WO2021217768A1 - 藻红蛋白免疫荧光探针制备方法 - Google Patents

藻红蛋白免疫荧光探针制备方法 Download PDF

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WO2021217768A1
WO2021217768A1 PCT/CN2020/092719 CN2020092719W WO2021217768A1 WO 2021217768 A1 WO2021217768 A1 WO 2021217768A1 CN 2020092719 W CN2020092719 W CN 2020092719W WO 2021217768 A1 WO2021217768 A1 WO 2021217768A1
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phycoerythrin
sulfhydryl
target protein
preparing
immunofluorescence
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张洋
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浙江正熙生物医药有限公司
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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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Electro-optical investigation, e.g. flow cytometers
    • 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
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • 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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • 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
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Electro-optical investigation, e.g. flow cytometers
    • G01N15/1434—Electro-optical investigation, e.g. flow cytometers using an analyser being characterised by its optical arrangement
    • G01N2015/144—Imaging characterised by its optical setup
    • 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
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks

Definitions

  • the invention belongs to the technical field of immunology, and specifically relates to a method for preparing a phycoerythrin immunofluorescence probe.
  • Phycoerythrin (P-phycoerythrin, PE for short) is a new type of fluorescent labeling reagent that is obtained by separation and purification from red algae and is currently commonly used. Under the excitation of a specific wavelength, phycobiliprotein can emit strong fluorescence, and its fluorescence intensity is 30-100 times that of fluorescein. It has good light absorption performance and high quantum yield, and has a wide excitation in the visible spectral region. And launch range.
  • phycoerythrin for fluorescence analysis has advantages that traditional chemical fluorescent dyes cannot match. For example: (1) It has a wide absorption spectrum in a wide pH range, and it is easier to select a suitable excitation wavelength to obtain high-efficiency fluorescence emission, and there is a specific fluorescence emission peak during excitation; (2) Absorbance and fluorescence quantum The yield is high, the fluorescence is strong and stable, and the sensitivity is high; (3) It has a small fluorescence background, which is not easy to quench, and the fluorescence storage period is long; (4) It has excellent water solubility and is easy to cross-link and combine with other molecules. Low heterosexual adsorption; (5) Pure natural marine organism extraction, without any toxic side effects, no radioactivity, and very safe operation and use.
  • PE labeling is often used to combine phycoerythrin with antibodies, biotin, avidin, immune proteins and other substances to prepare fluorescent probes.
  • fluorescence microscopy fluorescence immunoassay, two-color or multi-color fluorescence analysis, cancer cell surface antigen detection, protein and nucleic acid and other biological macromolecule analysis; it can also be used for immunodetection, fluorescence Clinical diagnosis and bioengineering technologies such as microscopy and flow cytometry.
  • the implementation steps of traditional PE labeling method are roughly as follows: (1) sulfhydryl the target protein; (2) activate PE with SMCC; (3) sulfhydryl target protein Cross-link with activated PE.
  • the implementation steps of the PE labeling method (phycoerythrin immunofluorescence probe labeling method, patent number 202010087828.0) developed by Zhengxi Biotechnology are roughly as follows: (1) sulfhydryl of PE; (2) using SMCC to activate the target protein; (3) ) Cross-link the thiolated PE with the activated target protein.
  • the disadvantage of the above-mentioned PE labeling method is that since the target protein and phycoerythrin carry both free amino groups and free sulfhydryl groups, when amine-sulfhydryl crosslinking agents such as SMCC are used for activation, the amine-sulfhydryl crosslinking agent will not only It binds to amino groups and also binds to sulfhydryl groups. This results in that when the protein activated by the amine-sulfhydryl crosslinker is crosslinked with sulfhydryl phycoerythrin, those on the protein activated by the amine-sulfhydryl crosslinker have been bound to the sulfhydryl group.
  • the cross-linking agent is difficult to cross-link with thiolated phycoerythrin again, resulting in less phycoerythrin bound to the target protein and poor labeling effect.
  • the obtained immunofluorescence probe produces weak positive signal and strong background signal. The noise ratio is low.
  • the purpose of the present invention is to provide a method for preparing a phycoerythrin immunofluorescence probe with high phycoerythrin labeling efficiency.
  • the prepared phycoerythrin immunofluorescence probe is subjected to immunodetection, the positive signal is strong and the signal-to-noise ratio is high. .
  • a method for preparing a phycoerythrin immunofluorescence probe includes the step of labeling phycoerythrin on the target protein; before the step of labeling phycoerythrin on the target protein, the phycoerythrin immunofluorescence
  • the probe preparation method also includes a step of blocking free sulfhydryl groups on the target protein or phycoerythrin.
  • the free sulfhydryl groups on the target protein or phycoerythrin are first sealed, and the free sulfhydryl groups on the target protein or phycoerythrin are freed from the target protein or phycoerythrin.
  • the subsequent amine-sulfhydryl crosslinking agent can only react with the amino group on the blocked target protein or blocked phycoerythrin, but will not react with the sulfhydryl group on the corresponding protein, thus ensuring the amine-sulfhydryl group
  • the maleimide groups of the cross-linking agent are all used to couple the sulfhydryl phycoerythrin or the target protein, thereby effectively improving the cross-linking efficiency of the phycoerythrin and the target protein, and improving the labeling of the target protein by the phycoerythrin Efficiency, and the obtained phycoerythrin immunofluorescence probe can also obtain a strong positive signal during immunoassay, which greatly improves the signal-to-noise ratio.
  • the free sulfhydryl groups on the target protein are blocked.
  • the phycoerythrin immunofluorescence probe obtained by blocking free sulfhydryl groups on the target protein has a stronger positive signal and a higher signal-to-noise ratio.
  • a method for preparing a phycoerythrin immunofluorescence probe includes the following steps:
  • the free sulfhydryl group on the target protein is first blocked and then activated by an amine-sulfhydryl crosslinking agent, so that the amine-sulfhydryl crosslinking agent can only react with the amino group on the target protein.
  • All the maleimide groups on the amine-sulfhydryl crosslinker are not involved in the reaction; when the activated target protein is crosslinked with the sulfhydryl phycoerythrin, the maleimide of the amine-sulfhydryl crosslinker on the target protein is activated
