Composition, reagent, kit, application of composition in detection of PCT and IL-6 and detection method of PCT and IL-6

Abstract

The invention relates to the field of clinical medical diagnosis, in particular to a composition, a reagent, a kit, application of the composition, the reagent and the kit in PCT and IL-6 detection and a PCT and IL-6 detection method. According to the invention, reagent components are screened and optimized, a composition (fluorescent probe) originally fixed on a test strip is stored in a dry form and then is directly mixed with a sample for immunochromatography detection of PCT and IL-6 combined detection, and test results show that the linear range of PCT detection is 0.05 ng / mL-100 ng / mL; the linear range of IL-6 detection is 1.5 pg / mL-5000 pg / mL, the coefficient of variation is 5% or below, repeatability is good, accuracy is high, the anti-interference capacity is good, and good clinical application prospects are achieved.

Description

Technical Field

[0001] The present invention relates to the field of clinical medical diagnosis, and particularly to compositions, reagents, test kits and their applications in the detection of PCT and IL-6, as well as the detection methods of PCT and IL-6. Background Art

[0002] PCT (procalcitonin) is a protein, and its level in plasma increases when there are severe bacterial, fungal, parasitic infections, as well as sepsis and multiple organ failure. PCT does not increase during autoimmune, allergic and viral infections. Localized limited bacterial infections, mild infections and chronic inflammation do not cause its increase. PCT reflects the activity degree of the systemic inflammatory response. Factors affecting the PCT level include the size and type of the infected organ, the type of bacteria, the degree of inflammation and the status of the immune response.

[0003] Interleukin-6 (IL-6 for short) is a multifunctional cytokine and plays an important role in the immune response, acute-phase response and hematopoiesis function of the human body. The increase of IL-6 usually indicates the presence of an inflammatory reaction in the human body, especially the inflammation caused by bacterial or viral infections. The level of IL-6 increases in direct proportion to the severity of the inflammation, so it can be used as an index to evaluate the severity of inflammation. In the intensive care unit (ICU), the detection of IL-6 can be used for the early assessment of the risk of sepsis and effectively predict the occurrence of sepsis. The increase of the IL-6 level is closely related to the severity and prognosis of sepsis patients.

[0004] The combined use of IL-6 and PCT can help distinguish between bacterial infections and viral infections. In bacterial infections, both IL-6 and PCT usually increase, while in viral infections, IL-6 may increase without the increase of PCT. PCT and IL-6 are commonly used inflammatory factor indicators in clinical practice, and the increase or decrease of their levels can reflect the changes of infectious diseases and provide more abundant information reference for the diagnosis of infectious diseases.

[0005] Currently, there are various methods for the combined detection of PCT and IL-6, mainly fluorescence immunoassay and chemiluminescence assay. The chemiluminescence reagents are expensive and not conducive to popularization at the grass-roots level. Currently, with the ordinary fluorescence immunoassay, the upper limit of the linear range of PCT is mostly only 100 ng / mL that can be achieved by chemiluminescence assay. Especially when the PCT antibody activity is very high, the signal value at the low end of the linearity will increase rapidly, resulting in difficulty in achieving a linearity of 100 ng / mL; moreover, the sensitivity after the combination of PCT and IL-6 still needs to be further improved compared with the single detection.

[0006] Therefore, the sensitivity and detection range of the combined detection based on IL-6 and PCT need to be further improved. It is particularly necessary to develop a new reagent combination and detection method 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, their applications in PCT and IL-6 detection, and a detection method for PCT and IL-6.

[0008] The present invention provides a composition, which includes a PCT antibody labeled with quantum dot microspheres, an IL-6 antibody labeled with quantum dot microspheres, a goat anti-chicken IgY antibody labeled with quantum dot microspheres, a PCT capture antibody, and a blocker;

[0009] In the present invention, in the PCT antibody labeled with quantum dot microspheres, the PCT antibody is PCT monoclonal antibody 2, which is purchased from Fapon Biotech Co., Ltd., product number: PCT-REAB-G1-015;

[0010] In the present invention, the capture antibody is PCT monoclonal antibody 3, which is purchased from Fapon Biotech Co., Ltd., product number: PCT-Ab4#;

[0011] In the present invention, the IL-6 antibody labeled with quantum dot microspheres is IL-6 monoclonal antibody 2, which is purchased from Hytide Biotech Co., Ltd., product number: L152;

[0012] In the present invention, the capture antibody and the PCT antibody labeled with quantum dot microspheres are screened; in group 1 of Example 2 of the present invention, the PCT capture antibody is replaced with PCT monoclonal antibody 1. The test results show that the linear range of PCT detection does not change much; however, the repeatability of PCT decreases; the sensitivity decreases accordingly.

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

[0014] The present invention optimizes and screens the goat anti-chicken IgY antibody labeled with quantum dot microspheres, replaces the goat anti-chicken IgY antibody labeled with quantum dot microspheres with a goat anti-rabbit IgG labeled with quantum dot microspheres, and adaptively changes the quality control antibody to rabbit IgG; the test results show that the stability of using the goat anti-chicken IgY antibody is better.

[0015] In the present invention, the blocker includes an active blocker and a passive blocker; in the present invention, the blocker is screened, and the active blocker with the Meridian, product number: A66800H is replaced with blocker 2, which is purchased from Phoenix Biotech, product number: HIER-R-013, and is a passive blocker; the test results show that the detection linearity is good, but the sensitivity of PCT using the passive blocker is reduced, and the precision of PCT and IL-6 is reduced.

[0016] In the present invention, the activation solution used in the preparation of the PCT antibody labeled with quantum dot microspheres, the IL-6 antibody labeled with quantum dot microspheres, and / or the goat anti-chicken IgY antibody labeled with quantum dot microspheres includes: 10 mM MES in which the total added mass of EDC and NHS is 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 10 mM MES containing EDC and NHS, and the total added mass of EDC and NHS is 2000 to 3000 times the mass of the quantum dot microspheres, specifically, 2000 is optimal; 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;

[0018] In the preparation of the IL-6 antibody labeled with quantum dot microspheres, the activation solution used is 10 mM MES containing EDC and NHS, and the total added mass of EDC and NHS is 2000 to 3000 times the mass of the quantum dot microspheres, specifically, 2400 is optimal; 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 goat anti-chicken IgY antibody labeled with quantum dot microspheres, the activation solution used is 10 mM MES containing EDC and NHS, the total added mass of EDC and NHS is 800 times the mass of the quantum dot microspheres, the mass ratio of EDC to NHS is 1:1; the mass ratio of quantum dots to antibody is 1:400.

