A kit for detecting nipah virus antigen and application thereof

Abstract

The application provides a kit for detecting nipah virus antigen and application thereof, and belongs to the technical field of biotechnology.The kit comprises quantum dot microsphere labeled nipah virus N protein specific antibody, quantum dot microsphere labeled nipah virus G protein specific antibody, loading liquid and detection test paper.The kit can set double detection lines for nipah virus N protein and G protein on the same detection test paper, realize synchronous combined detection of double targets, avoid false negative and missed detection risk caused by antigen expression abundance difference in different infection stages of viruses, different expression time and improper sampling time in single target detection mode, and significantly improve the overall detection rate of samples.The kit is simple in operation, rapid in detection, does not need large and precise instruments, can realize on-site and real-time qualitative and quantitative detection of samples, is suitable for large-scale rapid screening in primary field and epidemic area, and has good practical application value and popularization prospect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a kit for detecting Nipah virus antigen and its application. Background Technology

[0002] Nipah virus (NiV) is a highly pathogenic zoonotic RNA virus belonging to the genus Hennipahvirus in the family Paramyxoviridae. It can be transmitted to humans through intermediate hosts such as bats, causing severe respiratory infections and neurological symptoms, posing a significant threat to public health. Because early symptoms of Nipah virus infection are atypical and there is a risk of human-to-human transmission, rapid, sensitive, and accurate early diagnosis is crucial for controlling the spread of the epidemic and reducing mortality.

[0003] Currently, Nipah virus detection methods mainly include nucleic acid amplification technologies (such as RT-PCR), enzyme-linked immunosorbent assay (ELISA), and immunochromatography. While RT-PCR offers high sensitivity and specificity, it relies on specialized laboratory equipment, is cumbersome to operate, and has a long testing cycle, making it unsuitable for rapid on-site screening. ELISA is also complex and time-consuming, making it unsuitable for point-of-care testing. Immunochromatography (such as colloidal gold test strips), due to its ease of operation, speed, efficiency, and lack of the need for specialized personnel and equipment, is considered an ideal technology for rapid on-site testing and has significant application potential in emergency epidemic prevention and control.

[0004] However, there are currently no approved rapid immunochromatographic assays for Nipah virus, and the relevant technologies are still under exploration. The development of colloidal gold chromatographic test strips still faces numerous technical challenges. In single-target detection mode, if the expression level of the target antigen in the sample is below the detection limit, or if the sample collection time happens to miss the peak expression period of the target antigen, false negative results are highly likely, significantly reducing detection sensitivity and failing to meet the needs of early detection and screening. At the signal amplification level, the optical signal intensity of colloidal gold markers is limited, resulting in insufficient detection capability for low-concentration antigens and a high detection limit. In the early stages of Nipah virus infection, when the viral load in patients is low, the antigen concentration in the sample is far below the detection limit of the colloidal gold test strip, making effective detection difficult and easily leading to missed diagnoses of early infection, delaying epidemic prevention and control and clinical intervention.

[0005] Furthermore, the accuracy and stability of immunochromatographic assays are closely related to the formulation of the loading solution and the sample pad treatment solution. In existing technologies, parameters such as osmotic pressure, protein protectant concentration, and surfactant ratio of the loading solution lack targeted optimization, which can easily lead to loss of labeled antibody activity and uneven chromatography speed. On the other hand, an unreasonable sample pad treatment process may cause loss of antigen adsorption or increase in non-specific binding in the sample, further affecting the reliability and repeatability of the test results.

[0006] Therefore, developing a rapid detection technology for Nipah virus antigen based on dual-target joint recognition, which combines high sensitivity and high stability, and systematically solving key problems such as missed detection by single targets, insufficient sensitivity of colloidal gold labeling, and lack of optimization of process formulation, is of great practical significance and application value for improving the accuracy of early detection of Nipah virus and reducing the risk of epidemic transmission. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide a kit for detecting Nipah virus antigens. This kit uses quantum dot microsphere labeling technology to replace the traditional colloidal gold labeling system, enabling dual-target qualitative and quantitative detection of Nipah virus N and G proteins. This effectively reduces the risk of missed detection and improves detection sensitivity, making it suitable for early Nipah virus infection screening and auxiliary identification of infection stages.

[0008] The objective of this invention is achieved through the following technical solution: This invention provides a kit for detecting Nipah virus antigen, comprising: a Nipah virus N protein-specific antibody labeled with quantum dot microspheres, a Nipah virus G protein-specific antibody labeled with quantum dot microspheres, a sample loading solution, and a test strip.

[0009] Preferably, the Nipah virus N protein-specific antibody includes Nipah virus N protein-specific monoclonal antibody Ab1; the Nipah virus G protein-specific antibody includes Nipah virus G protein-specific monoclonal antibody Ab3.

[0010] Preferably, the loading solution comprises: 0.025~0.1 mol / L pH7.5 Tris-HCl, 2~4wt% sucrose, 1~5wt% trehalose, 0.25~1wt% PEG-20000, 0.2~1wt% BSA, and 0.5~2wt% Tween-20; with a pH value of 7.0~8.5.

[0011] Preferably, the sample loading solution further includes 0.25~0.5wt% NaCl.

[0012] Preferably, the Nipah virus N protein-specific antibody labeled with quantum dot microspheres is obtained by covalently coupling 8-15 μg of N protein-specific antibody with activated quantum dot microspheres; The quantum dot microsphere-labeled Nipah virus G protein-specific antibody was obtained by covalently coupling 8-15 μg of G protein-specific antibody with activated quantum dot microspheres. When used for Nipah virus antigen detection, the Nipah virus N protein-specific antibody and the Nipah virus G protein-specific antibody labeled with quantum dot microspheres are diluted 1000 to 2000 times respectively using the sample loading solution.

[0013] Preferably, the detection lines of the test strip are coated with Nipah virus N protein-specific antibody and Nipah virus G protein-specific antibody, respectively; the Nipah virus N protein-specific antibody includes Nipah virus N protein-specific monoclonal antibody Ab2; the Nipah virus G protein-specific antibody includes Nipah virus G protein-specific monoclonal antibody Ab4.

[0014] Preferably, the concentration of the antibody used to apply the detection line is 0.5~1.5 mg / mL.

[0015] Preferably, the method for processing the sample pad of the test strip includes: Immerse the sample pad in the treatment solution to ensure it is fully absorbed. The treatment solution comprises: 0.005~0.05M PB solution, pH 7.4, containing 0.25~1.5wt% BSA, 0.25~1wt% Tween-20 and 0.5~2wt% trehalose.

[0016] This invention provides the application of the kit described above in the detection of Nipah virus antigen for non-diagnostic purposes.

[0017] This invention provides a method for detecting Nipah virus antigen for non-diagnostic purposes, comprising: The Nipah virus N protein-specific antibody and the Nipah virus G protein-specific antibody labeled with quantum dot microspheres were diluted with the sample loading solution and then mixed with the sample to be tested and incubated. The incubated sample was then dropped onto the test strip for chromatography. The presence of Nipah virus in the sample was determined based on the signal intensity, or the Nipah virus was quantitatively detected.

