Composition, reagent, kit and use thereof in PCT and IL-6 detection and PCT and IL-6 detection method

Abstract

The present invention relates to the field of clinical medical diagnosis, and in particular to compositions, reagents, kits, and their applications in PCT and IL-6 detection, as well as methods for detecting PCT and IL-6. The present invention screens and optimizes the reagent components, and directly mixes the composition (fluorescent probe) originally fixed on the test strip with the sample after storage in a dry form. The test results show that the linear range of the PCT detection of the present invention is 0.05ng / mL to 100ng / mL; the linear range of IL-6 detection is 1.5pg / mL to 5000pg / mL, and the coefficient of variation is below 5%. It has good repeatability, high accuracy, good anti-interference ability, and good clinical application prospects.

Description

Technical Field

[0001] The present invention relates to the field of clinical medical diagnosis, and in particular to a composition, a reagent, a kit, and their use in PCT and IL-6 detection, as well as a method for detecting PCT and IL-6. Background Art

[0002] PCT (procalcitonin) is a protein whose plasma levels increase in severe bacterial, fungal, or parasitic infections, as well as in sepsis and multi-organ failure. PCT levels are not elevated in autoimmune, allergic, or viral infections. Localized bacterial infections, minor infections, and chronic inflammation do not cause PCT levels to rise. PCT reflects the activity of the systemic inflammatory response. Factors influencing PCT levels include the size and type of the infected organ, the type of bacteria, the severity of inflammation, and the status of the immune response.

[0003] Interleukin-6 (IL-6) is a multifunctional cytokine that plays a vital role in the body's immune response, acute phase reaction, and hematopoiesis. Increased IL-6 levels often indicate an inflammatory response, particularly inflammation caused by bacterial or viral infection. Increased IL-6 levels are directly proportional to the severity of inflammation, making it a useful indicator for assessing the severity of inflammation. In the intensive care unit (ICU), IL-6 testing can be used to early assess the risk of sepsis and effectively predict its onset. Elevated IL-6 levels are closely associated with the severity and prognosis of sepsis patients.

[0004] IL-6 and PCT, when used together, can help differentiate between bacterial and viral infections. In bacterial infections, both IL-6 and PCT are typically elevated, whereas in viral infections, IL-6 may be elevated without an associated PCT increase. PCT and IL-6 are commonly used clinical indicators of inflammatory factors. Their increases and decreases can reflect changes in infectious diseases, providing richer information for diagnosis.

[0005] Currently, there are multiple methods for the combined detection of PCT and IL-6, primarily fluorescence immunoassay and chemiluminescence. The high cost of chemiluminescence reagents hinders widespread adoption. Currently, with conventional fluorescence immunoassays, the upper limit of the linear range for PCT is typically only achieved with chemiluminescence, which reaches 100 ng / mL. Especially when PCT antibody activity is high, the PCT signal at the lower end of the linear range increases rapidly, making it difficult to achieve a linear range of 100 ng / mL. Furthermore, the sensitivity of the combined PCT and IL-6 assays compared to either assay alone needs to be further improved.

[0006] Therefore, the sensitivity and detection range of the combined detection of IL-6 and PCT need to be further improved, and it is particularly necessary to develop new reagent combinations and detection methods for PCT and IL-6 detection with high sensitivity and suitable for high-concentration PCT detection. Summary of the Invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide a composition, a reagent, a kit and their use in the detection of PCT and IL-6, as well as a method for detecting PCT and IL-6.

[0008] The present invention provides a composition comprising a PCT antibody labeled with quantum dot microspheres, an IL-6 antibody labeled with quantum dot microspheres, a sheep anti-chicken IgY antibody labeled with quantum dot microspheres, a PCT capture antibody, and a blocking agent;

[0009] In the present invention, the PCT antibody labeled with quantum dot microspheres is PCT monoclonal antibody 2, which is purchased from Feipeng Bio, with the product number: PCT-REAB-G1-015;

[0010] In the present invention, the capture antibody is PCT monoclonal antibody 3, purchased from Feipeng Bio, product number: PCT-Ab4#;

[0011] In the present invention, the IL-6 antibody labeled with quantum dot microspheres is IL-6 monoclonal antibody 2, purchased from Haitide Biotechnology, product number: L152;

[0012] In the present invention, the capture antibody and PCT antibody labeled with quantum dot microspheres were screened; in Group 1 of Example 2 of the present invention, the PCT capture antibody was replaced with PCT monoclonal antibody 1. The test results showed that the linear range of PCT detection did not change much; however, the repeatability of PCT was reduced; and the sensitivity was correspondingly reduced.

[0013] In Group 3 of Example 2 of the present invention, the IL-6 antibody labeled with quantum dot microspheres was replaced with IL-6 monoclonal antibody 3, and the signal value at the lower limit of the linear range of IL-6 detection decreased, and the sensitivity was reduced.

[0014] The present invention optimizes and screens quantum dot microsphere-labeled sheep anti-chicken IgY antibodies, replaces the quantum dot microsphere-labeled sheep anti-chicken IgY antibodies with quantum dot microsphere-labeled sheep anti-rabbit IgG, and adaptively changes the quality control antibody to rabbit IgG; the test results show that the use of sheep anti-chicken IgY antibodies has better stability.

[0015] In the present invention, the blockers include active blockers and passive blockers; the present invention screened the blockers and replaced the active blocker of Meridian, item number: A66800H with blocker 2, which was purchased from Feipeng Bio, item number: HIER-R-013, and is a passive blocker; the test results showed that the detection linearity was good, but the sensitivity of PCT using the passive blocker was reduced, and the precision of PCT and IL-6 was reduced.

[0016] In the present invention, the activation solution used in the preparation of the quantum dot microsphere-labeled PCT antibody, the quantum dot microsphere-labeled IL-6 antibody and / or the quantum dot microsphere-labeled sheep anti-chicken IgY antibody includes: 10mM MES with a total added mass of EDC and NHS being 800 to 4000 times the mass of the quantum dot microspheres, wherein the mass ratio of EDC to NHS is 1:1.

[0017] Specifically, in the preparation of the PCT antibody labeled with quantum dot microspheres, the activation solution used is 10mM MES containing EDC and NHS, the total mass of the added EDC and NHS is 2000-3000 times the mass of the quantum dot microspheres, and specifically 2000 is optimal; the mass ratio of EDC and NHS is 1:1; in the PCT antibody labeled with quantum dot microspheres, the mass ratio of quantum dot microspheres to antibodies is 1:500;

[0018] In the preparation of the IL-6 antibody labeled with quantum dot microspheres, the activation solution used is 10mM MES containing EDC and NHS. The total mass of the added EDC and NHS is 2000-3000 times the mass of the quantum dot microspheres, with 2400 being the optimal amount. The mass ratio of EDC to NHS is 1:1. In the PCT antibody labeled with quantum dot microspheres, the mass ratio of quantum dot microspheres to antibody is 1:500.

[0019] In the preparation of the quantum dot microsphere-labeled sheep anti-chicken IgY antibody, the activation solution used is 10mM MES containing EDC and NHS, the total added mass of the EDC and NHS is 800 times the mass of the quantum dot microspheres, the mass ratio of the EDC and NHS is 1:1; the mass ratio of quantum dots to antibodies is 1:400.

[0020] In the present invention, the mass of the quantum dot microspheres is calculated based on the solid content (1‰), where the solid content refers to the proportion or mass fraction of the solid component in a substance or mixture.

[0021] The present invention optimizes the reagent components in the method for preparing antibodies labeled with quantum dot microspheres; specifically, the method includes: (1) screening of activation solution buffer components; the present invention respectively tries 10mM MES, 20mM MES, 50mM MES and 10mM Hepes, the test results showed that 10mM MES had the highest sensitivity; (2) the dosage of EDC and NHS in the activation solution was optimized, and the total added mass of EDC and NHS in the preparation of PCT antibody labeled with quantum dot microspheres was adjusted to 4000 times, 3000 times, 2000 times, or 1000 times the mass of quantum dot microspheres, and the total added mass of EDC and NHS in the preparation of IL-6 antibody labeled with quantum dot microspheres was adjusted to 4000 times, 3000 times, 2400 times, or 1200 times the mass of quantum dot microspheres. The test results showed that the total added mass of EDC and NHS in the preparation of PCT antibody was adjusted to 2000 times the mass of quantum dot microspheres, and the total added mass of EDC and NHS in the preparation of IL-6 antibody labeled with quantum dot microspheres was adjusted to 2400 times the mass of quantum dot microspheres, which resulted in a higher signal value at the low concentration point and higher sensitivity.

