WO2023104131A1 - 基于qd-lumabs的抗体定量检测的曲线建立方法、检测方法及其试剂盒 - Google Patents

基于qd-lumabs的抗体定量检测的曲线建立方法、检测方法及其试剂盒 Download PDF

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WO2023104131A1
WO2023104131A1 PCT/CN2022/137421 CN2022137421W WO2023104131A1 WO 2023104131 A1 WO2023104131 A1 WO 2023104131A1 CN 2022137421 W CN2022137421 W CN 2022137421W WO 2023104131 A1 WO2023104131 A1 WO 2023104131A1
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lumabs
antigen recognition
specific antigen
detection
curve
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金宗文
卫小元
罗擎颖
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深圳先进技术研究院
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/588—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with semiconductor nanocrystal label, e.g. quantum dots
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • the invention relates to the field of analysis and detection, in particular to a method for establishing a curve for quantitative detection of antibodies based on QD-LUMABS, a detection method and a kit thereof.
  • the mNeonG-LUMABS probe antibody detection technology developed in recent years is mainly composed of luciferase that catalyzes blue light emission (BRET donor), green fluorescent protein mNeonGreen (acceptor) and two antigen recognition epitopes. Due to the interaction between the N-terminal and C-terminal sequences of the probe, when the expressed probe is in the "close” state, the spatial position of luciferase and green fluorescent protein is drawn closer to produce efficient BRET; when there is a specific antibody corresponding to the probe When present, the probe is in an "open” state, and the BRET efficiency is reduced, enabling quantitative detection of antibodies.
  • BRET donor blue light emission
  • mNeonGreen acceptor
  • the existing LUMABS probes detected by BRET are all using luciferase (Nanoluc) which catalyzes blue light as the donor, and green fluorescent proteins (GFP, mNeonGreen, Clover4, etc.)
  • the distance between the peaks is about 40nm, the peaks partially overlap, the donor signal interferes with the acceptor signal, and the difference between the maximum excitation wavelength and the maximum emission wavelength of the fluorescent protein is only about 10 nm, resulting in low detection sensitivity, and the luciferase-fluorescent protein
  • the BRET pair can only detect a single component. Therefore, there is an urgent need for an antibody quantitative detection method that has high detection sensitivity and can realize multi-channel detection of the same sample and homogeneous detection of multiple components in the same sample.
  • the present invention proposes a QD-LUMABS-based quantitative antibody detection curve establishment method, detection method and kit thereof.
  • the present invention realizes the self-assembly of LUMABS and quantum dots (QDs) through a streptavidin-biotin system, and introduces QDs into the specific antigen recognition epitope-LUMABS probe.
  • QDs quantum dots
  • the invention provides a method for establishing a curve for quantitative antibody detection based on QD-LUMABS, comprising the following steps:
  • S1 Design and express a specific antigen recognition epitope-LUMABS fusion protein with BirATag;
  • BirATag is a sequence of 15 amino acids that can be specifically recognized by a biotin ligase;
  • the biotin ligase is BirA;
  • the fusion protein in the step S1 is specifically SH3-BirATag-specific antigen recognition epitope-helix-helix-specific antigen recognition epitope-luciferase-sp1 from N-terminal to C-terminal.
  • the luciferase is Nanoluc. Nanoluc catalyzes substrates with higher fluorescence intensity and better effect; at the same time, its molecular weight is smaller than other luciferases, which has little effect on protein structure and is more conducive to the expression of fusion proteins.
  • the specific antigen recognition epitope is HIV antigen recognition epitope.
  • the antigenic epitope of HIV is a commonly used antigenic epitope, and the method of the present invention is shown to be effective with its successfully established curve, but the specific antigen recognition epitope of the present invention is not limited to the antigenic epitope of HIV, any antigenic epitope can be used for Construct LUMABS probes and establish corresponding curves to determine the concentration of antibodies corresponding to specific antigen recognition epitopes.
  • the quantum dot-streptavidin compound is QD655-SA.
  • QD655-SA is a commercially available product.
  • QD655 is a commonly used QD, but any QD can be used in the present invention.
  • the present invention also provides a method for quantitative antibody detection using the specific antibody concentration-BRET efficiency working curve obtained by the curve establishment method, comprising the following steps:
  • reaction temperature is 37°C.
  • the reaction temperature is the optimum temperature for the antibody to recognize the epitope.
  • reaction time is 20-40 min, preferably 30 min.
  • the invention also provides an antibody quantitative detection kit, including quantum dot-specific antigen recognition epitope-LUMABS probe.
