A test strip for detecting IL-6 in peripheral blood and a preparation method thereof

Abstract

This invention discloses a lateral flow immunochromatographic test strip based on water-soluble CdSe / ZnS quantum dot fluorescent microspheres for the detection of interleukin-6 (IL-6) in peripheral blood, its preparation method, and its application. The technical field belongs to the field of biodetection technology. Existing lateral flow chromatography techniques suffer from problems such as narrow linear range, low specificity, poor accuracy, low sensitivity, and poor repeatability (interference). This invention addresses these shortcomings by applying the double-antibody sandwich method combined with the characteristics of CdSe / ZnS quantum dot fluorescent microspheres to achieve rapid, specific, accurate, sensitive, reproducible, and convenient combined detection of IL-6 in peripheral blood samples.

Description

Technical Field

[0001] This invention relates to the field of biodetection technology, and in particular to a test strip for peripheral blood interleukin-6 (IL-6) lateral flow immunochromatography based on water-soluble CdSe / ZnS quantum dot fluorescent microspheres, its preparation method and application. Background Technology

[0002] Interleukin-6 is a mediator of the immune system with various biological functions. It is also considered to be a B cell stimulating factor (BCSF), B cell stimulating factor 2 (BSF-2), hybridoma growth factor (HGF), hepatocyte stimulating factor (HSF), and cytolytic T cell differentiation factor (CDF). The cDNA translation product of interleukin-6 is a 212-amino acid polypeptide chain, which can be cleaved to form a mature protein of 184 amino acids. Depending on the degree of glycosylation at positions 73 and 172, as well as the degree of phosphorylation, the molecular weight of interleukin-6 ranges from 21.5 to 28 kDa.

[0003] 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 interleukin-6, including monocytes / macrophages, fibroblasts, endothelial cells, keratinocytes, mast cells, T cells, and many tumor cells. In vivo and in vitro, interleukin-6 acts as a differentiation factor for B cells and an activating factor for T cells. Together with interleukin-2 (IL-2), it promotes the differentiation of T cells into cytotoxic T cells and induces thymocyte proliferation. Upon stimulation by interleukin-4 (IL-4), the activation of interleukin-6 plays a crucial role in the differentiation of B cells into Ig-secreting plasma cells. Interleukin-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, interleukin-3 and interleukin-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, infection, or transplant rejection.

[0005] Current measurement methods have the following shortcomings:

[0006] Chinese Patent Application No. 202211197200.1 discloses a kit and detection method for interleukin-6 fluorescence immunochromatographic detection. This invention has the inherent defect of using ordinary fluorescence as a marker.

[0007] Chinese Patent Application No. 202010124553.3 discloses a method for improving the sensitivity of immunochromatographic markers and its application in interleukin-6 detection. This invention re-couples a labeled antibody-fluorescent microsphere conjugate, allowing the same antibody to carry more fluorescent microspheres, significantly increasing the intensity of the fluorescent substance on the detection line and improving reagent sensitivity. The fluorescent microspheres exhibit green fluorescence (excitation at 475nm, emission at 525nm). While this invention greatly improves product sensitivity, it has drawbacks, including a short fluorescence lifetime, susceptibility to quenching, instability, and low Stokes shift.

[0008] Chinese Patent Application No. 201610033851.5 discloses a method and kit for detecting interleukin-6. This invention involves first conjugating fluorescent latex microparticles to albumin, and then conjugating them to an interleukin-6 monoclonal antibody to prepare a probe pad. However, after the albumin and latex microparticles undergo one conjugation, the number of remaining functional groups on the latex microparticles is insufficient for conjugation with IL-6. Although this invention improves sensitivity, it affects the conjugation rate between the latex microparticles and the target antibody.

[0009] Chinese Patent Application No. 201910357257.5 discloses a time-resolved detection kit and method for the combined detection of IL-6 / PCT. This invention relates to a detection method using rare earth vanadate nanofluorescent labeling materials, taking advantage of the sensitivity of time-resolved fluorescence immunochromatography. However, this invention does not investigate IL-6 in peripheral blood.

[0010] As the core raw material of immunofluorescence, the labeling agent significantly affects its linearity, precision, accuracy, and sensitivity. Taking fluorescein isothiocyanate (FITC) as an example, it is a widely used fluorescent labeling agent. However, due to its short fluorescence lifetime, susceptibility to quenching, instability, low Stokes shift, and monochromatic excitation from a monochromatic source, FITC luminescent labeling has some shortcomings. Nevertheless, with the increasing demand for quantitative detection, more and more electronic identification devices are being integrated into test strip detection devices. Although these detection devices are diverse in design and can select different signal sources (such as magnetic or fluorescent signals), ultimately, it is still the signal labeling agent that converts the immune reaction into a signal. Therefore, finding better signal labeling agents to provide more sensitive and accurate quantitative detection for test strips is an urgent task that immunofluorescence test strips need to address.

[0011] Quantum dots, as a novel semiconductor nanomaterial, possess optical properties unmatched by traditional fluorescent dyes (such as broad excitation and narrow emission, tunable emission spectrum, good photochemical stability, high fluorescence intensity, long lifetime, and single-excitation multivariate emission). Therefore, they are widely used as fluorescent markers in biomedical fields such as immunodiagnostics, biosensing, cell imaging, in vivo imaging, and photodynamic therapy. Thus, combining high-quality quantum dots with lateral flow immunochromatography can achieve rapid and sensitive quantitative detection of large inflammatory biomarkers and small antigen molecules.

