. The technical field belongs to the technical field of biological detection, the existing lateral flow chromatography technology has the problems of narrow linear range, low specificity, poor accuracy, low sensitivity, poorer repeatability (interference) and the like, and aiming at the defects in the prior art, the technical scheme is as follows: a double-antibody sandwich method principle is combined with the characteristics of time-resolved fluorescent microspheres, and the time-resolved fluorescent microspheres are used for detecting the time-resolved fluorescent microspheres. Based on a lateral flow immunochromatography method, rapid, specific, accurate, sensitive, simple and convenient combined detection on PCT and IL-6 in peripheral blood is realized at the same time, and reproducibility is good.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and in particular to a test strip for lateral flow immunochromatography of peripheral blood procalcitonin (PCT) / interleukin-6 (IL-6) based on time-resolved fluorescent microsphere labeling, its preparation method and application. Background Technology

[0002] In 1990, Dr. Bohuon's team, a French oncologist, discovered the presence of procalcitonin (PCT) while testing calcitonin levels in patients with thyroid tumors. In 1991, it was found that not all lung injuries resulted in elevated PCT levels; only soldiers experiencing severe sepsis and / or septic shock showed significantly elevated PCT levels in their blood. Infections caused by bacteria, fungi, viruses, and parasites can all lead to PCT elevation. Dr. Bohuon keenly sensed a potential close relationship between PCT and sepsis, and in 1993, he first published "High serum procalcitonin concentration in patients with sepsis and infection" in *The Lancet*. That same year, his team first proposed that PCT could serve as a serum inflammatory marker for bacterial infection: during bacterial infection, liver macrophages and monocytes, lymphocytes in the lungs and intestines, and neuroendocrine cells produce large amounts of PCT under the influence of endotoxins, tumor necrosis factor-α (TNF-α), and IL-6; and it can be measured 3 hours after bacterial stimulation, reaching its peak value after 6–12 hours. In healthy individuals, serum PCT concentration is below 0.05 ng / mL; in the elderly, patients with chronic diseases, and less than 10% of healthy individuals, serum PCT concentration is above 0.05 ng / mL, reaching a maximum of 0.1 ng / mL, but generally not exceeding 0.3 ng / mL.

[0003] Interleukin-6 (IL-6) is an essential component of the complex cytokine network in the body. Besides influencing the proliferation and differentiation of B cells and T cells, it plays other important biological functions. Many different cell types are capable of synthesizing IL-6, including monocytes / macrophages, fibroblasts, endothelial cells, keratinocytes, mast cells, T cells, and many tumor cells. In vivo and in vitro, IL-6 acts as a differentiation factor for B cells and an activating factor for T cells. Together with IL-2, it promotes the differentiation of T cells into cytotoxic T cells and induces thymocyte proliferation. Upon stimulation with IL-4, IL-6 activation plays a crucial role in the differentiation of B cells into Ig-secreting plasma cells. IL-6 is a potent growth factor in various human myeloma types, with an active concentration less than 10 pg / mL. In the differentiation of hematopoietic stem cells, IL-3 and IL-6 exhibit synergistic effects in vitro.

[0004] Elevated levels of interleukin-6 in the blood can occur in the following conditions: sepsis, autoimmune diseases, lymphoma, acquired immunodeficiency syndrome (AIDS), alcoholic liver disease, and infections, or in cases of transplant rejection.

[0005] Inflammation is a complex defensive response of the body's tissues, which have a vascular system, to various traumatic stimuli. PCT and IL-6 are two representative biomarkers. IL-6 rises earlier than other cytokines and even earlier than PCT, reaching its peak within 2 hours and maintaining a long duration. Combined detection of IL-6 and PCT can avoid the errors in determining the type of infection by a single indicator, improve early diagnosis of infection, and help clinicians determine the patient's treatment plan in a timely manner, thereby improving the patient's treatment success rate, which has significant clinical value.

[0006] Time-resolved fluoroimmunoassay (TRFIA) is a novel detection method proposed by Soini and Hemmia of Finland in the 1970s. TRFIA uses rare earth ions as tracers to label proteins, peptides, hormones, antibodies, nucleic acid probes, or bioactive cells. These ions, along with chelating agents and enhancement solutions (some of which are unnecessary), undergo a specific affinity reaction in the reaction system (e.g., antigen-antibody immunoassay, biotin-avidin reaction, nucleic acid probe hybridization reaction, target cell-effect cell killing reaction, etc.). The fluorescence intensity of the final product is measured using a time-resolved fluorescence analyzer. Based on the ratio of fluorescence intensity to relative fluorescence intensity, the concentration of the analyte in the reaction system is estimated, achieving quantitative analysis. TRFIA, along with chemiluminescence and electrochemiluminescence immunoassay, is considered one of the three major ultrasensitive detection technologies, widely used in food testing, clinical medical testing, environmental monitoring, and biological research.

[0007] In recent years, much effort has been focused on developing technologies for analyte quantification. Numerous platforms are being pursued using different particles and various detection techniques. Colored particles, quantum dots, fluorescent latex particles, liposome-based probes, magnetic particles, and Raman-active labels are being developed by various researchers for integration with lateral flow immunochromatography (LFIA) to achieve analyte quantification. Each detection technique has its advantages and disadvantages. Absorbance-based detection techniques often lack ideal detection sensitivity, while fluorescence techniques typically require complex and expensive instrumentation. Lateral flow devices (LFDs) for magnetic field measurements cannot be sealed in plastic housings, making them unsuitable for off-field detection. Like traditional fluorescence, Raman detection techniques also require expensive instrumentation.

[0008] To date, the only commercially successful system capable of analyte quantification is based on fluorescence-based LFIA technology (e.g., the RAMP system, first developed by Response Biomedical in British Columbia, Canada). However, conventional fluorescence techniques can generate high background noise due to Tyndall, Rayleigh, or Raman scattering. High background noise can also be caused by the instrument's optics, the sample matrix (e.g., blood or serum), and the testing equipment itself. In particular, membrane-based immunochromatography or lateral flow assays often exhibit very high background fluorescence when measured using conventional fluorescence systems due to scattering caused by the membrane itself. This is exacerbated by light scattering due to the small Stokes shift (typically 10–30 nm) of conventional fluorophores and interference from the sample matrix. Nevertheless, this technology has been successfully commercialized for the detection of cardiac biomarkers such as troponin I and NT-proBNP. Despite its excellent performance, this technology still has some drawbacks, such as limited detection sensitivity.

[0009] Time-resolved fluorescence detection is considered to offer higher detection sensitivity than conventional fluorescence due to its lower background noise. Unlike standard methods that use only optical filters to separate target fluorescence from background light through wavelength differences, time-resolved fluorescence separates the fluorescence of interest from the background through lifetime differences. Essentially, this technique involves exciting a long-lifetime fluorescent tag with a short pulse of light, then waiting for a period of time to allow background and other unwanted fluorescence to decay to low levels before collecting the remaining long-lifetime fluorescence signal. The excitation, delay, and collection processes can be cycled and accumulated to further improve the signal-to-noise ratio. This allows for the elimination of short-lifetime signals, such as those from interfering components (e.g., blood) and scattered light, so that only the long-lifetime fluorescence of the tag can be read. The ability to eliminate background is particularly important for fluorescence measurements from side-flow test strips, as their membrane-based structure tends to highly scatter excitation light. Overall, time-resolved methods have been reported to improve detection sensitivity by two or more orders of magnitude compared to conventional fluorescence detection in liquid samples.

[0010] Current measurement methods have the following shortcomings:

[0011] Chinese Patent Application No. 202211435565.3 discloses an immunochromatographic test strip for the combined detection of PCT antibody / IL-6 antibody and its application. The embodiments of this application disclose the preparation method of the test strip and its process verification, screening, and optimization, but no clinical evaluation studies were conducted. Therefore, the specific clinical performance of the test strip cannot be verified. Furthermore, this invention uses traditional fluorescent microspheres, thus the method has shortcomings such as narrow linear range, poor sensitivity, precision, specificity, and high signal-to-noise ratio.