  • an antibody quantitative detection kit including quantum dot-specific antigen recognition epitope-LUMABS probe.
  • commonly used reagents such as double distilled water may also be included.
  • the present invention also provides the application of the detection kit in the quantitative detection of antibodies.
  • the method of the present invention inserts the C-terminus of SH3 in LUMABS into BirATag, BirA marks a single biotin on LUMABS, and the LUMABS with biotin is connected to QD through high affinity with streptavidin.
  • QD has a high quantum yield as a donor and can be excited by a single light source.
  • the present invention realizes the self-assembly of LUMABS and QD through the streptavidin-biotin system, and introduces QD into the specific antigen recognition epitope-LUMABS probe, so that The acceptor fluorescence emission wavelength of BRET (Bioluminescence Resonance Energy Transfer) pair is red-shifted, which can be fully separated from the donor fluorescence spectrum, so that the acceptor fluorescence signal is free from the interference of the donor fluorescence signal and the detection sensitivity is improved.
  • BRET Bioluminescence Resonance Energy Transfer
  • the LUMABS probes of the same antibody can be assembled with QDs of different emission wavelengths to realize multi-channel detection of the same sample.
  • the detection method of the present invention can construct multiple LUMABS with BirATag containing different antigen recognition epitopes, and assemble them with QD-streptavidin of different emission wavelengths respectively, so as to realize the homogeneous detection of multiple components in the same sample.
  • Fig. 1 is a schematic flow chart of the method for establishing the working curve of the present invention.
  • Fig. 2 is a schematic diagram of the detection antibody of the present invention.
  • Fig. 3 is a standard curve for detecting HIV antibodies by the method of the present invention.
  • Quantum dots are semiconductor nanomaterials, which are suitable for multi-component fluorescence detection of biomarkers due to their high quantum yield, tunable emission wavelength, narrow emission peak, and large Stokes shift. It can shorten the time required for analysis, save detection reagents, and reduce analysis costs, and has become an important frontier in the field of fluorescent biosensor development.
  • QDs can be excited by a single light source, which greatly simplifies the dependence of multicolor fluorescence detection on multiple light sources; QDs can also be completely independent of external excitation light sources, and are ideal energy acceptors in energy transfer analysis methods.
  • Biotin ligase BirA can specifically recognize a 15aa amino acid sequence (BirATag), and label biotin (Biotin) to lysine in it.
  • Streptavidin (SA) and Biotin have extremely strong binding ability, and the non-covalent binding Kd reaches 10 -15 mol/L, which can be applied to chemiluminescence, immunochromatography, biomolecular purification, and monoclonal antibody preparation , enzyme-linked immunosorbent assay and other biotechnology fields.
  • the fluorescent protein sequence at the LUMABS acceptor position is replaced by the BirATag sequence, and the LUMABS with Biotin can self-assemble with QD-SA after being catalyzed by BirA, and the QD is used as the acceptor of the LUMABS probe.
  • the present invention realizes the self-assembly of LUMABS and QD through the streptavidin-biotin system, and introduces QD into the specific antigen recognition epitope-LUMABS probe, so that the acceptor fluorescence emission wavelength of the BRET (bioluminescent energy resonance transfer) pair is red It can be fully separated from the donor fluorescence spectrum, so that the acceptor fluorescence signal is free from the interference of the donor fluorescence signal, and the detection sensitivity is improved.
  • BRET biologicalluminescent energy resonance transfer
  • the present invention designs a specific antigen recognition epitope-LUMABS fusion protein with BirATag, namely (N-terminal) SH3-BirATag-HIV epitopes-helix-helix-HIV epitopes-Nanoluc-sp1 (C-terminal), catalyzed by BirA with Biotin-specific antigen recognition epitope-LUMABS can self-assemble with SA-QD to form QD-specific antigen recognition epitope- LUMABS probe. Put the probe into a sample containing an antibody that recognizes a specific antigen epitope, the probe changes from a "close” state to an "open” state, and the BRET efficiency decreases. A standard curve is established by measuring the BRET efficiency in samples with different antibody concentrations. As shown in Figure 1, and the principle is shown in Figure 2, the quantitative detection of antibodies is realized.
  • HIV is used as an example for the specific antigen recognition epitope
  • Nanoluc is used as an example for luciferase
  • QD655-SA is used as an example for the QD-SA compound to demonstrate the curve establishment method and antibody quantitative detection method of the present invention.