[0012] Quantum dots possess unique luminescence mechanisms and modes, exhibiting unparalleled advantages over traditional organic luciferins. These advantages are primarily manifested in the following aspects:

[0013] First, the emission peak of quantum dots exhibits Gaussian symmetry, with a narrow half-width at half-maximum (HWHM) and a large Stokes shift. Traditional organic fluoresceins, on the other hand, have wide emission peaks and narrow excitation peaks, limiting their application in biosensoring. Quantum dots display the opposite spectral characteristics: a narrow HWHM across a wide wavelength range allows for single-light excitation with different colors, avoiding interference caused by color overlap. This characteristic gives quantum dots a significant advantage in biosensoring.

[0014] Second, quantum dots exhibit a higher quantum yield, with fluorescence efficiency approximately 100 times that of conventional organic fluorophores. The first absorption band of quantum dots is typically larger, with a typical molar absorptivity of (10,000–1,000,000) M⁻¹ cm⁻¹, while for dyes, the molar absorptivity at the dominant (long-wavelength) absorption maximum is approximately (25,000–250,000) M⁻¹ cm⁻¹. Furthermore, in most cases, the fluorescence quantum yield of appropriately passivated quantum dots is high in the visible light range (400–700 nm). In contrast, organic dyes exhibit moderate quantum yields in the visible light wavelength range.

[0015] Third, quantum dots exhibit stronger fluorescence stability. Organic luciferins are prone to fluorescence quenching during use, leading to instability and inaccuracy in detection. In contrast, quantum dots are photochemically stable and can withstand repeated excitations, thus allowing them to be used as fluorescent labels to attach to biomolecules and form long-lived fluorescent probes.

[0016] Fourth, compared to organic dyes, another advantageous feature of quantum dots is their very large two-photon interaction cross section.

[0017] Fifth, the fluorescence lifetime of organic dyes is approximately 1 ns in the visible light wavelength range, which is typically too short to effectively distinguish short-lived fluorescence interference from scattered excitation light. Quantum dots, on the other hand, have a relatively long lifetime (typically 5 to hundreds of ns), which can be used to directly distinguish signals from cell autofluorescence and scattered excitation light through time-gated measurements, thereby improving sensitivity.

[0018] Therefore, there is an urgent need to develop a peripheral blood IL-6 detection product based on quantum dot fluorescence immunochromatography, making it a powerful tool for inflammation detection.

[0019] To address some shortcomings in existing technologies, this invention provides a method for detecting IL-6 in peripheral blood based on an immunoassay platform using water-soluble CdSe / ZnS quantum dot fluorescent microspheres. Summary of the Invention

[0020] The purpose of this invention is to provide a quantum dot fluorescent immunoassay strip suitable for the quantitative detection of IL-6 in peripheral blood, its preparation method, and its application. By combining stable and highly sensitive quantum dot fluorescent microspheres with lateral flow immunochromatography, the IL-6 biomarker can be rapidly and accurately measured, thereby solving the aforementioned problems in the prior art.

[0021] The above-mentioned objectives of the present invention have been achieved by the following technical solutions disclosed in the present invention.

[0022] In one aspect, the present invention provides a quantum dot immunofluorescence test strip suitable for the quantitative detection of IL-6 in peripheral blood. The test strip includes 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 blood filter pad is immobilized with anti-human erythrocyte antibodies; the cellulose membrane has a detection line (T line) and a control line (C line), the T line is coated with IL-6 capture antibody 2, and the C line is coated with DNP mouse monoclonal antibody; the cellulose membrane conjugate pad has a combination of IL-6 labeled antibody 1 and DNP-BSA labeled with quantum dot fluorescent microspheres.

[0023] 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.

[0024] In another aspect, the present invention provides a method for preparing the above-mentioned test strip, the method comprising the following steps:

[0025] Step S1 - Labeling antibodies with quantum dot fluorescent microspheres, step S1 includes:

[0026] (1) Quantum dot fluorescent nanospheres were added to a microsphere labeling buffer, followed by the addition of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) to activate the microspheres;

[0027] (2) Then add monoclonal antibody IL-6 labeled antibody 1 to couple the microspheres with IL-6 labeled antibody 1 to obtain microsphere conjugate complex.

[0028] (3) Further add microsphere blocking liquid to achieve the blocking of microsphere coupling complex;

[0029] (4) The supernatant was removed by low-temperature centrifugation, and the solid precipitate was reconstituted with buffer solution to obtain IL-6 labeled antibody 1 with quantum dot fluorescent microspheres;

[0030] (5) Repeat steps (1)-(4) above, but use DNP-BSA instead of IL-6 labeled antibody 1 to obtain DNP-BSA conjugate labeled with quantum dot fluorescent microspheres.

[0031] 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;

[0032] Step S3 - The conjugate pad is coated with a labeling dilution solution containing antibody IL-6 labeled with quantum dot fluorescent microspheres and DNP-BSA conjugate labeled with quantum dot fluorescent microspheres;

[0033] The conjoint pad is coated;

[0034] Step S4 - Coat the cellulose membrane with a cellulose membrane coating solution containing IL-6 capture antibody 2 and DNP mouse monoclonal antibody;

[0035] Step S5 - Laminate and assemble the test strips.

[0036] In another aspect, the present invention relates to the clinical application of a lateral flow immunochromatographic test strip based on water-soluble CdSe / ZnS quantum dot fluorescent microspheres for the detection of interleukin-6 (IL-6) in peripheral blood.

[0037] In some implementations, the test kit includes the test strip, sample dilution buffer, desiccant, and instructions for use.

[0038] In some implementations, the sample dilution buffer is a phosphate buffer, such as 10 mM phosphate buffer.

[0039] In some embodiments, the desiccant is silica gel.

[0040] The quantum dot immunofluorescence test strip of the present invention, suitable for the quantitative detection of IL-6 in peripheral blood, has at least the following beneficial effects:

[0041] 1. The test strip of this invention adopts quantum dot lateral flow immunochromatography technology with CdSe / ZnS quantum dot microspheres labeled with antibodies, which combines quantum dot fluorescence with lateral flow immunochromatography. Compared with traditional fluorescent microsphere immunochromatographic test strips and colloidal gold test strips, quantum dot microsphere fluorescent test strips have stronger anti-interference performance for capillary blood samples, higher detection sensitivity, and better result precision.