[0022] In the present invention, the blocking agents used in the preparation of the quantum dot microsphere-labeled PCT antibody, quantum dot microsphere-labeled IL-6 antibody and / or quantum dot microsphere-labeled goat anti-chicken IgY antibody include CE510 and / or CE210; specifically CE510 and CE210.

[0023] In the present invention, the blocking agent used in the preparation of quantum dot microsphere-labeled PCT antibody, quantum dot microsphere-labeled IL-6 antibody and / or quantum dot microsphere-labeled sheep anti-chicken IgY antibody was optimized. The test results showed that the precision and sensitivity decreased when the blocking agent was changed to 10% BSA.

[0024] The IL-6 antibody labeled with quantum dot microspheres is used at a concentration of 0.26 μg / mL to 0.33 μg / mL, with 0.3 μg / mL being the optimal concentration.

[0025] The concentration of the PCT antibody labeled with quantum dot microspheres is 0.18 μg / mL to 0.25 μg / mL, with 0.2 μg / mL being the optimal concentration.

[0026] The use concentration of the quantum dot microsphere-labeled sheep anti-chicken IgY antibody is 0.016 μg / mL to 0.33 μg / mL, with 0.02 μg / mL being the optimal concentration.

[0027] The PCT capture antibody is used at a concentration of 0.03 μg / mL to 0.05 μg / mL, with 0.04 μg / mL being optimal.

[0028] The blocker is used at a concentration of 0.3 mg / mL to 0.55 mg / mL, with 0.42 mg / mL being the optimal concentration.

[0029] Taking quantum dot microsphere-labeled IL-6 antibody as an example, the present invention optimizes the use concentrations of quantum dot microsphere-labeled IL-6 antibody, quantum dot microsphere-labeled PCT antibody, and quantum dot microsphere-labeled sheep anti-chicken IgY antibody. Due to space limitations, only the optimization results of the IL-6 antibody concentration labeled with quantum dot microspheres are shown. In the present invention, the concentration of IL-6 antibody labeled with quantum dot microspheres is set to 12-24 μg / mL; the test results show that when the concentration of IL-6 antibody 2 labeled with quantum dot microspheres is 16-20 μg / mL, the sensitivity and linearity meet the requirements, but are optimal at 18 μg / mL, that is, the optimal concentration is 0.3 μg / mL. The detection linearity is good within the use concentration range of 0.2 μg / mL-0.23 μg / mL, but the sensitivity is low; the detection sensitivity is good within the use concentration range of 0.36 μg / mL-0.4 μg / mL, but the linearity is poor.

[0030] The present invention provides a reagent comprising the composition of the present invention and a drying buffer;

[0031] The drying buffer comprises: Tris, NaCl, Tween-20, trehalose, glycine, preservatives and protective agents;

[0032] The preservative includes Proclin 300 and / or sodium azide; in a specific embodiment of the present invention, it is Proclin 300.

[0033] The protective agent includes BSA and / or casein.

[0034] Furthermore, the drying buffer comprises: 15-25 mM Tris, 145-155 mM NaCl, 0.09-0.11 wt% Tween-20, 0.5-1.5 wt% BSA, 4.5-5.5 wt% protective agent, 0.8-1.2 wt% glycine and 0.015-0.025 wt% preservative.

[0035] In a specific embodiment of the present invention, the drying buffer comprises 20 mM Tris solution, 150 mM NaCl, 0.1 wt % Tween-20, 1 wt % BSA, 5 wt % trehalose, 1 wt % glycine and 0.02 wt % Proclin 300, pH 7.5;

[0036] The present invention optimizes the drying buffer, specifically including optimizing the buffer components and concentration. The present invention attempts to replace the buffer component with a 50mM Hepes solution, or optimize the components of the drying buffer, specifically, such as replacing Tween-20 with Tween-80, replacing BSA with casein, or replacing trehalose with sucrose, or optimize the ratio of the drying buffer components, specifically, optimizing the trehalose concentration within the range of 1wt%, 3wt%, 7wt%, and 9wt%. Test results show that stability is reduced after component replacement, and when the trehalose content is within the range of 3wt% to 5wt%, the protective effect on the antibody is optimal, and the reagent can be made more stable during storage, with 5wt% being the optimal concentration.

[0037] In the present invention, the reagent may be present in the form of a solution or in the form of a dry powder after drying, which is not limited by the present invention. Specifically, in a specific embodiment of the present invention, the composition and the drying buffer are mixed and dried using a drying technique, and then dissolved in a reagent, water, or liquid sample before use. Drying the reagent improves the stability of the reagent and makes it more suitable for storage and transportation of the reagent. Furthermore, the reagent solution described in the present invention may be present in liquid form, which is not limited by the present invention.

[0038] Specifically, the drying condition is 18-28° C., drying for 24 hours, and the humidity in the last 2 hours of drying is required to be ≤11%.

[0039] The present invention provides a kit comprising at least one of the reagents described herein and at least one of the following:

[0040] PCT coating antibody; and / or

[0041] IL-6 coated antibody; and / or

[0042] Chicken IgY antibody; and / or

[0043] Coating fluid; and / or

[0044] sample pad buffer;

[0045] The PCT coating antibody is PCT monoclonal antibody 1, purchased from Feipeng Bio, catalog number: PCT-REAB-G1-016;

[0046] The IL-6 coated antibody is IL-6 monoclonal antibody 1, purchased from Haitide Biotechnology, product number L395;

[0047] The coating solution includes: Na2HPO4, NaH2PO4, NaCl, trehalose and Proclin300;

[0048] Specifically: 14-18 mM Na2HPO4, 2-4.5 mM NaH2PO4, 145-155 mM NaCl, 4.5-5.5 wt% trehalose and 0.035-0.045 wt% Proclin300;

[0049] More specifically: 16 mM Na2HPO4•12H2O, 3.8 mM NaH2PO4•2H2O, 150 mM NaCl, 5 wt% trehalose, 0.04 wt% Proclin300;

[0050] The present invention screened the coating solution components, and the test results showed that the stability decreased after the reagent components and ratios were replaced.

[0051] The sample pad buffer is a borate buffer containing Tween-20, trehalose, mouse anti-human erythrocyte monoclonal antibody, and Proclin 300.

[0052] Furthermore, the sample pad buffer is a 20 mM borate buffer containing 0.45 wt% to 0.55 wt% Tween-20, 3 wt% to 5 wt% trehalose, 0.025 wt% to 0.035 wt% mouse anti-human erythrocyte monoclonal antibody, and 0.015 wt% to 0.025 wt% Proclin 300.

[0053] Furthermore, in a specific embodiment of the present invention, the sample pad buffer is a 20 mM borate buffer with a pH of 8.0, containing 0.5 wt % Tween-20, 5 wt % trehalose, 0.03 wt % mouse anti-human erythrocyte monoclonal antibody, and 0.02 wt % Proclin 300.

[0054] The concentration of the IL-6 coated antibody is 1.4 mg / mL to 1.8 mg / mL, with the optimal concentration being 1.8 mg / mL;

[0055] The concentration of the PCT coating antibody is 1.1 mg / mL to 1.5 mg / mL, and the optimal concentration is 1.5 mg / mL.

[0056] The concentration of the chicken IgY antibody is 0.5-1.1 mg / mL; preferably, the concentration of the chicken IgY antibody is 0.8 mg / mL.

[0057] The present invention adjusts the concentration of PCT and IL-6 coated antibodies. The test results show that the detection effects at the following two concentrations are not as good as those at 1.8 mg / mL IL-6 coated antibody concentration and 1.5 mg / mL PCT coated antibody concentration;

[0058] The concentration of PCT coating antibody is 1.1 mg / mL and the concentration of IL-6 coating antibody is 1.4 mg / mL; or

[0059] The concentration of the PCT coated antibody was 1.3 mg / mL, and the concentration of the IL-6 coated antibody in the second test line was 1.6 mg / mL.