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

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

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

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

[0024] The use concentration of the IL-6 antibody labeled with quantum dot microspheres is 0.26 μg / mL to 0.33 μg / mL; among which, 0.3 μg / mL is the optimal.

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

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

[0027] The use concentration of the PCT capture antibody is 0.03 μg / mL to 0.05 μg / mL, and 0.04 μg / mL is the optimal.

[0028] The use concentration of the blocker is 0.3 mg / mL to 0.55 mg / mL, and 0.42 mg / mL is optimal among them.

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

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

[0031] The drying buffer includes: Tris, NaCl, Tween-20, trehalose, glycine, preservative, and protective agent;

[0032] The preservative includes Proclin300 and / or sodium azide; in the specific embodiment of the present invention, it is Proclin300.

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

[0034] Furthermore, the drying buffer includes: 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 the specific embodiment of the present invention, the drying buffer includes 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% Proclin300, pH 7.5;

[0036] The present invention optimizes the drying buffer, specifically including the optimization of buffer components and concentrations; attempts to replace the buffer components with a 50 mM Hepes solution, or optimize the components of the drying buffer, specifically, for example, replacing Tween-20 with Tween-80, BSA with casein, or trehalose with sucrose, or optimizing the ratio of the components of the drying buffer, specifically, for example, optimizing the concentration of trehalose within the range of 1 wt%, 3 wt%, 7 wt%, and 9 wt%. The test results show that the stability decreases after the component replacement, and when the content of trehalose is within the range of 3 wt% to 5 wt%, the protective effect on the antibody is the best, which can make the reagent more stable during storage, and the optimal value is 5 wt%.

[0037] In the present invention, the reagent can exist in the form of a solution or in the form of a dry powder after drying, and the present invention does not limit this; specifically, in the specific embodiments of the present invention, the composition and the drying buffer are mixed and then dried using a drying technique, and are used after being dissolved with a reagent, water, or a liquid sample during specific use; drying the reagent makes the reagent more stable and more suitable for the storage and transportation of the reagent. At the same time, the reagent solution of the present invention can exist in a liquid form, and the present invention does not limit this.

[0038] Specifically, the drying conditions are 18 to 28 °C, drying for 24 hours, and the humidity requirement in the last 2 hours of drying is ≤ 11%.

[0039] The present invention provides a kit, which includes at least one of the reagents described in the present invention and at least one of the following:

[0040] PCT-coated antibody; and / or

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

[0042] Chicken IgY antibody; and / or

[0043] Coating solution; and / or

[0044] Sample pad buffer;

[0045] The PCT-coated antibody is PCT monoclonal antibody 1, purchased from Fapon Biotech, product number: PCT-REAB-G1-016;

[0046] The IL-6-coated antibody is IL-6 monoclonal antibody 1, purchased from Hytide Biotech, 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 components of the coating solution, and the test results showed that the stability decreased after the reagent components and ratios were changed.

[0051] The sample pad buffer is a boric acid buffer containing Tween-20, trehalose, mouse anti-human red blood cell monoclonal antibody, and Proclin300.

[0052] Further, the sample pad buffer is a 20 mM boric acid buffer containing 0.45 wt%~0.55 wt% Tween-20, 3 wt%~5 wt% trehalose, 0.025 wt%~0.035 wt% mouse anti-human red blood cell monoclonal antibody, and 0.015 wt%~0.025 wt% Proclin300.

[0053] Even further, in a specific embodiment of the present invention, the sample pad buffer is a 20 mM boric acid buffer with a pH of 8.0 containing 0.5 wt% Tween-20, 5 wt% trehalose, 0.03 wt% mouse anti-human red blood cell monoclonal antibody, and 0.02 wt% Proclin300.

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

[0055] The concentration of the PCT coated antibody is 1.1 mg / mL~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 adjusted the concentrations of the PCT and IL-6 coated antibodies, and the test results showed that the detection effects in the following two concentrations were not as good as those when the concentration of the IL-6 coated antibody was 1.8 mg / mL and the concentration of the PCT coated antibody was 1.5 mg / mL;

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

[0059] The concentration of the PCT-coated antibody is 1.3 mg / mL, and the concentration of the IL-6-coated antibody in the second test line is 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 the present invention, the composition (fluorescent probe) originally fixed on the test strip is stored in a centrifuge tube in a dry form, and the unlabeled detection antibody and quality control antibody are fixed to 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 quality control complex, greatly improving the sensitivity and precision of PCT and IL-6 detection. The CV can be controlled within 5%. The detection limit of PCT can reach 0.05 ng / mL, and the detection limit of IL-6 can reach 1.5 pg / mL. At the same time, an appropriate amount of PCT capture antibody is added to the fluorescent probe to improve the linear range of PCT, enabling the linear range of PCT to be from 0.05 ng / mL to 100 ng / mL; the present invention also tried conventional reagents with a conjugate pad. Different from this patent, the sample is not directly mixed with the fluorescent probe, but the components in the fluorescent probe are sprayed onto the conjugate pad. The test results show that the method of the present invention is more excellent for detection.

[0061] The kit described in the present invention further includes a test strip;

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

[0063] On the chromatography membrane, a first test line (2), a second test line (3), and a quality control line (4) are sequentially arranged from the end close to the sample pad towards the water absorption pad;

[0064] The first test line is fixed with a PCT-coated antibody;

[0065] The second test line is fixed with an IL-6-coated antibody;

[0066] The quality control line is fixed with a chicken IgY antibody.

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

[0068] I), the composition described in 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 by 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 an immunoassay method for PCT and / or IL-6 for non-diagnostic purposes, which specifically includes the following steps:

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

[0077] Step 2, Apply the mixture to the sample pad in the kit of the present invention for reaction to obtain a reacted test strip;

[0078] Step 2, Read the signal of the reacted test strip to obtain the detection result.