  • the imine groups are all used to react with the sulfhydryl group on the thiolated phycoerythrin, so that more phycoerythrin can be bound to the target protein, the phycoerythrin labeling efficiency is higher, and the obtained phycoerythrin immunofluorescence probe is When performing immunoassays, stronger positive signals can also be obtained, which further improves the signal-to-noise ratio.
  • the phycoerythrin does not need to be activated in advance, it is directly sulfhylated, and then cross-linked with the pre-activated target protein, which is not only easy to operate, but also can further improve the algae The signal-to-noise ratio when using the red protein immunofluorescence probe.
  • the present invention selects a method with relatively simple operation, that is, in step (1) of the method for preparing the phycoerythrin immunofluorescence probe, a sulfhydryl blocking agent is used to block the target protein.
  • the blocking treatment includes: mixing the target protein and the sulfhydryl blocking agent at a molar ratio of 1:10-1:100, and reacting at room temperature. -2 h.
  • the sealing treatment includes: mixing the target protein and the sulfhydryl sealing agent at a molar ratio of 1:40-1:60, and reacting at room temperature for 1-2 hours.
  • the room temperature referred to in the present invention refers to room temperature conditions above 25°C.
  • the sulfhydryl blocking agent includes at least one of N-ethylmaleimide and iodoacetamide (IAAm).
  • the sulfhydryl blocking agent is N-ethylmaleimide (NEM).
  • NEM N-ethylmaleimide
  • NEM can gently block free sulfhydryl groups under neutral conditions.
  • the sealing treatment includes: mixing the target protein and N-ethylmaleimide at a molar ratio of 1:50, and reacting at room temperature for 1.5 hours.
  • the blocking treatment reaction solution needs to be desalted to remove the unreacted sulfhydryl blocking agent remaining in the blocking treatment reaction solution to ensure that the final thiol blocking target protein has a higher purity.
  • the activation treatment includes: the sulfhydryl-blocked target protein obtained in step (1) and the amine-sulfhydryl cross-linking agent are in a molar ratio of 1: Mix well at 10-1:100 and react at room temperature for 1-2 h.
  • the amine-sulfhydryl crosslinker can only connect to the amino groups of the target protein.
  • the activation treatment includes: mixing the sulfhydryl-blocked target protein obtained in step (1) with the amine-sulfhydryl crosslinking agent at a molar ratio of 1:40-1:60, and reacting 1-2 at room temperature. h.
  • the amine-sulfhydryl crosslinking agent includes 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide Ester (SMCC), sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (Sulfo-SMCC), 3-(2-pyridinedimercapto) N-hydroxysuccinimide propionate (SPDP) or N-succinimidyl 6-(3-maleimidopropionamido)hexanoate (SMPH).
  • SMCC 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide Ester
  • Sulfo-SMCC sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate
  • SPDP 3-(2-pyridinedimercapto) N-hydroxysuccinimide
  • the activation treatment includes: mixing the sulfhydryl-blocked target protein obtained in step (1) with SMCC at a molar ratio of 1:20, and reacting at room temperature for 1.5 hours.
  • the activation reaction solution needs to be desalted after the activation treatment to remove the unreacted amine-sulfhydryl crosslinking agent remaining in the activation reaction solution.
  • the sulfhydryl treatment includes: the phycoerythrin and 2-iminosulfane hydrochloride (traut's regent) are molested 1:1: After mixing at 10-1:50, place at room temperature to avoid light and react for 1-2 h.
  • the sulfhydryl treatment includes: mixing phycoerythrin and 2-iminosulfane hydrochloride at a molar ratio of 1:10-1:20, and then reacting for 1.5 hours at room temperature in the dark.
  • the sulfhydryl treatment includes: mixing phycoerythrin and 2-iminosulfane hydrochloride at a molar ratio of 1:10, and then reacting for 1.5 h at room temperature in the dark.
  • the phycoerythrin needs to be desalted before the sulfhydryl treatment to prevent the free amino groups contained in the phycoerythrin from interfering with the sulfhydryl reaction; and after the sulfhydryl treatment, the reaction solution must also be desalted to remove The unreacted 2-iminosulfane hydrochloride remaining in the reaction solution is activated.
  • step (4) of the method for preparing phycoerythrin immunofluorescence probes the thiolated phycoerythrin obtained in step (3) is compared with the activated target protein obtained in step (2) at a molar ratio of 1:1-30: 1 After mixing, put it at room temperature and avoid light to react for 2-4 h.
  • step (4) the thiolated phycoerythrin obtained in step (3) and the activated target protein obtained in step (2) are mixed at a molar ratio of 1:1-10:1, and then placed at room temperature to avoid Light reaction for 2-4 h.
  • step (4) the thiolated phycoerythrin obtained in step (3) and the activated target protein obtained in step (2) are mixed at a molar ratio of 5:1, and then placed at room temperature and protected from light. h.
  • the target protein or free sulfhydryl groups on the phycoerythrin are first sealed before the conventional method steps are used to label and cross-link the phycoerythrin to the target protein.
  • the subsequent amine-sulfhydryl crosslinking agent can only react with the blocked target protein or the amino group on the blocked phycoerythrin, but will not react with the corresponding protein.
  • the sulfhydryl reaction can ensure that all the amino groups of the amine-sulfhydryl crosslinking agent are used to couple the sulfhydryl phycoerythrin or the target protein, thereby effectively improving the crosslinking efficiency of phycoerythrin and the target protein, and improving the phycoerythrin pair
  • the labeling efficiency of the target protein, and the obtained phycoerythrin immunofluorescence probe can also obtain a strong positive signal during immunoassay, which greatly improves the signal-to-noise ratio.
  • Figure 1 is a flow cytometric scatter diagram of mouse spleen cells co-stained with phycoerythrin immunofluorescence probe NEM-CD62L-PE and CD3-ifluor488 prepared in Example 1;
  • Figure 2 is a flow cytometric scatter diagram of mouse spleen cells co-stained with phycoerythrin immunofluorescence probe CD62L-PE and CD3-ifluor488 prepared in Comparative Example 1;
  • Figure 3 is a histogram of the detection results of phycoerythrin fluorescence signal intensity of mouse spleen cells stained by the phycoerythrin immunofluorescence probe NEM-CD62L-PE prepared in Example 1;
  • Fig. 4 is a histogram of the detection result of phycoerythrin fluorescence signal intensity of mouse spleen cells stained by the phycoerythrin immunofluorescence probe CD62L-PE prepared in Comparative Example 1;
  • Figure 5 is a superimposed comparison diagram of Figures 3 and 4;
  • Figure 6 is a scatter diagram of flow cytometry of mouse splenocytes co-stained with phycoerythrin immunofluorescence probe NEM-CD4-PE and CD3-ifluor488 prepared in Example 2;
  • Figure 7 is a flow cytometric scatter diagram of mouse spleen cells co-stained with phycoerythrin immunofluorescence probe CD4-PE and CD3-ifluor488 prepared in Comparative Example 2;