[0042] 2. Compared with other detection technologies, such as magnetic particle chemiluminescence, electrochemiluminescence, immunoturbidimetry, and enzyme-linked immunosorbent assay (ELISA), the immunofluorescence chromatography test strip solves the problem that large instruments cannot perform on-site and real-time detection, and also saves detection time and costs.

[0043] 3. This invention can accurately interpret the results through quantum dot fluorescence analysis technology, and can be automated, reducing human error and providing fast and accurate diagnostic results.

[0044] 4. The test strip of this invention is provided with a sample application hole and an observation window for observing the results, and the results are accurately and reliably determined according to the analytical instrument.

[0045] 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 diagnosis and rapid on-site diagnosis.

[0046] 6. This invention has good clinical value in non-disease diagnosis fields.

[0047] 7. This invention can detect peripheral blood. Attached Figure Description

[0048] 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:

[0049] Figure 1 This is a side view of the fluorescent immunoassay strip of the present invention.

[0050] Figure 2 This is a schematic diagram of the internal structure of the fluorescent detection card of the present invention after assembly.

[0051] Figure 3 This is a schematic diagram of the external structure of the fluorescent detection card of the present invention after assembly.

[0052] Figure 4 This is a diagram illustrating the preparation process of the product of this invention.

[0053] Figure 5A and 5B 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.

[0054] Figure 6A and 6B These are the IL-6Bland-Altman bias analysis curves of the method to be evaluated in this invention, and comparative methods A and B, respectively. Detailed Implementation

[0055] definition

[0056] 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 this open-ended expression also covers situations where the invention consists only of the stated components and method steps (i.e., it covers the closed-ended expression “consisting of…”).

[0057] As used throughout, a range is used as a shorthand to describe each and all values ​​within that range. Any value within a 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.

[0058] 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.

[0059] 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.

[0060] As used in this article, "fluorescent microspheres (FM)" refer to microspheres with fluorescent substances on their surface (including surface coatings) 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 (0.01~10μm), 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.

[0061] As used in this paper, the "Quantum Dot Microsphere (QDMS)" encapsulates several to hundreds of quantum dots within a single quantum dot, significantly enhancing luminescence intensity and amplifying the signal. Therefore, compared to traditional fluorescent microspheres and time-resolved fluorescent microspheres, quantum dot fluorescent microspheres offer the following advantages:

[0062] It has a wider Strokes shift: enabling it to operate over a wider spectral range;

[0063] Strong resistance to photobleaching: It can maintain stable fluorescence intensity under long-term light exposure;

[0064] Narrow and symmetrical emission peaks: exhibiting high spectral resolution;

[0065] High excitation overlap region: capable of inducing multiple colors of light under the same excitation light;

[0066] High luminescence intensity: 1000 times that of ordinary fluorescence, exhibiting higher sensitivity and lower background noise; as used in this article, "quantum dot fluorescent microsphere labeled antibody" refers to a biolabeling technology that, through steps such as washing and activating microspheres, specifically binds the target antibody to carboxyl-modified quantum dot fluorescent microspheres. The antibody can covalently bind to the active groups on the surface of the microspheres, thereby achieving microsphere-antibody coupling 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.

[0067] 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.

[0068] Quantum dot immunofluorescence test strips

[0069] In one aspect, the present invention provides a quantum dot immunofluorescence test strip suitable for the quantitative detection of IL-6 in peripheral blood. The test strip includes a base plate, a cellulose 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 blood filter pad is immobilized with anti-human erythrocyte antibodies; the cellulose membrane has a detection line (T line) and a control line (C line), the T line is coated with IL-6 capture antibody 2, and the C line is coated with DNP mouse monoclonal antibody; the cellulose membrane conjugate pad has a combination of IL-6 labeled antibody 1 labeled with quantum dot fluorescent microspheres and DNP-BSA labeled with quantum dot fluorescent microspheres.

[0070] In some embodiments, the top end of the blood filtration pad presses against and adheres to the bottom end of the glass fiber membrane conjugate pad, the top end of the glass fiber membrane conjugate pad presses against and adheres to the bottom end of the nitrocellulose membrane, and the top end of the nitrocellulose membrane is pressed against and adhered to the bottom end of the absorbent pad.

[0071] Preferably, the base plate is a PVC base plate, and more preferably, the PVC base plate is a long strip structure.

[0072] Preferably, the cellulose membrane is a nitrocellulose membrane (e.g., purchased from Sartorius CN140).

[0073] Preferably, the cellulose membrane conjugate pad is a glass cellulose membrane conjugate pad (e.g., purchased from Ahlstrom 8964).

[0074] Preferably, the blood filtration pad is a GF2 blood filtration membrane (e.g., purchased from Shanghai Jieyi Biotechnology Co., Ltd.).

[0075] Preferably, the absorbent pad is H5015 (e.g., purchased from Shanghai Jieyi Biotechnology Co., Ltd.).

[0076] Preparation of quantum dot immunofluorescence test strips

[0077] In another aspect, the present invention provides a method for preparing the above-mentioned test strip.

[0078] Experimental materials:

[0079] This invention uses CdSe / ZnS core-shell quantum dot fluorescent microspheres from Merck Chemical Company, USA, with an emission wavelength of 620 nm and catalog number 919497.

[0080] Antibody raw materials: IL-6 was purchased from Feipeng Biotechnology Co., Ltd., catalog numbers: FAB-B003-2F7 (detection antibody) and FAB-B003-7E5 (capture antibody).

[0081] Blocker: Fipeng Biotechnology Co., Ltd., Product No.: HIER-E-015.

[0082] RBC antibody: EastCoast Bio, USA, catalog number: HM1079.