[0079] Furthermore,

[0080] The conditions for the reaction are to react at 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. At the same time, a blocker is added to reduce the background, improve the effective signal value of the low-value sample concentration, and improve the sensitivity. The linear range of PCT reaches 0.05 ng / mL to 100 ng / mL. By adjusting the amounts of the IL-6 coated antibody and the labeled antibody, the linear range of IL-6 reaches 1.5 pg / mL to 5000 pg / mL. Compared with the conventional immunochromatographic reagent with a conjugate pad, this application has no conjugate pad and sample diluent. When testing, only pure water and the sample need to be added, and the operation is simple. Moreover, the sample is first mixed with the fluorescent probe, which can make the antigen-antibody fully bind, greatly improving the sensitivity and precision of the PCT and IL-6 detections. The fluorescent probe is dried at room temperature, greatly reducing the cost of reagent production, and enabling room-temperature storage and transportation.

[0083] Through the screening and optimization of reagent components, the composition (fluorescent probe) originally fixed on the test strip is stored in a dry form and directly mixed with the sample for immunochromatographic detection of combined detection of PCT and IL-6. The test results show that the linear range of PCT detection in 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%. It has good repeatability, high precision, good anti-interference ability, and has good clinical application prospects. Description of the Drawings

[0084] Figure 1 Figure showing the structural schematic diagram of the test strip in the quantum dot fluorescence immunochromatographic reagent for 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 test membrane (chromatographic membrane); 6. Absorbent paper (absorbent pad); 7. Bottom plate;

[0085] Figure 2 Figure showing the standard curve of PCT;

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

[0087] Figure 4 Figure showing the correlation analysis diagram of the test system plasma detection value and the Roche electrochemiluminescence PCT plasma detection value obtained from the detection of PCT plasma samples in the correlation detection of clinical samples;

[0088] Figure 5 Figure showing the correlation analysis diagram of the test system plasma detection value and the Roche electrochemiluminescence IL-6 plasma detection value obtained from the detection of IL-6 plasma samples in the correlation detection of clinical samples;

[0089] Figure 6 Figure showing the correlation analysis diagram of the test system whole blood detection value and the Roche electrochemiluminescence PCT plasma detection value obtained from the detection of PCT homologous whole blood samples in the correlation detection of clinical samples;

[0090] Figure 7 Figure showing the correlation analysis diagram of the test system whole blood detection value and the Roche electrochemiluminescence IL-6 plasma detection value obtained from the detection of IL-6 homologous whole blood samples in the correlation detection of clinical samples. Detailed Embodiments

[0091] The present invention provides compositions, reagents, kits and their applications in the detection of PCT and IL-6, as well as methods for detecting PCT and IL-6. Those skilled in the art can draw on the content of this article and appropriately improve process parameters to achieve them. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all 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 make changes, or appropriate changes and combinations, to the methods and applications in this article without departing from the content, spirit and scope of the present invention, so as to implement and apply the technology of the present invention.

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

[0093] The CV value refers to the coefficient of variation, which is the ratio of the standard deviation (SD) to the mean value (Mean), usually expressed as a percentage. The CV value is used to measure the stability and repeatability of the detection results.

[0094] The fluorescence probe of the present invention (the composition for the combined detection of PCT and IL-6) adopts the method of drying at room temperature, which greatly reduces the cost of reagent production and enables storage and transportation at room temperature. Compared with the conventional immunochromatographic reagent with a conjugate pad, this application has no conjugate pad and sample diluent. When testing, only pure water and the sample need to be added, and the operation is simple, which greatly improves the sensitivity and precision of the detection of PCT and IL-6. The detection limit of IL-6 can reach 1.5 pg / mL, and the upper limit of the linear range reaches 5000 pg / mL; the detection limit of PCT reaches 0.05 ng / mL, and the upper limit of the linear range reaches 100 ng / mL, and the CV is all below 5%.

[0095] Currently, for the ordinary fluorescence immunoassay, the CV requirement is below 15%. The linear range of IL-6 can reach [5 pg / mL - 1000 pg / mL], the detection limit of PCT reaches 0.5 ng / mL, and the upper limit of the linear range of PCT is mostly only 100 ng / mL for chemiluminescence immunoassay.

[0096] At the same time, an appropriate amount of PCT capture antibody and blocker are added to the fluorescence probe to improve the linear range of PCT. Finally, the reagent CV can be controlled within 5%, the linear range of PCT reaches 0.05 ng / mL - 100 ng / mL, and the linear range of IL-6 reaches 1.5 pg / mL - 5000 pg / mL.

[0097] A certain amount of PCT capture antibody is added to the fluorescence probe of the invention to improve the linear range. At the same time, a blocker is added to reduce the background, improve the effective signal value of the low-value sample concentration, and improve the sensitivity. The linear range of PCT reaches 0.05 ng / mL to 100 ng / mL. By adjusting the amounts of the IL-6 coated antibody and the labeled antibody, the linear range of IL-6 reaches 1.5 pg / mL to 5000 pg / mL. Compared with the conventional immunochromatographic reagent with a conjugate pad, this application does not have a conjugate pad and a sample diluent. When testing, only pure water and the sample need to be added, and the operation is simple. Moreover, the sample is first mixed with the fluorescence probe, which can make the antigen and antibody fully bind, greatly improving the sensitivity and precision of PCT and IL-6 detection. The fluorescence probe adopts the method of drying at room temperature, greatly reducing 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 coated antibody is PCT monoclonal antibody 1; the numbers 1, 2, or 3 are used to illustrate or distinguish the differences among the three PCT monoclonal antibodies; the differences are as follows:

[0099] The capture antibody - PCT monoclonal antibody 3 is purchased from Fapon Biotech, and the product number is: PCT-Ab4#;

[0100] In the PCT antibody labeled with quantum dot microspheres, the PCT antibody is PCT monoclonal antibody 2, which is purchased from Fapon Biotech, and the product number is: PCT-REAB-G1-015;

[0101] The PCT coated antibody is PCT monoclonal antibody 1, which is purchased from Fapon Biotech, and the product 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, and the IL-6 monoclonal antibody 2 is purchased from Hytest Biotech, and the product number is: L152;

[0103] The IL-6 coated antibody is IL-6 monoclonal antibody 1, which is purchased from Hytest Biotech, and the product number is: L395;

[0104] The quantum dot microsphere-labeled goat anti-chicken IgY antibody is purchased from Arista Biologicals, and the product number is ABGAC-0500;

[0105] The blocker 1 is purchased from Meridian, and the product number is: A66800H, which is an active blocker.