[0060] The kit described in the present invention can be used for the combined detection of PCT and IL-6. In this method, the composition (fluorescent probe) originally immobilized on a test strip is stored in a dry form in a centrifuge tube, and unlabeled detection and control antibodies are immobilized on the test strip. During sample testing, the composition (fluorescent probe) is dissolved in water and then mixed with the sample, allowing the sample to fully react with the fluorescently labeled PCT, IL-6 antibodies, and control complex. This significantly improves the sensitivity and precision of PCT and IL-6 detection, achieving a CV of less than 5% and a detection limit of 0.05 ng / mL for PCT and 1.5 pg / mL for IL-6. Furthermore, an appropriate amount of PCT capture antibody is added to the fluorescent probe to extend the linear range of PCT to 0.05 ng / mL to 100 ng / mL. The present invention also tested a conventional reagent with a conjugate pad. Unlike this patent, the sample is not directly mixed with the fluorescent probe, but rather the components of the fluorescent probe are sprayed onto the conjugate pad. Test results indicate that the method of the present invention is superior for detection.

[0061] The kit of the present invention further comprises a test strip;

[0062] The test strip comprises: a bottom plate (7), and a sample pad (1), a chromatography membrane (5), and a water-absorbing pad (6) fixed to the bottom plate (7) in sequence; and the sample pad (1), the chromatography membrane (5), and the water-absorbing pad are in contact with (6) in sequence;

[0063] The chromatographic membrane is provided with a first detection line (2), a second detection line (3) and a quality control line (4) in sequence from the end close to the sample pad to the end of the absorbent pad;

[0064] The first detection line is immobilized with a PCT-coated antibody;

[0065] The second detection line is immobilized with an IL-6 coated antibody;

[0066] Chicken IgY antibody is fixed on the quality control line.

[0067] The present invention provides the use of at least one of the following I to III) in the detection of PCT and / or IL-6:

[0068] 1), the composition of the present invention;

[0069] II), the reagent of the present invention;

[0070] III), the kit of the present invention.

[0071] The present invention provides a method for detecting PCT and / or IL-6 for non-diagnostic purposes, which is to detect PCT and / or IL-6 using at least one of the following i) to iii):

[0072] i), the composition of the present invention;

[0073] ii), the reagent of the present invention;

[0074] iii), the kit of the present invention.

[0075] The present invention provides a non-diagnostic immunoassay method for PCT and / or IL-6, which specifically comprises the following steps:

[0076] Step 1: mixing a sample with the composition of the present invention to obtain a mixture;

[0077] Step 2: applying the mixture to the sample pad in the kit of the present invention to react, thereby obtaining a reacted reagent strip;

[0078] Step 2: Read the signal of the test strip after the reaction to obtain the test result.

[0079] Further,

[0080] The reaction conditions are 18° C. to 28° C. for 10 to 20 minutes.

[0081] The sample includes whole blood, serum, and / or plasma.

[0082] The present invention adds a certain amount of PCT capture antibody to the fluorescent probe to improve the linear range, and at the same time adds a blocker to reduce the background, improve the effective signal value of low-value sample concentration, and improve the sensitivity. The linear range of PCT reaches 0.05ng / mL~100ng / mL. By adjusting the amount of IL-6 coated antibody and labeled antibody, the linear range of IL-6 reaches 1.5pg / mL~5000pg / mL. Compared with conventional immunochromatography reagents with conjugate pads, the present application does not have a conjugate pad and sample diluent. Only pure water and sample need to be added during testing, which is simple to operate. The sample is first mixed with the fluorescent probe, which can fully combine the antigen and antibody, greatly improving the sensitivity and precision of PCT and IL-6 detection. The fluorescent probe adopts a room temperature drying method, which greatly reduces the cost of reagent production and can be stored and transported at room temperature.

[0083] The present invention screens and optimizes the reagent components, stores the composition (fluorescent probe) originally fixed on the test strip in a dry form, and then directly mixes it with the sample for use in an immunochromatographic test for the combined detection of PCT and IL-6. Test results show that the linear range of PCT detection of the present invention is 0.05 ng / mL to 100 ng / mL; the linear range of IL-6 detection is 1.5 pg / mL to 5000 pg / mL, and the coefficient of variation is below 5%. The method has good repeatability, high precision, and good anti-interference ability, and has good clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 Schematic diagram of the structure of the test strip in the quantum dot fluorescent immunochromatographic reagent for the combined detection of PCT and IL-6 of the present invention; wherein: 1. sample pad; 2. first detection line 1; 3. second detection line 2; 4. quality control line; 5. NC detection membrane (chromatographic membrane); 6. absorbent paper (absorbent pad); 7. bottom plate;

[0085] Figure 2 Shown is the standard curve of PCT;

[0086] Figure 3 shows the standard curve of IL-6;

[0087] Figure 4 The figure shows the correlation analysis of the test system plasma test value and the Roche electrochemiluminescence PCT plasma test value obtained by testing PCT plasma samples in the correlation test of clinical samples;

[0088] Figure 5 The figure shows the correlation analysis diagram of the test system plasma test value obtained by testing IL-6 plasma samples in the correlation test of clinical samples and the Roche electrochemiluminescence IL-6 plasma test value;

[0089] Figure 6 The figure shows the correlation analysis of the whole blood test value of the test system obtained by testing PCT homologous whole blood samples in the correlation test of clinical samples and the Roche electrochemiluminescence PCT plasma test value;

[0090] Figure 7 The figure shows the correlation analysis chart of the whole blood test value of the test system obtained by testing IL-6 homologous whole blood samples in the correlation test of clinical samples and the Roche electrochemiluminescence IL-6 plasma test value. DETAILED DESCRIPTION

[0091] The present invention provides compositions, reagents, kits, and their use in PCT and IL-6 detection, as well as methods for detecting PCT and IL-6. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0092] In the present invention, the PCT antibody labeled with quantum dot microspheres is also referred to as the PCT monoclonal antibody 2 labeled with quantum dot microspheres; the IL-6 antibody labeled with quantum dot microspheres is also referred to as the IL-6 monoclonal antibody 2 labeled with quantum dot microspheres; the goat anti-chicken IgY antibody labeled with quantum dot microspheres is also referred to as the goat anti-chicken IgY antibody labeled with quantum dot microspheres;

[0093] CV, or the coefficient of variation, is the ratio of the standard deviation (SD) to the mean (mean), usually expressed as a percentage. CV is used to measure the stability and repeatability of test results.

[0094] The fluorescent probe (a composition for the combined detection of PCT and IL-6) of the present invention is dried at room temperature, significantly reducing reagent production costs and enabling room-temperature storage and transportation. Compared to conventional immunochromatographic reagents with conjugate pads, this application does not require a conjugate pad or sample diluent. Testing requires only the addition of pure water and sample, simplifying operation and significantly improving the sensitivity and precision of PCT and IL-6 detection. The detection limit for IL-6 is 1.5 pg / mL, with an upper limit of the linear range of 5000 pg / mL; the detection limit for PCT is 0.05 ng / mL, with an upper limit of the linear range of 100 ng / mL, and the CVs are both below 5%.

[0095] Currently, using ordinary fluorescent immunoassays, the CV is required to be below 15%, the linear range of IL-6 can be achieved [5pg / mL~1000pg / mL], the detection limit of PCT can be achieved to 0.5ng / mL, and the upper limit of the linear range of PCT is mostly only 100ng / mL by chemiluminescence methods.

[0096] At the same time, adding an appropriate amount of PCT capture antibody and blocking agent to the fluorescent probe improves the linear range of PCT. Ultimately, the reagent CV can be controlled within 5%, and the linear range of PCT reaches 0.05ng / mL to 100ng / mL, and the linear range of IL-6 reaches 1.5pg / mL to 5000pg / mL.

[0097] The invention adds a certain amount of PCT capture antibody to the fluorescent probe to improve the linear range, and at the same time adds a blocker to reduce the background, increase the effective signal value of low-value sample concentration, and improve the sensitivity. The linear range of PCT reaches 0.05ng / mL~100ng / mL. By adjusting the amount of IL-6 coated antibody and labeled antibody, the linear range of IL-6 reaches 1.5pg / mL~5000pg / mL. Compared with conventional immunochromatography reagents with conjugate pads, the present application does not have a conjugate pad and sample diluent. Only pure water and sample need to be added during testing, which is simple to operate. In addition, the sample is first mixed with the fluorescent probe, which can fully combine the antigen and antibody, greatly improving the sensitivity and precision of PCT and IL-6 detection. The fluorescent probe is dried at room temperature, which greatly reduces the cost of reagent production and can be stored and transported at room temperature.