[0083] Nitrocellulose membrane: Merck Chemicals CN140.

[0084] Blood filtration membrane: GF2 blood filtration membrane from Shanghai Jieyi Biotechnology Co., Ltd., product number JY-Q01.

[0085] Glass cellulose membrane sample pad: Ahlstrom 8964, item number: JY-BX102.

[0086] Glass cellulose membrane conjugate pad: Ahlstrom 8964, item number: JY-BX102.

[0087] PVC base plate: Shanghai Jieyi Biotechnology Co., Ltd., item number: JY-D103.

[0088] All other chemical auxiliaries are from Merck Chemicals.

[0089] Gold spraying film scrubbing instrument: Shanghai Jiening Biotechnology Co., Ltd., Model: XYZ3010 all-in-one machine.

[0090] Instrument: Quantum dot immunochromatographic analyzer (Novizan Quantum Dot Fluorescence Immunoassay Analyzer QD-S1200).

[0091] The method for preparing test strips according to the present invention includes the following steps:

[0092] Step S1 - Labeling antibodies with quantum dot fluorescent microspheres, step S1 includes:

[0093] (1) Quantum dot fluorescent nanospheres were added to a microsphere labeling buffer, followed by the addition of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) to activate the microspheres;

[0094] (2) Then add monoclonal antibody IL-6 labeled antibody 1 to couple the microspheres with IL-6 labeled antibody 1 to obtain microsphere conjugate complex.

[0095] (3) Further add microsphere blocking liquid to achieve the blocking of microsphere coupling complex;

[0096] (4) The supernatant was removed by low-temperature centrifugation, and the solid precipitate was reconstituted with buffer solution to obtain IL-6 labeled antibody 1 with quantum dot fluorescent microspheres;

[0097] (5) Repeat steps (1)-(4) above, but use DNP-BSA instead of IL-6 labeled antibody 1 to obtain DNP-BSA conjugate labeled with quantum dot fluorescent microspheres.

[0098] 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;

[0099] Step S3 - The conjugate pad is coated with a labeling dilution solution containing antibody IL-6 labeled with quantum dot fluorescent microspheres and DNP-BSA conjugate labeled with quantum dot fluorescent microspheres;

[0100] Step S4 - Coat the cellulose membrane with a cellulose membrane coating solution containing IL-6 capture antibody 2 and DNP mouse monoclonal antibody;

[0101] Step S5 - Laminate and assemble the test strips.

[0102] Step S1. Labeling antibodies with quantum dot fluorescent microspheres.

[0103] Pretreatment of quantum dot fluorescent microspheres

[0104] 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, the storage buffer can optionally be replaced with a microsphere activation buffer to remove surfactants and preservatives when necessary. The quantum dot fluorescent microspheres used in this invention do not require a washing step.

[0105] activation

[0106] Measure a certain amount of 50mM microsphere labeling buffer (2-(N-morpholino)ethanesulfonic acid buffer, MES buffer) into a centrifuge tube. Measure quantum dot fluorescent microspheres (1% solid content) into the centrifuge tube and mix well, so that the volume ratio of microspheres to MES buffer is 1:10 to 1:20. Simultaneously add a certain amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) (0.5–2 mg / mL each), shake to activate for 30–60 min, and then invert on a 30 °C oven for 30–60 min. After inversion, place the centrifuge tube containing the mixture into a centrifuge for centrifugation.

[0107] The microsphere labeling buffer was prepared as follows: the amount of MES was calculated to a final concentration of 50 mmol / L, weighed, thoroughly mixed with purified water and diluted to a final concentration, and the pH was adjusted to 7.00 ± 0.05.

[0108] Alternatively, a solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) can be prepared in advance, so that the final concentration of the pre-prepared solution is 0.5 mg / mL.

[0109] In some implementations, the volume ratio of quantum dot fluorescent microspheres to MES buffer is 1:10. The activator is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC). The activation time is 30 min.

[0110] mark

[0111] After centrifugation following the activation steps described above, discard the supernatant. Add coupling buffer to restore volume. Mix well and add 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.

[0112] 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 in an 8:2 ratio, and after dilution, a 20mM BS (borax-boric acid buffer solution) with pH=7.8±0.05 can be obtained.

[0113] 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.

[0114] In some embodiments, the reaction time between the activated quantum dot fluorescent microspheres and the dialyzed monoclonal antibody is 30 min, and after the reaction, the mixture is centrifuged at 14,000 rpm for 15 min and the supernatant is discarded.

[0115] Closed

[0116] 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 minutes. After vortexing, centrifuge to separate the microspheres.

[0117] The microsphere blocking solution contains 0.5% BSA, 20mM glycine, Tris-HCl, and pH=8.00±0.05.

[0118] After centrifugation, discard the supernatant, add the microsphere preservation solution, and mix well.

[0119] 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, and pH = 9.0 ± 0.05.

[0120] Label the marked microsphere solution and refrigerate until needed.

[0121] In some implementations, the food is stored overnight in a sealed refrigerator at 4°C.

[0122] In some implementations, the above centrifugation is performed in a centrifuge at 14,000 rpm for 15 minutes.

[0123] Internal control markings:

[0124] Repeat the above steps, but use DNP-BSA instead of IL-6 antibody.

[0125] Step S2. Pretreatment of the blood filtration pad and conjunctival pad.

[0126] Blood filtration pads and conjugate pads can be made of glass fiber and both require pretreatment before use.

[0127] 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. Optionally, the blood filtration pad can be wetted with saline and buffer solutions to reduce differences in sample density as it reaches the conjugation pad and cellulose membrane. Optionally, surfactants, hydrophilic polymers, and blocking agents can be added simultaneously to reduce nonspecific binding, increase the signal-to-noise ratio, and improve effective blocking.