[0106] The blocker 2 is purchased from Fapon Biotech, and the product number is: HIER-R-013, which is a passive blocker.

[0107] The fluorescent substance used for fluorescent labeling is carboxyl-modified quantum dot microspheres (Beijing Nano-Gold Biotechnology Co., Ltd., product number FM610C);

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

[0109] The test materials used in the present invention are all ordinary commercially available products and can be purchased on the market. The present invention will be further described below in conjunction with embodiments:

[0110] Example 1 Immunochromatographic reagent for combined detection of PCT and IL-6

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

[0112] The fluorescent probes for reagents for combined detection of PCT and IL-6 include: PCT monoclonal antibody 2 labeled with quantum dot microspheres (Fitzgerald Industries International, product number: PCT-REAB-G1-015), IL-6 monoclonal antibody 2 labeled with quantum dot microspheres (Haitai Biotech, product number: L152), goat anti-chicken IgY antibody labeled with quantum dot microspheres (Arista Biologicals, product number: ABGAC-0500), capture antibody - PCT monoclonal antibody 3 (Fitzgerald Industries International, product number: PCT-Ab4#), and blocking agent (Meridian, product number: A66800H);

[0113] (1) Preparation of PCT monoclonal antibody 2 labeled with quantum dot microspheres: Dilute a certain amount of quantum dot microspheres (solid content of 1‰) with an activation solution (10 mM MES containing EDC and NHS, pH 6.5) at a dilution ratio of 1:4, and activate at room temperature for 15 min. Among them, the addition amounts of EDC and NHS are 2000 times the mass of the quantum dot microspheres (calculated according to the solid content), and the mass ratio of EDC and NHS is 1:1. After the reaction, centrifuge the obtained solution to remove the supernatant, redissolve it with a coupling solution (20 mM boric acid buffer solution with 0.1 wt% Tween-20 at pH 7.0), and add PCT monoclonal antibody 2 (the mass ratio of quantum dots to antibody is 1:500), and react at room temperature for 2 h; after the reaction, add 5 μL of CE510 and 5 μL of CE210, and block at room temperature for 1 h. After centrifuging the blocked solution to remove the supernatant, add 50 μL of a drying buffer solution, and mix well by ultrasound to make the final concentration of PCT monoclonal antibody 2 labeled with quantum dot microspheres 0.5 mg / ml, and store it in the dark at 2-8°C.

[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 addition amounts of EDC and NHS are 2400 times the mass of the quantum dot microspheres, and the others are the same as those described for quantum dot microsphere-labeled PCT monoclonal antibody 2.

[0115] (3)Preparation of Quantum Dot Microsphere-Labeled Goat Anti-Chicken IgY: During the preparation process, the addition amounts of EDC and NHS are 800 times the mass of the quantum dot microspheres; the coupling solution is 20 mM boric acid buffer with 0.1 wt% Tween-20 and pH 8.0; the mass ratio of quantum dots to antibody is 1:400; 50 μL of dry buffer is added and mixed well by ultrasound to make the final concentration of quantum dot microsphere-labeled goat anti-chicken IgY 0.3 mg / mL, and the others are the same as those described for quantum dot microsphere-labeled PCT monoclonal antibody 2.

[0116] (4)Preparation of Dry 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% Proclin300.

[0117] (5)Preparation of Fluorescent Probe Solution: Appropriate amounts of quantum dot microsphere-labeled PCT monoclonal antibody 2, quantum dot microsphere-labeled IL-6 monoclonal antibody 2, quantum dot microsphere-labeled goat anti-chicken IgY antibody, and PCT capture antibody - monoclonal antibody 3 as well as blocker 1 are added to the dry buffer respectively 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 goat anti-chicken IgY antibody, 2.4 μg / mL PCT monoclonal antibody 3 (PCT capture antibody) and 25.2 mg / mL blocker;

[0118] (6)Subpackaging and Drying of Fluorescent Probe: Subpackage 5 μL of the fluorescent probe solution into corresponding centrifuge tubes. After each subpackaging, weigh the centrifuge tubes to confirm whether the difference between the weight of the centrifuge tube after subpackaging and the weight of the empty test tube before subpackaging exceeds ±0.5 μL. After confirming that the subpackaging volumes are consistent, transfer the test tubes to an electronic moisture-proof cabinet for drying, control the temperature at 18 - 28 °C, the drying time is 24 hours, and the humidity requirement in the last 2 hours is ≤11% to obtain the dried fluorescent probe.

[0119] II. Preparation of Reagent Strip

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

[0121] (1) Composition of coating solution: 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 test line and the second test line solution: Use coating solution (5wt% trehalose, 50mM phosphate solution at pH7.4) to dilute PCT monoclonal antibody 1 (PCT coating antibody, Feipeng Biotechnology, catalog number: PCT-REAB-G1-016) to 1.5mg / mL to prepare the first test line; use coating solution to dilute IL-6 monoclonal antibody 1 (IL-6 coating antibody, Hi-Tide Biotechnology, catalog number L395) to 1.8mg / mL to prepare the second test line.

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

[0124] (4) Preparation of reagent strips:

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

[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] Preparation of sample pad: Prepare sample pad buffer, which is prepared by dissolving 0.5wt% Tween-20, 5wt% trehalose, 0.03wt% mouse anti-human erythrocyte monoclonal antibody, and 0.02wt% Proclin300 in 20mM, pH 8.0 boric acid buffer. Soak the glass fiber in the sample pad buffer, take it out after five minutes, place it in a drying oven, set the temperature to 37°C, dry it for 24 hours, and cut it into the required size to obtain the sample pad.