[0012] Furthermore, Chinese Patent Application No. 202010688922.1 discloses a rapid quantitative PCR test strip for PCT / IL-6, its preparation method, and its application. This invention discloses a method for preparing the test strip (including antibody preparation), and in Example 3, only a Pearson correlation analysis was performed on plasma samples. However, this invention did not conduct a statistical study on PCT / IL-6 in peripheral blood, and the traditional fluorescent microsphere-labeled antibody used in this invention has shortcomings, such as a narrow linear range, poor sensitivity, precision, specificity, and high signal-to-noise ratio.

[0013] Chinese Patent Application No. 201910357257.5 discloses a time-resolved detection kit and method for combined IL-6 / PCT detection. However, this invention does not study PCT / IL-6 in peripheral blood.

[0014] Currently, the main detection methods for PCT / IL-6 include enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay (CLI), and immunochromatography. However, ELISA suffers from poor reproducibility and low sensitivity due to the susceptibility of enzyme activity to various factors in the reaction system. Chemiluminescence immunoassay is costly, requiring large-scale equipment and specialized personnel, hindering its widespread adoption. Immunochromatography utilizes fluorescent microspheres as markers to create detection cards, but each method has its own limitations, such as narrow linear range and insufficient sensitivity. Time-resolved fluorescence immunochromatography (TRFI) uses polymers containing lanthanide rare earth elements as fluorescent substances as markers, conjugated with protein antibodies to form antibody conjugates. Combining the double-antibody sandwich method with immunochromatography, it allows for the quantitative detection of target antigens. Due to the unique fluorescence properties of lanthanides, combined with time delay and spectral resolution techniques, TRFI is particularly effective. Time-resolved fluorescent microspheres, labeled with fluorescent complexes of europium, offer several advantages over traditional fluorescent microspheres: longer Stokes shift and lower background fluorescence signal, effectively enhancing chromatographic detection sensitivity; and a stronger fluorescence quantum yield, which helps to lower the detection limit.

[0015] Therefore, there is an urgent need to develop a rapid detection reagent for peripheral blood PCT / IL-6 with a wide linear range, good accuracy, good precision, high sensitivity, and low signal-to-noise ratio.

[0016] This invention is based on a time-resolved fluorescent microsphere containing europium chelates as a labeling material to achieve rapid detection of PCT / IL-6 in peripheral blood. Summary of the Invention

[0017] The purpose of this invention is to provide a time-resolved fluorescent immunoassay strip suitable for combined detection of PCT / IL-6 in peripheral blood, its preparation method, and its application, thereby solving the aforementioned problems existing in the prior art.

[0018] The above-mentioned objectives of the present invention have been achieved by the following technical solutions disclosed in the present invention.

[0019] In one aspect, the present invention provides a time-resolved immunofluorescence test strip suitable for combined detection of PCT / IL-6 in peripheral blood, comprising a base plate, a cellulose membrane (NC membrane), a cellulose membrane conjugate pad (conjugate pad), a blood filter pad (sample pad), and an absorbent pad, wherein the base plate is located at the bottom, and the blood filter pad, the cellulose membrane conjugate pad, the cellulose membrane, and the absorbent pad are arranged sequentially above the base plate; the cellulose membrane has: a detection line (T1) coated with PCT capture antibody 2, a detection line (T2) coated with IL-6 capture antibody 2, and a control line (C) coated with mouse anti-DNP monoclonal antibody; the cellulose membrane conjugate pad is coated with a combination of PCT-labeled antibody 1, IL-6-labeled antibody 1, and DNP-BSA labeled with fluorescent microspheres.

[0020] In some embodiments, the top end of the blood filtration pad presses against and adheres to the bottom end of the cellulose membrane conjugate pad, the top end of the cellulose membrane conjugate pad presses against and adheres to the bottom end of the cellulose membrane, and the top end of the cellulose membrane is pressed against and adhered to the bottom end of the absorbent pad.

[0021] In another aspect, the present invention provides a method for preparing the above-mentioned test strip, the method comprising the following steps:

[0022] Step S1 - Labeling antibodies with fluorescent microspheres, step S1 includes:

[0023] (1) Time-resolved fluorescent microspheres were added to microsphere labeling buffer, followed by the addition of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) to activate the microspheres;

[0024] (2) Then add PCT-labeled antibody 1 / IL-6-labeled antibody 1 to couple the microspheres with PCT-labeled antibody 1 / IL-6-labeled antibody 1 to obtain microsphere conjugate complex;

[0025] (3) Further add microsphere blocking liquid to achieve the blocking of microsphere coupling complex;

[0026] (4) Remove the supernatant by low-temperature centrifugation, and redissolve the solid precipitate with buffer solution to obtain time-resolved fluorescent microsphere labeled PCT-labeled antibody 1 / IL-6-labeled antibody 1;

[0027] (5) Repeat steps (1)-(4) above, but replace PCT-labeled antibody 1 / IL-6 anti-labeled antibody 1 with DNP-BSA to obtain time-resolved fluorescent microsphere-labeled DNP-BSA conjugate.

[0028] Step S2 - Pretreatment of the blood filter pad and conjunctival pad with the blood filter pad pretreatment solution and the conjunctival pad pretreatment solution respectively;

[0029] Step S3 - The conjugate pad is coated with a labeling dilution containing PCT-labeled antibody 1 / IL-6-labeled antibody 1 labeled with time-resolved fluorescent microspheres and DNP-BSA conjugate labeled with time-resolved fluorescent microspheres;

[0030] Step S4 - Coat the cellulose membrane with a nitrocellulose membrane coating solution containing PCT capture antibody 2 / IL-6 capture antibody 2 and DNP mouse monoclonal antibody;

[0031] Step S5 - Laminate and assemble the test strips.

[0032] In another aspect, the present invention provides the clinical application of the aforementioned time-resolved fluorescent immunoassay strip for the combined detection of procalcitonin (PCT) / interleukin-6 (IL-6) in peripheral blood.

[0033] In some implementations, the test kit includes the test strip, sample dilution buffer, desiccant, and instructions for use.

[0034] In some implementations, the sample dilution buffer is a phosphate buffer, such as 10 mM phosphate buffer.

[0035] In some embodiments, the desiccant is silica gel.

[0036] The test strip of the present invention for time-resolved combined detection of procalcitonin / interleukin-6 in peripheral blood has at least the following beneficial effects:

[0037] 1. This invention employs time-resolved lateral flow immunochromatography (RTIM) technology, which combines time-resolved fluorescence with lateral flow immunochromatography and applies it to peripheral blood testing. Compared with traditional fluorescent microsphere immunochromatographic test strips and colloidal gold test strips, the time-resolved fluorescent microsphere fluorescent test strip has stronger anti-interference performance, higher detection sensitivity, and better result precision for peripheral blood samples.

[0038] 2. Compared with other detection technologies, such as magnetic particle chemiluminescence, electrochemiluminescence, and immunoturbidimetry, this invention, combined with immunofluorescence chromatography test strips, not only solves the problem that large instruments cannot perform on-site and bedside immediate detection, but also saves detection time and costs.

[0039] 3. This invention can accurately interpret the PCT / IL-6 results in peripheral blood using time-resolved immunofluorescence analysis technology, and can be automated, reducing human error and providing rapid and accurate diagnostic results.

[0040] 4. The test strip of this invention is provided with a sample application hole and an observation window for observing the results. The results are determined by the analytical instrument, which is fast, accurate and reliable.

[0041] 5. This invention uses dry test strips, which are small in size, easy to transport and store at room temperature, can be individually packaged, and have low testing costs. They are suitable for rapid clinical testing and point-of-care testing.

[0042] 6. This invention has good clinical value in non-disease diagnosis fields.

[0043] 7. This invention can detect peripheral blood. Attached Figure Description

[0044] To more clearly illustrate the test strip of the present invention, its preparation, and its uses, corresponding figures are provided. A brief description of the figures follows:

[0045] Figure 1 This is a side view of the fluorescent immunoassay strip of the present invention.