[0098] The PCT antibody is PCT monoclonal antibody 2; the PCT capture antibody is PCT monoclonal antibody 3; the PCT coating antibody is PCT monoclonal antibody 1; the numbers 1, 2 or 3 are used to illustrate or distinguish the differences between the three PCT monoclonal antibodies; the differences are as follows:

[0099] Capture antibody-PCT monoclonal antibody 3, purchased from Feipeng Bio, catalog number: PCT-Ab4#;

[0100] The PCT antibody labeled with quantum dot microspheres is PCT monoclonal antibody 2, which was purchased from Feipeng Biotechnology with the product number: PCT-REAB-G1-015;

[0101] The PCT coating antibody was PCT monoclonal antibody 1, which was purchased from Feipeng Biotechnology, catalog number: PCT-REAB-G1-016;

[0102] The IL-6 antibody in the IL-6 antibody labeled with quantum dot microspheres is IL-6 monoclonal antibody 2, which was purchased from Haitide Biotechnology with the product number: L152;

[0103] IL-6 coating antibody, IL-6 monoclonal antibody 1, purchased from Haitide Biotechnology, catalog number: L395;

[0104] Quantum dot microsphere-labeled goat anti-chicken IgY antibody was purchased from Arista Biologicals, catalog number ABGAC-0500;

[0105] Blocker 1, purchased from Meridian, product number: A66800H, is an active blocker.

[0106] Blocker 2, purchased from Feipeng Biotechnology, product number: HIER-R-013, is a passive blocker.

[0107] The fluorescent substance used for fluorescent labeling was carboxyl-modified quantum dot microspheres (Beijing Nanogene Biotechnology Co., Ltd., Cat. No. FM610C);

[0108] Solid content refers to the proportion or mass fraction of solid components in a substance or mixture.

[0109] The test materials used in the present invention are all common commercial products and can be purchased on the market. The present invention is further described below with reference to the following examples:

[0110] Example 1 Immunochromatographic Reagent for Combined Detection of PCT and IL-6

[0111] 1. Preparation of fluorescent probes for combined detection of PCT and IL-6

[0112] Fluorescent probes for combined detection of PCT and IL-6, including: quantum dot microsphere-labeled PCT monoclonal antibody 2 (PhiPeng Biotechnology, Catalog No. PCT-REAB-G1-015), quantum dot microsphere-labeled IL-6 monoclonal antibody 2 (Haipide Biotechnology, Catalog No. L152), quantum dot microsphere-labeled goat anti-chicken IgY antibody (Arista Biologicals, Catalog No. ABGAC-0500), capture antibody-PCT monoclonal antibody 3 (PhiPeng Biotechnology, Catalog No. PCT-Ab4#), and blocking agent (Meridian, Catalog No. A66800H);

[0113] (1) Preparation of PCT monoclonal antibody 2 labeled with quantum dot microspheres: A certain amount of quantum dot microspheres (solid content of 1‰) was diluted with activation solution (10 mM MES containing EDC and NHS, pH 6.5) at a dilution ratio of 1:4 and activated at room temperature for 15 min. The amount of EDC and NHS added was 2000 times the mass of the quantum dot microspheres (calculated based on the solid content), and the mass ratio of EDC to NHS was 1:1. After the reaction, the resulting solution was centrifuged to remove the supernatant, re-dissolved with coupling solution (20 mM boric acid buffer containing 0.1 wt% Tween-20, pH 7.0), and PCT monoclonal antibody 2 was added (quantum dot to antibody mass ratio of 1:500) and reacted at room temperature for 2 hours. After the reaction, 5 μL CE510 and 5 μL CE210 were added, and the solution was blocked at room temperature for 1 hour. After the blocked solution was centrifuged to remove the supernatant, 50 μL drying buffer was added, and ultrasonic mixing was performed to make the final concentration of PCT monoclonal antibody 2 labeled with quantum dot microspheres 0.5 mg / ml. The solution was stored at 2-8°C in the dark.

[0114] (2) Preparation of quantum dot microsphere-labeled IL-6 monoclonal antibody 2: During the preparation of quantum dot microsphere-labeled IL-6 monoclonal antibody 2, the amount of EDC and NHS added was 2400 times the mass of the quantum dot microspheres. Other procedures were the same as those described for quantum dot microsphere-labeled PCT monoclonal antibody 2.

[0115] (3) Preparation of quantum dot microspheres labeled sheep anti-chicken IgY: During the preparation process, the amount of EDC and NHS added was 800 times the mass of the quantum dot microspheres; the coupling solution was 20 mM boric acid buffer with a pH of 8.0 containing 0.1 wt% Tween-20; the mass ratio of quantum dots to antibodies was 1:400; 50 μL of drying buffer was added and ultrasonically mixed to make the final concentration of quantum dot microspheres labeled sheep anti-chicken IgY 0.3 mg / mL. Other procedures were the same as those described for quantum dot microspheres labeled PCT monoclonal antibody 2.

[0116] (4) Preparation of drying buffer: Prepare 20 mM Tris solution, adjust the pH to 7.5 with hydrochloric acid, and add 150 mM NaCl, 0.1 wt% Tween-20, 1 wt% BSA, 5 wt% trehalose, 1 wt% glycine, and 0.02 wt% Proclin 300.

[0117] (5) Preparation of fluorescent probe solution: Add appropriate amounts of quantum dot microsphere-labeled PCT monoclonal antibody 2, quantum dot microsphere-labeled IL-6 monoclonal antibody 2, quantum dot microsphere-labeled sheep anti-chicken IgY antibody and PCT capture antibody-monoclonal antibody 3 and blocking agent 1 to the drying buffer to prepare a fluorescent probe solution containing 12 μg / mL quantum dot microsphere-labeled PCT antibody, 18 μg / mL quantum dot microsphere-labeled IL-6 monoclonal antibody 2, 1.2 μg / mL quantum dot microsphere-labeled sheep anti-chicken IgY antibody, 2.4 μg / mL PCT monoclonal antibody 3 (PCT capture antibody) and 25.2 mg / mL blocking agent;

[0118] (6) Packaging and drying of fluorescent probes: Pack 5 μL of the fluorescent probe solution into the corresponding centrifuge tubes. Weigh the centrifuge tubes after each packing. Confirm whether the difference between the weight of the centrifuge tube after packing and the weight of the empty test tube before packing exceeds ±0.5 μL. After confirming that the packing volume is consistent, transfer the test tubes to an electronic moisture-proof cabinet for drying. The temperature is controlled at 18~28℃ and the drying time is 24 hours. The humidity in the last 2 hours is required to be ≤11% to obtain a dried fluorescent probe.

[0119] 2. Reagent Strip Preparation

[0120] like Figure 1 , which is a schematic structural diagram of the test strip in the quantum dot fluorescent immunochromatographic reagent for the combined detection of PCT and IL-6 of the present invention.

[0121] (1) Coating solution composition: 16 mM Na2HPO4•12H2O, 3.8 mM NaH2PO4•2H2O, 150 mM NaCl, 5 wt% trehalose, 0.04 wt% Proclin300.

[0122] (2) Preparation of the first and second test line solutions: PCT monoclonal antibody 1 (PCT coated antibody, Feipeng Bio, catalog number: PCT-REAB-G1-016) was diluted with coating solution (5 wt% trehalose, 50 mM phosphate solution at pH 7.4) to 1.5 mg / mL to prepare the first test line; IL-6 monoclonal antibody 1 (IL-6 coated antibody, Haipeptide Bio, catalog number: L395) was diluted with coating solution to 1.8 mg / mL to prepare the second test line.

[0123] (3) Preparation of quality control solution: Dilute chicken IgY (Arista Biologicals, Catalog No.: AGCIG-0100) antibody to 0.8 mg / mL with coating solution to prepare the quality control solution.

[0124] (4) Preparation of reagent strips:

[0125] On the adhesive-backed base plate, the NC membrane was first pasted in an overlapping manner. The first test line (PCT test line) and the second test line (IL-6 test line) were drawn on the nitrocellulose NC membrane (chromatographic membrane) near the sample pad end. The quality control line (chicken IgY) was drawn near the end of the absorbent paper. The spacing between the first test line, the second test line, and the quality control line was 3 mm.