[0128] Reagent strip filming and cutting: Paste the sample pad and absorbent paper on both ends of the NC membrane. Paste the sample pad on the side close to the test line, press it on the NC membrane 1~2mm, and paste the absorbent paper on the side close to the quality control line, press it on the top of the NC membrane, and the other side is flush with the outer edge of the bottom plate. Set the width of the test strip cut by the cutter to 4mm, and confirm whether the cut surface is flat.

[0129] Assembly of the test strip:

[0130] Put it into a plastic shell. The sample pad is installed at the top of the shell, and the absorbent paper is installed at the end of the shell. Compact it with a shell press, and then put it into an aluminum foil bag together with the desiccant. Seal the bag and store it in a drying cabinet for standby.

[0131] III. Detection of the reagent for combined detection of PCT and IL-6

[0132] Testing of the standard curve and manufacturing of the IC card

[0133] (1) Test materials:

[0134] Dilute the PCT and IL-6 standards with calf serum respectively, and the concentrations are: 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] Add 200 μL of pure water to the dried fluorescent probe to obtain a liquid fluorescent probe reagent;

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

[0138] Take 100 μL of the sample mixture and add it to the sample addition hole of the test strip. React at room temperature for 15 minutes to obtain the reacted test strip; read T1 / C and T2 / C respectively through an immunochromatography (model: ACCABLE-T or ACCABLE-E) reader compatible with the test strip, and detect the concentration of each standard three times. The test standard curve data are as follows:

[0139] Table 1. PCT standard curve

[0140]

[0141] Table 2. IL-6 standard curve

[0142]

[0143] (3) Manufacturing of the IC card:

[0144] Using the concentration values of the standard products PCT and IL-6 as the abscissa respectively, the average value (T / C) of each concentration standard product and the concentration are used to make a standard curve, as Figure 2 and Figure 3 (The standard curve is obtained by using the ELISA Calc regression / fitting calculation program - Version 0.2, and selecting cubic spline interpolation), burning the IC card and importing it into the supporting instrument.

[0145] IV. Performance Detection

[0146] 1. Precision Test

[0147] (1) Test materials: Clinical samples are provided by relevant hospitals. The determined concentrations of PCT and IL-6 are: PCT concentrations of 1, 10, and 50 ng / mL, and the concentration of IL-6 is 10, 100, and 500 pg / mL.

[0148] (2) Test method: Use a fluorescence immunoassay analyzer (model: ACCABLE-T or ACCABLE-E) supporting the kit of the present application to perform the precision test. Using the test method in Example 1, test the clinical samples of PCT and IL-6 respectively, and repeat the detection 10 times for each item and each concentration.

[0149] (3) Analysis of test results:

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

[0151]

[0152] As can be seen from the data in Table 3, for the reagent for the combined detection of PCT and IL-6 of the present invention, the precision of both PCT and IL-6 is less than 5%. When compared with the chromatographic reagent with a conjugate pad, when the sample is diluted with a diluent and then added to the sample loading hole, the immune reaction occurs when the sample flows through the membrane material, and the reaction is not complete, resulting in poor precision. When testing this patent, only pure water needs to be added to the fluorescence probe, and then the sample is added and mixed evenly. The reaction is sufficient and uniform, significantly improving the precision of the reagent strip.

[0153] 2. Anti-interference ability

[0154] (1) Test materials: Clinical samples are provided by relevant hospitals. The determined concentrations of PCT and IL-6 are: PCT concentrations of 1 ng / mL and 10 ng / mL, and the concentration of IL-6 is 10 pg / mL and 500 pg / mL. Interference substances such as hemoglobin, triglyceride, and bilirubin are all purchased externally.

[0155] (2) Test method: Use a fluorescence immunoassay analyzer (model: ACCABLE-T or ACCABLE-E) compatible with the kit of the present application to conduct interference tests. Using the test method in Example 1, clinically relevant samples of PCT and IL-6 after adding different concentrations of interfering substances were tested respectively, with no interfering substance added to the control group.

[0156] (3) Analysis of test results:

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

[0158]

[0159] As can be seen from the data in Table 4, for the reagent for combined detection of PCT and IL-6 of the present invention, the relative deviation between the test results of different concentrations of interfering substances and the control group is within 10%, indicating that the addition of interfering substances such as hemoglobin (within the concentration range of 5 mg / mL), triglyceride (within the concentration range of 17 mmol / L), and bilirubin (within the concentration range of 3.5×10 2 μmol / L) has an acceptable impact on the test results of the reagent for the samples.

[0160] 3. Stability - Accelerated test

[0161] (1) Test materials: Clinically relevant 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.

[0162] (2) Test method: Use a fluorescence immunoassay analyzer (model: ACCABLE-T or ACCABLE-E) compatible with the kit of the present application to conduct stability tests. The reagent was accelerated at 37°C for 60 days, and samples of PCT and IL-6 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 and IL-6 stability - accelerated test results

[0166]

[0167] As shown in the results of Table 5, for the samples of low, medium, and high levels of PCT and IL-6 tested within 60 days of acceleration, the relative deviation from the test results before acceleration is within 10%, indicating that the stability of the reagent meets the requirements.

[0168] 4. Correlation detection of clinically relevant samples

[0169] (1) Test materials:

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

[0171] (2) Test methods:

[0172] A fluorescence immunoassay analyzer (model: ACCABLE-T or ACCABLE-E) compatible with the kit of this application was used to detect the correlation of clinical samples, and each sample was detected once. The specific test results are shown in Table 11.

[0173] (3) Analysis of test results:

[0174] According to the test method, 50 cases of samples calibrated by Roche electrochemiluminescence immunoassay were detected. Taking the plasma test results as the benchmark, the correlation between whole blood and plasma was analyzed. The test results are shown in Table 4.

[0175] Table 6. Correlation analysis

[0176]

[0177]

[0178] Taking the PCT and IL-6 concentrations detected by chemiluminescence immunoassay as the abscissa and the PCT and IL-6 plasma concentrations detected by this application as the ordinate, a scatter plot was drawn for correlation analysis. As Figure 4 、 Figure 5 shown. For the detection of 50 cases of clinical plasma samples, PCT was basically consistent with the electrochemiluminescence detection results in the range of 0.05 - 100 ng / mL, and the correlation coefficient R 2 = 0.9972 in the whole linear range; IL-6 was basically consistent with the electrochemiluminescence detection results in the range of 1.5 - 5000 pg / mL, and the correlation coefficient R 2 = 0.9946 in the whole linear range. R 2 were both greater than 0.98, indicating that the reagent prepared by this application had good consistency with electrochemiluminescence immunoassay in detecting PCT and IL-6 plasma, fully meeting the requirements of clinical trials.