[0018] The beneficial effects of this invention are: This invention provides a kit for detecting Nipah virus antigens, comprising: a Nipah virus N protein-specific antibody labeled with quantum dot microspheres, a Nipah virus G protein-specific antibody labeled with quantum dot microspheres, a sample loading solution, and a test strip. This invention uses quantum dot microspheres with excellent luminescent properties to replace traditional colloidal gold as the immunomarker, significantly improving the intensity of the detection fluorescence signal and detection sensitivity, lowering the detection limit, and effectively detecting samples with low viral load in the early stages of infection. It overcomes the shortcomings of traditional colloidal gold test strips, such as low sensitivity and easy false negatives. The kit can be configured with dual detection lines targeting both the Nipah virus N and G proteins on the same test strip, enabling simultaneous joint detection of both targets. Regardless of whether the sample is in the early stage of infection (high expression of N protein) or the middle or late stage of infection (high expression of G protein), it can effectively capture the target antigens, avoiding the risks of false negatives and false negatives caused by differences in antigen expression abundance, expression sequence, and improper sampling timing at different stages of viral infection under single-target detection mode, significantly improving the overall sample detection rate. Furthermore, by optimizing the sample loading solution formulation and sample pad treatment system, this invention effectively maintains the bioactivity of the labeled antibody, reduces non-specific adsorption, optimizes the chromatographic flow rate and system stability, reduces background interference, and improves the repeatability and accuracy of the test strip detection. The kit provided by this invention is simple to operate, rapid in detection, and requires no large-scale precision instruments for Nipah virus antigen detection. It enables on-site, real-time qualitative and quantitative detection of samples, has a wide range of applications, and is convenient for large-scale rapid screening in grassroots fields and epidemic areas, possessing good practical application value and promising prospects for promotion. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0020] Figure 1 This is a schematic diagram of the structure of a fluorescent immunochromatographic test strip for Nipah virus antigen; the combined test strip consists of an NC membrane, a sample pad, and absorbent paper assembled on a base plate; where 1-4 are the sample pad, absorbent paper, nitrocellulose membrane, and base plate, respectively. Figure 2 The results of screening for G protein antibody (T1 line) concentration and conjugate dilution factor; Figure 3 The results of screening for N protein antibody (T2 line) concentration and conjugate dilution factor; Figure 4 For testing G protein nasal swab simulated samples; Figure 5 Testing of N-protein nasal swab simulated samples; Figure 6 Results of G protein detection using sample solutions with different concentrations of NaCl; Figure 7Results of N protein detection using sample solutions with different concentrations of NaCl; Figure 8 This is the standard curve for G protein antigen detection; Figure 9 This is the standard curve for N protein antigen detection; Figure 10 This is a result of specific testing using the test strip; Figure 11 Images showing the specificity of the test strips; Figure 12 The results are from Experiment 1 in Example 4; in the initial screening of N protein antibodies, Ab1 is RDMV022 and Ab2 is RDMV023. Figure 13 The results are from Experiment 2 in Example 4; in the initial screening of N protein antibodies, Ab1 is RDMV023 and Ab2 is RDMV022. Figure 14 The results are from Experiment 1 in Example 5; in the initial screening of G protein antibodies, Ab3 is ACT-mAb-NiV-005 and Ab4 is ACT-mAb-NiV-006. Figure 15 The results are from Experiment 2 in Example 5; in the initial screening of G protein antibodies, Ab3 is ACT-mAb-NiV-006 and Ab4 is ACT-mAb-NiV-005. Detailed Implementation

[0021] This invention provides a kit for detecting Nipah virus antigen, comprising: a Nipah virus N protein-specific antibody labeled with quantum dot microspheres, a Nipah virus G protein-specific antibody labeled with quantum dot microspheres, a sample loading solution, and a test strip.

[0022] As an optional embodiment of the present invention, the Nipah virus N protein-specific antibody includes Nipah virus N protein-specific monoclonal antibody Ab1; the Nipah virus N protein-specific monoclonal antibody Ab1 is a mouse anti-Nipah virus N protein-specific monoclonal antibody Ab1, catalog number: RDMV022, purchased from Suzhou Renduan Biomedical Technology Co., Ltd. The Nipah virus G protein-specific antibody includes Nipah virus G protein-specific monoclonal antibody Ab3; the Nipah virus G protein-specific monoclonal antibody Ab3 is a mouse anti-Nipah virus G protein-specific monoclonal antibody Ab3, catalog number: ACT-mAb-NiV-005, purchased from Changzhou Zhongmei Xinxin Biotechnology Co., Ltd.

[0023] In this invention, the Nipah virus N protein-specific antibody labeled with quantum dot microspheres is obtained by covalently coupling 10 μg of N protein-specific antibody with activated quantum dot microspheres. The preparation method of the Nipah virus N protein-specific antibody labeled with quantum dot microspheres in this invention includes: mixing a quantum dot microsphere solution with a MES solution, then mixing this mixture with an EDC solution and an NHS solution to activate the carboxyl groups on the surface of the quantum dot microspheres, obtaining carboxyl-activated quantum dot microspheres (also referred to as activated quantum dot microspheres); and covalently coupling the carboxyl-activated quantum dot microspheres with the Nipah virus N protein-specific antibody to obtain the Nipah virus N protein-specific antibody labeled with quantum dot microspheres. In this invention, the concentration of the quantum dot microsphere solution can be 1 μmol / L; the concentration of the MES solution can be 20 mM, and the pH value can be 6.0. The volume ratio of the quantum dot microsphere solution to the MES solution can be 1:1. In this invention, the concentration of the EDC solution can be 20 mg / mL; the concentration of the NHS solution can be 20 mg / mL; and the volume ratio of the quantum dot microsphere-MES mixed solution to the EDC solution and the NHS solution can be 50:1:1. In this invention, the activation reaction of the carboxyl groups on the surface of the quantum dot microspheres can be carried out at 37°C for 15 min, preferably under light-protected conditions. After the activation reaction is completed, the resulting mixture is preferably centrifuged and washed to obtain carboxyl-activated quantum dot microspheres. In this invention, the relative centrifugal force can be 10000 rcf; and the centrifugation time can be 20 min. After centrifugation, the supernatant is discarded, and the microspheres are washed with MES solution; the concentration of the MES solution is 10 mM, pH 6.0. After obtaining the carboxyl-activated quantum dot microspheres, the microspheres are mixed with 10 μg of Nipah virus N protein-specific antibody. In this invention, the covalent coupling temperature can be 37°C, the time can be 1 hour, and the covalent coupling can be performed under oscillation conditions; the oscillation speed can be 800 rpm. After covalent coupling, this invention preferably further includes blocking, washing, and resuspending the Nipah virus N protein-specific antibody labeled with quantum dot microspheres. This invention does not specifically limit the methods for blocking, washing, and resuspending; any conventional methods in the art can be used.