  • Personnel can infer that using other antigen recognition epitopes, luciferase and QD-SA compounds can achieve the technical effect of the present invention.
  • Embodiment 1 Curve establishment method of the present invention
  • the amino acid sequence of SH3-BirATag-HIV epitopes-helix-helix-HIV epitopes-Nanoluc-sp1 fusion protein is shown in SEQ ID NO.1.
  • Bold K in BirA tag is Biotin labeling site.
  • the fusion protein was purified by nickel column, 10K ultrafiltration tube and stored in 1 ⁇ PBS buffer. The prepared fusion protein was biotin-labeled, then the fusion protein and QD655-SA were mixed at a molar ratio of 1:1, and reacted at room temperature for 30 min to prepare the QD655-HIV-LUMABS probe.
  • the LUMABS probes of the same antibody can be assembled with QDs of different emission wavelengths to realize multi-channel detection of the same sample.
  • Multiple LUMABS with BirATag containing different antigen recognition epitopes can also be constructed and assembled with QD-streptavidin of different emission wavelengths to realize the homogeneous detection of multiple components in the same sample.
  • Embodiment 3 The kit of antibody quantitative detection of the present invention
  • the kit includes quantum dot-specific antigen recognition epitope-LUMABS probe.
  • 100 pM quantum dots-specific antigen recognition epitope-LUMABS probe in the kit to 100 ⁇ L working volume and gradient concentration of specific antigen recognition epitope antibody, react at 37°C for 30 min, and establish a working curve; Then react the specific antibody of unknown concentration with the quantum dot-specific antigen recognition epitope-LUMABS probe, detect the fluorescence intensity of the solution, obtain the BRET efficiency, and calculate the specific antibody according to the established specific antibody concentration-BRET efficiency working curve concentration.
  • the specific antigen recognition epitope is a specific antigen recognition epitope commonly used in practice, and the corresponding antibody is a frequently used antibody that needs to be quantitatively determined.
  • the present invention discloses a QD-LUMABS-based quantitative antibody detection curve establishment method, detection method and kit thereof.
