WO2023284686A1 - 一种结合抗体的生物传感检测方法及检测系统 - Google Patents

一种结合抗体的生物传感检测方法及检测系统 Download PDF

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WO2023284686A1
WO2023284686A1 PCT/CN2022/104952 CN2022104952W WO2023284686A1 WO 2023284686 A1 WO2023284686 A1 WO 2023284686A1 CN 2022104952 W CN2022104952 W CN 2022104952W WO 2023284686 A1 WO2023284686 A1 WO 2023284686A1
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antibody
sample
tested
binding
fiber optic
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French (fr)
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卞素敏
萨万·默罕默德
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西湖大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands

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  • the present application relates to the technical field of optical fiber biosensing, in particular to a biosensing detection method and detection system combined with antibodies.
  • neutralizing antibodies are antibodies that make the virus lose its ability to bind to receptors, and binding antibodies only refer to antibodies that can bind to the surface area of the virus.
  • the monitoring of neutralizing antibodies against the new crown is of the greatest significance. It is an important indicator for evaluating the safety and efficacy of the new crown vaccine. At the same time, monitoring the binding antibody can help answer the infection history of the new crown virus and contribute to epidemiological research.
  • Biolayer interferometry is a technique that detects the surface response of a sensor by detecting shift changes in the interference spectrum.
  • a beam of visible light is emitted from the spectrometer, two reflection spectra and an interference spectrum will be formed at the two interfaces of the optical film at the end of the fiber sensor.
  • the red shift (unit: nm) of the spectrum is positively correlated with the concentration of terminal-specific binding molecules.
  • its signal intensity at low detection concentrations is very limited, so it is rarely used in quantitative analysis.
  • Novel coronavirus or COVID-19 referred to as the new crown
  • BLI biolayer interferometry, biological layer interference technology
  • BAbs Binding antibodies, Binding antibody
  • RBD Receptor binding domain, receptor binding region
  • S-ECD Extracellular Domain of Spike Protein, spike protein
  • ELISA Enzyme linked immunosorbent assay, enzyme-linked immunosorbent assay; 7 .DAB: 3,3'-diaminobenzidine tetrahydrochloride, 3,3'-diaminobenzidine tetrahydrochloride; 8.HRP: Horseradish Peroxidase, horseradish peroxidase; 9.Anti-RBD BAbs: binding antibody against RBD region , also known as anti-RBD binding antibody; 10. Anti-S-ECD BAbs: binding antibody against S-ECD region, also known as anti-S-ECD binding antibody; 11. Anti-RBDN501Y BAbs: binding antibody against RBD variant N501Y , also known as anti-RBD N501Y binding antibody.
  • the purpose of this application is to provide a biosensing detection method and detection system for binding antibodies.
  • an automated biosensing system for rapid monitoring of new crown binding antibodies can be obtained to help the development of new crown vaccines. and potency assessment.
  • the first aspect of the present application provides a biosensing detection method that combines antibodies, including:
  • Preparation of fiber optic probes immobilize biotinylated antigens on the surface of a streptavidin chip to obtain fiber optic probes.
  • the biotinylated antigens include biotinylated 2019-nCoV core proteins.
  • test sample and reference sample obtain the serum or dried blood spot whole blood of the subject to be tested, and use it as the sample to be tested after dilution; Add a reference antibody chimeric anti-RBD monoclonal antibody to obtain several reference samples with different antibody concentrations.
  • sample capture, antibody detection and signal amplification the following sample capture, antibody detection and signal amplification are performed on several reference samples with different antibody concentrations respectively, and the spectral shifts of several reference samples are obtained, specifically:
  • Sample capture contact the fiber optic probe with the reference sample, and the antigen on the fiber optic probe specifically captures the binding antibody against the antigen present in the reference sample; antibody detection: make the fiber optic probe The needle is in contact with the HRP-coupled mouse anti-human IgG antibody, and the binding antibody captured on the fiber optic probe specifically binds to the mouse anti-human IgG antibody; signal amplification: contact the fiber optic probe with the signal amplifier DAB , the HRP on the fiber optic probe combines with the DAB with high affinity to generate metal precipitation and obtain the spectral shift of the reference sample.
  • sample capture make the optical fiber probe contact with the sample to be tested, and the optical fiber probe
  • the antigen on the surface specifically captures the binding antibody against the antigen present in the sample to be tested
  • antibody detection the fiber optic probe is contacted with the mouse anti-human IgG antibody coupled with HRP, and the fiber optic probe captures
  • the binding antibody specifically binds to the mouse anti-human IgG antibody
  • signal amplification the optical fiber probe is contacted with the signal amplifier DAB, and the HRP on the optical fiber probe binds to the DAB with high affinity to generate metal precipitation , to obtain the spectral shift of the sample to be tested.
  • the biosensing detection method for binding antibodies also includes the first elution: after preparing the fiber optic probe, the antigen that is not fixed to the surface of the streptavidin chip is eluted; the second elution: at After sample capture, the bound antibody that is not specifically captured by the fiber optic probe is eluted; the third elution: after antibody detection, the HRP-conjugated mouse anti-human IgG antibody that is not bound to the bound antibody is eluted.
  • the biotinylated novel coronavirus core protein includes biotinylated RBD protein, biotinylated S-ECD protein or biotinylated RBD- N501Y protein;
  • the binding antibody includes binding antibody produced by RBD protein, binding antibody produced by S-ECD protein or binding antibody produced by RBD-N501Y protein.
  • the serum or dried blood spot whole blood sample of the test subject or healthy subject is diluted to 80-120 times.
  • the spectral shift of the reference sample and/or the sample to be tested is within the range of 15-25 nm.
  • the concentration of the HRP-coupled mouse anti-human IgG antibody is 0.067 ⁇ g/mL ⁇ 0.4 ⁇ g/mL.
  • the signal amplifier DAB is diluted 200 times to 33.3 times before contacting the fiber optic probe: the 200 times dilution is: 5 ⁇ L of DAB added into 1 mL of DAB substrate buffer; the 33.3-fold dilution is: 30 ⁇ L of DAB was added to 1 mL of DAB substrate buffer.
  • the concentration of the above-mentioned HRP-coupled mouse anti-human IgG antibody and the concentration of the signal amplifier DAB can be controlled to make the amplified detection signal value in the range of 15-25nm, preferably 20nm, which not only ensures sufficient signal strength, but also avoids optical fiber Fiber shedding during system operation due to excessive load on the tip surface.
  • the second aspect of the present application provides a biosensing detection system that binds antibodies, comprising:
  • Optical fiber probe preparation module used to immobilize biotinylated antigens to the surface of streptavidin chips to obtain optical fiber probes, and the biotinylated antigens include biotinylated 2019-nCoV core protein.