[0128] The function of the binding pad is to adsorb quantum dot fluorescent nanospheres-protein conjugates, maintaining their activity within their shelf life and enabling effective release as the sample flows through the binding pad. The performance of the binding pad can be effectively improved by using coating blocking reagents, protein stabilizers, and separation and release reagents.

[0129] Blood filter pad pretreatment

[0130] 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.

[0131] The blood filter pad pretreatment solution contains 20 mM borate-borax buffer (pH 7.80 ± 0.05), 2% PVP, 0.4% sodium caseinate, 2% Tritium-X100, 1% S9, 0.04% sodium azide, 0.2 mg / mL anti-rbc monoclonal antibody, and 1.0 mg / mL active heterophile antibody blocking agent.

[0132] In some implementations, the blood filtration pad uses a glass fiber membrane with a thickness of 18 mm.

[0133] The drying process involves drying at 37℃ for 12 hours.

[0134] Combined with pad pretreatment

[0135] 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.

[0136] The conjugate pad treatment solution contains 20 mM borate-borax buffer (pH 7.80 ± 0.05), 0.2% PEG2000, 1% PVP-10, 0.5% sodium casein, 0.5% S9, 0.05% Proclin 300, 2% sucrose, and 1.0 mg / ml active heterophile antibody blocker (pH 7.0 ± 0.05).

[0137] In some implementations, the glass cellulose membrane used for the conjugate pad containing the antibody mixture has a width of 11 mm.

[0138] Step S3. Coating of the binding pad: Solidification of quantum dot fluorescent nanosphere coupling conjugates

[0139] The quantum dot fluorescent nanosphere-labeled antibody conjugate and the internal control conjugate (quantum dot fluorescent nanosphere-labeled DNP-BSA conjugate) prepared above were resuspended and mixed by ultrasonication and then diluted with labeling diluent.

[0140] The labeled diluent contains 25 mM Tris-base, 0.1% Tween-20, 1% BSA, 0.1% ProClin 300, 3% trehalose, 2% sucrose, and pH=8.0±0.05.

[0141] 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 (quantum dot fluorescent nanosphere-labeled antibody conjugate) and the labeled internal control conjugate (quantum dot fluorescent nanosphere-labeled DNP-BSA conjugate) were calculated, respectively.

[0142] The volume of the quantum dot fluorescent nanosphere-labeled antibody conjugate = 0.2~0.6 × the volume required for the coating solution.

[0143] The volume of the quantum dot fluorescent nanosphere-labeled DNP-BSA conjugate is 0.01~0.03 × the volume required for the coating solution. The remaining volume of the coating solution is supplemented with labeling diluent.

[0144] 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.

[0145] In some embodiments, the quantum dot fluorescent microsphere-labeled IL-6 antibody, the quantum dot fluorescent microsphere-labeled DNP-BSA conjugate, and the labeling diluent are mixed in a certain proportion and then sprayed onto the sealed conjugate pad 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.

[0146] Step S4. Coating with cellulose membrane

[0147] First, prepare the cellulose membrane coating solution. Calculate the total volume of the coating solution, dilute IL-6 capture antibody 2 to a final concentration of 1–3 mg / mL, dilute the internal control conjugate (DNP-BSA) to a final concentration of 0.2–0.5 mg / mL, the final concentration of 20% trehalose is 1%, and the final concentration of 1% BSA is 0.1%. The remaining coating solution is replenished with 1×BS (pH=8.0).

[0148] 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. Spraying can be performed using a gold spray gun 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.

[0149] In some implementations, IL-6 capture antibody 2 and DNP mouse monoclonal antibody are taken separately and then sprayed onto the corresponding detection line and control line, respectively.

[0150] In some implementation schemes, IL-6 capture antibody 2 with a concentration of 1 mg / mL and DNP mouse monoclonal antibody with a concentration of 0.5 mg / mL are respectively streaked onto the detection line and control line of the nitrocellulose membrane at a speed of 1 μL / cm, a length of 300 mm, and a speed of 60 cm / min; the parameters are 37 °C for 12–16 h.

[0151] Step S5. Lamination and assembly of test strips

[0152] The test strip of this invention comprises 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 (5) in sequence by adhesive. The blood filtration pad (1) has a sample dispensing port.

[0153] The lamination position can be, for example, 1 mm from the edge of the coated cellulose membrane base plate's release paper, with the overlap between the liquid absorbent pad and the cellulose membrane base plate not exceeding 2 ± 0.5 mm. The conjugate pad should be placed face up against the upper edge of the filtration pad's release paper, with the overlap between the conjugate pad and the nitrocellulose membrane not exceeding 2 ± 0.5 mm. The filtration pad should be placed against the lower edge of the filtration pad's release paper. After assembly, the base plate is cut into test strips using a strip cutter. The test strips are inserted into the retaining position of the cartridge, and the top cover is closed to obtain the IL-6 test strip. For example, the base plate can be cut into 4 mm wide strips using a strip cutter.

[0154] Preparation of reagent calibrators

[0155] 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).

[0156] 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.

[0157] Applications of quantum dot immunofluorescence test strips

[0158] In another aspect, the present invention provides the use of the quantum dot immunofluorescence test strip in the preparation of a kit for detecting the levels of interleukin-6 (IL-6) in the peripheral blood of a patient.

[0159] In another aspect, the present invention provides a kit comprising the quantum dot immunofluorescence test strip.

[0160] In some implementations, the test kit includes the test strip, buffer solution, desiccant, and instructions for use.

[0161] In some implementations, the sample dilution buffer is a phosphate buffer, such as 10 mM phosphate buffer.

[0162] In some embodiments, the desiccant is silica gel.

[0163] 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.

[0164] While various embodiments of the invention have been described above, it should be understood that they are merely examples and not limitations. Many modifications to the disclosed embodiments can 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 to any of the embodiments described above.