[0179] Taking the PCT and IL-6 concentrations detected by chemiluminescence immunoassay as the abscissa and the PCT and IL-6 whole blood concentrations detected by this application as the ordinate, a scatter plot was drawn for correlation analysis. As Figure 6 、 Figure 7As shown in the figure. 50 clinical whole blood samples were detected. PCT was basically consistent with the electrochemiluminescence detection results in the range of 0.05 - 100 ng / mL, and the correlation coefficient R 2 = 0.9983; IL-6 was basically consistent with the electrochemiluminescence detection results in the range of 1.5 - 5000 pg / mL, and the correlation coefficient R 2 = 0.9958. The R 2 values of both reagents were greater than 0.98, indicating that the reagents prepared in this application had good consistency with the electrochemiluminescence method in detecting PCT and IL-6 in whole blood, meeting the clinical needs for detecting PCT and IL-6.

[0180] Example 2 Related Optimization of the Invention

[0181] I. Screening of PCT Capture Antibody and Blocking Agent in Fluorescent Probe Solution

[0182] The fluorescent probe solution was prepared as in Example 1, with the difference that the capture antibody in Group 1 was PCT monoclonal antibody 1; the blocking agent in Group 2 was replaced with blocking agent 2 (Fitebio, product number: HIER-R-013).

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

[0184] Table 7. Precision Test Results

[0185]

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

[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 change in the low-value repeatability of IL-6 in Example 1 and Group 1 is not significant, indicating that the PCT capture antibody selected in this patent can improve the precision of PCT and has no obvious interference on the precision of IL-6. The low-value repeatability of both PCT and IL-6 in Example 1 is better than that in Group 2, indicating that the blocker selected in this patent can improve the precision of both PCT and IL-6.

[0190] As can be seen from Table 8, the upper limit concentration point of the PCT linear range in the three groups of data is twice the concentration of the second-highest point, and the corresponding signal values of the three groups are also close to a 2-fold relationship, indicating that the linearity of PCT in the three groups meets the requirements. The signal value of 0 ng / mL of PCT in Example 1 is lower than that in Group 2, indicating that the blocker in this patent has a good effect and can effectively block the interference of heterophilic antibodies and reduce the background. The signal value of 0.05 ng / 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 the PCT capture antibody and blocker in the three groups of data have no effect on the linearity and sensitivity of IL-6. In this invention, a PCT capture antibody with relatively low activity is selected, and a specific blocker is added to block the interference of heterophilic antibodies, reduce the background, and increase the effective signal value at low concentrations, thereby improving the sensitivity and the precision at low concentrations.

[0191] II. 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: Yiboxin, product number: MIS04); the IL-6 antibody (coated antibody) on the second test line is IL-6 monoclonal antibody 4 (brand: Yiboxin, product number: MIS03) - Group 3.

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

[0194] Table 9. Screening Results of IL-6 Antibodies

[0195]

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

[0197] III. 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 the fluorescent probe of Group 4 is 12 μg / mL to 24 μg / mL (the concentrations of each component in the fluorescent probe solution, and the converted working concentrations are shown in the brackets in the following table). The specific grouping is shown in Table 10.

[0199] Using the reagents prepared in Example 1 and Group 4, the signal values of the 0 concentration samples and the lower limit concentration points of the linear range corresponding to PCT and IL-6 (PCT: 0.05 ng / mL, IL-6: 1.5 pg / mL) were tested respectively, as well as the upper limit concentration points and the second highest concentration points of the linear range corresponding to PCT and IL-6 (PCT: 100 ng / mL, IL-6: 5000 pg / mL; PCT: 50 ng / mL, IL-6: 2000 pg / mL) to verify the sensitivity and linearity of the reagent detection. The test results are shown in Table 10:

[0200] Table 10. Optimization of the concentration of quantum dot-labeled IL-6 antibody 2

[0201]

[0202] As can be seen from Table 10, when the quantum dot microsphere-labeled IL-6 antibody 2 is 12 μg / mL and 14 μg / mL, the signal values at the 1.5 pg / mL concentration point of IL-6 test are both lower than 0.01, and the tested signal values are too low, and the sensitivity is not as good as that of other concentration groups. The signal value ratios of the high point and the second highest point of IL-6 at 22 μg / mL and 24 μg / mL are both lower than 2, and the high-value linearity is not as good as that of other concentration groups. When the quantum dot microsphere-labeled IL-6 antibody 2 is 16 - 20 μg / mL, both the sensitivity and linearity meet the requirements. When the quantum dot-labeled IL-6 antibody 2 is 18 μg / mL, when testing the 1.5 pg / mL concentration point, the signal value is higher than that at 16 μg / mL; when testing the 5000 and 2000 pg / mL concentration points, the ratio of the signal values corresponding to the two concentration points in the 18 μg / mL concentration group is higher than that in the 20 μg / mL concentration group, and the change in the concentration of the quantum dot microsphere-labeled IL-6 antibody 2 has no effect on the reagent performance of PCT. Based on the above data, it is shown that when the amount of the quantum dot microsphere-labeled IL-6 antibody is 18 μg / mL, both the sensitivity and linearity are optimal.

[0203] IV. Screening of goat anti-chicken IgY and chicken IgY antibodies

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

[0205] Using the test strip and fluorescent probe prepared in Example 1, Group 5, after accelerating at 37°C for 30 days, samples of 10 ng / mL PCT and 500 pg / mL IL-6 were tested respectively to verify the stability of the reagent after acceleration. Clinical samples were provided by relevant hospitals, and all samples were calibrated by Roche electrochemiluminescence method. The test results are shown in the following table.