  • Sample capture, antibody detection and signal amplification module used to perform the following sample capture, antibody detection and signal amplification on several reference samples with different antibody concentrations respectively, to obtain the spectral shifts of several reference samples, specifically: Sample capture: contact the fiber optic probe with the reference sample, and the antigen on the fiber optic probe specifically captures the binding antibody against the antigen present in the reference sample; antibody detection: make the fiber optic probe The needle is in contact with the HRP-coupled mouse anti-human IgG antibody, and the binding antibody captured on the fiber optic probe specifically binds to the mouse anti-human IgG antibody; signal amplification: contact the fiber optic probe with the signal amplifier DAB , the HRP on the fiber optic probe combines with the DAB with high affinity to generate metal precipitation and obtain the spectral shift of the reference sample.
  • sample capture the optical fiber probe is brought into contact with the sample to be tested, and the optical fiber The antigen on the probe specifically captures the binding antibody against the antigen present in the sample to be tested
  • antibody detection the fiber optic probe is contacted with an HRP-coupled mouse anti-human IgG antibody, and the fiber optic probe The binding antibody captured on the above specifically binds to the mouse anti-human IgG antibody
  • signal amplification the fiber optic probe is contacted with the signal amplifier DAB, and the HRP on the fiber optic probe binds to the DAB with high affinity to generate The metal precipitates to obtain the spectral shift of the sample to be tested.
  • Standard curve establishment module used to establish the "binding antibody” according to the antibody concentration and spectral shift of the several reference samples, with the antibody concentration in the reference sample as the X axis and the relative spectral shift of the reference sample as the Y axis.
  • the antibody-binding biosensing detection system also includes: a first cleaning module: set in the optical fiber probe module, used for eluting antigens that are not fixed to the surface of the streptavidin chip; second cleaning Module: set in the sample capture, antibody detection and signal amplification module, used to elute the bound antibody not specifically captured by the fiber optic probe; the third cleaning module: set in the sample capture, antibody detection and signal amplification In the module, it is used to elute the HRP-conjugated mouse anti-human IgG antibody that has not bound to the bound antibody.
  • this application is aimed at the detection of new coronavirus binding antibodies, based on optical fiber biological layer interference technology, combined with effective signal amplification, to establish a fast (within 15min), high sensitivity (10ng/mL), simple and quantitative
  • the fully automatic optical biosensing detection method and detection system can be used for the detection of new crown-binding antibodies in human serum and the detection of new crown-binding antibodies in whole blood samples obtained by dry blood spot sampling.
  • the biotinylated antigen is immobilized on the surface of the SA chip (30-70s), to the sample capture (5min), to the detection antibody ( 5min) to DAB signal amplification (2min), and three washing steps, the entire detection process can be completed within 14.5min.
  • Fig. 1 is a detection principle diagram of the biosensing detection method combined with antibodies according to the present application.
  • Fig. 2 is a detection flowchart of the biosensing detection method for binding antibodies according to the present application.
  • Fig. 3 is a standard curve diagram of "concentration-relative shift" of binding antibodies according to an embodiment of the present application.
  • 1 streptavidin fiber
  • 2 biotinylated antigen, including biotinylated RBD, biotinylated S-ECD and biotinylated RBD-N501Y variant
  • 3 new crown-binding antibody
  • 4 HRP conjugate Linked mouse anti-human IgG antibody
  • 5 signal amplifier DAB.
  • the first embodiment of the present application provides a biosensing detection system that binds antibodies, the detection system comprising:
  • Optical fiber probe preparation module used to immobilize biotinylated antigens to the surface of streptavidin chips to obtain optical fiber probes, and the biotinylated antigens include biotinylated 2019-nCoV core protein.
  • Sample capture, antibody detection and signal amplification module used to perform the following sample capture, antibody detection and signal amplification on several reference samples with different antibody concentrations respectively, to obtain the spectral shifts of several reference samples, specifically: Sample capture: contact the fiber optic probe with the reference sample, and the antigen on the fiber optic probe specifically captures the binding antibody against the antigen present in the reference sample; antibody detection: make the fiber optic probe The needle is in contact with the HRP-coupled mouse anti-human IgG antibody, and the binding antibody captured on the fiber optic probe specifically binds to the mouse anti-human IgG antibody; signal amplification: contact the fiber optic probe with the signal amplifier DAB , the HRP on the fiber optic probe combines with the DAB with high affinity to generate metal precipitation and obtain the spectral shift of the reference sample.
  • sample capture the optical fiber probe is brought into contact with the sample to be tested, and the optical fiber The antigen on the probe specifically captures the binding antibody against the antigen present in the sample to be tested
  • antibody detection the fiber optic probe is contacted with an HRP-coupled mouse anti-human IgG antibody, and the fiber optic probe The binding antibody captured on the above specifically binds to the mouse anti-human IgG antibody
  • signal amplification the fiber optic probe is contacted with the signal amplifier DAB, and the HRP on the fiber optic probe binds to the DAB with high affinity to generate The metal precipitates to obtain the spectral shift of the sample to be tested.
  • Standard curve establishment module used to establish the "binding antibody” according to the antibody concentration and spectral shift of the several reference samples, with the antibody concentration in the reference sample as the X axis and the relative spectral shift of the reference sample as the Y axis.
  • the antibody-binding biosensing detection system further includes: a first cleaning module: set in the fiber optic probe module, used for eluting The antigen on the surface; the second cleaning module: set in the sample capture, antibody detection and signal amplification module, used to elute the bound antibody not specifically captured by the fiber optic probe; the third cleaning module: set in the sample In the capture, antibody detection, and signal amplification modules, it is used to elute HRP-conjugated mouse anti-human IgG antibody that is not bound to the bound antibody.
  • the second embodiment of the present application provides an antibody-binding biosensing detection method, comprising the following steps:
  • sample capture, antibody detection and signal amplification perform the following sample capture, antibody detection and signal amplification on several reference samples with different antibody concentrations respectively, to obtain the spectral shifts of several reference samples, specifically: sample capture: contacting the fiber optic probe with the reference sample, the antigen on the fiber optic probe specifically captures bound antibodies against the antigen present in the reference sample, after sample capture, eluting Conjugated antibody specifically captured by the needle; antibody detection: the fiber optic probe is contacted with an HRP-coupled mouse anti-human IgG antibody, and the bound antibody captured on the fiber optic probe specifically binds to the mouse anti-human IgG antibody , after antibody detection, eluting the mouse anti-human IgG antibody that is not combined with the HRP of the binding antibody; signal amplification: the fiber optic probe is contacted with the signal amplifier DAB, and the HRP on the fiber optic probe is in contact with the The above-mentioned DAB binds with high affinity, produces metal precipitation, and obtains the spectral shift of the reference sample
  • sample capture make the optical fiber probe contact with the sample to be tested, and the optical fiber probe
  • the antigen on the surface specifically captures the binding antibody against the antigen present in the sample to be tested
  • antibody detection the fiber optic probe is contacted with the mouse anti-human IgG antibody coupled with HRP, and the fiber optic probe captures
  • the binding antibody specifically binds to the mouse anti-human IgG antibody, and after antibody detection, the HRP-coupled mouse anti-human IgG antibody that is not combined with the binding antibody is washed out
  • signal amplification the fiber optic probe and signal amplification When contacted with DAB, the HRP on the optical fiber probe binds with the DAB with high affinity to produce metal precipitation, and obtain the spectral shift of the sample to be tested.