  • Example 8 is a histogram of the detection result of the phycoerythrin fluorescence signal intensity of mouse spleen cells stained with the phycoerythrin immunofluorescence probe NEM-CD4-PE prepared in Example 2;
  • Figure 9 is a histogram of the detection results of phycoerythrin fluorescence signal intensity of mouse spleen cells stained by the phycoerythrin immunofluorescence probe CD4-PE prepared in Comparative Example 2;
  • Fig. 10 is an overlay comparison diagram of Fig. 8 and Fig. 9;
  • Figure 11 is a flow cytometric scatter diagram of mouse spleen cells co-stained with phycoerythrin immunofluorescence probe NEM-CD8-PE and CD19-FITC prepared in Example 3;
  • Figure 12 is a flow cytometric scatter diagram of mouse spleen cells co-stained with phycoerythrin immunofluorescence probe CD8-PE and CD19-FITC prepared in Comparative Example 3;
  • Figure 13 is a graph showing the detection result of the phycoerythrin fluorescence signal intensity of mouse spleen cells stained with the phycoerythrin immunofluorescence probe NEM-CD8-PE prepared in Example 3;
  • Figure 14 is a graph showing the detection result of phycoerythrin fluorescence signal intensity of mouse spleen cells stained by the phycoerythrin immunofluorescence probe CD8-PE prepared in Comparative Example 3;
  • Fig. 15 is an overlay comparison diagram of Fig. 13 and Fig. 14.
  • an anti-mouse CD62L monoclonal antibody [MEL-14] (hereinafter referred to as CD62L antibody) is used as the target protein to introduce a method for preparing a phycoerythrin immunofluorescence probe.
  • the preparation method includes the following steps:
  • the NEM molecule binds to the free sulfhydryl group in the CD62L antibody molecule to block the free sulfhydryl group in the CD62L antibody molecule;
  • step (1.3) Transfer the reaction solution obtained in step (1.2) to the ultrafiltration centrifuge tube, and then add 500 ⁇ l PBS (containing 0.25mM EDTA) buffer, centrifuge at 12000g for 5 min and remove the filtrate, then add 500 ⁇ l PBS (containing 0.25mM EDTA) buffer to mix and centrifuge, repeat this operation 5 times;
  • desalting is used to remove as much NEM residues in the reaction solution that are not involved in the reaction to ensure that the NEM residues in the reaction solution obtained after desalting are at least 10-3 times the total amount of antibodies;
  • the succinyl ester in the SMCC molecule reacts with the primary amino group in the sulfhydryl blocking antibody molecule;
  • step (2.4) Transfer the reaction solution obtained in step (2.3) to the ultrafiltration centrifuge tube, and then add 500 ⁇ l PBS (containing 0.25mM EDTA) buffer, centrifuge at 12000g for 5 min and remove the filtrate, then add 500 ⁇ l PBS (containing 0.25mM EDTA) buffer to mix and centrifuge, repeat this operation 5 times;
  • desalting is used to remove as much as possible the residual SMCC in the reaction solution that is not involved in the reaction, to ensure that the residual amount of SMCC in the reaction solution obtained after desalting is at least 10-3 times the total amount of antibodies;
  • Example 2 Using the anti-mouse CD4 monoclonal antibody [GK1.5] as the target protein, the same method as in Example 1 was used to prepare the phycoerythrin immunofluorescence probe NEM-CD4-PE.
  • Example 2 With anti-mouse CD8 monoclonal antibody [53-6.7] as the target protein, the same method as in Example 1 was used to prepare the phycoerythrin immunofluorescence probe NEM-CD8-PE.
  • This comparative example uses the anti-mouse CD62L monoclonal antibody [MEL-14] as the target protein to introduce a phycoerythrin immunofluorescence probe labeling method.
  • the labeling method includes the following steps:
  • (1-1) Take out the SMCC from a low temperature state and place it in a room temperature environment. When the temperature of the bottle equals to room temperature, open the bottle cap to avoid condensation in the bottle;
  • CD62L antibody solution obtained in step (1) with the thiolated phycoerythrin solution obtained in step (2) according to a molar ratio of 1:3, that is, add 3 ⁇ l (10 mg/ml) thiolated phycoerythrin solution, and then placed at room temperature above 25°C to avoid light for 3h. After the reaction is completed, add 28 ⁇ l PBS (containing 0.25mM EDTA) buffer to the reaction system to make the phycoerythrin labeled The final concentration of CD62L antibody (ie CD62L-PE) is 0.25mg/ml. After the operation is completed, the reaction solution is placed at 4°C for long-term storage.
  • the phycoerythrin immunofluorescence probe CD4-PE was prepared by the same method as in Comparative Example 1.
  • mouse splenocytes stained by NEM-CD62L-PE have a stronger positive fluorescence signal in the R6 region (28845 vs 20178).
  • Figure 4 and Figure 5 compared with CD62L-PE, mouse spleen cells stained by NEM-CD62L-PE have higher PE fluorescence signal value, which is consistent with the detection results in Figure 1 and Figure 2. Are consistent.
  • phycoerythrin immunofluorescence probes NEM-CD4-PE and CD4-PE were co-stained with CD3-iFluor488 at the same concentration to 1million mouse spleen cells (add 0.25 ⁇ g labeled iFluor in each reaction system) Labeled antibody and 0.125 ⁇ g PE-labeled antibody), 15 min after staining, the fluorescence signal was detected by flow cytometer, and the detection was repeated for at least three groups with at least two samples in each group; the detection results are shown in Figure 6, Figure 7, Figure 8, and Figure 9. And shown in Figure 10.
  • mouse spleen cells stained by NEM-CD4-PE have a stronger positive fluorescence signal in the Q2 (upper right corner of the figure) area (53560 vs 40325).
  • phycoerythrin immunofluorescence probes NEM-CD8-PE and CD8-PE were co-stained with CD19-FITC at the same concentration to 1million mouse spleen cells (add 0.25 ⁇ g labeled FITC to each reaction system) Labeled antibody and 0.125 ⁇ g PE-labeled antibody), 15 min after staining, the fluorescence signal was detected by flow cytometer, and the detection was repeated for at least three groups with at least two samples in each group; the detection results are shown in Figure 11, Figure 12, Figure 13, and Figure 14. And shown in Figure 15.
  • mouse spleen cells stained by NEM-CD8-PE have a stronger positive fluorescence signal in the Q4 (lower left corner of the figure) area (19417). vs 14075).
  • the invention discloses a method for preparing a phycoerythrin immunofluorescence probe.
  • the method includes the step of labeling phycoerythrin on a target protein; before the step of labeling phycoerythrin on the target protein, the method It also includes a step of blocking free sulfhydryl groups on the target protein or phycoerythrin.
  • the target protein or the free sulfhydryl groups on the phycoerythrin are sealed first, and when the free sulfhydryl groups are blocked, the amine-sulfhydryl cross-linking
  • the agent can only react with the amino group on the target protein or phycoerythrin to be blocked, and will not react with the sulfhydryl group on the corresponding protein. This ensures that all the amino groups of the amine-sulfhydryl crosslinker are used to couple the sulfhydryl algae.
  • Red protein or target protein thereby effectively improving the labeling efficiency of phycoerythrin on the target protein, and the obtained phycoerythrin immunofluorescence probe has a higher signal-to-noise ratio during immunoassay.