[0165] 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.

[0166] Example 1

[0167] Preparation of Interleukin-6 Assay Kit (Quantum Dot Immunofluorescence Chromatography)

[0168] Experimental materials:

[0169] This invention uses CdSe / ZnS core-shell quantum dot fluorescent microspheres from Merck Chemical Company, USA, with an emission wavelength of 620 nm and catalog number 919497.

[0170] Antibody raw materials: IL-6 was purchased from Feipeng Biotechnology Co., Ltd., catalog numbers: FAB-B003-2F7 (detection antibody) and FAB-B003-7E5 (capture antibody).

[0171] Blocker: Fipeng Biotechnology Co., Ltd., Product No.: HIER-E-015.

[0172] RBC antibody: EastCoast Bio, USA, catalog number: HM1079.

[0173] Nitrocellulose membrane: Merck Chemicals CN140.

[0174] Blood filtration membrane: GF2 blood filtration membrane from Shanghai Jieyi Biotechnology Co., Ltd., product number JY-Q01.

[0175] Glass cellulose membrane sample pad: Ahlstrom 8964, item number: JY-BX102.

[0176] Glass cellulose membrane conjugate pad: Ahlstrom 8964, item number: JY-BX102.

[0177] PVC base plate: Shanghai Jieyi Biotechnology Co., Ltd., item number: JY-D103.

[0178] All other chemical auxiliaries are from Merck Chemicals.

[0179] Gold spraying film scrubbing instrument: Shanghai Jiening Biotechnology Co., Ltd., Model: XYZ3010 all-in-one machine.

[0180] Instrument: Quantum dot immunochromatographic analyzer (Novizan Quantum Dot Fluorescence Immunoassay Analyzer QD-S1200).

[0181] Preparation of various main solutions

[0182] (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;

[0183] (2) 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) solution: final concentration 0.5 mg / mL;

[0184] (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 an 8:2 ratio, and dilute tenfold to obtain 20mM BS (borax-boric acid buffer solution) pH=7.8.

[0185] (4) Microsphere blocking solution: 0.5% BSA, 20mM glycine, Tris-HCl, pH=8.00±0.05.

[0186] (5) Microsphere preservation solution: 25mM Tris-base, 0.1% Tween20, 0.15M NaCl, 1% BSA, 0.1% ProClin300, 3% trehalose, pH = 9.0±0.05.

[0187] (6) Label diluent: 25mM Tris-base, 0.1% Tween-20, 1% BSA, 0.1% ProClin 300, 3% trehalose, 2% sucrose, pH=8.0±0.05.

[0188] (7) Blood filter pad pretreatment solution: 20mM borate-borax buffer pH7.80±0.05, 2% PVP, 0.4% sodium caseinate, 2% Tritium-X100, 1% S9, 0.04% sodium azide, 0.2mg / ml anti-rbc monoclonal antibody, 1.0mg / ml active heterophile antibody blocking agent.

[0189] (8) Conjugate pad pretreatment solution: 20mM borate-borax buffer pH 7.80±0.05, 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.

[0190] Step S1. Quantum dot fluorescent microsphere labeling process (using quantum dot fluorescent microspheres to label antibodies)

[0191] activation

[0192] Measure 50 mM MES buffer into a centrifuge tube, then measure quantum dot fluorescent microspheres (1% solid content) into the same tube and mix well to achieve a microsphere to MES buffer volume ratio of 1:10. Simultaneously add the calculated EDC + NHS (0.5 mg / mL each). Activate by shaking for 20 min, then invert on a 30 ℃ oven for 30 min. After inversion, place the centrifuge tube containing the mixture into a centrifuge, balance it, and set the centrifuge parameters to 14000 rpm for 15 min.

[0193] mark

[0194] After centrifugation, remove the centrifuge tube and discard the supernatant. Add coupling buffer to restore the volume. Mix well and add IL-6-labeled antibody 1 or DNP-BSA, then incubate at room temperature with shaking for 30 minutes. After shaking, place the centrifuge tube containing the mixture into a centrifuge, balance the mixture, and set the centrifuge parameters to 14000 rpm for 15 minutes.

[0195] Closed

[0196] After centrifugation, remove the centrifuge tubes and discard the supernatant. Add the microsphere blocking solution, mix well and disperse, and vortex for 30 minutes. After vortexing, place the centrifuge tubes containing the mixture into a centrifuge, balance the mixture, set the centrifuge parameters, and centrifuge at 14000 rpm for 15 minutes.

[0197] After centrifugation, remove the centrifuge tube, discard the supernatant, add the microsphere preservation solution again, and mix well.

[0198] Label the marked microsphere solution and place it in the refrigerator for later use.

[0199] Configuration of internal control tags

[0200] Repeat the above steps, but use DNP-BSA instead of IL-6 labeled antibody 1.

[0201] Step S2. Pretreatment of blood filtration pads and conjunctival pads

[0202] The blood filtration pads and conjugate pads are made of fiberglass and require pretreatment before use.

[0203] The blood filter pad is the area for sample addition. It has a sample inlet for adding the sample. Because some samples differ significantly in pH, ionic strength, etc., 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. Simultaneously, surfactants, hydrophilic polymers, and blocking agents are added to reduce non-specific binding, increase the signal-to-noise ratio, and improve effective blocking.

[0204] The function of the binding pad is to adsorb quantum dot fluorescent nanospheres-protein conjugates, maintaining their activity within their shelf life and enabling effective release as the sample flows through the binding pad. The performance of the binding pad can be effectively improved by using coating blocking reagents, protein stabilizers, and separation and release reagents.

[0205] Blood filter pad pretreatment

[0206] 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.

[0207] Combined with pad pretreatment

[0208] 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.