[0046] Figure 2 This is a schematic diagram of the internal structure of the fluorescent detection card of the present invention after assembly.

[0047] Figure 3 This is a schematic diagram of the external structure of the assembled fluorescent dual-card of the present invention.

[0048] Figure 4 This is a diagram illustrating the preparation process of the product of this invention.

[0049] Figure 5A and 5B These are the PCT level regression analysis curves of the method to be evaluated in this invention, and comparative methods A and B, respectively. Figure 5C and 5D These are the Bland-Altman bias analysis curves of PCT for the method to be evaluated in this invention, and comparative methods A and B, respectively.

[0050] Figure 6A and 6B These are the IL-6 level regression analysis curves of the method to be evaluated in this invention, and comparative methods A and B, respectively. Figure 6C and 6D These are the Bland-Altman bias analysis curves of IL-6 for the method to be evaluated in this invention, and comparative methods A and B, respectively. Detailed Implementation

[0051] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention. Unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “described” used herein are also intended to include the plural forms. Furthermore, the open-ended expressions “comprising” and “including” are to be interpreted as potentially containing structural components or method steps not mentioned, but it should be noted that these open-ended expressions also cover situations where the invention consists only of the stated components and method steps (i.e., they cover the closed-ended expressions “consisting of…”).

[0052] As used throughout, a range is used as a shorthand to describe each and all values ​​within that range. Any value within the range, such as an integer value, a value incremented by one-tenth (when the range ends with one decimal place), or a value incremented by one-hundredth (when the range ends with two decimal places), can be chosen as the end of the range. For example, the range 0.1–10 is used to describe all values ​​within that 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 (in increments of 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.

[0053] All scientific and technical terms mentioned in this specification have the same meaning as commonly understood by those skilled in the art, and in case of conflict, the definitions in this specification shall prevail. To make the description of this invention easier to understand, some terms are explained below.

[0054] As used in this article, "fluorescence" refers to a substance that absorbs a certain wavelength of light while emitting a different wavelength. When a substance is irradiated by incident light of a certain wavelength, it will emit visible light of different wavelengths and intensities. When the excitation light is removed, the luminescence phenomenon will quickly disappear. This emitted light is called fluorescence. Fluorescent reagents are widely used signal tracers. Most of them are compounds containing benzene rings or heterocycles with conjugated double bonds. They can be used alone or in combination to form composite fluorescent dyes.

[0055] As used in this article, "fluorescent microspheres (FM)" refer to microspheres with fluorescent substances on their surface (including surface coating) or containing fluorescent substances within their internal structure (embedded or polymerized), which can emit fluorescence when excited by a certain energy. It is a type of functional microsphere carrying fluorescent molecules, made of materials such as silica or polyethylene, and its shape is generally spherical with a diameter in the nanometer to micrometer range (0.01–10 μm), but it can also be of any shape. As a special type of functional microsphere, it has important applications in many fields, especially in the biomedical field, due to its stable morphology, narrow particle size distribution, good monodispersity, and high luminescence efficiency. It is currently widely used in immunochromatography.

[0056] As used in this article, "time-resolved fluorescent microspheres" differ from traditional fluorescent microspheres in that they utilize rare-earth ions with longer fluorescence half-lives as markers (such as europium). Their fluorescence lifetime is several orders of magnitude longer than that of the background material. This characteristic allows for the measurement of the marker signal by delaying the measurement time or using a filter, waiting for the background fluorescence to fully decay before measurement. This effectively eliminates interference from non-specific fluorescence and improves sensitivity. Furthermore, the large Stokes shift (greater than 280 nm) of time-resolved fluorescent microspheres helps avoid overlap between excitation and emission light, further enhancing the specificity of product detection.

[0057] As used in this article, "time-resolved fluorescent microsphere-labeled antibody" refers to a biolabeling technology that specifically binds target antibodies to time-resolved fluorescent microspheres through steps such as washing and activating the microspheres. The antibody can covalently bind to the active groups on the surface of the microspheres, thereby achieving microsphere-antibody conjugation for immunochromatographic applications. It refers to polymer microspheres that emit fluorescence, formed by introducing target fluorescent materials into an organic-inorganic matrix through certain chemical or physical methods.

[0058] As used in this article, procalcitonin (PCT) is a glycoprotein composed of 116 amino acids and a molecular weight of 13,000, and is a precursor of calcitonin (CT) with no hormonal activity. PCT has a half-life of 25–30 hours and exhibits good stability in vitro and in vivo. Recent findings suggest a close correlation between elevated serum PCT and bacterial infection. In severe systemic infections, PCT levels can rise early, and decrease after infection control with antibiotic treatment. In patients with viral infections and localized bacterial infections without systemic manifestations, PCT levels are only slightly elevated. Therefore, PCT has been used as an important new indicator for monitoring severe systemic infections or sepsis.

[0059] As used in this article, interleukins are a class of cytokines produced by and acting on multiple cell types. Interleukin 6 (IL6) is an important member of the cytokine network and has wide applications in many clinical fields, such as regulating the growth and differentiation of various cells and immune responses. It participates in the pathological processes of various clinical diseases, including bacterial infections, neonatal sepsis, respiratory failure, systemic lupus erythematosus, enteritis, cardiovascular diseases, rheumatoid arthritis, and various acute and chronic inflammatory diseases. When the human body is stimulated by inflammation, IL-6 is secreted by T cells, B cells, monocytes, and giant cells, which then triggers an acute-phase response in the liver, promoting 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 rise when inflammation occurs.

[0060] Time-resolved fluorescent immunoassay strips

[0061] In one aspect, the present invention provides a test strip for time-resolved combined detection of procalcitonin / interleukin-6 in peripheral blood, comprising a base plate, a cellulose membrane, a cellulose membrane conjugate pad, a blood filtering pad, and an absorbent pad, wherein the base plate is located at the bottom, and the blood filtering pad, the cellulose membrane conjugate pad, the cellulose membrane, and the absorbent pad are arranged sequentially above the base plate; the cellulose membrane has: a detection line (T1) coated with PCT capture antibody 2, a detection line (T2) coated with IL-6 capture antibody 2, and a control line (C) coated with mouse anti-DNP monoclonal antibody; the cellulose membrane conjugate pad is coated with a combination of time-resolved fluorescent microsphere-labeled PCT-labeled antibody 1 and IL-6-labeled antibody 1, and time-resolved fluorescent microsphere-labeled DNP-BSA conjugate.

[0062] In some embodiments, the top end of the blood filtration pad presses against and adheres to the bottom end of the cellulose membrane conjugate pad, the top end of the cellulose membrane conjugate pad presses against and adheres to the bottom end of the cellulose membrane, and the top end of the cellulose membrane is pressed against and adhered to the bottom end of the absorbent pad.

[0063] Preferably, the base plate is a PVC base plate, and more preferably, the PVC base plate is a long strip structure.

[0064] Preferably, the cellulose membrane is a nitrocellulose membrane (e.g., purchased from Sartorius CN140).

[0065] Preferably, the cellulose membrane conjugate pad is a glass cellulose membrane conjugate pad (e.g., purchased from Ahlstrom 8964).

[0066] Preferably, the blood filtration pad is a GF2 blood filtration membrane (e.g., purchased from Shanghai Jieyi Biotechnology Co., Ltd.).

[0067] Preferably, the absorbent pad is H5015 (e.g., purchased from Shanghai Jieyi Biotechnology Co., Ltd.).

[0068] Preparation of time-resolved fluorescent immunoassay strips

[0069] In another aspect, the present invention provides a method for preparing a time-resolved fluorescent immunoassay strip.

[0070] Experimental materials:

[0071] This invention uses time-resolved fluorescent microspheres ( Time-resolved fluorescent microspheres (microspheres labeled with fluorescent complexes of europium) are from Merck Chemicals, USA. The microspheres have a particle size of 200 nm, an excitation wavelength of 365 nm, and an emission wavelength of 610 nm. Catalog number: 80380624.