Abstract

一种藻红蛋白免疫荧光探针制备方法，包括将藻红蛋白标记到目标蛋白上的步骤；在将藻红蛋白标记到目标蛋白上的步骤前，还包括对目标蛋白或藻红蛋白上的游离巯基作封闭处理的步骤。在采用常规的方法步骤将藻红蛋白标记、交联到目标蛋白上之前，先对目标蛋白或藻红蛋白上的游离巯基作封闭处理，当游离巯基被封闭后，胺-巯基交联剂只能与被封闭处理目标蛋白或藻红蛋白上的氨基反应，而不会与相应蛋白上的巯基反应，如此能够确保胺-巯基交联剂的氨基全部用于偶联被巯基化的藻红蛋白或目标蛋白，从而有效提高藻红蛋白对目标蛋白的标记效率，获得的藻红蛋白免疫荧光探针在进行免疫检测时具有较高的信噪比。

Description

藻红蛋白免疫荧光探针制备方法 技术领域
本发明属于免疫学技术领域，具体涉及一种藻红蛋白免疫荧光探针制备方法。
 背景技术
藻红蛋白(P-phycoerythrin，简称PE)是从红藻中分离纯化获得的、目前普遍使用的一种新型荧光标记试剂。在特定波长激发下，藻胆蛋白能发射强烈的荧光，其荧光强度是荧光素的30-100倍，具有很好的吸光性能和很高的量子产率，在可见光谱区有很宽的激发及发射范围。
将藻红蛋白用于荧光分析，具有传统化学荧光染料无法比拟的优越性。比如：（1）在较宽的pH范围内具有较宽的吸收光谱，比较容易选择合适的激发波长，从而得到高效荧光发射，且激发时有特异的荧光发射峰；（2）吸光度和荧光量子产率很高，荧光强而稳定，灵敏度高；（3）具有较小的荧光背景，不易淬灭，荧光保存期较长；（4）水溶性极佳，易与其他分子交联结合，非特异性吸附少；（5）纯天然海洋生物提取，无任何毒副作用，不含放射性，操作使用非常安全。
现有技术中常常采用PE标记法，将藻红蛋白与抗体、生物素、亲合素、免疫蛋白等物质结合，制成荧光探针。通过检测其发出的荧光，可以用于荧光显微检测、荧光免疫测定、双色或多色荧光分析、癌细胞表面抗原检测、蛋白质和核酸等生物大分子的分析；还可以用于免疫检测、荧光显微技术和流式细胞荧光测定等临床诊断及生物工程技术。
传统的PE标记法（如AnaTag™ R-PE Labeling Kit）的实施步骤大致是：（1）将目标蛋白巯基化；（2）采用SMCC对PE进行活化；（3）将巯基化后的目标蛋白与活化后的PE交联。
正熙生物开发的PE标记法(藻红蛋白免疫荧光探针标记方法，专利号202010087828.0)的实施步骤大致是: （1）将PE巯基化；（2）采用SMCC对目标蛋白进行活化；（3）将巯基化后的PE与活化后的目标蛋白交联。
现有技术中还有一种实施步骤（参见文献：赵亚杰，闭兰，孙可芳，端义坤，詹骞，金玉玲，吕兰君，张爱华. 荧光染料R-藻红蛋白标记小鼠抗人CD4单克隆抗体. 微生物学免疫学进展[J]. 2006，34（2）：32-34.）为：（1）采用SPDP和SMCC分别活化PE和目标蛋白；（2）将经SPDP活化后的PE巯基化；（3）将巯基化的PE与经SMCC活化的目标蛋白交联。
 技术问题
上述PE标记法的不足之处在于：由于目标蛋白和藻红蛋白上既携带游离氨基，又携带游离巯基，当采用SMCC等胺-巯基交联剂进行活化时，胺-巯基交联剂不仅会与氨基结合，也会与巯基结合，这就导致当被胺-巯基交联剂活化的蛋白与巯基化藻红蛋白交联时，被胺-巯基交联剂活化的蛋白上那些已与巯基结合的交联剂难以与巯基化藻红蛋白再发生交联，导致目标蛋白上结合的藻红蛋白少、标记效果差，获得的免疫荧光探针产生的阳性信号弱、背景信号强，检测时信噪比低。
 技术解决方案
本发明的发明目的是提供一种藻红蛋白标记效率高的藻红蛋白免疫荧光探针制备方法，制备获得的藻红蛋白免疫荧光探针在进行免疫检测时，阳性信号强、信噪比高。
为实现上述发明目的，本发明的技术方案如下：
一种藻红蛋白免疫荧光探针制备方法，包括将藻红蛋白标记到目标蛋白上的步骤；在所述的将藻红蛋白标记到目标蛋白上的步骤前，所述的藻红蛋白免疫荧光探针制备方法还包括对目标蛋白或藻红蛋白上的游离巯基作封闭处理的步骤。
本发明中，在采用常规的方法步骤将藻红蛋白标记、交联到目标蛋白上前，先对目标蛋白或藻红蛋白上的游离巯基作封闭处理，在目标蛋白或藻红蛋白上的游离巯基被封闭后，后续的胺-巯基交联剂只能与被封闭处理目标蛋白或被封闭处理藻红蛋白上的氨基反应，而不会与相应蛋白上的巯基反应，如此能够确保胺-巯基交联剂的马来酰亚胺基团全部用于偶联被巯基化的藻红蛋白或目标蛋白，从而有效提高藻红蛋白与目标蛋白的交联效率，提高藻红蛋白对目标蛋白的标记效率，而获得的藻红蛋白免疫荧光探针在进行免疫检测时，也能获得较强阳性信号，大大提高信噪比。
作为优选，在上述的藻红蛋白免疫荧光探针制备方法中，对目标蛋白上的游离巯基作封闭处理。与对藻红蛋白上的游离巯基作封闭处理相比，对目标蛋白上的游离巯基作封闭处理获得的藻红蛋白免疫荧光探针具有更强的阳性信号，信噪比更高。
即一种藻红蛋白免疫荧光探针制备方法，包括以下步骤：
（1）对目标蛋白上的游离巯基作封闭处理；
（2）采用胺-巯基交联剂对步骤（1）获得的巯基封闭目标蛋白作活化处理；
（3）对藻红蛋白作巯基化处理；
（4）将步骤（3）获得的巯基化藻红蛋白与步骤（2）获得的活化目标蛋白交联，获得所述的藻红蛋白免疫荧光探针。
本发明的藻红蛋白免疫荧光探针制备方法中，先封闭目标蛋白上的游离巯基再采用胺-巯基交联剂进行活化，如此胺-巯基交联剂只能与目标蛋白上的氨基反应，胺-巯基交联剂上的马来酰亚胺基团全部未参与反应；则当将活化目标蛋白与巯基化藻红蛋白交联时，活化目标蛋白上胺-巯基交联剂的马来酰亚胺基团全部用于与巯基化藻红蛋白上的巯基反应，如此目标蛋白上能够结合更多的藻红蛋白，藻红蛋白标记效率更高，而获得的藻红蛋白免疫荧光探针在进行免疫检测时，也能获得更强的阳性信号，进一步提高信噪比。
本发明的藻红蛋白免疫荧光探针制备方法中，藻红蛋白无需预先活化，直接对其作巯基化处理，而后与预先活化的目标蛋白交联即可，不仅操作简便，还能进一步提高藻红蛋白免疫荧光探针使用时的信噪比。
现有技术中，对蛋白上的游离巯基作封闭处理的方法较多。本发明选择操作较为简便的方法，即在上述的藻红蛋白免疫荧光探针制备方法的步骤（1）中，采用巯基封闭剂对目标蛋白作封闭处理。