[0209] Step S3. Coating of the conjugate pad (solid-state formation of quantum dot fluorescent nanosphere coupling conjugate)

[0210] The quantum dot fluorescent nanosphere-labeled antibody conjugate and the internal control conjugate (quantum dot fluorescent nanosphere-labeled DNP-BSA conjugate) prepared above were resuspended and mixed by ultrasonication and then diluted with labeling diluent.

[0211] Based on the required volume of the coating conjugate pad, the volumes of the labeled microsphere-conjugated antibody conjugate (quantum dot fluorescent nanosphere-labeled antibody conjugate) and the labeled internal control conjugate (quantum dot fluorescent nanosphere-labeled DNP-BSA conjugate) were calculated respectively.

[0212] The volume of the quantum dot fluorescent nanosphere-labeled antibody conjugate is 0.2–0.6 × the volume of the coating solution required.

[0213] The volume of the quantum dot fluorescent nanosphere labeled DNP-BSA conjugate is 0.01 to 0.03 times the volume required for the coating solution. The remaining volume of the coating solution is supplemented with label diluent.

[0214] Add the calculated solution to a centrifuge tube and mix thoroughly using a vortex mixer. Spray the solution 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 hours, the conjugate pad containing the antibody mixture is ready for use.

[0215] Step S4. Coating with nitrocellulose membrane

[0216] First, prepare the nitrocellulose membrane coating solution. Calculate the total volume of the coating solution, dilute IL-6 capture antibody 2 to a final concentration of 1 mg / mL, dilute the internal control conjugate (DNP-BSA) to a final concentration of 0.5 mg / mL, the 20% trehalose to a final concentration of 1%, and the 1% BSA to a final concentration of 0.1%. The remaining coating solution is replenished with 1×BS (pH=8.0).

[0217] Add the prepared nitrocellulose membrane coating solution to a centrifuge tube and mix thoroughly using a vortex mixer. Using a streak sprayer, apply the capture antibody and internal control antibody onto the nitrocellulose membrane at a spray 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.

[0218] Step S5. Lamination and assembly of test strips

[0219] like Figure 1 and 2 As shown, the filtration pad, conjunctival pad, nitrocellulose membrane, and absorbent pad are fixed to the PVC base plate in sequence using adhesive. The liquid absorbent pad is 1 mm away from the edge of the nitrocellulose membrane base plate's release liner. The overlap between the liquid absorbent pad and the nitrocellulose membrane base plate should not exceed 2 ± 0.5 mm. The conjunctival pad is placed face up against the upper edge of the filtration pad's release liner. The overlap between the conjunctival pad and the nitrocellulose membrane should not exceed 2 ± 0.5 mm. The filtration pad is placed against the lower edge of the filtration pad's release liner.

[0220] 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 IL-6 test strip.

[0221] Preparation of reagent calibrators

[0222] 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).

[0223] 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.

[0224] Example 2

[0225] Clinical evaluation

[0226] The evaluation methods were based on the Clinical and Laboratory Standards Institute (CLSI) EP9-A2 and EP9-A3 documents.

[0227] I. Test Products

[0228] The interleukin-6 (IL-6) assay strip (quantum dot fluorescence immunochromatography) was prepared according to Example 1.

[0229] II. Selection of Cases Participating in the Trial

[0230] From January to June 2024, samples were collected from 100 infected patients aged 18 to 90 years admitted to the ICU of Linfen People's Hospital. These patients included 58 males and 42 females. Homologous serum and peripheral blood samples were collected from each case.

[0231] III. Sample Collection

[0232] After the specimen collection is completed, test the following methods:

[0233] The method to be evaluated was: using interleukin-6 (IL-6) assay strips (quantum dot fluorescence immunochromatography) prepared according to the specific implementation method, the sample type was peripheral blood, the IL-6 level was detected and the test results were recorded;

[0234] Comparison Method A: The Interleukin-6 (IL-6) / Pseudomonas sulphurin (PSP) combined detection kit (time-resolved lateral flow immunoassay) developed by Shanxi Kangjianen Biotechnology Co., Ltd. was used. The sample type was serum, and the IL-6 level was detected and the results were recorded.

[0235] Comparison Method B: The Siemens Interleukin-6 (IL-6) Assay Kit (chemiluminescence method) was used to detect IL-6 levels in serum samples and record the results.

[0236] IV. Statistical Research

[0237] (1) Quantitative detection: Referring to the Clinical and Laboratory Standards Institute (CLSI) EP9-A3 document, IL-6 levels in 100 homologous peripheral blood and serum samples were measured in parallel with the method to be evaluated using two comparative methods (Method A and Method B). Scatter plots were drawn with the results of the comparative method as the X-axis and the results of the method to be evaluated as the Y-axis. The regression equation Y=a+bX was calculated, and the consistency and bias of different detection methods were analyzed using the Bland-Altman method.

[0238] (2) Expected bias at the medical decision level: Referring to the CLSIEP9-A2 document, the expected bias of the method to be evaluated and the comparison methods A and B at the IL-6 medical decision level (7.0 pg / mL) and their 95% confidence interval (CI) were obtained, and the relative expected bias of the three methods (expected bias / medical decision level × 100%) was calculated.

[0239] V. Test Results

[0240] 1. General Information

[0241] The test results for 100 subjects are shown in Table 1 below.

[0242] Table 1: Test results for IL-6 (unit: pg / mL)

[0243]

[0244]

[0245]

[0246] 2. Results Analysis

[0247] (1) Regression analysis

[0248] In 100 samples, the slope of the regression equation for the comparison method A and the IL-6 method to be evaluated in this invention is 1.0011, and the intercept is 2.0096. The regression equation for the method to be evaluated in this invention and the comparison method A is Y = 1.0011X + 2.0096 (R²). 2 =0.9999). The slope of the regression equation for the IL-6 level of the comparison method B and the method to be evaluated in this invention is 1.0006, and the intercept is 1.327. The regression equation for the method to be evaluated in this invention and the comparison method B is Y=1.0006X+1.327(R). 2 =0.9999). See also Figure 5A , Figure 5B .