  • the spectral relative shift of the spectral shift of the sample to be tested - the spectral shift of the reference sample with zero antibody concentration.
  • the biotinylated new coronavirus core protein includes biotinylated RBD protein, biotinylated S-ECD protein or biotinylated RBD-N501Y protein;
  • the binding antibody includes the binding antibody produced by RBD protein , the binding antibody produced by the S-ECD protein or the binding antibody produced by the RBD-N501Y protein.
  • the serum or dried blood spot whole blood sample of the test subject or healthy subject is diluted to 80-120 times.
  • the concentration of the mouse anti-human IgG antibody coupled with horseradish peroxidase is 0.067 ⁇ g/mL ⁇ 0.4 ⁇ g/mL; the signal amplifier DAB is diluted 200-fold before contacting with the optical fiber probe.
  • the 200-fold dilution is: 5 ⁇ L DAB is added to 1 mL of DAB substrate buffer; the 33.3-fold dilution is: 30 ⁇ L DAB is added to 1 mL of DAB substrate buffer; the above-mentioned horseradish peroxide
  • concentration of the enzyme-coupled mouse anti-human IgG antibody and the concentration of the signal amplifier DAB can be controlled so that the amplified detection signal value is in the range of 15-25nm, preferably 20nm, which not only ensures sufficient signal strength, but also avoids optical fiber tip
  • the surface is caused by the optical fiber falling off during the operation of the system due to the heavy load.
  • Anti-RBD BAbs the detection of anti-RBD binding antibodies
  • the detection process is briefly described as follows (abbreviations or abbreviations for some of the detection systems and detection methods that have been described in the previous section).
  • sample to be tested and the reference sample prepare the sample to be tested: dilute the real serum sample 100 times: add 5 ⁇ L of serum to 495 ⁇ L of the diluent, mix gently and ready for use.
  • the diluent is: 10mM PBS+0.1%BSA+0.02%Tween20, pH 7.4.
  • Preparation of reference samples Human chimeric monoclonal antibody against RBD region (MA-RBD-S309, commercially available) was used as the reference antibody of the binding antibody. Add MA-RBD-S309 to 100-fold diluted serum to obtain 6 reference samples (concentrations are 0, 10, 50, 100, 500, 1000 ng/mL, respectively).
  • Sample capture, antibody detection and signal amplification perform the following sample capture, antibody detection and signal amplification on several reference samples with different antibody concentrations respectively, to obtain the spectral shifts of several reference samples, specifically: sample capture: contacting the fiber optic probe with the reference sample, and the antigen on the fiber optic probe specifically captures the binding antibody against the antigen present in the reference sample; antibody detection: making the fiber optic probe and HRP The coupled mouse anti-human IgG antibody is contacted, and the binding antibody captured on the optical fiber probe is specifically combined with the mouse anti-human IgG antibody; signal amplification: the optical fiber probe is contacted with the signal amplifier DAB, and the The HRP on the fiber optic probe binds to the DAB with high affinity to produce metal precipitation and obtain the spectral shift of the reference sample.
  • sample capture make the optical fiber probe contact with the sample to be tested, and the optical fiber probe
  • the antigen on the surface specifically captures the binding antibody against the antigen present in the sample to be tested
  • antibody detection the fiber optic probe is contacted with the mouse anti-human IgG antibody coupled with HRP, and the fiber optic probe captures
  • the binding antibody specifically binds to the mouse anti-human IgG antibody
  • signal amplification the optical fiber probe is contacted with the signal amplifier DAB, and the HRP on the optical fiber probe binds to the DAB with high affinity to generate metal precipitation , to obtain the spectral shift of the sample to be tested.
  • DAB 3,3′-Diaminobenzidine
  • DAB Enhanced Liquid Substrate System Tetrahydrochloride
  • Cat. No.: D3939-1SET Manufacturer: Sigma-Aldrich (Shanghai) Trading Co.Ltd: Sigma-Aldrich (Shanghai) Trading Co. Ltd.
  • the signal amplifier Before contacting the signal amplifier DAB with the fiber optic probe, the signal amplifier was diluted 200 times: 5 ⁇ L DAB was added to 1 mL of DAB substrate buffer, and horseradish peroxidase-coupled mouse antibody
  • concentration of human IgG antibody is 0.067 ⁇ g/mL ⁇ 0.4 ⁇ g/mL, which can make effective signal amplification and control the maximum signal value at about 20nm, which not only ensures sufficient signal strength, but also avoids system operation due to excessive load on the surface of the fiber tip When the optical fiber falls off.
  • the antibody content of different concentrations in the sample can be visually displayed through specific binding and signal amplification.
  • Fig. 1 is a detection schematic diagram of the biosensing detection method combined with an antibody according to the present application
  • Fig. 2 is a detection flow chart of the biosensing detection method combined with an antibody according to the present application.
  • the detection process is concerned: from biotinylated antigen immobilization on the surface of the SA chip (30-70s), to sample capture (5min), to detection antibody (5min) to DAB signal amplification (2min), and three washing steps, the entire detection The process can be completed within 14.5 minutes.
  • the X-axis is plotted with the reference antibody MA-RBD S309 concentration ( ⁇ g/mL), and the Y-axis is plotted with the relative displacement (nm) of each reference sample), to establish the binding antibody " Concentration-relative shift" combined with the standard curve, as shown in Figure 3.
  • Spectral relative shift (nm) of the sample to be tested spectral shift (nm) of the sample to be tested – spectral shift (nm) of the reference sample with zero antibody concentration
  • the detection concentration range of the binding antibody of the sample to be tested is 0-100 ⁇ g/mL, and the quantitative concentration range is set as: 1-100 ⁇ g/mL.
  • the formula for calculating the concentration ( ⁇ g/mL) of the bound antibody in the sample is:
  • 100 represents the 100-fold dilution of the sample
  • 1000 represents the concentration unit converted from ng/mL to ⁇ g/mL
  • X represents the concentration of bound antibody in the sample
  • Bmax represents the maximum specific binding constant in the nonlinear regression standard curve
  • Kd represents the equilibrium dissociation constant in the nonlinear regression standard curve
  • Y represents the spectral relative shift (nm) of the sample to be tested.
  • the method of the present application can also be used for anti-S-ECD binding antibody (abbreviation: Anti-S-ECD BAbs) and anti-RBD variant protein (RBD-N501Y) binding antibody (abbreviation: Anti-RBD N501Y BAbs) Specific and highly sensitive detection.
  • Anti-S-ECD BAbs anti-S-ECD BAbs
  • RBD-N501Y anti-RBD variant protein binding antibody
  • Anti-RBD N501Y BAbs anti-RBD N501Y BAbs
  • the antibody-binding biosensing detection method and detection system provided by this application provide an in vitro diagnostic platform with clinical applicability, which can be applied to rapid evaluation of antibodies in healthy people after vaccination, and to test the efficacy of vaccines. Effectiveness; it can also be applied to the rapid assessment of the history of infection of the new coronavirus in the population or individual. In addition, the system has the potential to be applied to national incidence screening of other pandemic diseases as well.