[0206] Table 11. Screening of Goat Anti-Chicken IgY and Chicken IgY Antibodies

[0207]

[0208] As can be seen from Table 11, the increase in the test results of Group 5 after accelerating for 30 days exceeded 10% compared with that before acceleration. The relative deviation of the test results after acceleration in Example 1 was within 10%, and the result was better than that of Group 5. The data shows that the paired combination of goat anti-chicken IgY and chicken IgY selected in this patent is better in terms of reagent stability, making the T / C of the reagent in this patent more stable.

[0209] V. Screening of Buffer Components in Activation Solution

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

[0211] Using the fluorescent probes and test strips prepared from Groups 6 to 8 in Example 1, the signal values of the 0-concentration samples and the lower limit concentration of the linear range (PCT: 0.05 ng / mL, IL-6: 1.5 pg / mL) samples corresponding to PCT and IL-6 were tested 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, there were certain signal values when testing the 0-concentration samples in Groups 6 and 7, indicating that there was a certain aggregation of the labeled quantum dot-labeled antibodies, and the greater the MES concentration, the more obvious the aggregation. The signal value of the lower limit concentration point of the linear range in Group 8 was lower than that in Example 1, indicating that the activation solution in this patent had a certain promoting effect on sensitivity, and the aggregation of quantum dot-labeled antibodies was not likely to occur at the activation solution concentration in this patent.

[0215] VI. Optimization of the Dosages of EDC and NHS in the Activation Solution

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

[0217] In the preparation process of PCT antibody 2 labeled with Group 10 quantum dot microspheres, the addition amounts of EDC and NHS are 3000 times the mass of the quantum dot microspheres; in the preparation process of IL-6 antibody 2 labeled with quantum dot microspheres, the addition amounts of EDC and NHS are 3000 times the mass of the quantum dot microspheres.

[0218] In the preparation process of PCT monoclonal antibody 2 labeled with Group 11 quantum dot microspheres, the addition amounts of EDC and NHS are 1000 times the mass of the quantum dot microspheres; in the preparation process of IL-6 monoclonal antibody 2 labeled with quantum dot microspheres, the addition amounts of EDC and NHS are 1200 times the mass of the quantum dot microspheres.

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

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

[0221]

[0222] It can be seen from Table 13 that the data in Example 1 and Group 10 are better than those in other groups. In Group 9, the signal values of the 0 concentration sample and the lower limit concentration sample of PCT and IL-6 cannot be separated, indicating that too high an amount of the activator leads to a certain aggregation of the labeled quantum dot-labeled antibody; in Group 11, the signal values of PCT and the lower limit concentration point of the linear range are very low, and almost no signal value can be measured. Combining the above two points, it shows that when the addition amounts of EDC and NHS in the IL-6 activation process are 2400 - 3000 times the mass of the quantum dot microspheres, and when the addition amounts of EDC and NHS in the PCT activation process are 2000 - 3000 times the mass of the quantum dot microspheres, the sensitivity of the reagent is optimal.

[0223] VII. Screening of the blocking agent

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

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

[0226] Table 14. Screening Precision of Blocking Agent

[0227]

[0228] The reagent prepared in Example 1 and Group 12 was used to test the signal values of the samples corresponding to 0 concentration and the lower limit concentration of the linear range of PCT and IL-6 (PCT: 0.05 ng / mL, IL-6: 1.5 pg / mL) 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] It can be seen from the data in Table 14 that the repeatability of PCT and IL-6 in Example 1 is better than that in Group 12. It can be seen from the data in Table 15 that 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 10 wt% BSA in Group 12. The quantum dot microspheres after blocking in Example 1 can more effectively prevent the non-specific adsorption of subsequent antibodies, reduce the background signal and background interference, and improve the precision and sensitivity of the low value of the reagent.

[0232] VIII. Screening of Each Component in the Drying Buffer

[0233] Groups 13 to 16 were for the screening of the concentrations of each component in the drying buffer. The preparation of the drying buffer was the same 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; the difference in Group 16 was that trehalose was replaced with sucrose.

[0234] The test strips and fluorescent probes prepared in Example 1 and Groups 13 to 16 were used to detect samples of 10 ng / mL PCT and 500 pg / mL IL-6 to verify the accuracy of the reagent. Clinical samples were provided by relevant hospitals, and all samples were calibrated by Roche electrochemiluminescence method. The detection results are shown in the following table.

[0235] Table 16. Screening of Each Component in the Drying Buffer

[0236]

[0237] As can be seen from Table 16, the PCT and IL-6 accuracy results of Example 1 and Group 15 are better than those of other groups, indicating that both BSA and casein can be used as components of the drying protective agent. Comparing the accuracy results of Example 1 and Group 15, Example 1 is better than Group 15, indicating that the combination of the components in the drying buffer in Example 1 is optimal, and the antibody protection effect during the drying process is better, maintaining the activity of the antibody.

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

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

[0240] Using the test strips and fluorescent probes prepared in Example 1 and Group 17, the reagents were accelerated at 37 degrees for 30 days, and samples with a concentration of 10 ng / mL PCT and 500 pg / mL IL-6 were tested to verify the stability of the reagents. Clinical samples were provided by relevant hospitals, and the samples were all valued by Roche electrochemiluminescence method. The test results are shown in Table 17.

[0241] Table 17. Screening of drying buffers

[0242]

[0243] It can be seen from the data in Table 17 that the relative deviations of the accelerated test results of Example 1 and the 3wt% trehalose concentration group compared with those before acceleration are better than those of the other groups, indicating that when the trehalose content in the drying buffer is in the range of 3wt% to 5wt%, 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 solution components. The coating solutions in Groups 18 and 19 were prepared as in Example 1, except that the phosphate base solution in the coating solution of Group 18 was replaced with TRIS; the difference in Group 19 was that the base solution in the coating solution was Hepes.

[0246] The reagents prepared in Example 1 and Groups 18-19 were used to test samples of 10 ng / mL PCT and 500 pg / mL IL-6 to verify the effect of different coating solutions on the accuracy of the reagents. The clinical samples were provided by relevant hospitals, and the samples were all valued by Roche electrochemiluminescence method. The test results are shown in the following table.

[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 of other groups, and the data shows that the base solution phosphate buffer in the coating solution of this patent has the best effect.