  • the present invention realizes the self-assembly of LUMABS and quantum dots (QDs) through the streptavidin-biotin system, introduces QDs into the specific antigen recognition epitope-LUMABS probe, and makes the acceptor of BRET (bioluminescence resonance energy transfer) pair
  • BRET bioluminescence resonance energy transfer

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Abstract

本发明公开了一种基于QD-LUMABS的抗体定量检测的曲线建立方法、检测方法及其试剂盒。本发明通过链霉亲和素-生物素体系实现LUMABS与量子点（QD）自组装，在特定抗原识别表位-LUMABS探针中引入QD，使BRET（生物发光能量共振转移）对的受体荧光发射波长红移，可与供体荧光光谱充分分离开，使受体荧光信号免受供体荧光信号干扰，提高检测灵敏度，还能够实现同一样品的多通道检测和同一样品中多组分均相检测。

Description

基于QD-LUMABS的抗体定量检测的曲线建立方法、检测方法及其试剂盒 技术领域
本发明涉及分析检测领域，特别涉及一种基于QD-LUMABS的抗体定量检测的曲线建立方法、检测方法及其试剂盒。
 背景技术
近几年发展起来的mNeonG-LUMABS探针抗体检测技术，主要由催化发蓝光的荧光素酶（BRET供体）、绿色荧光蛋白mNeonGreen（受体）和两个抗原识别表位构成。由于探针N端与C端序列的相互作用，表达的探针处于“close”状态时，荧光素酶与绿色荧光蛋白空间位置被拉近发生高效的BRET；当有探针对应的特异性抗体存在时，探针呈“open”状态，BRET效率降低，实现对抗体的定量检测。
 技术问题
现有的以BRET方式检测的LUMABS探针，都是以催化发蓝光的荧光素酶（Nanoluc）为供体，绿色荧光蛋白（GFP、mNeonGreen、Clover4等）为受体，二者的发射主峰仅相距约40nm，峰图部分重叠，供体信号对受体信号造成干扰，且荧光蛋白的最大激发波长与最大发射波长仅相差约10 nm，导致检测的灵敏度不高，而且荧光素酶-荧光蛋白的BRET对仅能做单一组分的检测。因此目前亟需一种检测灵敏度高并且能实现同一样品的多通道检测和同一样品中多组分均相检测的抗体定量检测方法。
 技术解决方案
针对现有技术中的缺陷，本发明提出了一种基于QD-LUMABS的抗体定量检测的曲线建立方法、检测方法及其试剂盒。本发明通过链霉亲和素-生物素体系实现LUMABS与量子点（QD）自组装，在特定抗原识别表位-LUMABS探针中引入QD。
本发明提供一种基于QD-LUMABS的抗体定量检测的曲线建立方法，包括如下步骤：
S1：设计并表达带有BirATag的特定抗原识别表位-LUMABS融合蛋白；所述BirATag为生物素连接酶可特异性识别的一段15个氨基酸的序列；所述生物素连接酶为BirA；
S2：将S1的融合蛋白加上生物素标记；
S3：将S2得到的带有生物素标记的融合蛋白与量子点-链霉亲和素化合物反应，得到量子点-特定抗原识别表位-LUMABS探针；
S4：将S3得到的所述量子点-特定抗原识别表位-LUMABS探针和梯度浓度的所述特定抗原识别表位的抗体反应，加入LUMABS探针底物，检测溶液的供体通道和受体通道荧光强度，计算获得BRET效率（受体通道信号/供体通道信号），建立特定抗体浓度-BRET效率工作曲线。
进一步的，所述步骤S1的所述融合蛋白由N端到C端具体为SH3-BirATag-特定抗原识别表位-helix-helix-特定抗原识别表位-荧光素酶-sp1。
进一步的，所述荧光素酶为Nanoluc。Nanoluc催化底物所发荧光强度高，效果更好；同时其分子量较其他荧光素酶更小，对蛋白结构的影响小，更利于融合蛋白的表达。
进一步的，所述特定抗原识别表位为HIV的抗原识别表位。HIV的抗原表位为常用的抗原表位，用其成功建立曲线说明本发明的方法有效，但是本发明的特定抗原识别表位并不限于HIV的抗原表位，任何抗原表位都可以用来构建LUMABS探针，建立相应的曲线，来测定特定抗原识别表位对应的抗体的浓度。
进一步的，所述量子点-链霉亲和素化合物为QD655-SA。QD655-SA为市售可以直接买到的产品。QD655为常用的QD，但是本发明可使用的QD可为任意一种QD。
本发明还提供一种使用所述的曲线建立方法所获得的特定抗体浓度-BRET效率工作曲线来进行抗体定量检测的方法，包括如下步骤：
将未知浓度的特定抗体与所述量子点-特定抗原识别表位-LUMABS探针反应，检测溶液的供体通道和受体通道荧光强度，并计算获得BRET效率，根据已建立的特定抗体浓度-BRET效率工作曲线计算出所述特定抗体的浓度。
进一步的，所述反应的温度为37℃。反应温度为抗体识别抗原表位的最适温度。
进一步的，所述反应的时间为20-40 min，优选为30 min。
本发明还提供一种抗体定量检测试剂盒，包括量子点-特定抗原识别表位-LUMABS探针。除此之外，还可以包括双蒸水等常用的试剂。
本发明还提供所述的检测试剂盒在抗体定量检测中的应用。
 有益效果