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Abstract

一种结合抗体的生物传感检测方法及检测系统，基于光纤生物层干涉技术，结合有效的信号放大，建立快速、高灵敏性、简便、可定量的全自动光学生物传感检测方法和检测系统，可用于对人血清中的新冠结合抗体的检测和以干血斑取样方法获得的全血样本中的新冠结合抗体的检测。

Description

一种结合抗体的生物传感检测方法及检测系统
相关申请的交叉引用
本申请基于申请号为“202110784727.3”、申请日为2021年07月12日的中国专利申请提出，并要求该中国专利申请的优先权，该中国专利申请的全部内容在此以引入方式并入本申请。
技术领域
本申请涉及光纤生物传感技术领域，特别涉及一种结合抗体的生物传感检测方法及检测系统。
背景技术
为防止疫情进一步蔓延，新冠疫苗研发成为全球药物研究领域的热点。在疫苗接种的过程中，体内会产生大量的特异性抗体，包括中和抗体与结合抗体。中和抗体是使病毒失去结合受体的能力的抗体，结合抗体仅指可以结合病毒表面区域的抗体。临床上对新冠中和抗体的监测意义最为重大，它是评价新冠疫苗安全效用的重要指标，同时，对结合抗体进行监测可以帮助回答新冠病毒的感染史，助于流行病学研究。国内外现有针对新冠结合抗体的检测主要是ELISA和试纸条，前者精确但耗时耗力，后者简便快速、但无法提供准确的定量信息。国内外现有针对新冠中和抗体的检测是基于活细胞的病毒中和测试实验，需要严格的生物安全三级实验室的操作环境、操作程序复杂。因此，临床上亟需兼具高灵敏性、简便与快速的自动化生物传感体系，允许在常规实验室对单个或者高通量样本进行快速监测。
生物层干涉技术是一种通过检测干涉光谱的位移变化来检测传感器 表面反应的技术。当一束可见光从光谱仪射出后，在光纤传感器末端的光学膜层的两个界面会形成两束反射光谱，并形成一束干涉光谱。光谱的红移(单位：nm)大小与末端特异性结合分子的浓度呈正相关。但是其在低检测浓度下呈现的信号强度非常有限，因此很少用于定量分析中。
在本申请中用到的一些缩略语、英文和关键术语定义如下：1.新型冠状病毒或COVID-19，简称新冠；2.BLI：biolayer interferometry，生物层干涉技术；3.BAbs：Binding antibodies，结合抗体；4.RBD：Receptor binding domain，受体结合区域；5.S-ECD：Extracellular Domain of Spike Protein，刺突蛋白；6.ELISA：Enzyme linked immunosorbent assay，酶联免疫吸附剂测定法；7.DAB：3,3’-diaminobenzidine tetrahydrochloride，3,3’-二氨基联苯胺四盐酸；8.HRP：Horseradish Peroxidase，辣根过氧化物酶；9.Anti-RBD BAbs：针对RBD区域的结合抗体，又称抗RBD结合抗体；10.Anti-S-ECD BAbs：针对S-ECD区域的结合抗体，又称抗S-ECD结合抗体；11.Anti-RBDN501Y BAbs：针对RBD变异体N501Y的结合抗体，又称抗RBD N501Y结合抗体。
发明内容
本申请的目的在于提供一种结合抗体的生物传感检测方法及检测系统，通过对生物层干涉技术有效的信号放大，获得针对新冠结合抗体快速监测的自动化生物传感体系，助力新冠疫苗的研发与效价评估。
为解决上述技术问题，本申请的第一方面提供一种结合抗体的生物传感检测方法，包括：
(1)制备光纤探针：将生物素化的抗原固定至链霉亲和素芯片表面，得到光纤探针，所述生物素化的抗原包括生物素化的新冠病毒核心蛋白。
(2)获得待测样本和参照样本：获得待测者的血清或干血斑全血，稀释后，作为待测样本；获得健康者的血清或干血斑全血若干份，稀释后， 分别添加参照抗体嵌合式抗RBD单克隆抗体，得到不同抗体浓度的若干个参照样本。
(3)样本捕获、抗体检测和信号放大：对所述不同抗体浓度的若干个参照样本分别进行如下样本捕获、抗体检测和信号放大，得到若干个参照样本的光谱位移，具体为：
样本捕获：使所述光纤探针与所述参照样本接触，所述光纤探针上的抗原特异性捕获所述参照样本中存在的针对所述抗原的结合抗体；抗体检测：使所述光纤探针与HRP偶联的鼠抗人IgG抗体接触，所述光纤探针上捕获的结合抗体与所述鼠抗人IgG抗体特异性结合；信号放大：使所述光纤探针与信号放大剂DAB接触，所述光纤探针上的HRP与所述DAB高亲和力结合，产生金属沉淀，得到参照样本的光谱位移。
对所述待测样本进行如下样本捕获、抗体检测和信号放大，得到待测样本的光谱位移，具体为：样本捕获：使所述光纤探针与所述待测样本接触，所述光纤探针上的抗原特异性捕获所述待测样本中存在的针对所述抗原的结合抗体；抗体检测：使所述光纤探针与HRP偶联的鼠抗人IgG抗体接触，所述光纤探针上捕获的结合抗体与所述鼠抗人IgG抗体特异性结合；信号放大：使所述光纤探针与信号放大剂DAB接触，所述光纤探针上的HRP与所述DAB高亲和力结合，产生金属沉淀，得到待测样本的光谱位移。
(4)建立标准曲线：基于所述若干个参照样本的抗体浓度和光谱位移，以参照样本中的抗体浓度为X轴，以参照样本的光谱相对位移为Y轴，建立结合抗体“浓度-相对位移”标准曲线，其中，参照样本的光谱相对位移＝参照样本的光谱位移–零抗体浓度的参照样本的光谱位移。
(5)获得待测样本的结合抗体浓度：根据所述结合抗体“浓度-相对位移”标准曲线以及待测样本的光谱相对位移，获得待测样本的结合抗体浓度，其中，待测样本的光谱相对位移＝待测样本的光谱位移–零抗体浓度 的参照样本的光谱位移。
本申请提供的结合抗体的生物传感检测方法，还包括第一次洗脱：在制备光纤探针之后，洗脱未固定至链霉亲和素芯片表面的抗原；第二次洗脱：在样本捕获之后，洗脱未被光纤探针特异性捕获的结合抗体；第三次洗脱：在抗体检测之后，洗脱未与结合抗体结合的HRP偶联的鼠抗人IgG抗体。
优选地，本申请提供的结合抗体的生物传感检测方法中，所述生物素化的新冠病毒核心蛋白包括生物素化的RBD蛋白、生物素化的S-ECD蛋白或生物素化的RBD-N501Y蛋白；所述结合抗体包括RBD蛋白所产生的结合抗体、S-ECD蛋白所产生的结合抗体或RBD-N501Y蛋白所产生的结合抗体。