[0072] Antibody raw materials: PCT was purchased from Feipeng Biotechnology Co., Ltd., catalog number: PCT-Ab7# (detection antibody 1), PCT-Ab4# (capture antibody 2); IL-6 was purchased from Feipeng Biotechnology Co., Ltd., catalog number: FAB-B003-2F7 (detection antibody 1), FAB-B003-7E5 (capture antibody 2).

[0073] Blocker: Fipeng Biotechnology Co., Ltd., Product No.: HIER-E-015.

[0074] RBC antibody: EastCoast Bio, USA, catalog number: HM1079.

[0075] Nitrocellulose membrane: Merck Chemicals CN140.

[0076] Blood filtration membrane: GF2 blood filtration membrane from Shanghai Jieyi Biotechnology Co., Ltd., product number JY-Q01.

[0077] Glass cellulose membrane sample pad: Ahlstrom 8964, item number: JY-BX102.

[0078] Glass cellulose membrane conjugate pad: Ahlstrom 8964, item number: JY-BX102.

[0079] PVC base plate: Shanghai Jieyi Biotechnology Co., Ltd., item number: JY-D103.

[0080] All other chemical auxiliaries are from Merck Chemicals.

[0081] Gold spraying film scrubbing instrument: Shanghai Jiening Biotechnology Co., Ltd., Model: XYZ3010 all-in-one machine.

[0082] Instrument: ROFI HANDYT time-resolved immunoassay analyzer.

[0083] The method for preparing time-resolved fluorescent immunoassay strips of the present invention includes the following steps.

[0084] Step S1 - Labeling antibodies with fluorescent microspheres, step S1 includes:

[0085] (1) Time-resolved fluorescent microspheres were added to microsphere labeling buffer, followed by the addition of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) to activate the microspheres;

[0086] (2) Then add PCT-labeled antibody 1 / IL-6-labeled antibody 1 to couple the microspheres with PCT-labeled antibody 1 / IL-6-labeled antibody 1 to obtain microsphere conjugate complex;

[0087] (3) Further add microsphere blocking liquid to achieve the blocking of microsphere coupling complex;

[0088] (4) Remove the supernatant by low-temperature centrifugation, and redissolve the solid precipitate with buffer solution to obtain time-resolved fluorescent microsphere labeled PCT-labeled antibody 1 / IL-6-labeled antibody 1;

[0089] (5) Repeat steps (1)-(4) above, but use DNP-BSA instead of PCT-labeled antibody 1 / IL-6-labeled antibody 1 to obtain time-resolved fluorescent microsphere-labeled DNP-BSA conjugate.

[0090] Step S2 - Pretreatment of the blood filter pad and conjunctival pad with the blood filter pad pretreatment solution and the conjunctival pad pretreatment solution respectively;

[0091] Step S3 - The conjugate pad is coated with a labeling dilution containing PCT-labeled antibody 1 / IL-6-labeled antibody 1 labeled with time-resolved fluorescent microspheres and DNP-BSA conjugate labeled with time-resolved fluorescent microspheres;

[0092] Step S4 - Coat the cellulose membrane with a nitrocellulose membrane coating solution containing PCT capture antibody 2, IL-6 capture antibody 2 and DNP mouse monoclonal antibody;

[0093] Step S5 - Laminate and assemble the test strips.

[0094] Step S1. Antibody is labeled using time-resolved fluorescent microspheres.

[0095] Pretreatment of time-resolved fluorescent microspheres

[0096] Since the storage buffer for fluorescent nanospheres typically contains surfactants and preservatives, these substances can affect the coupling efficiency between the microspheres and proteins. Therefore, it is necessary to replace the storage buffer with a microsphere labeling buffer to remove surfactants and preservatives. Thus, the fluorescent microspheres can be pretreated by centrifugation if necessary. The suspended fluorescent nanospheres are thoroughly mixed using a vortex mixer. 100 μL (1 mg) of the 1% solids microsphere suspension is transferred to a 1.5 mL centrifuge tube and centrifuged at 14000 rpm for 15 min at 4°C. The supernatant is carefully removed to obtain the pretreated fluorescent microspheres. The time-resolved microspheres used in this invention are wash-free and do not require this step.

[0097] activation

[0098] A certain amount of 50 mM microsphere labeling buffer (2-(N-morpholino)ethanesulfonic acid buffer, MES buffer) was measured into a centrifuge tube. Time-resolved microspheres (1% solid content) were then measured into the centrifuge tube and mixed thoroughly, ensuring a microsphere to microsphere labeling buffer volume ratio of 1:10 to 1:20. Simultaneously, a certain amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) (20 mg / mL each) were added. The mixture was shaken for 30–60 min and then inverted in a 30°C oven for 30–60 min. After inversion, the centrifuge tube containing the mixture was centrifuged.

[0099] The microsphere labeling buffer is prepared as follows: Calculate the total volume required for the microsphere labeling buffer, add MES(g) = 0.009762 × the prepared volume (mL) according to the required volume, mix thoroughly with purified water and bring to a final volume, and adjust the pH to 7.00 ± 0.05.

[0100] In some implementations, the activator is 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), and the activation time is 30 min.

[0101] mark

[0102] After centrifugation following the activation steps described above, discard the supernatant. Add coupling buffer to restore volume. Mix well and add PCT-labeled antibody 1 / IL-6-labeled antibody 1. Incubate at room temperature with shaking for 30–60 min. After shaking, place the centrifuge tube containing the mixture into a centrifuge for centrifugation.

[0103] The coupling buffer solution is prepared as follows: First, prepare a 0.2M boric acid (H3BO3) solution and a 0.05M borax (NaB4O7·7H2O) solution. Then, according to the pH requirement, mix the prepared 0.2M boric acid solution and 0.05M borax solution at an 8:2 ratio. After dilution, a 20mM BS (borax-boric acid buffer solution) with pH = 7.8 ± 0.05 is obtained.

[0104] In some implementations, the order of adding EDC and antibody is a two-step labeling method, that is, after adding EDC to activate the surface carboxyl groups, centrifuge to remove the supernatant, reconstitute, and then connect the antibody.

[0105] In some embodiments, the reaction time between the activated time-resolved fluorescent microspheres and the dialyzed monoclonal antibody is 30 min. After the reaction, the mixture is centrifuged at 14,000 rpm for 15 min, and the supernatant is discarded.

[0106] Closed

[0107] After centrifugation following the labeling steps described above, discard the supernatant, add the microsphere blocking solution, mix well and disperse, and vortex for 30–60 min. After vortexing, centrifuge to separate the microspheres.

[0108] The microsphere blocking solution contains: 10% BSA at a final concentration of 2%, 5% casein at a final concentration of 0.1%, and 20% Tween at a final concentration of 0.3%. The remaining amount of the diluent is supplemented with 0.05M Tris-HCl, and the pH is 8.00±0.05.

[0109] After centrifugation, discard the supernatant, add the microsphere preservation solution, and mix well.

[0110] The microsphere preservation solution contains: 25 mM Tris-base, 0.1% Tween 20, 0.15 M NaCl, 1% BSA, 0.1% ProClin 300, 3% trehalose, pH = 9.0 ± 0.05.

[0111] Label the marked microsphere solution and refrigerate until needed.

[0112] In some implementations, the food is stored overnight in a sealed refrigerator at 4°C.

[0113] In some implementations, the above centrifugation is performed in a centrifuge at 14,000 rpm for 15 minutes.

[0114] Internal control markings:

[0115] Repeat the above steps, but use DNP-BSA instead of PCT-labeled antibody 1 / IL-6-labeled antibody 1. Step S2. Filter Pretreatment of blood pads and conjunctival pads

[0116] The blood filtration pads and conjugate pads are made of fiberglass and require pretreatment before use.

[0117] The blood filter pad is the area where samples are added. Because some samples differ significantly in factors such as pH and ionic strength, the blood filter pad requires strong pretreatment and filtration capabilities. By wetting the blood filter pad with saline and buffer solutions, the differences in sample density when reaching the conjugate release pad and detection zone can be reduced. Optional addition of surfactants, hydrophilic polymers, and blocking agents can further reduce non-specific binding, increase the signal-to-noise ratio, and improve effective blocking.