作为优选，在上述的藻红蛋白免疫荧光探针制备方法中，所述的封闭处理包括：将目标蛋白与巯基封闭剂按摩尔比1:10-1:100混匀，置于室温下反应1-2 h。
作为优选，所述的封闭处理包括：将目标蛋白与巯基封闭剂按摩尔比1: 40-1:60混匀，置于室温下反应1-2 h。
若无特殊说明，本发明中所指的室温是指25℃以上的室温条件。
在上述的藻红蛋白免疫荧光探针制备方法中，所述的巯基封闭剂包括N-乙基马来酰亚胺和碘乙酰胺(IAAm)中的至少一种。
作为优选，所述的巯基封闭剂为N-乙基马来酰亚胺（NEM）。NEM在中性条件下可以温和地封闭游离巯基。
作为进一步优选，所述的封闭处理包括：将目标蛋白与N-乙基马来酰亚胺按摩尔比1:50混匀，置于室温下反应1.5 h。
作为优选，封闭处理完成后还需对封闭处理反应液作脱盐处理，以去除封闭处理反应液中残留的未反应的巯基封闭剂，确保最终获得的巯基封闭目标蛋白具有较高的纯度。
在上述的藻红蛋白免疫荧光探针制备方法的步骤（2）中，所述的活化处理包括：将步骤（1）获得的巯基封闭目标蛋白与胺-巯基交联剂以摩尔比为1:10-1:100混匀，置于室温下反应1-2 h。当目标蛋白上的游离巯基被封闭后，胺-巯基交联剂只能连接到目标蛋白的氨基上。
作为优选，所述的活化处理包括：将步骤（1）获得的巯基封闭目标蛋白与胺-巯基交联剂以摩尔比为1:40-1:60混匀，置于室温下反应1-2 h。
在上述的藻红蛋白免疫荧光探针制备方法中，所述的胺-巯基交联剂包括4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺酯（SMCC）、磺基琥珀酰亚胺基-4-（N-马来酰亚胺甲基）环己烷-1-羧酸酯（Sulfo-SMCC）、3-(2-吡啶二巯基)丙酸N-羟基琥珀酰亚胺酯（SPDP）或N-琥珀酰亚胺基 6-(3-马来酰亚胺基丙酰胺基)己酸酯（SMPH）。
作为优选，所述的活化处理包括：将步骤（1）获得的巯基封闭目标蛋白与SMCC以摩尔比为1:20混匀，置于室温下反应1.5 h。
同样地，作为优选，活化处理后也需对活化反应液作脱盐处理，以去除活化反应液中残留的未反应的胺-巯基交联剂。
在上述的藻红蛋白免疫荧光探针制备方法的步骤（3）中，所述的巯基化处理包括：将藻红蛋白与2-亚氨基硫烷盐酸盐（traut’s regent）按摩尔比1:10-1:50混匀后，置于室温下避光反应1-2 h。
作为优选，所述的巯基化处理包括：将藻红蛋白与2-亚氨基硫烷盐酸盐按摩尔比1:10-1:20混匀后，置于室温下避光反应1.5 h。
作为进一步优选，所述的巯基化处理包括：将藻红蛋白与2-亚氨基硫烷盐酸盐按摩尔比1:10混匀后，置于室温下避光反应1.5 h。
同样地，作为优选，巯基化处理前，需要对藻红蛋白作脱盐处理，防止藻红蛋白中含有的游离氨基干扰巯基化反应；而巯基化处理后也需对反应液作脱盐处理，以去除活化反应液中残留的未反应的2-亚氨基硫烷盐酸盐。
在上述的藻红蛋白免疫荧光探针制备方法的步骤（4）中，将步骤（3）获得的巯基化藻红蛋白与步骤（2）获得的活化目标蛋白按摩尔比1:1-30:1混匀后，置于室温下避光反应2-4 h。
作为优选，步骤（4）中，将步骤（3）获得的巯基化藻红蛋白与步骤（2）获得的活化目标蛋白按摩尔比1:1-10:1混匀后，置于室温下避光反应2-4 h。
作为进一步优选，步骤（4）中，将步骤（3）获得的巯基化藻红蛋白与步骤（2）获得的活化目标蛋白按摩尔比5:1混匀后，置于室温下避光反应3 h。
 有益效果
与现有技术相比，本发明的有益效果体现在：
本发明的藻红蛋白免疫荧光探针制备方法中，在采用常规的方法步骤将藻红蛋白标记、交联到目标蛋白上前，先对目标蛋白或藻红蛋白上的游离巯基作封闭处理，在目标蛋白或藻红蛋白上的游离巯基被封闭后，后续的胺-巯基交联剂只能与被封闭处理目标蛋白或被封闭处理藻红蛋白上的氨基反应，而不会与相应蛋白上的巯基反应，如此能够确保胺-巯基交联剂的氨基全部用于偶联被巯基化的藻红蛋白或目标蛋白，从而有效提高藻红蛋白与目标蛋白的交联效率，提高藻红蛋白对目标蛋白的标记效率，而获得的藻红蛋白免疫荧光探针在进行免疫检测时，也能获得较强阳性信号，大大提高信噪比。
 附图说明
图1为实施例1制备的藻红蛋白免疫荧光探针NEM-CD62L-PE与CD3-ifluor488共染小鼠脾细胞的流式检测散点图；
图2为对比例1制备的藻红蛋白免疫荧光探针CD62L-PE与CD3-ifluor488共染小鼠脾细胞的流式检测散点图；
图3为被实施例1制备的藻红蛋白免疫荧光探针NEM-CD62L-PE染色的小鼠脾细胞的藻红蛋白荧光信号强度检测结果直方图；
图4为被对比例1制备的藻红蛋白免疫荧光探针CD62L-PE染色的小鼠脾细胞的藻红蛋白荧光信号强度检测结果直方图；
图5为图3和图4的叠置比对图；
图6为实施例2制备的藻红蛋白免疫荧光探针NEM-CD4-PE与CD3-ifluor488共染小鼠脾细胞的流式检测散点图；
图7为对比例2制备的藻红蛋白免疫荧光探针CD4-PE与CD3-ifluor488共染小鼠脾细胞的流式检测散点图；
图8为被实施例2制备的藻红蛋白免疫荧光探针NEM-CD4-PE染色的小鼠脾细胞的藻红蛋白荧光信号强度检测结果直方图；
图9为被对比例2制备的藻红蛋白免疫荧光探针CD4-PE染色的小鼠脾细胞的藻红蛋白荧光信号强度检测结果直方图；
图10为图8和图9的叠置比对图；
图11为实施例3制备的藻红蛋白免疫荧光探针NEM-CD8-PE与CD19-FITC共染小鼠脾细胞的流式检测散点图；
图12为对比例3制备的藻红蛋白免疫荧光探针CD8-PE与CD19-FITC共染小鼠脾细胞的流式检测散点图；
图13为被实施例3制备的藻红蛋白免疫荧光探针NEM-CD8-PE染色的小鼠脾细胞的藻红蛋白荧光信号强度检测结果图；
图14为被对比例3制备的藻红蛋白免疫荧光探针CD8-PE染色的小鼠脾细胞的藻红蛋白荧光信号强度检测结果图；
图15为图13和图14的叠置比对图。
 本发明的最佳实施方式
下面结合附图和具体实施方式对本发明的技术方案做进一步详细说明。
 