优选地，本申请提供的结合抗体的生物传感检测方法中，对所述待测者或健康者的血清或干血斑全血样本进行的稀释，为稀释至80～120倍。
优选地，本申请提供的结合抗体的生物传感检测方法中，所述参照样本和/或待测样本的光谱位移在15～25nm范围内。
优选地，本申请提供的结合抗体的生物传感检测方法中，所述HRP偶联的鼠抗人IgG抗体的浓度为0.067μg/mL～0.4μg/mL。
优选地，本申请提供的结合抗体的生物传感检测方法中，所述信号放大剂DAB与所述光纤探针接触前进行200倍稀释～33.3倍稀释：所述200倍稀释为：5μL DAB添加至1mL的DAB底物缓冲液中；所述33.3倍稀释为：30μL DAB添加至1mL的DAB底物缓冲液中。
上述HRP偶联的鼠抗人IgG抗体的浓度和信号放大剂DAB的浓度控制，可使放大后的检测信号值在15～25nm范围内，优选为20nm，既保证足够的信号强度、又避免光纤尖端表面由于负载过重而造成系统运行时的光纤脱落。
本申请的第二方面提供一种结合抗体的生物传感检测系统，包括：
(1)光纤探针制备模块：用于将生物素化的抗原固定至链霉亲和素芯片表面，得到光纤探针，所述生物素化的抗原包括生物素化的新冠病毒核心蛋白。
(2)参照样本和待测样本获得模块：用于获得待测者的血清或干血斑全血，稀释后，作为待测样本；用于获得健康者的血清或干血斑全血若干份，稀释后，分别添加参照抗体嵌合式抗RBD单克隆抗体(简称MA-RBD S309)，得到不同抗体浓度的若干个参照样本。
(3)样本捕获、抗体检测和信号放大模块：用于对所述不同抗体浓度的若干个参照样本分别进行如下样本捕获、抗体检测和信号放大，得到若干个参照样本的光谱位移，具体为：样本捕获：使所述光纤探针与所述参照样本接触，所述光纤探针上的抗原特异性捕获所述参照样本中存在的针对所述抗原的结合抗体；抗体检测：使所述光纤探针与HRP偶联的鼠抗人IgG抗体接触，所述光纤探针上捕获的结合抗体与所述鼠抗人IgG抗体特异性结合；信号放大：使所述光纤探针与信号放大剂DAB接触，所述光纤探针上的HRP与所述DAB高亲和力结合，产生金属沉淀，得到参照样本的光谱位移。
用于对所述待测样本进行如下样本捕获、抗体检测和信号放大，得到待测样本的光谱位移，具体为：样本捕获：使所述光纤探针与所述待测样本接触，所述光纤探针上的抗原特异性捕获所述待测样本中存在的针对所述抗原的结合抗体；抗体检测：使所述光纤探针与HRP偶联的鼠抗人IgG抗体接触，所述光纤探针上捕获的结合抗体与所述鼠抗人IgG抗体特异性结合；信号放大：使所述光纤探针与信号放大剂DAB接触，所述光纤探针上的HRP与所述DAB高亲和力结合，产生金属沉淀，得到待测样本的光谱位移。
(4)标准曲线建立模块：用于根据所述若干个参照样本的抗体浓度和光谱位移，以参照样本中的抗体浓度为X轴，以参照样本的光谱相对位 移为Y轴，建立结合抗体“浓度-相对位移”标准曲线，其中，参照样本的光谱相对位移＝参照样本的光谱位移–零抗体浓度的参照样本的光谱位移。
(5)待测样本的结合抗体浓度获得模块：用于根据所述结合抗体“浓度-相对位移”标准曲线以及待测样本的光谱相对位移，获得待测样本的结合抗体浓度，其中，待测样本的光谱相对位移＝待测样本的光谱位移–零抗体浓度的参照样本的光谱位移。
本申请提供的结合抗体的生物传感检测系统还包括：第一清洗模块：设于所述光纤探针模块中，用于洗脱未固定至链霉亲和素芯片表面的抗原；第二清洗模块：设于所述样本捕获、抗体检测和信号放大模块中，用于洗脱未被光纤探针特异性捕获的结合抗体；第三清洗模块：设于所述样本捕获、抗体检测和信号放大模块中，用于洗脱未与结合抗体结合的HRP偶联的鼠抗人IgG抗体。
相对于现有技术而言，本申请针对新冠病毒结合抗体检测，基于光纤生物层干涉技术，结合有效的信号放大，建立快速(15min内)、高灵敏性(10ng/mL)、简便、可定量的全自动光学生物传感检测方法和检测系统，可用于对人血清中的新冠结合抗体的检测和以干血斑取样方法获得的全血样本中的新冠结合抗体的检测。
基于本申请所提供的结合抗体的生物传感检测方法及检测系统，其检测流程中，从生物素化抗原固定于SA芯片表面(30～70s)、到样本捕获(5min)、到检测抗体(5min)至DAB信号放大(2min)、以及三次清洗步骤，整个检测过程可于14.5min内完成。
附图说明
图1为根据本申请的结合抗体的生物传感检测方法的检测原理图。
图2为根据本申请的结合抗体的生物传感检测方法的检测流程图。
图3为根据本申请实施方式的结合抗体“浓度-相对位移”标准曲线图。
其中，1：链霉亲和素光纤；2：生物素化抗原，包括生物素化RBD、生物素化S-ECD与生物素化RBD-N501Y变异体；3：新冠结合抗体；4：HRP偶联的鼠抗人IgG抗体；5：信号放大剂DAB。
具体实施方式
为使本申请的目的、技术方案和优点更加清楚，下面将对本申请的各实施方式进行详细的阐述。然而，本领域的普通技术人员可以理解，在本申请各实施方式中，为了使读者更好地理解本申请而提出了许多技术细节。但是，即使没有这些技术细节和基于以下各实施方式的种种变化和修改，也可以实现本申请所要求保护的技术方案。
本申请的第一实施方式提供一种结合抗体的生物传感检测系统，该检测系统包括：
(1)光纤探针制备模块：用于将生物素化的抗原固定至链霉亲和素芯片表面，得到光纤探针，所述生物素化的抗原包括生物素化的新冠病毒核心蛋白。
(2)参照样本和待测样本获得模块：用于获得待测者的血清或干血斑全血，稀释后，作为待测样本；用于获得健康者的血清或干血斑全血若干份，稀释后，分别添加参照抗体嵌合式抗RBD单克隆抗体，得到不同抗体浓度的若干个参照样本。
(3)样本捕获、抗体检测和信号放大模块：用于对所述不同抗体浓度的若干个参照样本分别进行如下样本捕获、抗体检测和信号放大，得到若 干个参照样本的光谱位移，具体为：样本捕获：使所述光纤探针与所述参照样本接触，所述光纤探针上的抗原特异性捕获所述参照样本中存在的针对所述抗原的结合抗体；抗体检测：使所述光纤探针与HRP偶联的鼠抗人IgG抗体接触，所述光纤探针上捕获的结合抗体与所述鼠抗人IgG抗体特异性结合；信号放大：使所述光纤探针与信号放大剂DAB接触，所述光纤探针上的HRP与所述DAB高亲和力结合，产生金属沉淀，得到参照样本的光谱位移。