[0249] (2) Bland-Altman bias analysis

[0250] Bland-Altman bias analysis showed that the bias for IL-6 levels ranged from -9.53 to 7.32. The smallest bias was observed between the evaluated method and comparison method A (-9.53), with 5% (5 / 100) of the data falling outside the maximum permissible error range (i.e., 1.96 standard deviations). The smallest bias was also observed between the evaluated method and comparison method B (-8.52), with 7% (7 / 100) of the data falling outside the maximum permissible error range (i.e., 1.96 standard deviations). See the bias analysis curves below. Figure 6A , Figure 6B .

[0251] 3. Expected bias of the method to be evaluated in this invention at the medical decision level.

[0252] When using Method A as a reference, the expected bias of the IL-6 level at the medical decision level (7 pg / mL) measured by the method to be evaluated according to the present invention is 2.0173 (95% CI: 1.288358533~2.74624) pg / mL. This 95% CI includes the allowable error, indicating that the bias is acceptable, suggesting that the method to be evaluated according to the present invention is comparable to the serum IL-6 detection results of Method A. When using Method B as a reference, the expected bias of the IL-6 level at the medical decision level measured by the method to be evaluated according to the present invention is 1.3312 (95% CI: 0.513013~2.14939) pg / mL. This 95% CI also includes the allowable error, indicating that the bias is acceptable, suggesting that the method to be evaluated according to the present invention is comparable to the serum IL-6 detection results of Method B. The expected bias between Method A and Method B is 0.7081 (95% CI: -0.13695 to 1.55315) 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 2.

[0253] Table 2. Bias at IL-6 medical decision levels for the three methodologies

[0254]

[0255] 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 method for preparing a test strip for detecting IL-6 in peripheral blood, characterized in that, The method includes the following steps: Step S1 - Labeling antibodies with CdSe / ZnS quantum dot fluorescent microspheres, step S1 includes: (1) Quantum dot fluorescent nanospheres were added to microsphere labeling buffer, followed by the addition of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide to activate the microspheres; (2) Then add monoclonal antibody IL-6 labeled antibody 1 to couple the microspheres with IL-6 labeled antibody 1 to obtain microsphere conjugate complex; (3) Further add microsphere blocking solution to achieve the blocking of microsphere coupling complex; (4) The supernatant was removed by low-temperature centrifugation, and the solid precipitate was reconstituted with buffer solution to obtain IL-6 labeled antibody 1 labeled with CdSe / ZnS quantum dot fluorescent microspheres; (5) Repeat steps (1)-(4) above, but use DNP-BSA instead of IL-6 to label antibody 1 to obtain DNP-BSA conjugate labeled with CdSe / ZnS quantum dot fluorescent microspheres. Step S2 - The filtration pad and conjunctival pad are pretreated with filtration pad pretreatment solution and conjunctival pad pretreatment solution, respectively. The filtration pad pretreatment solution contains 20mM borate-borax buffer (pH 7.80±0.05), 2% PVP, 0.4% sodium caseinate, 2% Tritium-X100, 1% S9, and 0.04% sodium azide. The conjunctival pad pretreatment solution contains 20mM borate-borax buffer (pH 7.80±0.05), 0.2% PEG2000, 1% PVP-10, 0.5% sodium caseinate, 0.5% S9, 0.05% Proclin300, 2% sucrose, and pH 7.0±0.

05. The filtration pad is immobilized with anti-human erythrocyte antibodies. Step S3 - The conjugate pad is coated with a label dilution solution containing IL-6 labeled antibody 1 labeled with CdSe / ZnS quantum dot fluorescent microspheres and DNP-BSA conjugate labeled with CdSe / ZnS quantum dot fluorescent microspheres; Step S4 - Take IL-6 capture antibody 2 and DNP mouse monoclonal antibody respectively and spray them onto the test line and control line; Step S5 - Laminate and assemble the test strips.

2. The method according to claim 1, characterized in that, In step S1, CdSe / ZnS quantum dot fluorescent microspheres are used as quantum dot fluorescent nanospheres, MES buffer solution is used as the labeling buffer for CdSe / ZnS quantum dot fluorescent microspheres, and the volume ratio of microspheres to MES buffer is 1:10 to 1:20; 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is used as an activator.

3. The method according to claim 1, 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, and pH=8.0±0.

05.

4. A test strip prepared by the method according to any one of claims 1 to 3, comprising: The components include a blood filtration pad (1), a cellulose membrane conjugate pad (2), a cellulose membrane (3), an absorbent pad (4), and a base plate (5). 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 an IL-6 detection line (T line) and a DNP mouse monoclonal antibody control line (C line). The cellulose membrane conjugate pad (2) contains a combination of IL-6-labeled antibody 1 labeled with CdSe / ZnS quantum dot fluorescent microspheres and DNP-BSA conjugate labeled with CdSe / ZnS quantum dot fluorescent microspheres; and The blood filter pad (1) is fixed with anti-human red blood cell antibodies.

5. The test strip according to claim 4, characterized in that, The top end of the blood filtration pad (1) presses against the bottom end of the cellulose membrane conjugate pad (2) and is attached thereto. The top end of the cellulose membrane conjugate pad (2) presses against the bottom end of the cellulose membrane (3) and is attached thereto. The top end of the cellulose membrane (3) is pressed against the bottom end of the absorbent pad (4) and is attached thereto.

6. The test strip according to claim 4, characterized in that, The IL-6 level was tested using peripheral blood samples.

7. Use of the test strip according to any one of claims 4 to 6 in the preparation of a assay kit for detecting IL-6 in the peripheral blood of a patient.

8. The use according to claim 7, characterized in that, The test was performed 10 minutes after the sample was added.

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