[0126] The marking volume of the test line and the quality control line was 1 μL / cm, and the marking speed was 5 cm / s. After marking, the sample was placed in an oven and dried at 50°C for 48 hours.

[0127] Sample pad preparation: Prepare a sample pad buffer consisting of 0.5 wt% Tween-20, 5 wt% trehalose, 0.03 wt% mouse anti-human erythrocyte monoclonal antibody, and 0.02 wt% Proclin 300 dissolved in 20 mM boric acid buffer, pH 8.0. Soak the glass fiber in the sample pad buffer for five minutes, remove it from the oven, set to 37°C, and dry it for 24 hours. Cut it to the desired size to obtain the sample pad.

[0128] Applying and Cutting the Test Strips: Attach a sample pad and absorbent paper to each end of the NC membrane. Place the sample pad on the side closest to the test line, pressing 1-2 mm above the NC membrane. Place the absorbent paper on the side closest to the control line, pressing it above the NC membrane, with the other side flush with the outer edge of the base plate. Set the test strip cutter to a 4 mm width and verify that the cut surface is smooth.

[0129] Assembly of the reagent strips:

[0130] Put it into a plastic shell, put the sample pad on the top of the shell, put the absorbent paper on the end of the shell, compact it with a shell press, then put it into an aluminum foil bag together with the desiccant, seal it and store it in a drying cabinet for later use.

[0131] 3. Detection of reagents for combined detection of PCT and IL-6

[0132] Standard curve testing and IC card manufacturing

[0133] (1) Test materials:

[0134] PCT and IL-6 standards were diluted with calf serum at the following concentrations: PCT: 0, 0.05, 0.5, 1, 10, 50.0, 100 ng / mL; IL-6: 0, 1.5, 5, 50, 250, 500, 2000, 5000 pg / mL.

[0135] (2) Test method:

[0136] Take 200 μL of pure water and add the dried fluorescent probe to obtain a liquid fluorescent probe reagent;

[0137] 100 μL of the standard was mixed with the liquid fluorescent probe reagent to obtain a sample mixture, wherein the concentration of the quantum dot microsphere-labeled PCT monoclonal antibody 2 was 0.2 μg / mL, the concentration of the quantum dot microsphere-labeled IL-6 monoclonal antibody 2 was 0.3 μg / mL, the concentration of the quantum dot microsphere-labeled goat anti-chicken IgY antibody was 0.02 μg / mL, the concentration of the capture antibody-PCT monoclonal antibody 3 was 0.04 μg / mL, and the concentration of the blocking agent was 0.42 mg / mL;

[0138] Add 100 μL of the sample mixture to the sample well of the test strip and react at room temperature for 15 minutes to obtain the reacted test strip. Read the T1 / C / D and T2 / C / D values ​​using the immunochromatographic reader (model: ACCABLE-T or ACCABLE-E) that comes with the test strip. Each standard concentration is measured three times. The test curve data is as follows:

[0139] Table 1. PCT standard curve

[0140]

[0141] Table 2. IL-6 standard curve

[0142]

[0143] (3) Making IC cards:

[0144] The concentration values ​​of the standard substances PCT and IL-6 are used as the horizontal axis, and the average value (T / C) of each concentration standard substance is plotted against the concentration to make a standard curve, such as Figure 2 and Figure 3 (The standard curve was prepared using the ELISA Calc regression / fitting calculation program - version 0.2, selecting cubic spline difference), burned into the IC card, and imported into the supporting instrument.

[0145] 4. Performance Testing

[0146] 1. Precision test

[0147] (1) Experimental materials: Clinical samples were provided by relevant hospitals. The fixed concentrations of PCT and IL-6 were: PCT concentrations of 1, 10, and 50 ng / mL, and IL-6 concentrations of 10, 100, and 500 pg / mL, respectively.

[0148] (2) Test Method: Precision testing was performed using a fluorescent immunoassay analyzer (model: ACCABLE-T or ACCABLE-E) compatible with the test kit of this application. Clinical samples for PCT and IL-6 were tested using the test method described in Example 1, with each test item and concentration repeated 10 times.

[0149] (3) Analysis of test results:

[0150] Table 3. PCT and IL-6 precision test results

[0151]

[0152] As shown in Table 3, the combined PCT and IL-6 detection reagent of the present invention achieves precision of less than 5% for both PCT and IL-6. This compares to chromatographic reagents with conjugate pads, where the sample is diluted with a diluent before being added to the wells. The immune reaction occurs as the sample flows through the membrane, resulting in an incomplete reaction and poor precision. The present patented test method simply requires adding pure water to the fluorescent probe and then adding the sample for thorough mixing, resulting in a complete and uniform reaction and significantly improved precision.

[0153] 2. Anti-interference ability

[0154] (1) Test materials: Clinical samples were provided by relevant hospitals. The fixed concentrations of PCT and IL-6 were: PCT concentrations were 1 ng / mL and 10 ng / mL, and IL-6 concentrations were 10 pg / mL and 500 pg / mL, respectively. Interfering substances such as hemoglobin, triglycerides, and bilirubin were purchased externally.

[0155] (2) Test Method: Interference tests were performed using a fluorescent immunoassay analyzer (model: ACCABLE-T or ACCABLE-E) compatible with the test kit of this application. Using the test method described in Example 1, clinical samples of PCT and IL-6 were tested after the addition of different concentrations of interfering substances. No interfering substances were added to the control group.

[0156] (3) Analysis of test results:

[0157] Table 4. PCT and IL-6 anti-interference detection results

[0158]

[0159] As shown in Table 4, the relative deviations of the interfering substances at different concentrations tested by the reagent for combined detection of PCT and IL-6 of the present invention and the control group were all within 10%, indicating that the addition of hemoglobin (within the concentration range of 5 mg / mL), triglycerides (within the concentration range of 17 mmol / L), and bilirubin (3.5×10 2 The effects of interfering substances such as (in the concentration range of 100 μmol / L) on the results of reagent test samples are all within an acceptable range.

[0160] 3. Stability-accelerated test

[0161] (1) Experimental materials: Clinical samples were provided by relevant hospitals. The low, medium, and high concentrations of PCT and IL-6 were: PCT concentrations of 1, 10, and 50 ng / mL, and IL-6 concentrations of 10, 500, and 2500 pg / mL, respectively.

[0162] (2) Test method: A stability test was conducted using a fluorescent immunoassay analyzer (model: ACCABLE-T or ACCABLE-E) compatible with the test kit of this application. The reagent was accelerated at 37°C for 60 days, and PCT and IL-6 samples were tested to verify the stability of the reagent.

[0163] (3) Analysis of test results:

[0164] The stability test data are as follows:

[0165] Table 5. PCT, IL-6 stability - accelerated test results

[0166]

[0167] As shown in Table 5, the relative deviations of the low, medium, and high levels of PCT and IL-6 tested within 60 days of acceleration were all within 10% compared with the results before acceleration, indicating that the stability of the reagents met the requirements.

[0168] 4. Correlation testing of clinical samples

[0169] (1) Test materials:

[0170] Clinical samples were provided by relevant hospitals, including 50 cases of PCT and IL-6. All samples were valued by Roche electrochemiluminescence method, including plasma and homologous whole blood. The distribution range of PCT content was 0.05~100ng / mL, and the distribution range of IL-6 content was 1.5~5000pg / mL.

[0171] (2) Test method:

[0172] The correlation test of clinical samples was performed using a fluorescent immunoassay analyzer (model: ACCABLE-T or ACCABLE-E) that is compatible with the kit of this application, with each sample tested once. The specific test results are shown in Table 11.

[0173] (3) Analysis of test results:

[0174] Fifty Roche electrochemiluminescence (ECL) samples were tested according to the test method. The correlation between whole blood and plasma was analyzed using the plasma test results as a benchmark. The test results are shown in Table 4.