实施例1
本实施例以抗小鼠CD62L单克隆抗体[MEL-14]（以下简称CD62L抗体）作为目标蛋白，介绍一种藻红蛋白免疫荧光探针制备方法，该制备方法包括以下步骤：
（1）对目标蛋白上的游离巯基作封闭处理；
具体包括：
（1.1）将50mM的NEM从低温状态下拿出后，先放置于室温环境中，待瓶子温度平衡至室温时再将瓶盖打开，以免瓶子内出现冷凝水；
（1.2）向200 μg CD62L抗体中加入1.4 μl  50mM NEM溶液（n(Ab)：n(NEM)≈1：50），混匀后，置于25℃以上室温反应1.5 h；
在本步骤中，NEM分子与CD62L抗体分子中的游离巯基结合，封闭CD62L抗体分子中的游离巯基；
（1.3）将步骤（1.2）获得的反应液移至超滤离心管内，再加入500μl  PBS（含有0.25mM EDTA）缓冲液，12000 g离心5 min并除去滤液，再加入500μl  PBS（含有0.25mM EDTA）缓冲液混匀离心，重复此操作5次；
本步骤通过脱盐处理以尽可能地除去反应液中未参与反应的NEM残余，确保脱盐后获得的反应液中，NEM残余量至少为抗体总量的10-3倍以上；
（1.4）收集脱盐后的巯基封闭抗体溶液，将巯基封闭抗体溶液体积定容为40 μl （5mg/ml）；
（2）采用胺-巯基交联剂对步骤（1）获得的巯基封闭目标蛋白作活化处理；
具体包括：
（2.1）将SMCC从低温状态下拿出后放置于室温环境中，待瓶子温度平衡至室温时再将瓶盖打开，以免瓶子内出现冷凝水；
（2.2）称取适量SMCC溶解于适量DMSO内，配置成浓度为10mg/ml的母液；将母液按5 μl /管分装，保存于-20℃下；每次使用时新融化一支，不要反复冻融使用，使用后剩余试剂请丢弃；
（2.3）向每40μl （5mg/ml）巯基封闭抗体溶液中加入0.8 μl（10mg/ml） SMCC溶液（n(Ab)：n(SMCC)≈1：20），置于25℃以上室温反应1.5 h；
本步骤中，SMCC分子中的琥珀酰酯与巯基封闭抗体分子中的伯氨基反应结合；
（2.4）将步骤（2.3）获得的反应液移至超滤离心管内，再加入500μl  PBS（含有0.25mM EDTA）缓冲液， 12000 g离心5 min并除去滤液，再加入500μl  PBS（含有0.25mM EDTA）缓冲液混匀离心，重复此操作5次；
本步骤通过脱盐处理以尽可能地除去反应液中未参与反应的SMCC残余，确保脱盐后获得的反应液中，SMCC残余量至少为抗体总量的10-3倍以上；
（2.5）收集脱盐后的活化抗体溶液，将活化抗体溶液体积定容为80 μl （2mg/ml）；
（3）对藻红蛋白作巯基化处理；
具体包括：
（3.1）藻红蛋白悬浊液预处理；
包括：
（a）将试剂管中的藻红蛋白悬浊液（浓度为5mg/ml）用涡旋振荡器震荡均匀，而后取出0.5g（约100μl）置于1.5ml离心管内，在12000g下离心5 min，并用移液枪完全吸走上清液，注意不要吸走藻红蛋白沉淀；
（b）取100μl PBS（含有0.25mM EDTA）缓冲液，加入离心管内充分溶解离心管底部的藻红蛋白沉淀，在12000g下离心5 min，吸取藻红蛋白上清液；
（c）将藻红蛋白上清液置于50kd超滤离心管内，再加入400μl PBS（含有0.25mM EDTA）缓冲液混匀，在12000g下离心5 min，除去滤出液；再加入500μl PBS（含有0.25mM EDTA）缓冲液混匀离心，重复此操作3次后，收集脱盐后的藻红蛋白溶液定容到50μl，得到浓度为10mg/ml 的藻红蛋白溶液；
（3.2）称取适量traut’s regent，溶解于适量去离子水中，配置成2 mg/ml（14mM）母液；将母液按5 μl/管分装并保存于-20℃下，每次使用时新融化一支，不要反复冻融使用，使用后的剩余试剂请丢弃
（3.3）向每50μl 藻红蛋白溶液（10mg/ml）中加入2.3 μl的traut’s溶液（n(PE)：n(traut’s)≈1：10），混匀后置于25℃以上室温下避光反应1.5 h；
（3.4）反应完成后，将反应液移至超滤离心管内，再加入500μl PBS（含有0.25mM EDTA）缓冲液，于5 min、12000 g下离心并除去滤液，再加入500μl PBS（含有0.25mM EDTA）缓冲液混匀离心；重复此操作5次；
（3.5）收集脱盐后的巯基化藻红蛋白溶液，检测藻红蛋白浓度，将巯基化藻红蛋白溶液体积定容至40μl（浓度为10mg/ml）；
（4）将步骤（3）获得的巯基化藻红蛋白与步骤（2）获得的活化目标蛋白交联，获得本实施例的藻红蛋白免疫荧光探针NEM-CD62L-PE；
具体包括：
（4.1）取步骤（2）获得的活化抗体溶液5μl （2mg/ml）与步骤（3）获得的巯基化藻红蛋白溶液5μl （10mg/ml）混匀（n(Ab)：n(PE)=1：5），而后置于25℃以上室温下避光反应3h；
（4.2）反应完成后，向反应体系内加入30μl PBS（含有0.25mM EDTA）缓冲液，使经藻红蛋白标记的CD62L抗体（即NEM-CD62L-PE）终浓度为0.25mg/ml，操作完成后将反应液放置于4℃长期保存。
 本发明的实施方式
实施例2
以抗小鼠CD4单克隆抗体[GK1.5]为目标蛋白，采用与实施例1相同的方法，制备藻红蛋白免疫荧光探针NEM-CD4-PE。
 