[0024] In this invention, the Nipah virus G protein-specific antibody labeled with quantum dot microspheres is obtained by covalently coupling 8-15 μg of G protein-specific antibody with activated quantum dot microspheres; the G protein-specific antibody can also be 8, 9, 10, 11, 12, 13, 14, or 15 μg. The preparation method of the Nipah virus G protein-specific antibody labeled with quantum dot microspheres in this invention includes: mixing a quantum dot microsphere solution with a MES solution, then mixing it with an EDC solution and an NHS solution to activate the carboxyl groups on the surface of the quantum dot microspheres, obtaining carboxyl-activated quantum dot microspheres; and covalently coupling the carboxyl-activated quantum dot microspheres with the Nipah virus G protein-specific antibody to obtain the Nipah virus G protein-specific antibody labeled with quantum dot microspheres. The specific method is the same as that for the Nipah virus N protein-specific antibody labeled with quantum dot microspheres, and will not be repeated here.

[0025] As an optional embodiment of the present invention, when used for detection, the Nipah virus N protein-specific antibody labeled with quantum dot microspheres and the Nipah virus G protein-specific antibody labeled with quantum dot microspheres are diluted by 1000 to 2000 times, respectively, or by 1000, 1500 or 2000 times, respectively, using a sample loading solution.

[0026] In an optional embodiment of the present invention, the loading solution comprises: 0.025~0.1 mol / L Tris-HCl, 2~4 wt% sucrose, 1~5 wt% trehalose, 0.25~1 wt% PEG-20000, 0.2~1 wt% BSA, and 0.5~2 wt% Tween-20; the pH value is 7.0~8.5. In an optional embodiment of the present invention, the concentration of Tris-HCl in the loading solution can be 0.025~0.1 mol / L, or 0.025, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, or 0.1 mol / L; the pH value of Tris-HCl can be 7.0~8.5, or 7.5. In an optional embodiment of the present invention, the concentration of sucrose in the loading solution can be 2~4 wt%, or 2, 3, or 4 wt%. In an optional embodiment of the present invention, the concentration of trehalose in the loading solution can be 1-5 wt%, or 1, 2, 3, 4, or 5 wt%. In an optional embodiment of the present invention, the concentration of PEG-20000 in the loading solution can be 0.25-1 wt%, or 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1 wt%. In an optional embodiment of the present invention, the concentration of BSA in the loading solution can be 0.2-1 wt%, or 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 wt%. As an optional embodiment of the present invention, the amount of Tween-20 in the loading solution can be 0.5~2wt%, or it can be 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2wt%.

[0027] As an optional embodiment of the present invention, the loading solution further includes 0.25~0.5wt% NaCl, which can also be 0.25, 0.3, 0.35, 0.4, 0.45 or 0.5wt%.

[0028] The loading solution provided by this invention uses a pH 7.5 Tris-HCl base buffer system. Sucrose, trehalose, and BSA synergistically protect the activity of quantum dot-labeled antibodies, preventing their denaturation and inactivation. Combined with PEG-20000 and Tween-20, it can effectively inhibit non-specific adsorption, reduce background interference, and regulate the chromatographic flow rate. With the addition of appropriate NaCl to regulate ionic strength, it optimizes the antigen-antibody specific binding environment. It is compatible with the simultaneous detection of Nipah virus N and G proteins as dual targets, with high detection sensitivity and good stability.

[0029] In an optional embodiment of the present invention, the detection lines of the test strip are coated with Nipah virus N protein-specific antibodies and Nipah virus G protein-specific antibodies, respectively. The Nipah virus N protein-specific antibody includes Nipah virus N protein-specific monoclonal antibody Ab2; the Nipah virus N protein-specific monoclonal antibody Ab2 is a mouse anti-Nipah virus N protein-specific monoclonal antibody Ab2, catalog number: RDMV023, purchased from Suzhou Renduan Biomedical Technology Co., Ltd.; the Nipah virus G protein-specific antibody includes Nipah virus G protein-specific monoclonal antibody Ab4; the Nipah virus G protein-specific monoclonal antibody Ab4 is a mouse anti-Nipah virus G protein-specific monoclonal antibody Ab4, catalog number: ACT-mAb-NiV-006, purchased from Changzhou Zhongmei Xinxin Biotechnology Co., Ltd. In an optional embodiment of the present invention, the concentration of the antibody used for the stripping of the detection lines can be 0.5~1.5 mg / mL, or 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 mg / mL. As an optional embodiment of the present invention, the scratching speed can be 1 µL / cm. In the present invention, the T1 line can be a Nipah virus G protein-specific antibody; the T2 line can be a Nipah virus N protein-specific antibody. As an optional embodiment of the present invention, the control line of the test strip is coated with goat anti-mouse IgG polyclonal antibody; the goat anti-mouse IgG polyclonal antibody is purchased from Changzhou Zhongmei Xinxin Biotechnology Co., Ltd., model: ACT-Ag-IgG(M)-001. The scratching antibody concentration of the control line can be 0.5 mg / mL; the scratching speed can be 1 µL / cm.

[0030] As an optional embodiment of the present invention, the method for processing the sample pad of the test strip includes: immersing the sample pad in a processing solution, fully absorbing the processing solution, and then drying it; the processing solution includes: 0.005~0.05M PB solution, pH 7.4, containing 0.25~1.5wt% BSA, 0.25~1wt% Tween-20, and 0.5~2wt% trehalose. As an optional embodiment of the present invention, the processing solution may be: 0.01M PB solution, pH 7.4, containing 0.5wt% BSA, 0.05wt% Tween-20, and 1wt% trehalose.

[0031] The kit provided by this invention enables the joint quantitative detection of Nipah virus N and G proteins. The sensitivity of the method is enhanced by fluorescent labeling, achieving sensitivities of 5 pg / mL for N protein and 20 pg / mL for G protein antigens. This kit utilizes quantum dot labeling technology instead of traditional colloidal gold, leveraging the high fluorescence quantum yield and excellent photostability of quantum dots to improve detection sensitivity by two orders of magnitude, enabling the detection of lower concentrations of antigens. This invention combines the high sensitivity of quantum dots with the speed and simplicity of chromatography, significantly improving detection sensitivity while maintaining the advantage of rapid 15-minute detection. It bridges the performance gap between rapid on-site testing and laboratory diagnostics, making it particularly suitable for primary healthcare institutions and emergency on-site testing scenarios.

[0032] This invention provides the application of the kit described above in the detection of Nipah virus antigen for non-diagnostic purposes. This invention uses quantum dot microspheres with excellent luminescent properties to replace traditional colloidal gold as the immunomarker, significantly improving the intensity of the detection fluorescence signal and detection sensitivity, lowering the detection limit, and effectively detecting samples with low viral load in the early stages of infection. This overcomes the shortcomings of traditional colloidal gold test strips, such as low sensitivity and easy false negatives. The kit can be configured with dual detection lines targeting both the Nipah virus N and G proteins on the same test strip, achieving simultaneous joint detection of dual targets. This avoids the risks of false negatives and false negatives caused by differences in antigen expression abundance, expression sequence, and improper sampling timing at different stages of viral infection under single-target detection mode, significantly improving the overall sample detection rate. Furthermore, this invention optimizes the sample loading solution formulation and sample pad treatment system, effectively maintaining the biological activity of the labeled antibody, reducing non-specific adsorption, optimizing the chromatographic flow rate and system stability, reducing background interference, and improving the repeatability and accuracy of the test strip detection.