[0118] The function of the conjugate pad is to adsorb europium chelate fluorescent nanospheres-protein conjugates, so that the europium chelate fluorescent nanospheres-protein conjugates can maintain their activity within the shelf life and be effectively released when the sample flows through the conjugate pad. The performance of the conjugate pad can be effectively improved by coating blocking reagents, protein stabilizers and using separation and release reagents.

[0119] Blood filter pad pretreatment

[0120] Cut the blood filtration pad into strips, then soak them in the blood filtration pad pretreatment solution for a period of time, drain them, and place them in a forced-air drying oven to dry for later use.

[0121] The pretreatment solution for the blood filtration pad contains: 20 mM Tris solution, 1% PVP-10, 0.5% casein, 0.5% NaCl, 0.1% Tween-20, 0.1% Proclin 300, 0.2 mg / mL anti-rbc monoclonal antibody, 1.0 mg / mL active heterophile antibody blocking agent, pH = 7.0 ± 0.05.

[0122] In some implementations, the blood filtration pad uses a glass fiber membrane with a thickness of 18 mm.

[0123] Combined with pad pretreatment

[0124] Cut the conjoint pad into strips, soak them in the conjoint pad pretreatment solution for a period of time, drain them, and then place them in a forced-air drying oven to dry for later use.

[0125] The conjugate pad treatment solution comprises: 20 mM sodium tetraborate solution, 0.2% PEG2000, 1% PVP-10, 0.5% sodium caseinate, 0.5% S9, 0.05% Proclin 300, 2% sucrose, 1.0 mg / ml active heterophile antibody blocking agent, pH = 7.0 ± 0.05.

[0126] In some implementations, the glass cellulose membrane used for the conjugate pad containing the antibody mixture has a width of 11 mm.

[0127] Step S3. Coating of the binding pad: Solidification of europium chelate fluorescent nanosphere coupling conjugate

[0128] The time-resolved fluorescent nanosphere-labeled antibody conjugate and internal control conjugate (time-resolved fluorescent nanosphere-labeled DNP-BSA conjugate) prepared above were resuspended and mixed by sonication, and then diluted with labeling diluent.

[0129] The labeled diluent contains: 25 mM Tris-base, 0.1% Tween-20, 1% BSA, 0.1% ProClin300, 3% trehalose, 2% sucrose, pH = 8.0 ± 0.05.

[0130] Next, the binding pad coating solution was prepared. Based on the required volume for coating the binding pad, the volumes of the labeled microsphere-conjugated antibody conjugate (time-resolved fluorescent nanosphere-labeled antibody conjugate) and the labeled internal control conjugate (time-resolved fluorescent nanosphere-labeled DNP-BSA conjugate) were calculated, where:

[0131] The volume of the time-resolved fluorescent nanosphere-labeled antibody conjugate = 0.4 × the volume of coating solution required; the volume of the time-resolved fluorescent nanosphere-labeled DNP-BSA conjugate = 0.01 × the volume of coating solution required.

[0132] To increase volume

[0133] The remaining volume of the coating solution was replenished with the marked diluent.

[0134] The prepared binding pad coating solution was added to a centrifuge tube and thoroughly mixed. The mixture was then sprayed onto the prepared blocking binding pad and dried to obtain a binding pad containing the antibody mixture. Mixing can be performed using a vortex mixer, and spraying can be carried out using a gold spraying apparatus at a speed of 8 μL / cm, a length of 300 mm, and a speed of 60 cm / min. The drying temperature can be, for example, 37°C, and the drying time can be, for example, 12–16 h.

[0135] In some embodiments, the time-resolved fluorescent microsphere-labeled PCT-labeled antibody 1 / IL-6-labeled antibody 1, the time-resolved fluorescent microsphere-labeled DNP-BSA conjugate, and the labeling diluent are mixed in a certain proportion and then sprayed onto the sealed conjugate pads using a gold spraying apparatus at a spraying volume of 8 μL / cm, a length of 300 mm, and a speed of 60 cm / min; the drying process is carried out at 37°C for 12–16 h.

[0136] Step S4. Coating with cellulose membrane

[0137] First, prepare the cellulose membrane coating solution. Calculate the total volume of the coating solution, and dilute PCT capture antibody 2 and IL-6 capture antibody 2 to a final concentration of 1–3 mg / mL. Dilute the internal control conjugate (DNP mouse monoclonal antibody) to a final concentration of 0.2 mg / mL. ～0.5 mg / mL, 20% trehalose final concentration 1%, 1% BSA final concentration 0.1%, the remaining coating solution was replenished with 1×BS (pH=8.0).

[0138] The prepared cellulose membrane coating solution was added to a centrifuge tube and thoroughly mixed. The mixture was then sprayed onto the cellulose membrane and dried for later use. A vortex mixer can be used for mixing, and a gold spraying apparatus can be used for spraying at a speed of 1 μL / cm, a length of 300 mm, and a speed of 60 cm / min. The drying temperature is, for example, 60℃, and the drying time is 12–16 h.

[0139] In some implementations, PCT capture antibody 2, IL-6 capture antibody 2, and DNP mouse monoclonal antibody are taken and then coated onto the corresponding detection lines and control lines, respectively.

[0140] Step S5. Lamination and assembly of test strips

[0141] The test strips used in this study consist of five parts: a base plate, a cellulose membrane, an absorbent pad, a blood filtering pad, and a conjugate pad. For example... Figure 1 As shown, the blood filtration pad (1), conjugation pad (2), cellulose membrane (3), and absorbent pad (4) are fixed to the base plate in sequence by adhesive. A sample application port is located above the blood filtration pad.

[0142] The liquid absorbent pad should be 1 mm away from the edge of the coated nitrocellulose membrane base plate's release liner, and the overlap between the liquid absorbent pad and the nitrocellulose membrane base plate should not exceed 2 ± 0.5 mm. The conjugate pad should be placed face up against the upper edge of the release liner of the blood filtration pad, and the overlap between the conjugate pad and the nitrocellulose membrane should not exceed 2 ± 0.5 mm. The blood filtration pad should be placed against the lower edge of the release liner of the blood filtration pad. After assembly, cut the base plate into test strips using a strip cutter, insert the test strips into the retaining position of the cartridge, and close the top cover to obtain the dual assay kit. For example, the base plate can be cut into 4 mm wide strips using a strip cutter.

[0143] Preparation of reagents and calibrators

[0144] Preparation of calibrators: Human negative serum samples were used to prepare a high-concentration stock solution by adding antigen and assigning it for verification. The stock solution was then diluted to produce samples at different concentration points (covering the linear range).

[0145] Each concentration point of the calibrator was measured five times. The measured fluorescence signal value was used as the Y-axis and the concentration value as the X-axis. A standard curve was developed by fitting the scatter plot to the Logistic curve (four parameters) for the determination of sample concentration.

[0146] Applications of time-resolved immunofluorescence test strips

[0147] In another aspect, the present invention provides the use of the time-resolved immunofluorescence test strip in the preparation of a kit for detecting the levels of procalcitonin and interleukin-6 in the peripheral blood of a patient.

[0148] In another aspect, the present invention provides a kit comprising the time-resolved immunofluorescence test strip.

[0149] In some implementations, the test kit includes the test strip, sample dilution buffer, desiccant, and instructions for use.

[0150] In some implementations, the sample dilution buffer is a phosphate buffer, such as 10 mM phosphate buffer.

[0151] In some embodiments, the desiccant is silica gel.

[0152] In some implementations, during testing, only 15 μL of capillary blood and 100 μL of diluent need to be added to the sample application port of the test strip. After 10 minutes of chromatography, the test strip can be matched with a specific immunoassay analyzer to read the results.

[0153] It should be noted that, using the test strip of the present invention, both PCT and IL-6 can be detected simultaneously using the same sample and the same test strip.