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

[0251] Groups 20 and 21 are for comparison of the adjustment of PCT and IL-6 coating antibody concentrations; different from Example 1, in Group 20, the concentration of PCT monoclonal antibody 1 (PCT coating antibody) in the first test line is 1.1 mg / mL, and the concentration of IL-6 monoclonal antibody 1 (IL-6 coating antibody) in the second test line is 1.4 mg / mL.

[0252] In Group 21, the concentration of PCT monoclonal antibody 1 (PCT coating antibody) in the first test line is 1.3 mg / mL, and the concentration of IL-6 monoclonal antibody 1 (IL-6 coating antibody) in the second test line is 1.6 mg / mL.

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

[0254] Table 19. Precision Results of Adjustment of PCT and IL-6 Coating Antibody Concentrations

[0255]

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

[0257] Table 20. Linearity Results of Adjustment of 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 coating antibody concentration increases, the signal values corresponding to the concentrations also increase, indicating that increasing the concentration of the coating antibody on the test line can increase the signal values of the upper limit concentration of the entire linear range, thereby improving the high-value precision.

[0260] XI. Comparison with Reagents with Conjugate Pads

[0261] In Group 22, for the adjustment of the reagent strip structure, a binding pad with a width of 8 mm was pasted between the sample pad and the NC membrane of the reagent strip (the binding pad was treated by conventional methods), and the components in the fluorescent probe of Example 1 were sprayed onto the binding pad so that the final concentrations of the components remained unchanged.

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

[0263] Table 21. Precision results of comparative tests with reagents having a binding pad

[0264]

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

[0266] Table 22. Sensitivity results of comparative tests with reagents having a binding pad

[0267]

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

[0269] The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can still be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A composition, characterized in that, Including PCT antibody labeled with quantum dot microspheres, IL-6 antibody labeled with quantum dot microspheres, goat anti-chicken IgY antibody labeled with quantum dot microspheres, PCT capture antibody and blocker; The PCT antibody has the trade name of Fapon Biotech and the catalog number of PCT-REAB-G1-015; The PCT capture antibody has the trade name of Fapon Biotech and the catalog number of PCT-Ab4#; The IL-6 antibody has the trade name of Hytest Biotech and the catalog number of L152; The blocker includes an active blocker.

2. The composition according to claim 1, wherein In the preparation of the PCT antibody labeled with quantum dot microspheres, the activation solution used is 10 mM MES containing EDC and NHS, and the total added mass of EDC and NHS is 2000 - 3000 times the mass of the quantum dot microspheres; In the preparation of the IL-6 antibody labeled with quantum dot microspheres, the activation solution used is 10 mM MES containing EDC and NHS, and the total added mass of EDC and NHS is 2400 - 3000 times the mass of the quantum dot microspheres; In the preparation of the goat anti-chicken IgY antibody, the activation solution used is 10 mM MES containing EDC and NHS, and the total added mass of EDC and NHS is 800 times the mass of the quantum dot microspheres.

3. The composition according to claim 1, wherein The blocking agent used in the preparation of the PCT antibody labeled with quantum dot microspheres, the IL-6 antibody labeled with quantum dot microspheres and / or the goat anti-chicken IgY antibody labeled with quantum dot microspheres includes CE510 and / or CE210.

4. The composition according to any one of claims 1 - 3, wherein The use concentration of the IL-6 antibody labeled with quantum dot microspheres is 0.26 μg / mL - 0.33 μg / mL; The use concentration of the PCT antibody labeled with quantum dot microspheres is 0.18 μg / mL - 0.25 μg / mL; The use concentration of the goat anti-chicken IgY antibody labeled with quantum dot microspheres is 0.016 μg / mL - 0.33 μg / mL; The use concentration of the PCT capture antibody is 0.03 μg / mL - 0.05 μg / mL; The use concentration of the blocker is 0.3 mg / mL - 0.55 mg / mL.

5. A reagent, characterized in that, Including the composition according to any one of claims 1 - 4 and a drying buffer; The drying buffer includes: Tris, NaCl, Tween-20, trehalose, glycine, preservative and protective agent; The protective agent includes BSA and / or casein.

6. The reagent according to claim 5, wherein The drying buffer includes: 15 - 25 mM Tris, 145 - 155 mM NaCl, 0.09 - 0.11 wt% Tween-20, 0.5 - 1.5 wt% BSA, 3 wt% - 5 wt% trehalose, 0.8 - 1.2 wt% glycine and 0.015 - 0.025 wt% preservative.

7. Kit, characterized in that, Including: At least one of PCT coated antibody, IL-6 coated antibody, chicken IgY antibody, coating solution and / or sample pad buffer solution and the reagent according to claim 5 or 6; The commercial name of the PCT-coated antibody is Fapon Biotech, and the product number is PCT-REAB-G1-016; The commercial name of the IL-6-coated antibody is Hiteche Biotech, and the product number is L395; The coating solution includes: Na2HPO4, NaH2PO4, NaCl, trehalose, and Proclin300; The sample pad buffer is a boric acid buffer containing Tween-20, trehalose, mouse anti-human red blood cell 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.

8. The kit according to claim 7, wherein It also includes a test strip; The test strip includes: a bottom plate (7), a sample pad (1), a chromatographic membrane (5), and an absorbent pad (6) that are sequentially fixed to the bottom plate (7); and the sample pad (1), the chromatographic membrane (5), and the absorbent pad (6) are in contact in sequence; On the chromatographic membrane, a first detection line (2), a second detection line (3), and a quality control line (4) are sequentially arranged from the end close to the sample pad to the absorbent pad; The first detection line is fixed with a PCT-coated antibody; The second detection line is fixed with an IL-6-coated antibody; The quality control line is fixed with a chicken IgY antibody.

9. Use of at least one of the following I) to III) in the detection of PCT and / or IL-6: I), the composition according to any one of claims 1 to 4; II), the reagent according to claim 5 or 6; III), the kit according to claim 7 or 8. A method for detecting PCT and / or IL-6, characterized in that, Detect PCT and / or IL-6 by using at least one of the following i) to iii): i), the composition according to any one of claims 1 to 4; ii), the reagent according to claim 5 or 6; iii), the kit according to claim 7 or 8.

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