[0175] Table 6. Correlation analysis

[0176]

[0177]

[0178] The PCT and IL-6 concentrations detected by chemiluminescence were used as the horizontal axis, and the PCT and IL-6 plasma concentrations detected by this application were used as the vertical axis. A scatter plot was drawn to conduct correlation analysis. Figure 4 、 Figure 5 50 clinical plasma samples were tested, and the PCT results in the range of 0.05~100ng / mL were basically consistent with those of electrochemiluminescence detection, with the correlation R 2 =0.9972; the IL-6 detection results were basically consistent with those of electrochemiluminescence detection in the range of 1.5~5000pg / mL, and the correlation R 2 =0.9946. R 2 All of them are greater than 0.98, indicating that the reagent prepared in this application has good consistency with the electrochemiluminescence method in detecting PCT and IL-6 plasma, which fully meets the requirements of clinical trials.

[0179] The PCT and IL-6 concentrations detected by chemiluminescence were used as the horizontal axis, and the PCT and IL-6 whole blood concentrations detected by this application were used as the vertical axis. A scatter plot was drawn and correlation analysis was performed. Figure 6 、 Figure 750 clinical whole blood samples were tested, and the PCT results in the range of 0.05~100ng / mL were basically consistent with those of electrochemiluminescence detection, with a correlation R 2 =0.9983; the results of IL-6 in the range of 1.5~5000pg / mL were basically consistent with those of electrochemiluminescence detection, and the correlation R 2 =0.9958. R of the two reagents 2 All of them are greater than 0.98, indicating that the reagent prepared in the present application has good consistency with the electrochemiluminescence method in detecting PCT and IL-6 in whole blood, and meets the needs of clinical detection of PCT and IL-6.

[0180] Example 2 Optimization of the present invention

[0181] 1. Screening of PCT capture antibodies and blockers in fluorescent probe solution

[0182] The fluorescent probe solution was prepared as in Example 1, except that the capture antibody in Group 1 was PCT monoclonal antibody 1; and the blocker in Group 2 was replaced with blocker 2 (Feipeng Bio, product number: HIER-R-013).

[0183] The reagents prepared in Example 1, Group 1, and Group 2 were used to test samples containing 0.5 ng / mL PCT and 50 pg / mL IL-6 to verify the precision of the reagents. Clinical samples were provided by relevant hospitals and were assayed using Roche electrochemiluminescence. The test results are shown in Table 7.

[0184] Table 7. Precision test results

[0185]

[0186] The reagents prepared in Example 1, Group 1, and Group 2 were used to test the signal values ​​of samples at a concentration of 0 and the lower limit of the linear range (PCT: 0.05 ng / mL, IL-6: 1.5 pg / mL) for PCT and IL-6, as well as the upper limit of the linear range (PCT: 100 ng / mL, IL-6: 5000 pg / mL) and the next highest concentration (PCT: 50 ng / mL, IL-6: 2000 pg / mL) for PCT and IL-6 to verify the sensitivity and linearity of the reagent detection. The test results are shown in Table 8 below:

[0187] Table 8. Sensitivity and linearity test results

[0188]

[0189] As can be seen from the data in Table 7, the low-value repeatability of PCT in Example 1 is better than that in Group 1, while the low-value repeatability of IL-6 in Example 1 and Group 1 does not change much, indicating that the PCT capture antibody selected in this patent can improve the precision of PCT and has no significant interference with the precision of IL-6. The low-value repeatability of PCT and IL-6 in Example 1 is better than that in Group 2, indicating that the blocker selected in this patent has an improving effect on the precision of both PCT and IL-6.

[0190] It can be seen from Table 8 that the upper limit concentration point of the PCT linear range of the three sets of data is 2 times the concentration of the second highest point, and the corresponding three sets of signal values ​​are also close to a 2-fold relationship, indicating that the linearity of the three sets of PCT meets the requirements. The signal value of PCT 0ng / mL in Example 1 is lower than that in Group 2, indicating that the blocker of this patent is effective and can effectively block the interference of heterophilic antibodies and reduce background. The signal value of 0.05ng / mL in Example 1 is higher than that in Group 1, indicating that the capture antibody in this patent can minimize the impact on sensitivity while improving linearity. The changes in PCT capture antibodies and blockers in the three sets of data have no effect on the linearity and sensitivity of IL-6. The present invention selects a PCT capture antibody with lower activity, and adds a specific blocker to block the interference of heterophilic antibodies, reduce background, and increase the effective signal value of low concentrations, thereby improving sensitivity and precision of low concentrations.

[0191] 2. Screening of IL-6 Antibodies

[0192] Different from Example 1, the IL-6 antibody labeled with quantum dot microspheres is IL-6 monoclonal antibody 3 (brand Yibaixin, product number MIS04); the IL-6 antibody (coated antibody) on the second detection line is IL-6 monoclonal antibody 4 (brand Yibaixin, product number MIS03) - Group 3.

[0193] Using the test strips and fluorescent probes prepared in Example 1 and Group 3, the signal values ​​of samples with a concentration of 0 and the lower limit of the linear range (PCT: 0.05 ng / mL, IL-6: 1.5 pg / mL) corresponding to PCT and IL-6 were tested to verify the sensitivity of the reagent detection. The test results are shown in the following table:

[0194] Table 9. Screening results of IL-6 antibodies

[0195]

[0196] As can be seen from Table 9, the signal value of the reagent produced in Example 1 when testing the lower limit concentration point of the linear range of IL-6 is higher than that of Group 3, indicating that the antibody pairing in this patent is better than the antibody pair in Group 3 and has a certain effect on improving the sensitivity of IL-6.

[0197] 3. Screening the concentration of quantum dot-labeled IL-6 antibody 2 in the fluorescent probe

[0198] The difference from Example 1 is that the concentration of quantum dot-labeled IL-6 antibody 2 in Group 4 fluorescent probe is 12 μg / mL~24 μg / mL (the concentration of each component in the fluorescent probe solution, converted to the working concentration as shown in the brackets in the table below). The specific grouping is shown in Table 10.

[0199] The reagents prepared in Example 1 and Group 4 were used to test the signal values ​​of samples at zero concentration and the lower limit of the linear range (PCT: 0.05 ng / mL, IL-6: 1.5 pg / mL) for PCT and IL-6, as well as the upper limit of the linear range (PCT: 100 ng / mL, IL-6: 5000 pg / mL) and the second highest concentration (PCT: 50 ng / mL, IL-6: 2000 pg / mL) for PCT and IL-6, to verify the sensitivity and linearity of the reagent detection. The test results are shown in Table 10:

[0200] Table 10. Concentration optimization of quantum dot labeled IL-6 antibody 2

[0201]

[0202] As shown in Table 10, when the quantum dot microsphere-labeled IL-6 antibody 2 was tested at concentrations of 12 μg / mL and 14 μg / mL, the signal value at the 1.5 pg / mL IL-6 concentration was below 0.01, indicating that the signal value was too low and the sensitivity was lower than that of other concentration groups. At 22 μg / mL and 24 μg / mL, the ratio of the highest to the second highest IL-6 signal value was less than 2, indicating that the high-value linearity was lower than that of other concentration groups. When the quantum dot microsphere-labeled IL-6 antibody 2 was tested at concentrations of 16 to 20 μg / mL, both sensitivity and linearity met the requirements. When the concentration of quantum dot-labeled IL-6 antibody 2 was 18 μg / mL, the signal value was higher than 16 μg / mL when testing the 1.5 pg / mL concentration point; when testing the 5000 and 2000 pg / mL concentration points, the ratio of the signal values ​​corresponding to the two concentration points, the 18 μg / mL concentration group was higher than the 20 μg / mL concentration group, and the change in the concentration of IL-6 antibody 2 labeled with quantum dot microspheres had no effect on the reagent performance of PCT. The above data show that when the amount of IL-6 antibody labeled with quantum dot microspheres is 18 μg / mL, the sensitivity and linearity are optimal.

[0203] 4. Screening of Sheep Anti-Chicken IgY and Chicken IgY Antibodies

[0204] The difference between Group 5 and Example 1 is that the goat anti-chicken IgY labeled with quantum dot microspheres is changed to goat anti-rabbit IgG; the antibody on the quality control line is rabbit IgG.

[0205] Test strips and fluorescent probes were prepared using Example 1 and Group 5. After accelerating at 37°C for 30 days, samples containing 10 ng / mL PCT and 500 pg / mL IL-6 were tested to verify the stability of the reagents after acceleration. Clinical samples were provided by relevant hospitals and assayed using Roche electrochemiluminescence. The test results are shown in the table below.