[0033] This invention provides a method for detecting Nipah virus antigen for non-diagnostic purposes, using the kit described above, comprising the following steps: diluting Nipah virus N protein-specific antibodies and Nipah virus G protein-specific antibodies labeled with quantum dot microspheres using a sample loading solution, mixing them with the sample to be tested, and incubating them; then adding the incubated sample to a test strip for chromatography; and determining whether the sample contains Nipah virus or performing quantitative detection of Nipah virus based on the signal intensity. As an optional embodiment of this invention, the incubation temperature can be 37°C; the incubation time can be 2 min; and the chromatography time can be 13-15 min, or 14 min. This invention does not specifically limit the method for detecting the signal intensity; any conventional testing method in the art can be used.

[0034] N protein is an internal nucleocapsid protein with low antigenic epitope exposure in its native conformation; G protein is a surface glycoprotein, highly glycosylated and with a complex conformation. The optimal antibody recognition environments (pH, ionic strength, buffer system) for both conflict, and the strong hydrophobicity of G protein easily leads to non-specific adsorption. Sharing a solid-phase carrier will interfere with the purity of the N protein detection signal, making it difficult to achieve effective detection simultaneously under the same reaction conditions. Existing technologies mostly employ a single-target independent detection strategy.

[0035] During the replication of Nipah virus in vivo, the N and G proteins exhibit differences in expression timing, antigenic fluctuations, immune masking, and sample stability, rather than simply a difference in early or late expression. The N protein is highly expressed in the early stages but is easily cleared as the immune response initiates, posing a risk of false negatives in the mid-to-late stages. The G protein has a narrow expression window and is insufficiently detected in samples with low viral loads, but its antigenic conservation and specificity are significantly superior to the N protein during the viral maturation and release phase, compensating for late-stage detection deficiencies. This invention constructs a dual-target synergistic detection system for both the N and G proteins, overcoming the technical bottleneck of single biomarkers being unable to adapt to the detection of samples throughout the entire disease course, and overcoming the inherent false negatives and false negatives of single-protein detection, significantly improving the detection accuracy of samples throughout the entire disease course.

[0036] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0037] The following technical solutions involve reagent-related information: The dengue virus antigen, Ebola virus antigen, Chikungunya virus antigen, yellow fever virus antigen, and influenza A virus antigen were purchased from Changzhou Zhongmei Xinxin Biotechnology Co., Ltd.

[0038] In the following examples, Examples 1-3: mouse anti-Nipah virus N protein specific monoclonal antibodies Ab1 (catalog number: RDMV022) and Ab2 (catalog number: RDMV023) were purchased from Suzhou Renduan Biomedical Technology Co., Ltd.; Nipah virus N protein was purchased from Suzhou Renduan Biomedical Technology Co., Ltd. (catalog number: RDPV016); mouse anti-Nipah virus G protein specific monoclonal antibodies Ab3 (catalog number: ACT-mAb-NiV-005) and Ab4 (catalog number: ACT-mAb-NiV-006) were purchased from Changzhou Sino-American Xinxin Biotechnology Co., Ltd.; and Nipah virus G protein was purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd. (catalog number: 40980-V08H).

[0039] The carboxylated quantum dot microspheres were purchased from Beijing Nanokin Biotechnology Co., Ltd., model: FM610C, specification: 1mL / vial.

[0040] Goat anti-mouse IgG polyclonal antibody, purchased from Changzhou Zhongmei Xinxin Biotechnology Co., Ltd., model: ACT-Ag-IgG(M)-001, specification: 2.0mg / vial.

[0041] N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide hydrochloride (EDC) were purchased from Beijing Bailingwei Technology Co., Ltd. Dimethyl sulfoxide (DMSO) was purchased from Beijing Innocare Technology Co., Ltd. 2-(N-morpholine)ethanesulfonic acid (MES) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Polyethylene glycol-20000 (PEG-20000), sucrose, and bovine serum albumin (BSA) were purchased from Sinopharm Chemical Reagent Co., Ltd. Anhydrous trehalose was purchased from Shanghai TCI Chemical Industry Development Co., Ltd. Tween-20 was purchased from Sigma-Aldrich, Inc., USA.

[0042] The main instruments in the specific embodiments of this invention include: The automatic gold spraying and film drawing instrument (model: WRF-HPY001) was purchased from Haining Weierfen Automation Equipment Co., Ltd.; the microcomputer automatic chopping machine (model: ZQ2000) and CNC strip cutting machine (model: CTS300) were both purchased from Shanghai Jinbiao Biotechnology Co., Ltd.; the dry fluorescence immunoassay analyzer (model: FIC-S100) was purchased from Suzhou Hemai Precision Instrument Co., Ltd.; the fully automatic gel imaging instrument (model: Fusion FX Spectra) was produced by VILBER LOURMAT of France; and the small ultrasonic cleaner (model: JP-3800S) was purchased from Shenzhen Jiemeng Cleaning Equipment Co., Ltd.

[0043] All experiments were performed in triplicate, and results are expressed as mean ± standard deviation.

[0044] Example 1 A Nipah virus antigen quantitative detection kit, the preparation method and detection method of which are as follows: This includes the preparation of the test strip: a schematic diagram of the test strip structure is shown below. Figure 1 As shown.

[0045] 1. Preparation of quantum dot microsphere labeled antibodies: Nipah virus N protein antibody and G protein antibody were coupled to the surface of quantum dot microspheres using an activated ester method.

[0046] First, mix 25 μL of quantum dot (QB) microsphere (QBs) solution (1 μmol / L) with 25 μL of MES solution (20 mM, pH 6.0), then add 1 μL of EDC solution (20 mg / mL) and 1 μL of NHS solution (20 mg / mL), and activate at 37°C in the dark for 15 minutes. Centrifuge at 10000 rcf for 20 minutes, discard the supernatant, and resuspend the precipitate in 25 μL of MES solution (10 mM, pH 6.0), vortex to mix. Next, add 10 μg of Nipah virus N protein-specific monoclonal antibody Ab1 or Nipah virus G protein-specific monoclonal antibody Ab3, and incubate at 37°C and 800 rpm in the dark for 1 hour. Add 25 μL of 10% BSA solution, vortex to mix, block at 37°C in the dark for 30 minutes, centrifuge at 8000 rcf for 15 minutes, discard the supernatant, and add 50 µL of borate buffer (5 Resuspend once in 5 mM (pH 8.0) and 1% BSA solution, wash once, centrifuge at 8000 rcf for 15 minutes, and discard the supernatant; finally, add 25 µL borate buffer (5 mM, pH 8.0) and 1% BSA solution to resuspend, thus obtaining the quantum dot microsphere-labeled Nipah virus N protein antibody (abbreviated as QBs-Ab1) and the quantum dot microsphere-labeled Nipah virus G protein antibody (abbreviated as QBs-Ab3), and store at 4℃ for later use.

[0047] 2. Sample pad treatment: Prepare the sample pad treatment solution: 0.01M PB solution, pH 7.4, containing 0.5% BSA, 0.05% Tween-20, and 1% trehalose. Completely immerse the sample pad in the treatment solution, allowing it to fully absorb the solution, then place it in an oven at 37°C and dry overnight.