[0154] While various embodiments of the invention have been described above, it should be understood that they are provided by way of example only and not as limitations. Many changes to the disclosed embodiments may be made in accordance with the disclosure herein without departing from the spirit or scope of the invention. Therefore, the breadth and scope of the invention should not be limited by any of the embodiments described above.

[0155] All references mentioned herein are incorporated herein by reference. All publications and patent documents cited in this application are incorporated herein by reference for all purposes, and are cited as if they were individually cited.

[0156] Example 1

[0157] Preparation of Procalcitonin (PCT) / Interleukin-6 Assay Kit (Time-Resolved Lateral Flow Immunochromatography) test materials material:

[0158] This invention uses time-resolved fluorescent microspheres ( Time-resolved fluorescent microspheres (microspheres labeled with fluorescent complexes of europium) are from Merck Chemicals, USA. The microspheres have a particle size of 200 nm, an excitation wavelength of 365 nm, and an emission wavelength of 610 nm. Catalog number: 80380624.

[0159] Antibody raw materials: PCT was purchased from Feipeng Biotechnology Co., Ltd., catalog number: PCT-Ab7# (detection antibody 1), PCT-Ab4# (capture antibody 2); IL-6 was purchased from Feipeng Biotechnology Co., Ltd., catalog number: FAB-B003-2F7 (detection antibody 1), FAB-B003-7E5 (capture antibody 2).

[0160] Blocker: Fipeng Biotechnology Co., Ltd., Product No.: HIER-E-015.

[0161] RBC antibody: EastCoast Bio, USA, catalog number: HM1079.

[0162] Nitrocellulose membrane: Merck Chemicals CN140.

[0163] Blood filtration membrane: GF2 blood filtration membrane from Shanghai Jieyi Biotechnology Co., Ltd., product number JY-Q01.

[0164] Glass cellulose membrane sample pad: Ahlstrom 8964, item number: JY-BX102.

[0165] Glass cellulose membrane conjugate pad: Ahlstrom 8964, item number: JY-BX102.

[0166] PVC base plate: Shanghai Jieyi Biotechnology Co., Ltd., item number: JY-D103.

[0167] All other chemical auxiliaries are from Merck Chemicals.

[0168] Gold spraying film scrubbing instrument: Shanghai Jiening Biotechnology Co., Ltd., Model: XYZ3010 all-in-one machine.

[0169] Instrument: ROFI HANDYT time-resolved immunoassay analyzer.

[0170] All other raw materials involved in the implementation case can be purchased on the market.

[0171] Preparation of various main solutions

[0172] (1) Microsphere labeling buffer (50mM MES pH 7.0): Calculate the amount of MES to a final concentration of 50mmol / L, weigh it, mix it thoroughly with purified water and dilute it to a final concentration, and adjust the pH to 7.00±0.05;

[0173] (2) 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) solution: final concentration 20 mg / mL;

[0174] (3) Coupling buffer: 20mM BS (borax boric acid buffer solution) pH 7.80±0.05. First, prepare 0.2M boric acid (H3BO3) solution and 0.05M borax (NaB4O7.7H2O) solution. Weigh 12.37g of boric acid (H3BO3) and dissolve it in 1L of water. Weigh 19.07g of borax (NaB4O7.7H2O) and dissolve it in 1L of water. Then, according to the pH requirement, mix the prepared 0.2M boric acid solution and 0.05M borax solution in a ratio of 8:2. After diluting tenfold, you can obtain 20mM BS (borax boric acid buffer solution) pH = 7.8.

[0175] (4) Microsphere blocking solution: 10% BSA final concentration 2%, 5% casein final concentration 0.1%, 20% Tween final concentration 0.3%, the remaining dilution is supplemented with 0.05M Tris-HCl, pH=8.00±0.05;

[0176] (5) Microsphere preservation solution: 25mM Tris-base, 0.1% Tween 20, 0.15M NaCl, 1% BSA, 0.1% ProClin 300, 3% trehalose, pH=9.0±0.05;

[0177] (6) Labeled dilution: 25mM Tris-base, 0.1% Tween-20, 1% BSA, 0.1% ProClin300, 3% trehalose, 2% sucrose, pH=8.0±0.05;

[0178] (7) Blood filtration pad pretreatment solution: 20mM Tris solution, 1% PVP-10, 0.5% casein, 0.5% NaCl, 0.1% Tween-20, 0.1% Proclin 300, pH=7.0,±0.05;

[0179] (8) Pretreatment solution for conjugated pads: 20mM sodium tetraborate solution, 0.2% PEG2000, 1% PVP-10, 0.5% sodium caseinate, 0.5% S9, 0.05% Proclin 300, 2% sucrose, pH=7.0, ±0.05.

[0180] Step S1. Time-resolved fluorescent microsphere labeling procedure (using time-resolved fluorescent microspheres to label antibodies)

[0181] activation

[0182] Measure 450 μL of 50 mM MES buffer (pH 7.0) and pour it into a centrifuge tube. Measure 50 μL of time-resolved fluorescent microspheres (1% solid content) (the volume ratio of microspheres to MES buffer is 1:10) and add them to the centrifuge tube to mix and disperse.

[0183] Add the calculated EDC and NHS (20 mg / mL each), measuring 4 μL and 4 μL respectively. Shake and activate for 30 min, then invert in a 30°C oven for 30 min. After inversion, place the centrifuge tube containing the mixture into a centrifuge. After balancing, set the centrifuge parameters and centrifuge at 14000 rpm for 15 min.

[0184] mark

[0185] After centrifugation, remove the centrifuge tube and discard the supernatant. Add 500 μL of coupling buffer to restore the volume, mix well, add 0.01 mg of antibody, and incubate at room temperature with shaking for 30 min. After shaking, place the centrifuge tube containing the mixture into a centrifuge. After balancing, set the centrifuge parameters and centrifuge at 14000 rpm for 15 min.

[0186] Closed

[0187] After centrifugation, remove the centrifuge tube and discard the supernatant. Add 500 μL of microsphere blocking solution, mix well, and vortex for 30 min. After vortexing, place the centrifuge tube containing the mixture into a centrifuge. After balancing, set the centrifuge parameters and centrifuge at 14000 rpm for 15 min.

[0188] After centrifugation, remove the centrifuge tube and discard the supernatant. Add 500 μL of microsphere preservation solution and mix well.

[0189] Label the marked microsphere solution and place it in the refrigerator for later use.

[0190] Preparation of internal control markers

[0191] Repeat the above steps, but use DNP-BSA instead of PCT-labeled antibody 1 / IL-6-labeled antibody 1. Step S2. Pretreatment of the filtration pad and conjugate pad.

[0192] The blood filtration pads and conjugate pads are made of fiberglass and require pretreatment before use.

[0193] The blood filtration pad is the area where samples are added. Because some samples differ significantly in factors such as pH and ionic strength, the blood filtration pad requires strong pretreatment and filtration capabilities. By wetting the blood filtration pad with saline and buffer solutions, the differences in sample density when reaching the conjugate release pad and detection zone can be reduced. Simultaneously, the addition of surfactants, hydrophilic polymers, and blocking agents reduces non-specific binding, increases the signal-to-noise ratio, and improves effective blocking.

[0194] The function of the conjugate pad is to adsorb europium chelate fluorescent nanospheres-protein conjugates, so that the europium chelate fluorescent nanospheres-protein conjugates can maintain their activity within the shelf life and be effectively released when the sample flows through the conjugate pad. The performance of the conjugate pad can be effectively improved by coating blocking reagents, protein stabilizers and using separation and release reagents.

[0195] Blood filter pad pretreatment

[0196] Cut the blood filtration pads into strips of 18mm (width) * 300mm (length), soak them in the blood filtration pad pretreatment solution for 30 minutes, drain them, and then dry them in a 37℃ forced-air drying oven for 12 hours for later use.

[0197] Combined with pad pretreatment

[0198] After cutting the conjoint pad into strips of 11mm (width) * 300mm (length), soak them in the conjoint pad pretreatment solution for 30 minutes, drain them, and then dry them in a 37℃ forced-air drying oven for 12 hours for later use.