[0206] Table 11. Screening of sheep anti-chicken IgY and chicken IgY antibodies

[0207]

[0208] As can be seen from Table 11, the test results of Group 5 after 30 days of acceleration increased by more than 10% compared to those before acceleration. The relative deviation of the test results of Example 1 after acceleration was within 10%, which is better than that of Group 5. The data shows that the sheep anti-chicken IgY and chicken IgY pairing selected in this patent has better reagent stability, making the reagent T / C of this patent more stable.

[0209] 5. Screening of activation solution buffer components

[0210] Groups 6, 7, and 8 were used for screening of activation solution components. The activation solution was prepared as in Example 1, except that in Group 6, the MES content of the activation solution was 20 mM; in Group 7, the MES content of the activation solution was 50 mM; and in Group 8, the MES content of the activation solution was replaced with Hepes at a content of 10 mM.

[0211] The fluorescent probes and test strips prepared from Groups 6 to 8 in Example 1 were used to test the signal values ​​of samples with a concentration of 0 and the lower limit of the linear range (PCT: 0.05 ng / mL, IL-6: 1.5 pg / mL) for PCT and IL-6, respectively, to verify the sensitivity of the reagent detection. The test results are shown in the following table:

[0212] Table 12. Screening of activation solution components

[0213]

[0214] As can be seen from Table 12, the samples tested at 0 concentration in Groups 6 and 7 all showed certain signal values, indicating that the labeled quantum dot-labeled antibodies exhibited some aggregation, with the aggregation becoming more pronounced at higher MES concentrations. The signal value at the lower limit of the linear range in Group 8 was lower than that in Example 1, demonstrating that the activation solution of this patent has a certain effect on improving sensitivity and that aggregation of quantum dot-labeled antibodies is less likely to occur at this concentration.

[0215] 6. Optimization of EDC and NHS dosage in activation solution

[0216] Different from Example 1, during the preparation of PCT antibody 2 labeled with quantum dot microspheres in Group 9, the amount of EDC and NHS added was 4000 times the mass of the quantum dot microspheres; during the preparation of IL-6 antibody 2 labeled with quantum dot microspheres, the amount of EDC and NHS added was 4000 times the mass of the quantum dot microspheres.

[0217] During the preparation of PCT antibody 2 labeled with quantum dot microspheres in group 10, the amount of EDC and NHS added was 3000 times the mass of the quantum dot microspheres; during the preparation of IL-6 antibody 2 labeled with quantum dot microspheres, the amount of EDC and NHS added was 3000 times the mass of the quantum dot microspheres.

[0218] During the preparation of PCT monoclonal antibody 2 labeled with quantum dot microspheres in group 11, the amount of EDC and NHS added was 1000 times the mass of the quantum dot microspheres; during the preparation of IL-6 monoclonal antibody 2 labeled with quantum dot microspheres, the amount of EDC and NHS added was 1200 times the mass of the quantum dot microspheres.

[0219] The reagents prepared in Example 1, Group 9, Group 10, and Group 11 were used to test the signal values ​​of samples with a concentration of 0 and the lower limit of the linear range (PCT: 0.05 ng / mL, IL-6: 1.5 pg / mL) for PCT and IL-6, respectively, to verify the sensitivity of the reagent detection. The test results are shown in the following table:

[0220] Table 13. Optimization of EDC and NHS dosage in activation solution

[0221]

[0222] As can be seen from Table 13, the data in Example 1 and Group 10 are superior to those in the other groups. In Group 9, the signal values ​​for the 0 concentration sample and the sample at the lower limit of the linear range for PCT and IL-6 are inseparable, indicating that the excessive amount of activator causes some aggregation of the labeled quantum dot-labeled antibody. In Group 11, the signal values ​​for PCT and the lower limit of the linear range are both very low, with almost no signal values ​​being detected. These two points indicate that the sensitivity of the reagent is optimal when the amount of EDC and NHS added during IL-6 activation is 2400-3000 times the mass of the quantum dot microspheres, and when the amount of EDC and NHS added during PCT activation is 2000-3000 times the mass of the quantum dot microspheres.

[0223] 7. Screening of sealants

[0224] Group 12 was for the screening of blocking agents. The preparation process of PCT monoclonal antibody 2 labeled with quantum dot microspheres was the same as that in Example 1, except that 10 wt % BSA was used as the blocking agent.

[0225] The reagents prepared in Example 1 and Group 12, respectively, were used to test samples containing 0.5 ng / mL PCT and 50 pg / mL IL-6 to verify the precision of the reagents. Clinical samples were provided by relevant hospitals and were assayed using Roche electrochemiluminescence. The test results are shown in Table 14.

[0226] Table 14. Screening precision of blocking agents

[0227]

[0228] The reagents prepared in Example 1 and Group 12 were used to test the signal values ​​of samples with a concentration of 0 and the lower limit of the linear range (PCT: 0.05 ng / mL, IL-6: 1.5 pg / mL) for PCT and IL-6, respectively, to verify the sensitivity of the reagent detection. The test results are shown in Table 13 below:

[0229] Table 15. Sensitivity of blocking agent screening

[0230]

[0231] As can be seen from the data in Table 14, the repeatability of PCT and IL-6 in Example 1 is better than that in Group 12. As can be seen from the data in Table 15, the sensitivity of PCT and IL-6 in Example 1 is better than that in Group 12, indicating that the blocking effect of the blocking agent in Example 1 is better than that of 10 wt% BSA in Group 12. The quantum dot microspheres blocked in Example 1 can more effectively prevent the nonspecific adsorption of subsequent antibodies, reduce background signals and background interference, and improve the precision and sensitivity of the low-level reagent.

[0232] 8. Screening of components in drying buffer

[0233] Groups 13 to 16 were screened for the concentration of each component in the drying buffer. The drying buffer was prepared as in Example 1, except that the solution in Group 13 was replaced with Hepes solution; the difference in Group 14 was that Tween-20 was replaced with Tween-80; the difference in Group 15 was that BSA was replaced with casein; and the difference in Group 16 was that trehalose was replaced with sucrose.

[0234] Using the test strips and fluorescent probes prepared in Example 1 and Groups 13-16, samples containing 10 ng / mL PCT and 500 pg / mL IL-6 were tested to verify the accuracy of the reagents. Clinical samples were provided by relevant hospitals and were assayed using Roche electrochemiluminescence. The test results are shown in the table below.

[0235] Table 16. Screening of components in drying buffer

[0236]

[0237] As can be seen in Table 16, the PCT and IL-6 accuracy results for Example 1 and Group 15 were superior to those for the other groups, indicating that both BSA and casein can be used as components of the drying protectant. Comparing the accuracy results for Example 1 and Group 15, Example 1 was superior to Group 15, indicating that the combination of components in the drying buffer in Example 1 was optimal, providing better protection for the antibodies during the drying process and maintaining antibody activity.

[0238] 9. Optimization of Trehalose Concentration in Drying Buffer

[0239] Group 17 is for screening the concentration of trehalose in the drying buffer. The preparation of the drying buffer is the same as in Example 1, except that the concentration of trehalose in the drying buffer is: 1 wt%, 3 wt%, 7 wt%, and 9 wt%.

[0240] Using the test strips and fluorescent probes prepared in Example 1 and Group 17, the reagents were accelerated at 37°C for 30 days and tested with samples containing 10 ng / mL PCT and 500 pg / mL IL-6 to verify the reagent's stability. Clinical samples were provided by relevant hospitals and were assayed using Roche electrochemiluminescence. The test results are shown in Table 17.

[0241] Table 17. Screening of drying buffers

[0242]

[0243] As can be seen from the data in Table 17, the relative deviations of the accelerated test results of Example 1 and the 3 wt % trehalose concentration group compared to those before acceleration were better than those of the other groups, indicating that when the trehalose content in the drying buffer is in the range of 3 wt % to 5 wt %, the protective effect on the antibody is optimal, and the reagent can be made more stable during storage.

[0244] 10. Screening of coating fluid components

[0245] Groups 18 and 19 were used for screening of coating fluid components. The coating fluids in Groups 18 and 19 were prepared as in Example 1, except that the phosphate base fluid in the coating fluid of Group 18 was replaced with TRIS; the difference in Group 19 was that the base fluid in the coating fluid was Hepes.