[0048] 3. Test strip assembly: Nitrocellulose membranes (NC membranes) were adhered to the substrate. Using an automated gold-spraying membrane scribing instrument, Nipah virus N protein capture antibody Ab2 (diluted to 0.8 mg / mL, T2 line) and G protein capture antibody Ab4 (diluted to 0.8 mg / mL, T1 line) were coated onto the NC membrane as detection lines, and goat anti-mouse IgG polyclonal antibody (diluted to 0.5 mg / mL) was coated onto the NC membrane as the control line (C line). The membrane was scribed at a speed of 1 µL / cm. After scribing, the plate was placed in an oven at 37°C for 2 hours to dry. The sample pads and absorbent paper were then assembled onto the substrate and cut into 3 mm wide strips using a CNC strip cutter. These strips were then dried, protected from light, and stored for later use.

[0049] 4. Detection method: Take an appropriate amount of the preserved N and G protein antibody conjugates and dilute them 1500-fold with loading buffer (Tris-HCl, with added Tween-20, sugar, BSA, and PEG-20000). Then, add N and G protein antigens to prepare positive samples; samples without antigens serve as negative controls. Incubate these mixtures at 37°C for 2 minutes in a constant temperature shaker. Add 60 µL of the incubated sample to the sample pad on the test card. After chromatography for 13-15 minutes, place the test card into a dry fluorescence immunoassay analyzer to read the results.

[0050] 5. Detection Principle When a sample containing Nipah virus N and G proteins is mixed with the diluted conjugate of the loading solution, Ab1 / Ab3 binds to the N / G protein antigens in the sample to form a QBs-Ab1 / Ab3-Ag complex. The mixture containing the complex is added to the sample pad and chromatography upwards to the T2 / T1 line. The capture antibody binds to the N / G protein antigens in the complex, forming a QBs-Ab1 / Ab3-Ab2 / Ab4 complex that deposits at the T2 / T1 line. The remaining complex continues to precipitate upwards, and Ab1 / Ab3 in the complex reacts with goat anti-mouse IgG and deposits at the C line. Fifteen minutes after sample addition, under UV light, if both the T1 / T2 and C lines show red fluorescent bands, the result is positive; if only the C line shows a fluorescent band, the result is negative; if no fluorescent band appears at the C line, the test strip is invalid. Combined with a dual-channel fluorescence immunoassay analyzer, N and G proteins can be quantified simultaneously.

[0051] Example 2 System optimization 1.1 Optimization of sample loading solution composition The size and content of each component in the loading solution directly affect the antigen-antibody specific binding efficiency, the stability of quantum dot conjugates, and chromatographic flowability, thus significantly altering the detection signal intensity and background noise level. This experiment uses the absolute signal mean of Nipah virus G protein (T1 line) and N protein (T2 line) as the core detection indicators, and optimizes the key components of the loading solution through orthogonal experiments to obtain the best detection performance.

[0052] The experiment optimized the pH value, sucrose, PEG-20000, and Tween-20 content of the main influencing factors of the loading solution to obtain the optimal ratio. Other components of the loading solution were 3% trehalose, 0.5% BSA, and a Tris-HCl ion concentration of 0.05 mol / L. During the optimization of the loading solution components, the dilution factor for both G and N protein antibody conjugates was 2000-fold. That is, two conjugates were added to the loading solution so that both conjugates were diluted 2000-fold. The concentrations of T1 and T2 antibodies were both 0.5 mg / mL. The antigen sample used was 50 ng / mL of G protein and 50 ng / mL of N protein.

[0053] Detection method: The N protein and G protein antibody conjugate was diluted with loading buffer. Then, G protein and N protein were added to a final concentration of 50 ng / mL. A sample without the antigen was used as a negative control. The mixture was incubated at 37°C for 2 minutes using a shaker. 60 µL of the incubated sample was then added to the sample pad on the test card. After chromatography for 15 minutes, the test card was placed in a dry fluorescence immunoassay analyzer to read the results.

[0054] Except for the factors described, the other components or methods of the kit are the same as in Example 1.

[0055] The specific orthogonal experimental design and results are shown in Table 1. According to the detection results, the order of influence on the mean absolute signals (i.e., the difference between the signal value and the background value) of T1 and T2 is pH > sucrose > Tween-20 > PEG-20000. The optimal loading solution ratio is: 0.05 mol / L pH7.5 Tris-HCl, containing 3% sucrose, 3% trehalose, 0.25% PEG-20000, 0.5% BSA, and 1% Tween-20.

[0056] Where k is the mean of the detected signal at the current level of the current factor. For example, k1 in the pH column corresponds to the mean of the three detected signals at the current level of 6.5 for the current factor pH, i.e., the mean of the total signal values ​​of experiments 1, 2, and 3. K1 = (2249 + 2542 + 2068) / 3 = 2286.33. R is the range, which is the difference between the mean of the maximum signal and the mean of the minimum signal at the current factor. For example, the calculation method for R for pH is: R = 3552.00 - 2205.33 = 1346.67.

[0057] Table 1. Orthogonal experimental design and detection results of the sample loading solution

[0058] Note: Absolute signal mean = (absolute signal of T1 line + absolute signal of T2 line) / 2, where the absolute signal is the signal value of the detected antigen minus the background signal (i.e., the signal of the negative control).

[0059] 1.2 Screening based on T-line concentration and coupling agent dilution factor Based on the orthogonal experimental results in section 1.1, and using corresponding optimized parameters, the antibody concentrations for the T1 and T2 lines of the combined test strip were set to 0.5, 0.8, and 1.0 mg / mL, respectively, and the dilution factors for the N and G protein antibody conjugates were set to 1000, 1500, and 2000 times. The combined test strip was then used to detect the corresponding N and G protein antigens.

[0060] Experiments 1-3: The conjugates were diluted 1000 times, and the concentrations at the T line were 0.5, 0.8, and 1.0 mg / mL, respectively. Experiments 4-6: The conjugates were diluted 1500 times, and the concentrations at the T line were 0.5, 0.8, and 1.0 mg / mL, respectively. Experiments 7-9: The conjugates were diluted 2000 times, and the concentrations at the T line were 0.5, 0.8, and 1.0 mg / mL, respectively.

[0061] The experiment was conducted as follows: N protein and G protein antibody conjugates were diluted with loading buffer at the set dilution ratios (1000 / 1500 / 2000 times); simultaneously, the concentrations of the T1 (G protein antibody) and T2 (N protein antibody) lines were set to 0.5, 0.8, and 1.0 mg / mL, respectively, to prepare a combined test strip. Subsequently, positive samples were prepared by adding N protein antigen to a final concentration of 50 ng / mL and G protein antigen to 50 ng / mL, with samples without antigen serving as negative controls. The mixture was incubated at 37°C for 2 minutes in a constant temperature shaker. 60 µL of the incubated sample was added to the sample pad of the test strip, and after chromatography for 15 minutes, the test strip was placed in a dry fluorescence immunoassay analyzer to read the fluorescence signal values ​​and background values ​​of the T1 and T2 lines.

[0062] All experiments were performed in triplicate, and results are expressed as mean ± standard deviation.

[0063] Test results as follows Figures 2-3 As shown.