实施例3
以抗小鼠CD8单克隆抗体[53-6.7]为目标蛋白，采用与实施例1相同的方法，制备藻红蛋白免疫荧光探针NEM-CD8-PE。
 
对比例1
本对比例以抗小鼠CD62L单克隆抗体[MEL-14]作为目标蛋白，介绍一种藻红蛋白免疫荧光探针标记方法，该标记方法包括以下步骤：
（1）采用胺-巯基交联剂活化CD62L抗体；
具体包括：
（1-1）将SMCC从低温状态下拿出后放置于室温环境中，待瓶子温度平衡至室温时再将瓶盖打开，以免瓶子内出现冷凝水；
（1-2）称取适量SMCC溶解于适量DMSO内，配置成10mg/ml母液；母液按5 μl/管分装，并保存于-20℃下，每次使用时新融化一支，不要反复冻融使用，使用后剩余的试剂请丢弃。
（1-3）配置浓度为4mg/mL的CD62L抗体溶液，向每50μl CD62L抗体溶液中加入1.6μl 10mg/ml SMCC溶液，混匀后，置于25℃以上室温下反应1.5 h，使SMCC分子中琥珀酰酯与CD62L分子中伯氨基反应结合；
（1-4）反应完成后，将反应液移至超滤离心管内，再加入500μl PBS（含有0.25mM EDTA）缓冲液，于5 min、12000 g下离心并除去滤液，再加入500μl PBS（含有0.25mM EDTA）缓冲液混匀离心；重复此操作1-5次，使反应液中未反应SMCC的参与量降至至少为抗体总量的10-3倍以上，尽可能较多稀释；
（1-5）收集脱盐后的高浓度活化抗体蛋白溶液，测量CD62L抗体浓度，将活化CD62L抗体溶液的体积进行定容，将其浓度调整至2mg/ml。
（2）对藻红蛋白作巯基化处理，获得巯基化的藻红蛋白；
具体包括：
（2-1）对藻红蛋白作脱盐处理；
具体为：
（2-1-1）将试剂管中的藻红蛋白悬浊液（浓度为5mg/ml）用涡旋振荡器震荡均匀，而后取出0.5g（约100μl）置于1.5mL离心管内，在12000g下离心5 min，并用移液枪完全吸走上清液，注意不要吸走藻红蛋白沉淀；
（2-1-2）取100μl PBS（含有0.25mM EDTA）缓冲液，加入离心管内充分溶解离心管底部的藻红蛋白沉淀，在12000g下离心5 min，吸取藻红蛋白上清液；
（2-1-3）将藻红蛋白上清液置于50kd超滤离心管内，再加入400μl PBS（含有0.25mM EDTA）缓冲液混匀，在12000g下离心5 min，除去滤出液；再加入500μl PBS（含有0.25mM EDTA）缓冲液混匀离心，重复此操作5次后，收集脱盐后的藻红蛋白溶液定容到50μl，得到浓度为10mg/mL 的藻红蛋白溶液。
（2-2）对脱盐后的藻红蛋白作巯基化处理；
具体为：
（2-2-1）称取适量traut’s regent，溶解于适量去离子水中，配置成2 mg/ml（14mM）母液；将母液按5 μl/管分装并保存于-20℃下，每次使用时新融化一支，不要反复冻融使用，使用后的剩余试剂请丢弃。
（2-2-2）向每50μl 藻红蛋白溶液（10mg/mL）中加入4.6 μl的traut’s溶液，混匀后置于25℃以上室温下，避光反应1.5 h；
（2-2-3）反应完成后，将反应液移至超滤离心管内，再加入500μl PBS（含有0.25mM EDTA）缓冲液，于5 min、12000 g下离心并除去滤液，再加入500μl PBS（含有0.25mM EDTA）缓冲液混匀离心；重复此操作5次；
（2-2-4）收集脱盐后的巯基化藻红蛋白溶液，检测藻红蛋白浓度，巯基化藻红蛋白溶液体积定容，使其浓度为10mg/ml。
（3）将巯基化的藻红蛋白与活化后的目标蛋白交联，获得本对比例的藻红蛋白免疫荧光探针CD62L-PE；
具体包括：
将步骤（1）获得的活化CD62L抗体溶液与步骤（2）获得的巯基化藻红蛋白溶液按照摩尔比1:3混匀，即在5μl（2mg/ml）活化CD62L抗体溶液中加入3μl（10 mg/ml）巯基化藻红蛋白溶液，而后置于25℃以上室温下避光反应3h，反应完成后向反应体系内加入28μl PBS（含有0.25mM EDTA）缓冲液，使经藻红蛋白标记的CD62L抗体（即CD62L-PE）终浓度为0.25mg/ml，操作完成后将反应液放置于4℃长期保存。
 