[0199] Step S3. Coating of the conjugate pad (solidification of europium storage fluorescent nanosphere coupling conjugate)

[0200] The europium chelate fluorescent nanosphere-labeled antibody conjugate and the internal control conjugate (europium chelate fluorescent nanosphere-labeled DNP-BSA conjugate) prepared above were resuspended and mixed by ultrasonication, and then diluted with labeling diluent.

[0201] Based on the required volume of the binding pad coating solution, the volumes of the labeled microsphere-conjugated antibody conjugate (time-resolved fluorescent nanosphere-labeled antibody conjugate) and the labeled internal control conjugate (time-resolved fluorescent nanosphere-labeled DNP-BSA conjugate) were calculated respectively.

[0202] The volume of the time-resolved fluorescent nanosphere-labeled antibody conjugate = 0.4 × the volume of the coating solution required.

[0203] The volume of the time-resolved fluorescent nanosphere-labeled DNP-BSA conjugate is 0.01 × the volume required for the coating solution. The remaining volume of the coating solution is supplemented with microsphere diluent.

[0204] Add the calculated solution to a centrifuge tube, mix thoroughly using a vortex mixer, and then spray it onto the sealed conjugate pad using a gold sprayer at a spray volume of 8 μL / cm, a length of 300 mm, and a speed of 60 cm / min. After drying at 37°C for 12 h, the conjugate pad containing the antibody mixture is obtained for later use.

[0205] Step S4. Coating with nitrocellulose membrane

[0206] First, prepare the nitrocellulose membrane coating solution. Calculate the total volume of the coating solution, dilute the PCT antibody capture 2 / IL-6 antibody capture 2 to a final concentration of 1 mg / mL, dilute the internal control concentration to a final concentration of 0.5 mg / mL, add 20% trehalose to a final concentration of 1%, add 1% BSA to a final concentration of 0.1%, and replenish the remaining coating solution with 1×BS (pH=8.0).

[0207] Add the calculated solution to a centrifuge tube, mix thoroughly using a vortex mixer, and then use a gold spraying apparatus to spray the capture antibody and internal control antibody onto the NC membrane at a spraying rate of 1 μL / cm, a length of 300 mm, and a speed of 60 cm / min. Dry at 60°C for 12 h for later use.

[0208] Step S5. Lamination and assembly of test strips

[0209] like Figure 1 and 2 As shown, the filtration pad (1), conjunctival pad (2), nitrocellulose membrane (3), and absorbent pad (4) are fixed to the PVC base plate in sequence using adhesive. The liquid absorbent pad is 1 mm away from the edge of the release paper on the coated nitrocellulose membrane base plate during lamination. The overlap between the liquid absorbent pad and the nitrocellulose membrane base plate does not exceed 2 ± 0.5 mm. The conjunctival pad is placed face up against the upper edge of the release paper on the filtration pad. The overlap between the conjunctival pad and the nitrocellulose membrane does not exceed 2 ± 0.5 mm. The filtration pad is placed against the lower edge of the release paper on the filtration pad.

[0210] After assembly, cut the PVC board into 4mm wide test strips using a strip cutter, insert the test strips into the fixed position of the housing, and close the top cover to obtain the dual assay kit.

[0211] Preparation of reagents and calibrators

[0212] Preparation of calibrators: Human negative serum samples were used to prepare a high-concentration stock solution by adding antigen and assigning it for verification. The stock solution was then diluted to produce samples at different concentration points (covering the linear range).

[0213] Each concentration point of the calibrator was measured five times. The measured fluorescence signal value was used as the Y-axis and the concentration value as the X-axis. A standard curve was developed by fitting the scatter plot to the Logistic curve (four parameters) for the determination of sample concentration.

[0214] Example 2

[0215] Clinical evaluation

[0216] The evaluation methods were based on the Clinical and Laboratory Standards Institute (CLSI) EP9-A3 document.

[0217] I. Test Products

[0218] The procalcitonin (PCT) / interleukin-6 (IL-6) assay kit (time-resolved lateral flow immunochromatography) was prepared according to Example 1.

[0219] II. Selection of Cases Participating in the Trial

[0220] From January to June 2024, samples were collected from 100 infected patients aged 23 to 91 years admitted to the ICU of Linfen People's Hospital. These patients were over 18 years old. There were 61 males and 39 females. Homologous serum and peripheral blood samples were collected from each case.

[0221] III. Sample Collection

[0222] After the specimen collection is completed, test the following methods:

[0223] The method to be evaluated in this invention is as follows: a procalcitonin (PCT) / interleukin-6 (IL-6) assay kit (time-resolved lateral flow immunochromatography) prepared according to a specific implementation is used. The sample type is peripheral blood. The PCT / IL-6 level is detected and the test results are recorded.

[0224] Comparison Method A: Roche Procalcitonin (PCT) Assay Kit (electrochemiluminescence assay) and Interleukin-6 (IL-6) Assay Kit (electrochemiluminescence assay), with serum as the sample type, to detect PCT / IL-6 levels and record the results;

[0225] Comparison Method B: Procalcitonin (PCT) assay reagent (fluorescent immunochromatography) and interleukin-6 (IL-6) assay reagent (fluorescent immunochromatography) from Guangzhou Wondfo Biotech Co., Ltd., with serum as the sample type, PCT / IL-6 levels were measured and the results were recorded.

[0226] IV. Statistical Processing

[0227] (1) Quantitative detection: Referring to the Clinical and Laboratory Standards Institute (CLSI) EP9-A3 document, two comparative methods (Method A and Method B) were used to measure the PCT and IL-6 levels of 100 homologous peripheral blood and serum samples in parallel with the method to be evaluated. Scatter plots were drawn with the detection results of the comparative methods as the X-axis and the detection results of the method to be evaluated in this invention as the Y-axis. The regression equation Y = a + bX was calculated, and the consistency and bias of different detection methods were analyzed by applying the Bland-Altman method.

[0228] (2) Expected bias at the medical decision level: Referring to the CLSI EP9-A2 document, the expected bias and 95% confidence interval (CI) of the method to be evaluated and the comparison method at the PCT medical decision level (0.05 ng / mL) and the IL-6 medical decision level (7.0 pg / mL) were obtained, and the relative expected bias of the two methods (expected bias / medical decision level × 100%) was calculated.

[0229] V. Test Results

[0230] 1. General Information

[0231] The study included 100 participants. The test results are shown in Table 1 and Table 2 below.

[0232] Table 1: Test results for PCT (unit: ng / mL)

[0233]

[0234]

[0235]

[0236] Table 2: Test results for IL-6 (unit: pg / mL)

[0237]

[0238]

[0239]

[0240] 2. Results Analysis

[0241] (1) Regression Analysis

[0242] In the 100 samples, the slope of the regression equation for method A and the PCT method under evaluation was 0.9993, and the intercept was -0.0791. The regression equation for the method under evaluation and the comparison method in this invention is Y = 0.9993X - 0.0791.

[0243] (R 2 =0.9999); The slope of the PCT level regression equation for Method B and the method to be evaluated in this invention is 1.0018, and the intercept is -0.05. The regression equation for the method to be evaluated in this invention and the comparison method is Y = 1.0018X - 0.05(R). 2 =0.9998). See also Figure 5A , 5BThe slope of the regression equation for Method A and the IL-6 method to be evaluated in this invention is 1, and the intercept is -0.0438. The regression equation for the method to be evaluated in this invention and the comparison method is Y = 1X - 0.0438(R). 2 =1); The slope of the regression equation for method B and the IL-6 method to be evaluated in this invention is 1.0001, and the intercept is 0.0741. The regression equation for the method to be evaluated in this invention and the comparison method is Y = 1.0001X + 0.0741(R). 2 =1). See also Figure 6A , 6B .