综上，与现有技术相比，本发明达到了以下技术效果：
1. 本发明的方法将LUMABS中SH3的C端插入BirATag，BirA将单个生物素定点标记到LUMABS上，带生物素的LUMABS通过与链霉亲和素的高亲和力连接上QD。
2. 本发明的检测方法中QD作为供体具有高量子产率，可以用单一光源激发。
3. 同样以催化发蓝光的荧光素酶为供体时，本发明通过链霉亲和素-生物素体系实现LUMABS与QD自组装，在特定抗原识别表位-LUMABS探针中引入QD，使BRET（生物发光能量共振转移）对的受体荧光发射波长红移，可与供体荧光光谱充分的分离开，使受体荧光信号免受供体荧光信号干扰，提高检测灵敏度。
4. 本发明的检测方法中同一抗体的LUMABS探针可组装上不同发射波长的QD，实现同一样品的多通道检测。
5. 本发明的检测方法可构建多个带BirATag的含不同抗原识别表位的LUMABS，分别与不同发射波长的QD-链霉亲和素组装，实现同一样品中多组分均相检测。
 附图说明
为了更清楚地说明本发明实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本发明的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为本发明的工作曲线建立方法的流程示意图。
图2为本发明的检测抗体示意图。
图3为本发明的方法检测HIV抗体的标准曲线。
 本发明的实施方式
为了使本技术领域的人员更好地理解本发明方案，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分的实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例，都应当属于本发明保护的范围。
量子点（QD）是一种半导体纳米材料，因其具有高量子产率、可调发射波长、发射峰窄、大斯托克斯位移等特点，适用于生物标记物的多组分荧光检测，可缩短分析所需的时间，节省检测试剂，降低分析成本，成为荧光生物传感器开发领域的重要前沿。QD可以用单一光源激发，大大的简化多色荧光检测对多种光源的依赖性；QD还可以完全不受制于外部激发光源，在能量转移分析方法中是非常理想的能量受体。
生物素连接酶BirA可特异性识别一段15aa的氨基酸序列（BirATag），并将生物素（Biotin）标记到其中的赖氨酸上。链霉亲和素（SA）和Biotin有极强的结合能力，非共价结合力的Kd达到10 -15 mol/L，能够应用于化学发光、免疫层析、生物分子纯化、单克隆抗体制备、酶联免疫吸附实验等生物技术领域。用BirATag序列取代LUMABS受体位置上的荧光蛋白序列，经BirA催化后带Biotin的LUMABS即可与QD-SA进行自组装，将QD作为该LUMABS探针的受体。
本发明通过链霉亲和素-生物素体系实现LUMABS与QD自组装，在特定抗原识别表位-LUMABS探针中引入QD，使BRET（生物发光能量共振转移）对的受体荧光发射波长红移，可与供体荧光光谱充分分离开，使受体荧光信号免受供体荧光信号干扰，提高检测灵敏度。
本发明通过设计带BirATag的特定抗原识别表位-LUMABS融合蛋白，即（N端）SH3-BirATag-HIV epitopes-helix-helix-HIV epitopes-Nanoluc-sp1（C端），经BirA催化后带Biotin的特定抗原识别表位-LUMABS即可与SA-QD进行自组装，形成QD-特定抗原识别表位-LUMABS探针。将探针放入含有特定抗原识别表位的抗体的样品中，探针由“close”状态转变成“open”状态，BRET效率降低，通过测定不同抗体浓度样品中的BRET效率建立标准曲线，流程如图1所示，原理如图2所示，实现对抗体的定量检测。
以下实施例中特定抗原识别表位以HIV为例，荧光素酶以Nanoluc为例，QD-SA化合物以QD655-SA为例，来展示本发明的曲线建立方法和抗体定量检测方法，本领域技术人员可以推及使用其他的抗原识别表位、荧光素酶和QD-SA化合物均能实现本发明的技术效果。
实施例1  本发明的曲线建立方法
在pCold I质粒中插入融合蛋白（N端）SH3-BirATag-HIV epitopes-helix- helix-HIV epitopes-Nanoluc-sp1（C端）的编码序列，将重组质粒经热激转化入BL21感受态细胞中。转化后筛选出的阳性克隆菌经扩增培养后，按1：100接种到200 ml含Amp的LB培养基中，37℃ 200 rpm培养至OD 600 = 0.6，加入0.1 mM IPTG在15℃ 200 rpm条件下诱导表达融合蛋白。SH3-BirATag-HIV epitopes-helix- helix-HIV epitopes-Nanoluc-sp1融合蛋白的氨基酸序列如SEQ ID NO.1所示。BirA tag中加粗的K为Biotin标记位点。融合蛋白经镍柱纯化，10K超滤管纯化，保存在1×PBS缓冲液中。对制备得到的融合蛋白进行生物素标记，之后按摩尔比1：1混合融合蛋白和QD655-SA，室温反应30 min制备QD655-HIV-LUMABS探针。在100 μL工作体积中加入100 pM QD655-HIV- LUMABS探针和梯度浓度的HIV抗体，37℃反应30 min，检测溶液的供体通道和受体通道荧光强度，计算获得BRET效率（受体通道信号/供体通道信号），建立HIV抗体浓度-BRET效率工作曲线，如图3所示，该工作曲线的R 2 = 0.999，表明拟合度非常好，本发明的曲线建立方法有效。