对比例2
以抗小鼠CD4单克隆抗体[GK1.5]为目标蛋白，采用与对比例1相同的方法制备藻红蛋白免疫荧光探针CD4-PE。
 
对比例3
以抗小鼠CD8单克隆抗体[53-6.7]为目标蛋白，采用与对比例1相同的方法，制备藻红蛋白免疫荧光探针CD8-PE。
 
取藻红蛋白免疫荧光探针NEM-CD62L-PE、CD62L-PE，按相同的浓度分别与CD3-iFluor488对1million小鼠脾细胞进行共染（每个反应体系中加入0.25μg标记的iFluor标记抗体与0.125μg PE标记抗体），染色15 min后通过流式细胞仪检测荧光信号，重复检测至少三组，每组至少两个样品；检测结果如图1、图2、图3、图4和图5所示。
如图1和图2所示，与CD62L-PE相比，被NEM-CD62L-PE染色的小鼠脾细胞在R6区域具有更强的阳性荧光信号（28845 vs 20178）。如图3、图4和图5所示，与CD62L-PE相比，被NEM-CD62L-PE染色的小鼠脾细胞具有更高的PE荧光信号值，这与图1和图2的检测结果是相一致的。
 
同样地，取藻红蛋白免疫荧光探针NEM-CD4-PE、CD4-PE按相同的浓度分别与CD3-iFluor488对1million小鼠脾细胞进行共染（每个反应体系中加入0.25μg标记的iFluor标记抗体与0.125μg PE标记抗体），染色15 min后通过流式细胞仪检测荧光信号，重复检测至少三组，每组至少两个样品；检测结果如图6、图7、图8、图9和图10所示。
如图6和图7所示，与CD4-PE相比，被NEM-CD4-PE染色的小鼠脾细胞在Q2（图右上角）区域具有更强的阳性荧光信号（53560 vs 40325）。
如图8、图9和图10所示，与CD4-PE相比，被NEM-CD4-PE染色的小鼠脾细胞具有更高的PE荧光信号值，这与图6和图7的检测结果是相一致的。
 
同样地，取藻红蛋白免疫荧光探针NEM-CD8-PE、CD8-PE按相同的浓度分别与CD19-FITC对1million小鼠脾细胞进行共染（每个反应体系中加入0.25μg标记的FITC标记抗体与0.125μg PE标记抗体），染色15 min后通过流式细胞仪检测荧光信号，重复检测至少三组，每组至少两个样品；检测结果如图11、图12、图13、图14和图15所示。
如图11和图12所示，与CD8-PE相比，被NEM-CD8-PE染色的小鼠脾细胞在Q4（图左下角）区域具有更强的阳性荧光信号（19417 vs 14075）。
如图13、图14和图15所示，与CD8-PE相比，被NEM-CD8-PE染色的小鼠脾细胞具有更高的PE荧光信号值，这与图11和图12的检测结果是相一致的。
 工业实用性
本发明公开了一种藻红蛋白免疫荧光探针制备方法，该方法包括将藻红蛋白标记到目标蛋白上的步骤；在所述的将藻红蛋白标记到目标蛋白上的步骤前，该方法还包括对目标蛋白或藻红蛋白上的游离巯基作封闭处理的步骤。本发明在采用常规的方法步骤将藻红蛋白标记、交联到目标蛋白上前，先对目标蛋白或藻红蛋白上的游离巯基作封闭处理，当游离巯基被封闭后，胺-巯基交联剂只能与被封闭处理目标蛋白或藻红蛋白上的氨基反应，而不会与相应蛋白上的巯基反应，如此能够确保胺-巯基交联剂的氨基全部用于偶联被巯基化的藻红蛋白或目标蛋白，从而有效提高藻红蛋白对目标蛋白的标记效率，获得的藻红蛋白免疫荧光探针在进行免疫检测时具有较高的信噪比。

Claims (10)

  1. 一种藻红蛋白免疫荧光探针制备方法，包括将藻红蛋白标记到目标蛋白上的步骤；其特征在于，在所述的将藻红蛋白标记到目标蛋白上的步骤前，所述的藻红蛋白免疫荧光探针制备方法还包括对目标蛋白或藻红蛋白上的游离巯基作封闭处理的步骤。
  2. 在此处键入权利要求项2如权利要求1所述的藻红蛋白免疫荧光探针制备方法，其特征在于，包括以下步骤：
    （1）对目标蛋白上的游离巯基作封闭处理；
    （2）采用胺-巯基交联剂对步骤（1）获得的巯基封闭目标蛋白作活化处理；
    （3）对藻红蛋白作巯基化处理；
    （4）将步骤（3）获得的巯基化藻红蛋白与步骤（2）获得的活化目标蛋白交联，获得所述的藻红蛋白免疫荧光探针。
  3. 如权利要求2所述的藻红蛋白免疫荧光探针制备方法，其特征在于，步骤（1）中，采用巯基封闭剂对目标蛋白作封闭处理。
  4. 如权利要求3所述的藻红蛋白免疫荧光探针制备方法，其特征在于，所述的封闭处理包括：将目标蛋白与巯基封闭剂按摩尔比1:10-1:100混匀，置于室温下反应1-2 h。
  5. 如权利要求3所述的藻红蛋白免疫荧光探针制备方法，其特征在于，所述的巯基封闭剂包括N-乙基马来酰亚胺和碘乙酰胺中的至少一种。
  6. 如权利要求5所述的藻红蛋白免疫荧光探针制备方法，其特征在于，所述的巯基封闭剂为N-乙基马来酰亚胺。
  7. 如权利要求1-6中任意一项所述的藻红蛋白免疫荧光探针制备方法，其特征在于，步骤（2）中，所述的活化处理包括：将步骤（1）获得的巯基封闭目标蛋白与胺-巯基交联剂以摩尔比为1:10-1:100混匀，置于室温下反应1-2 h。
  8. 如权利要求2所述的藻红蛋白免疫荧光探针制备方法，其特征在于，所述的胺-巯基交联剂包括4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺酯、磺基琥珀酰亚胺基-4-（N-马来酰亚胺甲基）环己烷-1-羧酸酯、3-(2-吡啶二巯基)丙酸N-羟基琥珀酰亚胺酯或N-琥珀酰亚胺基 6-(3-马来酰亚胺基丙酰胺基)己酸酯。
  9. 如权利要求1-6中任意一项所述的藻红蛋白免疫荧光探针制备方法，其特征在于，步骤（3）中，所述的巯基化处理包括：将藻红蛋白与2-亚氨基硫烷盐酸盐按摩尔比1:10-1:50混匀后，置于室温下避光反应1-2 h。
  10. 如权利要求1-6中任意一项所述的藻红蛋白免疫荧光探针制备方法，其特征在于，步骤（4）中，将步骤（3）获得的巯基化藻红蛋白与步骤（2）获得的活化目标蛋白按摩尔比1:1-30:1混匀后，置于室温下避光反应2-4 h。
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