[0244] (2) Bland-Altman bias analysis

[0245] Bland-Altman bias analysis showed that the biases of the three methods in detecting PCT levels ranged from -0.62 to 0.75. The method under evaluation in this invention had the smallest bias (-0.28) compared to comparative method A, with 6% (6 / 100) of the data falling outside the maximum permissible error range (i.e., 1.96 times the standard deviation). The method under evaluation in this invention also had the smallest bias (-0.62) compared to comparative method B, with 6% (6 / 100) of the data falling outside the maximum permissible error range (i.e., 1.96 times the standard deviation). See also Figure 5C , 5D The bias at the IL-6 level ranged from -2.23 to 1.62. The method under evaluation in this invention showed the smallest bias (-2.23) compared to comparative method A, with 5% (5 / 100) of the data falling outside the maximum permissible error range (i.e., 1.96 times the standard deviation). The method under evaluation in this invention showed the smallest bias (-1.34) compared to comparative method B, with 6% (6 / 100) of the data falling outside the maximum permissible error range (i.e., 1.96 times the standard deviation). See the bias analysis chart. Figure 6C , 6D .

[0246] 3. Expected bias of the method under evaluation at the medical decision level

[0247] (1) When using Method A as a reference, the expected bias of the PCT level of the method under evaluation at the medical decision level (0.05 ng / mL) is -0.079135 (95% CI: -0.1396 to -0.01867) ng / mL. This 95% CI includes the allowable error, indicating that the bias is acceptable and suggesting that the results of the method under evaluation are comparable to those of Method APCT. When using Method B as a reference, the expected deviation of the method under evaluation at the medical decision level is -0.04991 (95% CI: -0.12884 to 0.02902) ng / mL. This 95% CI also includes the allowable error, indicating that the bias is acceptable and suggesting that the results of the serum PCT test of the method under evaluation are comparable to those of Method B. The expected bias between Method A and Method B is -0.02613 (95% CI: -0.084198 to 0.03194) ng / mL. This 95% CI also includes the allowable error, indicating that the bias is acceptable. The detection results of the two methods are comparable, as shown in Table 3.

[0248] (2) When using Method A as a reference, the expected bias of the method to be evaluated for IL-6 levels at the medical decision level (7 pg / mL) is -0.0438 (95% CI: -0.13971 to 0.05211) pg / mL. This 95% CI includes the allowable error, indicating that the bias is acceptable and suggesting that the method to be evaluated is comparable to the serum IL-6 detection results of Method A. When using Method B as a reference, the expected deviation of the method to be evaluated for IL-6 levels at the medical decision level is 0.0748 (95% CI: 0.001097 to 0.148503) pg / mL. This 95% CI also includes the allowable error, indicating that the bias is acceptable and suggesting that the method to be evaluated is comparable to the serum IL-6 detection results of Method B. The expected bias between Method A and Method B is -0.1183 (95% CI: -0.212557 to 0.02404) pg / mL. This 95% CI also includes the allowable error, indicating that the bias is acceptable. The detection results of the two methods are comparable, as shown in Table 4.

[0249] Table 3. Bias at the PCT medical decision level for the three methodologies

[0250]

[0251] Table 4. Bias at IL-6 medical decision levels in three methodologies

[0252]

[0253] While various embodiments of the invention have been described above, it should be understood that they are provided by way of example only and not as limitations. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications will fall within the scope of the invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A test strip for time-resolved combined detection of procalcitonin / interleukin-6 in peripheral blood, comprising a blood filtering pad (1), a cellulose membrane conjugate pad (2), a cellulose membrane (3), an absorbent pad (4), and a base plate (5), wherein, The bottom plate (5) is located at the bottommost point, and above the bottom plate (5) are arranged in sequence a blood filtration pad (1), a cellulose membrane conjugate pad (2), a cellulose membrane (3) and an absorbent pad (4); The cellulose membrane (3) has: a detection line (T1) coated with PCT capture antibody 2, a detection line (T2) coated with IL-6 capture antibody 2, and a control line (C) coated with mouse anti-DNP monoclonal antibody; The cellulose membrane conjugate pad (2) is coated with a combination of time-resolved fluorescent microsphere-labeled PCT-labeled antibody 1 / IL-6-labeled antibody 1 and time-resolved fluorescent microsphere-labeled DNP-BSA conjugate.

2. The test strip according to claim 1, wherein the top end of the blood filtering pad (1) presses against and adheres to the bottom end of the cellulose membrane conjugate pad (2), the top end of the cellulose membrane conjugate pad (2) presses against and adheres to the bottom end of the cellulose membrane (3), and the top end of the cellulose membrane (3) is pressed against and adhered to the bottom end of the absorbent pad (4).

3. The test strip according to claim 1, wherein the test strip is used to simultaneously detect two items, PCT and IL-6, in the same sample.

4. A method for preparing a test strip according to any one of claims 1-3, the method comprising the following steps: Step S1 - Labeling antibodies with fluorescent microspheres, step S1 includes: (1) Time-resolved fluorescent microspheres were added to microsphere labeling buffer, followed by the addition of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) to activate the microspheres; (2) Then add PCT-labeled antibody 1 / IL-6-labeled antibody 1 to couple the microspheres with PCT-labeled antibody 1 / IL-6-labeled antibody 1 to obtain microsphere conjugate complex; (3) Further add microsphere blocking liquid to achieve the blocking of microsphere coupling complex; (4) Remove the supernatant by low-temperature centrifugation, and redissolve the solid precipitate with buffer solution to obtain time-resolved fluorescent microsphere labeled PCT-labeled antibody 1 / IL-6-labeled antibody 1; (5) Repeat steps (1)-(4) above, but use DNP-BSA instead of PCT-labeled antibody 1 / IL-6-labeled antibody 1 to obtain time-resolved fluorescent microsphere-labeled DNP-BSA conjugate. Step S2 - Pretreatment of the blood filter pad and conjunctival pad with the blood filter pad pretreatment solution and the conjunctival pad pretreatment solution respectively; Step S3 - The conjugate pad is coated with a labeling dilution containing PCT-labeled antibody 1 / IL-6-labeled antibody 1 labeled with time-resolved fluorescent microspheres and DNP-BSA conjugate labeled with time-resolved fluorescent microspheres; Step S4 - Coat the cellulose membrane with a cellulose membrane coating solution containing PCT capture antibody 2, IL-6 capture antibody 2 and DNP mouse monoclonal antibody; Step S5 - Laminate and assemble the test strips.

5. The method according to claim 4, characterized in that, In step S1, 200 nm time-resolved fluorescent microspheres containing europium chelates are used as time-resolved fluorescent microspheres, 2-(N-morpholino)ethanesulfonic acid buffer (MES buffer) is used as the time-resolved fluorescent microsphere labeling buffer, and the volume ratio of microspheres to MES buffer is 1:10 to 1:20; 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) is used as the activator.

6. The method according to claim 4, characterized in that, In step S2, the pretreatment solution for the blood filtration pads includes: 20 mM Tris solution, 1% PVP-10, 0.5% casein, 0.5% NaCl, 0.1% Tween-20, 0.1% Proclin 300, pH = 7.0 ± 0.05; the pretreatment solution for the conjugation pads includes: 20 mM sodium tetraborate solution, 0.2% PEG2000, 1% PVP-10, 0.5% sodium casein, 0.5% S9, 0.05% Proclin 300, 2% sucrose, pH = 7.0 ± 0.

05.

7. The method according to claim 4, characterized in that, In step S3, the labeled diluent used contains: 25 mM Tris-base, 0.1% Tween-20, 1% BSA, 0.1% ProClin 300, 3% trehalose, 2% sucrose, pH = 8.0 ± 0.

05.

8. The method according to claim 4, characterized in that, In step S4, PCT capture antibody 2, IL-6 capture antibody 2 and DNP mouse monoclonal antibody are taken and sprayed onto the corresponding detection line and control line, respectively.

9. Use of the test strip according to any one of claims 1-3 in the preparation of a assay kit for detecting procalcitonin and interleukin-6 in peripheral blood of patients.

10. The use according to claim 9, characterized in that, The test was performed 10 minutes after the sample was added.

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