实施例 2  通过构建好的特定抗体浓度-BRET效率工作曲线进行抗体定量检测
将未知浓度的特定抗体与量子点-特定抗原识别表位-LUMABS探针反应，检测溶液的荧光强度，获得BRET效率，根据已经建立的特定抗体浓度-BRET效率工作曲线计算出所述特定抗体的浓度。
此外，本发明的检测方法中同一抗体的LUMABS探针可组装上不同发射波长的QD，实现同一样品的多通道检测。还可构建多个带BirATag的含不同抗原识别表位的LUMABS，分别与不同发射波长的QD-链霉亲和素组装，实现同一样品中多组分均相检测。
实施例 3  本发明的抗体定量检测的试剂盒
所述试剂盒包括量子点-特定抗原识别表位-LUMABS探针。使用时，在100 μL工作体积中加入100 pM 试剂盒中的量子点-特定抗原识别表位-LUMABS探针和梯度浓度的特定抗原识别表位的抗体，37℃反应30 min，建立工作曲线；随后将未知浓度的特定抗体与量子点-特定抗原识别表位-LUMABS探针反应，检测溶液的荧光强度，获得BRET效率，根据已经建立的特定抗体浓度-BRET效率工作曲线计算出所述特定抗体的浓度。特定抗原识别表位为实际中常用的特定抗原识别表位，其对应的抗体为经常使用的需要进行定量测定的抗体。
综合以上实施例，本发明公开了一种基于QD-LUMABS的抗体定量检测的曲线建立方法、检测方法及其试剂盒。本发明通过链霉亲和素-生物素体系实现LUMABS与量子点（QD）自组装，在特定抗原识别表位-LUMABS探针中引入QD，使BRET（生物发光能量共振转移）对的受体荧光发射波长红移，可与供体荧光光谱充分分离开，使受体荧光信号免受供体荧光信号干扰，提高检测灵敏度，还能够实现实现同一样品的多通道检测和同一样品中多组分均相检测。
以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
 序列表自由内容
SEQ ID NO.1：
SH3-BirA tag-HIV epitope-helix-helix-HIV epitope-Nluc-sp1
MASDDNFIYKAKALYPYDADDDDAYEISFEQNEILQVSDIEGRWWKARRANGETGIIPSNYVQLIDGPEEMHRGGSGLNDIFEAQ KIEWHESGGSGGELDRWEKIRLRPGGSGGSGGSGGSGGSGGSGAEAAAKEAAAKEAAAKEAAAKEAAAKEAAAKAGSGGSGGSGGSGGSGGSGAEAAAKEAAAKEAAAKEAAAKEAAAKEAAAKAGSGGSGGSGGSGGSGGSGGELDRWEKIRLRPGGSVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILASSGGGSIRSKPLPPLPVTG

Claims (10)

  1. 一种基于QD-LUMABS的抗体定量检测的曲线建立方法，其特征在于，包括如下步骤：
    S1：设计并表达带有BirATag的特定抗原识别表位-LUMABS融合蛋白；所述BirATag为生物素连接酶可特异性识别的一段15个氨基酸的序列；所述生物素连接酶为BirA；
    S2：将S1的融合蛋白加上生物素标记；
    S3：将S2得到的带有生物素标记的融合蛋白与量子点-链霉亲和素化合物反应，得到量子点-特定抗原识别表位-LUMABS探针；
    S4：将S3得到的所述量子点-特定抗原识别表位-LUMABS探针和梯度浓度的所述特定抗原识别表位的抗体反应，加入LUMABS探针底物，检测溶液的供体通道和受体通道荧光强度，计算获得BRET效率，建立特定抗体浓度-BRET效率工作曲线。
  2. 根据权利要求1所述的曲线建立方法，其特征在于，所述步骤S1的所述融合蛋白由N端到C端具体为SH3-BirATag-特定抗原识别表位-helix-helix-特定抗原识别表位-荧光素酶-sp1。
  3. 根据权利要求2所述的曲线建立方法，其特征在于，所述荧光素酶为Nanoluc。
  4. 根据权利要求2所述的曲线建立方法，其特征在于，所述特定抗原识别表位为HIV的抗原识别表位。
  5. 根据权利要求1所述的曲线建立方法，其特征在于，所述量子点-链霉亲和素化合物为QD655-SA。
  6. 一种使用权利要求1所述的曲线建立方法所获得的特定抗体浓度-BRET效率工作曲线来进行抗体定量检测的方法，其特征在于，包括如下步骤：
    将未知浓度的特定抗体与所述量子点-特定抗原识别表位-LUMABS探针反应，检测溶液的供体通道和受体通道荧光强度，并计算获得BRET效率，根据权利要求1建立的特定抗体浓度-BRET效率工作曲线计算出所述特定抗体的浓度。
  7. 根据权利要求6所述的抗体定量检测的方法，其特征在于，所述反应的温度为37℃。
  8. 根据权利要求6所述的抗体定量检测的方法，其特征在于，所述反应的时间为20-40 min。
  9. 一种抗体定量检测试剂盒，其特征在于，包括量子点-特定抗原识别表位-LUMABS探针。
  10. 权利要求9所述的检测试剂盒在抗体定量检测中的应用。
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