[0064] At each dilution factor, the signal value of both detection lines increased with increasing T-line concentration, and the increase was greater at 0.8 mg / mL than at 1.0 mg / mL. Therefore, a T-line concentration of 0.8 mg / mL was selected. At this concentration, the signal increase was largest at a conjugate dilution factor of 1500, so a dilution factor of 1500 was chosen for the conjugate. In summary, the concentrations of both T1 and T2 lines for the combined detection strip were selected as 0.8 mg / mL, and the dilution factor for both protein antibody conjugates was selected as 1500.

[0065] 1.3 Screening by Simulated Sample Dilution Factor Since there have been no cases of Nipah virus infection in China and no clinical samples were available, this study used simulated samples to evaluate the performance of the reagent kit.

[0066] Based on the orthogonal experimental results in 1.1 and the optimal parameters for screening the T-line concentration and coupling dilution factor in 1.2, corresponding optimized parameters were used to screen the dilution factor of simulated samples.

[0067] Nasal swabs were collected from healthy individuals. Each swab was eluted with 0.5 mL of sample loading solution to obtain a nasal swab sample. The N and G protein antibody conjugate was diluted 1500-fold using the nasal swab samples and 2-fold and 5-fold diluted samples, and N and G protein antigens were added to a final concentration of 50 ng / mL. A control group without elution of nasal swabs was also prepared. The combined test strips were used to test the above samples. The test results are as follows: Figures 4-5 As shown, experiments 1-4 are respectively the unwashed nasal swab control, nasal swab sample, 2-fold dilution of nasal swab sample, and 5-fold dilution of nasal swab sample.

[0068] The results show that the G protein (T1) and N protein (T2) simulated sample experiments were very effective when directly eluted from nasal swabs. Further dilution of the nasal swab samples did not significantly increase the detection signal.

[0069] 1.4 Nasal swab elution with added NaCl Because the current detection system has a relatively high background value, a certain amount of NaCl is added during nasal swab elution to maintain osmotic pressure and reduce matrix interference.

[0070] Based on the results of the orthogonal experiments in 1.1, the selection of optimal parameters for T-line concentration and coupling agent dilution factor in 1.2, and the selection of dilution factor for simulated samples in 1.3, corresponding optimized parameters were adopted. Nasal swabs were collected from healthy individuals. Each swab was eluted with 0.5 mL of sample loading solution to obtain nasal swab samples. Sodium chloride was added to the sample loading solution for elution of the nasal swabs at concentrations of 0.1%, 0.25%, 0.5%, and 0.9%, respectively. The eluted nasal swabs were then directly diluted 1500-fold with N and G protein antibody conjugates, and N and G protein antigens were added to a final concentration of 50 ng / mL. The samples were then tested using a combined test strip. The test results are as follows: Figures 6-7 As shown, experiments 1-4 represent NaCl concentrations of 0.1%, 0.25%, 0.5%, and 0.9%, respectively.

[0071] The experimental results show that for the G protein detection line (T1), the signal value decreases as the NaCl content increases, and the background signal also decreases accordingly. For the N protein detection line (T2), changes in NaCl within the current experimental range have little impact on the signal value, although the background value decreases somewhat. Considering both the signal value and the background signal, a NaCl content of 0.25% was chosen.

[0072] Example 3 A method for preparing a Nipah virus antigen detection kit, comprising the following steps: 1. Preparation of quantum dot microsphere labeled antibodies: Nipah virus N protein antibody and G protein antibody were coupled to the surface of quantum dot microspheres using an activated ester method.

[0073] First, mix 25 μL of quantum dot (QB) microsphere (QBs) solution (1 μmol / L) with 25 μL of MES solution (20 mM, pH 6.0), then add 1 μL of EDC solution (20 mg / mL) and 1 μL of NHS solution (20 mg / mL), and activate at 37°C in the dark for 15 minutes. Centrifuge at 10000 rcf for 20 minutes, discard the supernatant, and resuspend the precipitate in 25 μL of MES solution (10 mM, pH 6.0), vortex to mix. Next, add 10 μg of Nipah virus N protein-specific monoclonal antibody Ab1 or / and Nipah virus G protein-specific monoclonal antibody Ab3, and incubate at 37°C and 800 rpm in the dark for 1 hour. Add 25 μL of 10% BSA solution, vortex to mix, block at 37°C in the dark for 30 minutes, centrifuge at 8000 rcf for 15 minutes, discard the supernatant, and add 50 µL of borate buffer (5 Resuspend once in 5 mM (pH 8.0) and 1% BSA solution, wash once, centrifuge at 8000 rcf for 15 minutes, and discard the supernatant; finally, add 25 µL borate buffer (5 mM, pH 8.0) and 1% BSA solution to resuspend, thus obtaining the quantum dot microsphere-labeled Nipah virus N protein antibody (abbreviated as QBs-Ab1) and the quantum dot microsphere-labeled Nipah virus G protein antibody (abbreviated as QBs-Ab3), and store at 4℃ for later use.

[0074] 2. Sample pad treatment: Prepare the sample pad treatment solution: 0.01M PB solution, pH 7.4, containing 0.5% BSA, 0.05% Tween-20, and 1% trehalose. Completely immerse the sample pad in the treatment solution, allowing it to fully absorb the solution, then place it in an oven at 37°C and dry overnight.

[0075] 3. Test strip assembly: A nitrocellulose membrane (NC membrane) was adhered to the substrate. Using an automated gold-spraying membrane scribing device, Nipah virus N protein capture antibody Ab2 (diluted to 0.8 mg / mL, T2 line) and G protein capture antibody Ab4 (diluted to 0.8 mg / mL, T1 line) were coated onto the NC membrane as detection lines. Goat anti-mouse IgG polyclonal antibody (diluted to 0.5 mg / mL) was coated onto the NC membrane as a control line (C line). The membrane was scribed at a speed of 1 µL / cm. After scribing, the plate was dried in an oven at 37°C for 2 hours. The sample pad and absorbent paper were then assembled onto the substrate and cut into 3 mm wide strips using a CNC strip cutter. These strips were then dried, protected from light, and stored for later use. The assembled Nipah virus N / G protein antigen combined test strip was obtained.

[0076] 4. Sensitivity testing: Take an appropriate amount of the preserved N and G protein antibody conjugates and dilute them 1500 times with loading buffer (0.05 mol / L pH 7.5, Tris-HCl, with 1% Tween-20, 3% sucrose, 0.5% BSA, 3% trehalose, and 0.25% PEG-20000). Then add different concentrations of N and G protein antigens (0.005, 0.025, 0.1, 0.5, 2.5, 10, 25, 100, and 250 ng / mL; N and G mixed antigens are added to the diluted conjugates at the same concentration simultaneously, and then loaded after mixing). Incubate these mixtures at 37°C for 2 minutes in a constant temperature shaker. Add 60 µL of the incubated sample to the sample pad on the test card. After chromatography for 15 minutes, place the test card into a dry fluorescence immunoassay analyzer to read the results.

[0077] A standard curve is plotted based on the detected signal values; the G protein standard curve is shown below. Figure 8 As shown, the equation of the curve is: y = 184.7 + (7841 - 184.7) / (1 + 10) ((4.493-x)×1.483) ), R 2 =0.9960. The calculated LOD is 20 pg / mL, with a visual LOD of 100 pg / mL. N protein standard curve as follows... Figure 9 As shown, the equation of the curve is: y = 278.3 + (39093 - 278.3) / (1 + 10) ((4.965 -x)×1.054) ), R 2 =0.9931. The calculated LOD is 5 pg / mL, of which the visual LOD is 25 pg / mL.