[0246] Using the reagents prepared in Example 1 and Groups 18-19, samples containing 10 ng / mL PCT and 500 pg / mL IL-6 were tested to verify the effect of different coating solutions on the reagent's accuracy. Clinical samples were provided by relevant hospitals and were assayed using Roche electrochemiluminescence. The test results are shown in the table below.

[0247] Table 18. Screening accuracy results of coating solution components

[0248]

[0249] As can be seen from the table, the accuracy in Example 1 is better than that in other groups. The data show that the base solution phosphate buffer in the coating solution of this patent has the best effect.

[0250] 11. Adjustment of PCT and IL-6 Coating Antibody Concentrations

[0251] Groups 20 and 21 are comparisons of the concentration adjustments of PCT and IL-6 coated antibodies; different from Example 1, in Group 20, the concentration of PCT monoclonal antibody 1 (PCT coated antibody) in the first test line was 1.1 mg / mL, and the concentration of IL-6 monoclonal antibody 1 (IL-6 coated antibody) in the second test line was 1.4 mg / mL.

[0252] The concentration of PCT monoclonal antibody 1 (PCT coated antibody) in the first test line in group 21 was 1.3 mg / mL, and the concentration of IL-6 monoclonal antibody 1 (IL-6 coated antibody) in the second test line was 1.6 mg / mL.

[0253] The reagents prepared in Example 1, Groups 20 and 21 were used to test samples containing 80 ng / mL PCT and 4000 pg / mL IL-6 to verify the precision of the reagents in detecting high values ​​of PCT and IL-6. The test results are shown in Table 19.

[0254] Table 19. Precision results adjusted for PCT and IL-6 coating antibody concentrations

[0255]

[0256] The reagents prepared in Example 1, Groups 20 and 21 were used to test the upper limit concentration point of the linear range of PCT and IL-6 (PCT: 100 ng / mL, IL-6: 5000 pg / mL) and the second highest concentration point (PCT: 50 ng / mL, IL-6: 2000 pg / mL) to verify the linearity of the reagent detection. The test results are shown in Table 20.

[0257] Table 20. Linearity results adjusted for PCT and IL-6 coating antibody concentrations

[0258]

[0259] As can be seen from Tables 19 and 20, the high-value precision and linearity of PCT and IL-6 in Example 1 are better than those of the other two groups. As the concentration of the coated antibody increases, the signal value of the corresponding concentration also increases. This shows that increasing the concentration of the coated antibody on the detection line can increase the signal value of the upper limit concentration of the entire linear range, thereby improving the high-value precision.

[0260] 11. Comparison with reagents with conjugate pads

[0261] Group 22 is an adjustment of the reagent strip structure. An 8 mm wide conjugate pad (the conjugate pad is treated using conventional methods) is pasted between the sample pad and the NC membrane of the reagent strip, and the components of the fluorescent probe in Example 1 are sprayed onto the conjugate pad to keep the final concentration of each component unchanged.

[0262] The reagents prepared in Example 1 and Group 22 were used to test samples containing 0.5 ng / mL PCT and 50 pg / mL IL-6 to verify the reagent's detection precision. Clinical samples were provided by relevant hospitals and were assayed using Roche electrochemiluminescence. The test results are shown in Table 21.

[0263] Table 21. Precision results compared with reagents with conjugate pads

[0264]

[0265] The reagents prepared in Example 1 and Group 22 were used to test the signal values ​​of samples with a concentration of 0 and the lower limit of the linear range (PCT: 0.05 ng / mL, IL-6: 1.5 pg / mL) for PCT and IL-6, respectively, to verify the sensitivity of the reagent detection. The test results are shown in Table 22 below:

[0266] Table 22. Comparison of sensitivity test results with reagents with conjugate pads

[0267]

[0268] As can be seen from Tables 21 and 22, the precision and sensitivity of PCT and IL-6 in Example 1 are better than those in Group 22, indicating that the performance indicators of the reagent produced by this patent are better than those of the reagent with a conjugate pad.

[0269] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A composition for the combined detection of PCT and IL-6, characterized in that: Including quantum dot microsphere-labeled PCT antibody, quantum dot microsphere-labeled IL-6 antibody, quantum dot microsphere-labeled sheep anti-chicken IgY antibody, PCT capture antibody and blocking agent; The PCT antibody is traded under the name of Feipeng Bio, with the product number being PCT-REAB-G1-015; The PCT capture antibody is produced by Feipeng Biotechnology and its product number is PCT-Ab4#; The trade name of the IL-6 antibody is Haitide Bio, and the product number is L152; The blockers include active blockers; In the preparation of the PCT antibody labeled with the quantum dot microspheres, the activation solution used is 10 mM MES containing EDC and NHS, and the total mass of the added EDC and NHS is 2000-3000 times the mass of the quantum dot microspheres; In the preparation of the IL-6 antibody labeled with the quantum dot microspheres, the activation solution used was 10 mM MES containing EDC and NHS, and the total mass of the added EDC and NHS was 2400-3000 times the mass of the quantum dot microspheres; In the preparation of the sheep anti-chicken IgY antibody, the activation solution used is 10mM MES containing EDC and NHS, and the total mass of the added EDC and NHS is 800 times the mass of the quantum dot microspheres; The IL-6 antibody labeled with quantum dot microspheres is used at a concentration of 0.26 μg / mL to 0.33 μg / mL; The concentration of the PCT antibody labeled with quantum dot microspheres is 0.18 μg / mL to 0.25 μg / mL; The quantum dot microspheres-labeled sheep anti-chicken IgY antibody is used at a concentration of 0.016 μg / mL to 0.33 μg / mL; The PCT capture antibody is used at a concentration of 0.03 μg / mL to 0.05 μg / mL; The blocker is used at a concentration of 0.3 mg / mL to 0.55 mg / mL.

2. The composition according to claim 1, characterized in that The blocking agents used in the preparation of the quantum dot microsphere-labeled PCT antibody, the quantum dot microsphere-labeled IL-6 antibody and / or the quantum dot microsphere-labeled goat anti-chicken IgY antibody include CE510 and / or CE210.

3. A reagent for the combined detection of PCT and IL-6, characterized in that: Comprising the composition according to claim 1 or 2 and a drying buffer; The drying buffer comprises: 15-25 mM Tris, 145-155 mM NaCl, 0.09-0.11 wt% Tween-20, 0.5-1.5 wt% BSA, 3-5 wt% trehalose, 0.8-1.2 wt% glycine and 0.015-0.025 wt% preservative.

4. A kit for the combined detection of PCT and IL-6, characterized in that: include: At least one of a PCT coating antibody, an IL-6 coating antibody, a chicken IgY antibody, a coating solution and / or a sample pad buffer, and the reagent of claim 3; The trade name of the PCT coating antibody is Feipeng Bio, and the product number is PCT-REAB-G1-016; The IL-6 coated antibody is produced by Haitide Biotechnology with a product number of L395. The coating solution includes: Na2HPO4, NaH2PO4, NaCl, trehalose and Proclin300; The sample pad buffer is a borate buffer containing Tween-20, trehalose, mouse anti-human erythrocyte monoclonal antibody and Proclin300; The concentration of the IL-6 coated antibody is 1.4 mg / mL to 1.6 mg / mL; The concentration of the PCT coated antibody is 1.1 mg / mL to 1.8 mg / mL.

5. The kit according to claim 4, characterized in that Also included are test strips; The test strip comprises: a bottom plate (7), and a sample pad (1), a chromatography membrane (5), and a water-absorbing pad (6) fixed to the bottom plate (7) in sequence; and the sample pad (1), the chromatography membrane (5), and the water-absorbing pad (6) are in contact with each other in sequence; The chromatographic membrane is provided with a first detection line (2), a second detection line (3) and a quality control line (4) in sequence from the end close to the sample pad to the end of the absorbent pad; The first detection line is immobilized with a PCT-coated antibody; The second detection line is immobilized with an IL-6 coated antibody; Chicken IgY antibody is fixed on the quality control line.

6. Use of at least one of the following I to III) in the combined detection of PCT and IL-6: 1), the composition according to claim 1 or 2; II), the reagent according to claim 3; III), the kit according to claim 4 or 5.

7. A combined detection method of PCT and IL-6, characterized in that: The combined detection of PCT and IL-6 is performed using at least one of the following i) to iii): i), the composition according to claim 1 or 2; ii), the reagent according to claim 3; iii) The kit according to claim 4 or 5.

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