用于对所述待测样本进行如下样本捕获、抗体检测和信号放大，得到待测样本的光谱位移，具体为：样本捕获：使所述光纤探针与所述待测样本接触，所述光纤探针上的抗原特异性捕获所述待测样本中存在的针对所述抗原的结合抗体；抗体检测：使所述光纤探针与HRP偶联的鼠抗人IgG抗体接触，所述光纤探针上捕获的结合抗体与所述鼠抗人IgG抗体特异性结合；信号放大：使所述光纤探针与信号放大剂DAB接触，所述光纤探针上的HRP与所述DAB高亲和力结合，产生金属沉淀，得到待测样本的光谱位移。
(4)标准曲线建立模块：用于根据所述若干个参照样本的抗体浓度和光谱位移，以参照样本中的抗体浓度为X轴，以参照样本的光谱相对位移为Y轴，建立结合抗体“浓度-相对位移”标准曲线，其中，参照样本的光谱相对位移＝参照样本的光谱位移–零抗体浓度的参照样本的光谱位移。
(5)待测样本的结合抗体浓度获得模块：用于根据所述结合抗体“浓度-相对位移”标准曲线以及待测样本的光谱相对位移，获得待测样本的结合抗体浓度，其中，待测样本的光谱相对位移＝待测样本的光谱位移–零抗体浓度的参照样本的光谱位移。
在本申请的部分实施方式中，所述结合抗体的生物传感检测系统还包括：第一清洗模块：设于所述光纤探针模块中，用于洗脱未固定至链霉亲和 素芯片表面的抗原；第二清洗模块：设于所述样本捕获、抗体检测和信号放大模块中，用于洗脱未被光纤探针特异性捕获的结合抗体；第三清洗模块：设于所述样本捕获、抗体检测和信号放大模块中，用于洗脱未与结合抗体结合的HRP偶联的鼠抗人IgG抗体。
基于第一实施方式提供的结合抗体的生物传感检测系统，本申请的第二实施方式提供一种结合抗体的生物传感检测方法，包括如下步骤：
(1)制备光纤探针：将生物素化的抗原固定至链霉亲和素芯片表面，得到光纤探针，所述生物素化的抗原包括生物素化的新冠病毒核心蛋白；在制备光纤探针之后，洗脱未固定至链霉亲和素芯片表面的抗原。
(2)获得参照样本和待测样本：获得待测者的血清或干血斑全血，稀释后，作为待测样本；获得健康者的血清或干血斑全血若干份，稀释后，分别添加参照抗体嵌合式抗RBD单克隆抗体，得到不同抗体浓度的若干个参照样本。
(3)样本捕获、抗体检测和信号放大：对所述不同抗体浓度的若干个参照样本分别进行如下样本捕获、抗体检测和信号放大，得到若干个参照样本的光谱位移，具体为：样本捕获：使所述光纤探针与所述参照样本接触，所述光纤探针上的抗原特异性捕获所述参照样本中存在的针对所述抗原的结合抗体，在样本捕获之后，洗脱未被光纤探针特异性捕获的结合抗体；抗体检测：使所述光纤探针与HRP偶联的鼠抗人IgG抗体接触，所述光纤探针上捕获的结合抗体与所述鼠抗人IgG抗体特异性结合，在抗体检测之后，洗脱未与结合抗体结合的HRP偶联的鼠抗人IgG抗体；信号放大：使所述光纤探针与信号放大剂DAB接触，所述光纤探针上的HRP与所述DAB高亲和力结合，产生金属沉淀，得到参照样本的光谱位移。
对所述待测样本进行如下样本捕获、抗体检测和信号放大，得到待测样本的光谱位移，具体为：样本捕获：使所述光纤探针与所述待测样本接触， 所述光纤探针上的抗原特异性捕获所述待测样本中存在的针对所述抗原的结合抗体；抗体检测：使所述光纤探针与HRP偶联的鼠抗人IgG抗体接触，所述光纤探针上捕获的结合抗体与所述鼠抗人IgG抗体特异性结合，在抗体检测之后，洗脱未与结合抗体结合的HRP偶联的鼠抗人IgG抗体；信号放大：使所述光纤探针与信号放大剂DAB接触，所述光纤探针上的HRP与所述DAB高亲和力结合，产生金属沉淀，得到待测样本的光谱位移。
(4)建立标准曲线：基于所述若干个参照样本的抗体浓度和光谱位移，以参照样本中的抗体浓度为X轴，以参照样本的光谱相对位移为Y轴，建立结合抗体“浓度-相对位移”标准曲线，其中，参照样本的光谱相对位移＝参照样本的光谱位移–零抗体浓度的参照样本的光谱位移。
(5)获得待测样本的结合抗体浓度：用于根据所述结合抗体“浓度-相对位移”标准曲线以及待测样本的光谱相对位移，获得待测样本的结合抗体浓度，其中，待测样本的光谱相对位移＝待测样本的光谱位移–零抗体浓度的参照样本的光谱位移。
其中，所述生物素化的新冠病毒核心蛋白包括生物素化的RBD蛋白、生物素化的S-ECD蛋白或生物素化的RBD-N501Y蛋白；所述结合抗体包括RBD蛋白所产生的结合抗体、S-ECD蛋白所产生的结合抗体或RBD-N501Y蛋白所产生的结合抗体。
其中，所述待测者或健康者的血清或干血斑全血样本稀释至80～120倍。
其中，所述辣根过氧化物酶偶联的鼠抗人IgG抗体的浓度为0.067μg/mL～0.4μg/mL；所述信号放大剂DAB与所述光纤探针接触前进行200倍稀释～33.3倍稀释：所述200倍稀释为：5μL DAB添加至1mL的DAB底物缓冲液中；所述33.3倍稀释为：30μL DAB添加至1mL的DAB底物缓冲液中；上述辣根过氧化物酶偶联的鼠抗人IgG抗体的浓度和信号放大剂 DAB的浓度控制，可使放大后的检测信号值在15～25nm范围内，优选为20nm，既保证足够的信号强度、又避免光纤尖端表面由于负载过重而造成系统运行时的光纤脱落。
检测示例
本实施例以抗RBD结合抗体(Anti-RBD BAbs)检测为例，简述检测过程如下(对已在前面部分中阐述过的检测系统及检测方法的部分内容进行简写或略写)。
(1)制备光纤探针：使用商业可购买的链霉亲和素(streptavidin，下称SA)芯片作为探针，将生物素化的抗原RBD固定至SA芯片表面。生物素化操作程序可借鉴市场上可购买的商业化生物素试剂盒。
(2)获得待测样本和参照样本：制备待测样本：将真实血清样本稀释100倍：5μL血清添加至495μL稀释液中，轻轻混匀即可备用。稀释液为：10mM PBS+0.1％BSA+0.02％Tween20,pH 7.4。制备参照样本：采用针对RBD区域的人源型嵌合式单克隆抗(MA-RBD-S309，商业可购买)作为结合性抗体的参照抗体。添加MA-RBD-S309于100倍稀释的血清中，得到6个参照样本(浓度分别为0、10、50、100、500、1000ng/mL)。