[0078] 5. Specific detection The assembled Nipah virus N / G protein antigen combined test strip was used to detect Nipah virus N and G protein antigens at concentrations of 50 ng / mL (i.e., a mixture of 50 ng / mL N protein antigen and 50 ng / mL G protein antigen), as well as 100 ng / mL dengue virus antigen, 100 ng / mL Ebola virus antigen, 100 ng / mL Chikungunya virus antigen, 100 ng / mL yellow fever virus antigen, and 100 ng / mL influenza A virus antigen. After chromatography for 15 minutes, the Nipah virus N / G protein antigen test result was positive, while the other viral antigen test results were negative. Figure 10 This indicates that the prepared combined detection strip has good specificity and no cross-reactivity with other related pathogens. Figure 11 This is an image obtained using a gel imaging system after sample chromatography on the test strip.

[0079] 6. Quantitative accuracy of simulated samples The prepared combined test strips were used to quantitatively detect nasal swab samples. The concentrations of N protein simulated samples were set to 0.025, 25, and 250 ng / mL, and the concentrations of G protein simulated samples were set to 0.1, 10, and 100 ng / mL.

[0080] Nasal swabs were collected from healthy individuals. Each swab was eluted with 0.5 mL of sample loading solution to obtain nasal swab samples. 0.25% sodium chloride was added to the sample loading solution used for elution. The eluted nasal swabs were then directly diluted 1500-fold with N and G protein antibody conjugates. The N protein simulated sample concentrations were set to 0.025, 25, and 250 ng / mL, respectively; the G protein simulated sample concentrations were set to 0.1, 10, and 100 ng / mL, respectively. The samples were then tested using a combined test strip.

[0081] The sample concentrations were calculated based on the standard curve, and the results are shown in Table 2.

[0082] Table 2 Quantitative Results of the Combined Detection Test Strip

[0083] Note: The mean fluorescence signal is the average of the three detection signals.

[0084] It can be seen that the quantitative concentrations of N and G protein simulated samples prepared by the test strips are basically consistent with the actual concentrations, and the recovery rate is between 91% and 108%, which shows the accuracy of the quantitative results.

[0085] Example 4 Initial screening for N protein antibodies Specifically, it was divided into two experimental groups: Experiment 1: Nipah virus N protein-specific monoclonal antibody Ab1 was selected as RDMV022; Nipah virus N protein-specific monoclonal antibody Ab2 was selected as RDMV023 as a group of experiments to detect the fluorescence signal value of N protein.

[0086] Implementation 2: Nipah virus N protein-specific monoclonal antibody Ab1 was selected as RDMV023; Nipah virus N protein-specific monoclonal antibody Ab2 was selected as RDMV022 as a set of experiments to detect the fluorescence signal value of N protein.

[0087] The fluorescence signal values ​​were detected in Experiments 1 and 2 using the following methods.

[0088] 1. Preparation of quantum dot microsphere labeled antibodies: Nipah virus N protein antibody was conjugated to the surface of quantum dot microspheres using an activated ester method.

[0089] First, mix 25 μL of quantum dot (QB) microsphere (QBs) solution (1 μmol / L) with 25 μL of MES solution (20 mM, pH 6.0), then add 1 μL of EDC solution (20 mg / mL) and 1 μL of NHS solution (20 mg / mL), and activate at 37°C in the dark for 15 minutes. Centrifuge at 10000 rcf for 20 minutes, discard the supernatant, and resuspend the precipitate in 25 μL of MES solution (10 mM, pH 6.0), vortex to mix. Next, add 10 μg of Nipah virus N protein-specific monoclonal antibody Ab1, and incubate at 37°C and 800 rpm in the dark for 1 hour. Add 25 μL of 10% BSA solution, vortex to mix, block at 37°C in the dark for 30 minutes, centrifuge at 8000 rcf for 15 minutes, discard the supernatant, and add 50 µL of borate buffer (5 Resuspend and wash once with 1% BSA solution (5 mM, pH 8.0), centrifuge at 8000 rcf for 15 minutes, and discard the supernatant; finally, add 25 µL of borate buffer (5 mM, pH 8.0) and 1% BSA solution to resuspend, and obtain the quantum dot microsphere-labeled Nipah virus N protein antibody (abbreviated as QBs-Ab1), which is stored at 4℃ for later use.

[0090] 2. Sample pad treatment: Prepare the sample pad treatment solution: 0.01M PB solution, pH 7.4, containing 0.5% BSA, 0.05% Tween-20, and 1% trehalose. Completely immerse the sample pad in the treatment solution, allowing it to fully absorb the solution, then place it in an oven at 37°C and dry overnight.

[0091] 3. Test strip assembly: Nitrocellulose membranes (NC membranes) were adhered to the substrate. Using an automated gold-spraying membrane scribing instrument, Nipah virus N protein capture antibody Ab2 (diluted to 0.5 mg / mL, T line) was coated onto the NC membrane as the detection line, and goat anti-mouse IgG polyclonal antibody (diluted to 0.5 mg / mL) was coated onto the NC membrane as the control line (C line). The membranes were scribed at a speed of 1 µL / cm. After scribing, the plate was placed in an oven at 37°C for 2 hours to dry. The sample pads and absorbent paper were then assembled onto the substrate and cut into 3 mm wide strips using a CNC strip cutter. These strips were then dried, protected from light, and stored for later use.

[0092] 4. Detection method: Take an appropriate amount of the stored N protein antibody-conjugate and dilute it 2000-fold with loading buffer (0.05 mol / L Tris-HCl, containing 3% sucrose, 3% trehalose, 0.5% PEG-20000, 0.5% BSA, and 1% Tween-20), with the pH of the loading buffer set to 5.5, 6.5, and 7.5, respectively. Then, add N protein antigen to prepare positive samples, with the amount of N protein antigen added set at 100 ng / mL and 50 ng / mL, respectively. Samples without antigen serve as negative controls. Incubate these mixtures in a constant temperature shaker at 37°C for 2 minutes. Add 60 µL of the incubated sample to the sample pad on the test card, and after chromatography for 15 minutes, place the test card into a dry fluorescence immunoassay analyzer to read the results.

[0093] The results of Experiment 1 are as follows Figure 12 As shown; the results of Experiment 2 are as follows. Figure 13 As shown.

[0094] Depend on Figures 12-13 It can be seen that in the initial screening experiment of N protein antibody, by comparing the sample solutions with three pH values, it can be seen that when Ab1:RDMV022 and Ab2:RDMV023 are combined, a larger effective signal can be obtained, and conversely, the background signal is larger.

[0095] Example 5 Initial screening for G protein antibodies Specifically, it was divided into two experimental groups: Experiment 1: Nipah virus G protein-specific monoclonal antibody Ab3 (ACT-mAb-NiV-005) and Nipah virus G protein-specific monoclonal antibody Ab4 (ACT-mAb-NiV-006) were selected as one experimental group to detect the fluorescence signal value of G protein.