(3)样本捕获、抗体检测和信号放大：对所述不同抗体浓度的若干个参照样本分别进行如下样本捕获、抗体检测和信号放大，得到若干个参照样本的光谱位移，具体为：样本捕获：使所述光纤探针与所述参照样本接触，所述光纤探针上的抗原特异性捕获所述参照样本中存在的针对所述抗原的结合抗体；抗体检测：使所述光纤探针与HRP偶联的鼠抗人IgG抗体接触，所述光纤探针上捕获的结合抗体与所述鼠抗人IgG抗体特异性结合；信号放大：使所述光纤探针与信号放大剂DAB接触，所述光纤探针上的HRP与所述DAB高亲和力结合，产生金属沉淀，得到参照样本的光谱位移。
对所述待测样本进行如下样本捕获、抗体检测和信号放大，得到待测 样本的光谱位移，具体为：样本捕获：使所述光纤探针与所述待测样本接触，所述光纤探针上的抗原特异性捕获所述待测样本中存在的针对所述抗原的结合抗体；抗体检测：使所述光纤探针与HRP偶联的鼠抗人IgG抗体接触，所述光纤探针上捕获的结合抗体与所述鼠抗人IgG抗体特异性结合；信号放大：使所述光纤探针与信号放大剂DAB接触，所述光纤探针上的HRP与所述DAB高亲和力结合，产生金属沉淀，得到待测样本的光谱位移。
在上述样本捕获、抗体检测和信号放大步骤中，信号放大剂DAB的商业可够渠道："3,3′-二氨基联苯胺(DAB)增强型液体底物系统四盐酸盐"，货号：D3939-1SET；厂家：Sigma-Aldrich(Shanghai)Trading Co.Ltd：西格玛奥德里奇(上海)贸易有限公司。在将信号放大剂DAB与所述光纤探针接触前，对信号放大剂进行200倍稀释：5μL DAB添加至1mL的DAB底物缓冲液中，同时使辣根过氧化物酶偶联的鼠抗人IgG抗体的浓度为0.067μg/mL～0.4μg/mL，可使有效的信号放大控制最大信号值在20nm左右，既保证足够的信号强度、又避免光纤尖端表面由于负载过重而造成系统运行时的光纤脱落。样本中不同浓度的抗体含量可经过特异性结合与信号放大来直观的显示。
图1为根据本申请的结合抗体的生物传感检测方法的检测原理图；图2为根据本申请的结合抗体的生物传感检测方法的检测流程图。就检测流程而言：从生物素化抗原固定于SA芯片表面(30～70s)、到样本捕获(5min)、到检测抗体(5min)至DAB信号放大(2min)、以及三次清洗步骤，整个检测过程可于14.5min内完成。
(4)建立标准曲线：基于所述若干个参照样本的抗体浓度和光谱位移，以参照样本中的抗体浓度为X轴，以参照样本的光谱相对位移为Y轴，建立结合抗体“浓度-相对位移”标准曲线，其中，参照样本的光谱相对位移＝参照样本的光谱位移–零抗体浓度的参照样本的光谱位移。
参照样本的抗体浓度和光谱位移、相对位移如下表1所示：
表1
Figure PCTCN2022104952-appb-000001
基于以上100倍稀释健康人血清基质下获得的数据，以参照抗体MA-RBD S309浓度(μg/mL)绘制X轴，以各参照样本的相对位移(nm)绘制Y轴)，建立结合抗体“浓度-相对位移”结合标准曲线，如图3所示。
获得待测样本的结合抗体浓度：根据所述结合抗体“浓度-相对位移”标准曲线以及待测样本的光谱相对位移，获得待测样本的结合抗体浓度。
首先，计算得到待测样本的光谱相对位移：
待测样本的光谱相对位移(nm)＝待测样本的光谱位移(nm)–零抗体浓度的参照样本的光谱位移(nm)
结合所建立的结合抗体“浓度-相对位移”标准曲线，考虑到100倍稀释，待测样本的结合抗体检测浓度范围为0-100μg/mL，定量浓度范围设定为：1-100μg/mL。样本中结合抗体的浓度(μg/mL)计算公式为：
X＝Bmax*Y/(Kd-Y)*100/1000
＝548.9*待测样本的光谱相对位移/(29.35-待测样本的光谱相对位移)*0.1
＝54.89*待测样本的光谱相对位移/(29.35-待测样本的光谱相对位 移)
其中：100代表样本的100倍稀释，1000代表浓度单位从ng/mL转化为μg/mL，
X代表样本中结合抗体的浓度；
Bmax代表为非线性回归标准曲线中的最大特异性结合常数；
Kd代表非线性回归标准曲线中的平衡解离常数；
Y代表待测样本的光谱相对位移(nm)。
以两个真实血清样本为例，参照以上检测流程，得到以下数据，进而计算结合抗体浓度，如下表2所示：
表2
待测血清样本 位移(nm) 相对位移(nm) 浓度计算(μg/mL)
样本1 11.78 11.78–1.78＝10.00 54.89*10.00/(29.35-10.00)＝28.37
样本2 6.48 6.48–1.78＝4.70 54.89*4.70/(29.35-4.70)＝10.47
除此之外，本申请的方法还可以用于对抗S-ECD结合抗体(简称：Anti-S-ECD BAbs)与抗RBD变异蛋白(RBD-N501Y)结合抗体(简称：Anti-RBD N501Y BAbs)的特异性与高灵敏性检测。在100倍稀释的血清基质中，该技术对三种结合性抗体的检测线为10ng/mL。
本申请所提供的结合抗体的生物传感检测方法及检测系统为临床提供了一项具有临床适用价值的体外诊断平台，可应用于对健康人群接种疫苗后体内的抗体进行快速评估，测试疫苗的有效性；也可应用于人群或者个体新冠病毒感染史的快速评估。此外，该体系同样有潜力应用于其它大流行疾病的全国性发病率筛查。
本领域的普通技术人员可以理解，上述各实施方式是实现本申请的具体实施例，而在实际应用中，可以在形式上和细节上对其作各种改变，而不偏离本申请的精神和范围。

Claims (10)

  1. 一种结合抗体的生物传感检测方法，其包括：
    (1)制备光纤探针：
    将生物素化的抗原固定至链霉亲和素芯片表面，得到光纤探针，所述生物素化的抗原包括生物素化的新冠病毒核心蛋白；
    (2)获得参照样本和待测样本：
    获得待测者的血清或干血斑全血，稀释后，作为待测样本；
    获得健康者的血清或干血斑全血若干份，稀释后，分别添加参照抗体嵌合式抗RBD单克隆抗体，得到不同抗体浓度的若干个参照样本；
    (3)样本捕获、抗体检测和信号放大：
    对所述不同抗体浓度的若干个参照样本分别进行如下样本捕获、抗体检测和信号放大，得到若干个参照样本的光谱位移，具体为：
    样本捕获：使所述光纤探针与所述参照样本接触，所述光纤探针上的抗原特异性捕获所述参照样本中存在的针对所述抗原的结合抗体；抗体检测：使所述光纤探针与HRP偶联的鼠抗人IgG抗体接触，所述光纤探针上捕获的结合抗体与所述鼠抗人IgG抗体特异性结合；信号放大：使所述光纤探针与信号放大剂DAB接触，所述光纤探针上的HRP与所述DAB高亲和力结合，产生金属沉淀，得到参照样本的光谱位移；