[0096] Implementation 2: Nipah virus G protein-specific monoclonal antibody Ab3 was selected as ACT-mAb-NiV-006; Nipah virus G protein-specific monoclonal antibody Ab4 was selected as ACT-mAb-NiV-005 as a group of experiments to detect the fluorescence signal value of G protein.

[0097] The fluorescence signal values ​​were detected in Experiments 1 and 2 using the following methods.

[0098] 1. Preparation of quantum dot microsphere labeled antibodies: Nipah virus G protein antibody was conjugated to the surface of quantum dot microspheres using an activated ester method.

[0099] First, mix 25 μL of quantum dot (QB) microsphere (QBs) solution (1 μmol / L) with 25 μL of MES solution (20 mM, pH 6.0), then add 1 μL of EDC solution (20 mg / mL) and 1 μL of NHS solution (20 mg / mL), and activate at 37°C in the dark for 15 minutes. Centrifuge at 10000 rcf for 20 minutes, discard the supernatant, and resuspend the precipitate in 25 μL of MES solution (10 mM, pH 6.0), vortex to mix. Next, add 10 μg of Nipah virus G protein-specific monoclonal antibody Ab3, and incubate at 37°C and 800 rpm in the dark for 1 hour. Add 25 μL of 10% BSA solution, vortex to mix, block at 37°C in the dark for 30 minutes, centrifuge at 8000 rcf for 15 minutes, discard the supernatant, and add 50 µL of borate buffer (5 Resuspend and wash once with 1% BSA solution (5 mM, pH 8.0), centrifuge at 8000 rcf for 15 minutes, and discard the supernatant; finally, add 25 µL of borate buffer (5 mM, pH 8.0) and 1% BSA solution to resuspend, and obtain the quantum dot microsphere-labeled Nipah virus G protein antibody (abbreviated as QBs-Ab3), which is stored at 4℃ for later use.

[0100] 2. Sample pad treatment: Prepare the sample pad treatment solution: 0.01M PB solution, pH 7.4, containing 0.5% BSA, 0.05% Tween-20, and 1% trehalose. Completely immerse the sample pad in the treatment solution, allowing it to fully absorb the solution, then place it in an oven at 37°C and dry overnight.

[0101] 3. Test strip assembly: Nitrocellulose membranes (NC membranes) were adhered to the substrate. Using an automated gold-spraying membrane scribing system, Nipah virus G protein capture antibody Ab4 (diluted to 0.5 mg / mL, T line) was coated onto the NC membrane as the detection line, and goat anti-mouse IgG polyclonal antibody (diluted to 0.5 mg / mL) was coated onto the NC membrane as the control line (C line). The membranes were scribed at a speed of 1 µL / cm. After scribing, the plate was placed in an oven at 37°C for 2 hours to dry. The sample pads and absorbent paper were then assembled onto the substrate and cut into 3 mm wide strips using a CNC strip cutter. These strips were then dried, protected from light, and stored for later use.

[0102] 4. Detection method: Take an appropriate amount of the stored G protein antibody-conjugate and dilute it 2000-fold with loading buffer (0.05 mol / L Tris-HCl, containing 3% sucrose, 3% trehalose, 0.5% PEG-20000, 0.5% BSA, and 1% Tween-20), with the pH of the loading buffer set to 5.5, 6.5, and 7.5, respectively. Then, add G protein antigen to prepare positive samples, with the addition amounts set to 100 ng / mL and 50 ng / mL, respectively. Samples without antigen serve as negative controls. Incubate these mixtures in a constant temperature shaker at 37°C for 2 minutes. Add 60 µL of the incubated sample to the sample pad on the test card. After chromatography for 15 minutes, place the test card into a dry fluorescence immunoassay analyzer to read the results.

[0103] The results of Experiment 1 are as follows Figure 14 As shown; the results of Experiment 2 are as follows. Figure 15 As shown.

[0104] Depend on Figures 14-15 It can be seen that in the initial screening experiment of G protein antibodies, by comparing the sample solutions with three pH values, it can be seen that when Ab3:ACT-mAb-NiV-005 and Ab4:ACT-mAb-NiV-006 are combined, a larger effective signal can be obtained; otherwise, the background signal is larger.

[0105] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A kit for detecting Nipah virus antigen, characterized in that, include: Quantum dot microsphere-labeled Nipah virus N protein-specific antibody, quantum dot microsphere-labeled Nipah virus G protein-specific antibody, sample loading solution, and test strip.

2. The reagent kit according to claim 1, characterized in that, The Nipah virus N protein-specific antibody includes Nipah virus N protein-specific monoclonal antibody Ab1; the Nipah virus G protein-specific antibody includes Nipah virus G protein-specific monoclonal antibody Ab3.

3. The reagent kit according to claim 1, characterized in that, The loading solution comprises: 0.025~0.1 mol / L Tris-HCl, 2~4wt% sucrose, 1~5wt% trehalose, 0.25~1wt% PEG-20000, 0.2~1wt% BSA, and 0.5~2wt% Tween-20; with a pH of 7.0~8.

5.

4. The reagent kit according to claim 3, characterized in that, The sample loading solution also includes 0.25~0.5wt% NaCl.

5. The reagent kit according to claim 1, characterized in that, The quantum dot microsphere-labeled Nipah virus N protein-specific antibody was obtained by covalently coupling 8-15 μg of N protein-specific antibody with activated quantum dot microspheres. The quantum dot microsphere-labeled Nipah virus G protein-specific antibody was obtained by covalently coupling 8-15 μg of G protein-specific antibody with activated quantum dot microspheres. When used for Nipah virus antigen detection, the Nipah virus N protein-specific antibody and the Nipah virus G protein-specific antibody labeled with quantum dot microspheres are diluted 1000 to 2000 times respectively using the sample loading solution.

6. The kit according to claim 1, characterized in that, The test strip's detection lines are coated with Nipah virus N protein-specific antibodies and Nipah virus G protein-specific antibodies, respectively; the Nipah virus N protein-specific antibodies include Nipah virus N protein-specific monoclonal antibody Ab2; and the Nipah virus G protein-specific antibodies include Nipah virus G protein-specific monoclonal antibody Ab4.

7. The reagent kit according to claim 6, characterized in that, The concentration of the antibody applied to the detection line is 0.5~1.5 mg / mL.

8. The kit according to claim 6 or 7, characterized in that, The method for processing the sample pad of the test strip includes: Immerse the sample pad in the treatment solution to ensure it is fully absorbed. The treatment solution comprises: 0.005~0.05M PB solution, pH 7.4, containing 0.25~1.5wt% BSA, 0.25~1wt% Tween-20 and 0.5~2wt% trehalose.

9. The use of the kit according to any one of claims 1 to 8 in the detection of Nipah virus antigen for non-diagnostic purposes.

10. A method for detecting Nipah virus antigen for non-diagnostic purposes, characterized in that, The kit according to any one of claims 1 to 8 comprises the following steps: The Nipah virus N protein-specific antibody and the Nipah virus G protein-specific antibody labeled with quantum dot microspheres were diluted with the sample loading solution and then mixed with the sample to be tested and incubated. The incubated sample was then dropped onto the test strip for chromatography. The presence of Nipah virus in the sample was determined based on the signal intensity, or the Nipah virus was quantitatively detected.

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