    对所述待测样本进行如下样本捕获、抗体检测和信号放大，得到待测样本的光谱位移，具体为：
    样本捕获：使所述光纤探针与所述待测样本接触，所述光纤探针上的抗原特异性捕获所述待测样本中存在的针对所述抗原的结合抗体；抗体检测：使所述光纤探针与HRP偶联的鼠抗人IgG抗体接触，所述光纤探针上捕获 的结合抗体与所述鼠抗人IgG抗体特异性结合；信号放大：使所述光纤探针与信号放大剂DAB接触，所述光纤探针上的HRP与所述DAB高亲和力结合，产生金属沉淀，得到待测样本的光谱位移；
    (4)建立标准曲线：
    基于所述若干个参照样本的抗体浓度和光谱位移，以参照样本中的抗体浓度为X轴，以参照样本的光谱相对位移为Y轴，建立结合抗体“浓度-相对位移”标准曲线，其中，
    参照样本的光谱相对位移＝参照样本的光谱位移–零抗体浓度的参照样本的光谱位移；
    (5)获得待测样本的结合抗体浓度：
    根据所述结合抗体“浓度-相对位移”标准曲线以及待测样本的光谱相对位移，获得待测样本的结合抗体浓度，其中，
    待测样本的光谱相对位移＝待测样本的光谱位移–零抗体浓度的参照样本的光谱位移。
  2. 根据权利要求1所述的结合抗体的生物传感检测方法，还包括：
    第一次洗脱：在制备光纤探针之后，洗脱未固定至链霉亲和素芯片表面的抗原；
    第二次洗脱：在样本捕获之后，洗脱未被光纤探针特异性捕获的结合抗体；
    第三次洗脱：在抗体检测之后，洗脱未与结合抗体结合的HRP偶联的鼠抗人IgG抗体。
  3. 根据权利要求1所述的结合抗体的生物传感检测方法，其中：
    所述生物素化的新冠病毒核心蛋白包括生物素化的RBD蛋白、生物素化的S-ECD蛋白或生物素化的RBD-N501Y蛋白；
    所述结合抗体包括RBD蛋白所产生的结合抗体、S-ECD蛋白所产生的结合抗体或RBD-N501Y蛋白所产生的结合抗体。
  4. 根据权利要求1所述的结合抗体的生物传感检测方法，其中，对所述待测者或健康者的血清或干血斑全血样本进行的稀释，为稀释至80～120倍。
  5. 根据权利要求1所述的结合抗体的生物传感检测方法，其特征在于，所述参照样本和/或待测样本的光谱位移在15～25nm范围内。
  6. 根据权利要求5所述的结合抗体的生物传感检测方法，其中，所述HRP偶联的鼠抗人IgG抗体的浓度为0.067μg/mL～0.4μg/mL。
  7. 根据权利要求5所述的结合抗体的生物传感检测方法，其中，所述信号放大剂DAB与所述光纤探针接触前进行200倍稀释～33.3倍稀释：
    所述200倍稀释为：5μL DAB添加至1mL的DAB底物缓冲液中；
    所述33.3倍稀释为：30μL DAB添加至1mL的DAB底物缓冲液中。
  8. 一种结合抗体的生物传感检测系统，包括：
    (1)光纤探针制备模块：
    用于将生物素化的抗原固定至链霉亲和素芯片表面，得到光纤探针，所述生物素化的抗原包括生物素化的新冠病毒核心蛋白；
    (2)参照样本和待测样本获得模块：
    用于获得待测者的血清或干血斑全血，稀释后，作为待测样本；
    用于获得健康者的血清或干血斑全血若干份，稀释后，分别添加参照抗体嵌合式抗RBD单克隆抗体，得到不同抗体浓度的若干个参照样本；
    (3)样本捕获、抗体检测和信号放大模块：
    用于对所述不同抗体浓度的若干个参照样本分别进行如下样本捕获、抗 体检测和信号放大，得到若干个参照样本的光谱位移，具体为：
    样本捕获：使所述光纤探针与所述参照样本接触，所述光纤探针上的抗原特异性捕获所述参照样本中存在的针对所述抗原的结合抗体；抗体检测：使所述光纤探针与HRP偶联的鼠抗人IgG抗体接触，所述光纤探针上捕获的结合抗体与所述鼠抗人IgG抗体特异性结合；信号放大：使所述光纤探针与信号放大剂DAB接触，所述光纤探针上的HRP与所述DAB高亲和力结合，产生金属沉淀，得到参照样本的光谱位移；
    用于对所述待测样本进行如下样本捕获、抗体检测和信号放大，得到待测样本的光谱位移，具体为：
    样本捕获：使所述光纤探针与所述待测样本接触，所述光纤探针上的抗原特异性捕获所述待测样本中存在的针对所述抗原的结合抗体；抗体检测：使所述光纤探针与HRP偶联的鼠抗人IgG抗体接触，所述光纤探针上捕获的结合抗体与所述鼠抗人IgG抗体特异性结合；信号放大：使所述光纤探针与信号放大剂DAB接触，所述光纤探针上的HRP与所述DAB高亲和力结合，产生金属沉淀，得到待测样本的光谱位移；
    (4)标准曲线建立模块：
    用于根据所述若干个参照样本的抗体浓度和光谱位移，以参照样本中的抗体浓度为X轴，以参照样本的光谱相对位移为Y轴，建立结合抗体“浓度-相对位移”标准曲线，其中，
    参照样本的光谱相对位移＝参照样本的光谱位移–零抗体浓度的参照样本的光谱位移；
    (5)待测样本的结合抗体浓度获得模块：
    用于根据所述结合抗体“浓度-相对位移”标准曲线以及待测样本的光谱相对位移，获得待测样本的结合抗体浓度，其中，
    待测样本的光谱相对位移＝待测样本的光谱位移–零抗体浓度的参照样本的光谱位移。
  9. 根据权利要求8所述的结合抗体的生物传感检测系统，还包括：
    第一清洗模块：设于所述光纤探针模块中，用于洗脱未固定至链霉亲和素芯片表面的抗原；
    第二清洗模块：设于所述样本捕获、抗体检测和信号放大模块中，用于洗脱未被光纤探针特异性捕获的结合抗体；
    第三清洗模块：设于所述样本捕获、抗体检测和信号放大模块中，用于洗脱未与结合抗体结合的HRP偶联的鼠抗人IgG抗体。
  10. 根据权利要求8所述的结合抗体的生物传感检测系统，其中：
    所述生物素化的新冠病毒核心蛋白包括生物素化的RBD蛋白、生物素化的S-ECD蛋白或生物素化的RBD-N501Y蛋白；
    所述结合抗体包括RBD蛋白所产生的结合抗体、S-ECD蛋白所产生的结合抗体或RBD-N501Y蛋白所产生的结合抗体。
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