CN112098640B - Fluorescent protein and/or coupled protein monoclonal antibody marking method and kit thereof - Google Patents

Fluorescent protein and/or coupled protein monoclonal antibody marking method and kit thereof Download PDF

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CN112098640B
CN112098640B CN202010972671.XA CN202010972671A CN112098640B CN 112098640 B CN112098640 B CN 112098640B CN 202010972671 A CN202010972671 A CN 202010972671A CN 112098640 B CN112098640 B CN 112098640B
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protein
monoclonal antibody
fluorescent protein
reaction
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CN112098640A (en
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张洋
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Zhejiang Zhengxi Biotechnology Co ltd
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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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • 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
    • 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
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • 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
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/7051—T-cell receptor (TcR)-CD3 complex
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70514—CD4
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70517—CD8

Abstract

The invention provides a fluorescent protein and/or conjugated protein monoclonal antibody labeling method and a kit thereof. Firstly, desalting fluorescent protein and/or coupled protein, adopting NEM to seal sulfydryl of the fluorescent protein and/or coupled protein, and carrying out cross-linking reaction on the fluorescent protein and/or coupled protein-NEM and S-SMCC; simultaneously, S-LC-SPDP and the monoclonal antibody are adopted for crosslinking, TCEP reduces the disulfide bond of the monoclonal antibody, and the monoclonal antibody is sulfhydrylated; finally, the thiolated antibody is cross-linked with NEM-fluorescent protein and/or conjugated protein-S-SMCC. The invention realizes the directional coupling of the fluorescent protein and/or the coupled protein and the monoclonal antibody, improves the cross-linking efficiency of the fluorescent protein and/or the coupled protein and the monoclonal antibody label, further improves the specificity of the fluorescent protein and/or the coupled protein and the monoclonal antibody label, and enhances the fluorescent signal.

Description

Fluorescent protein and/or coupled protein monoclonal antibody marking method and kit thereof
Technical Field
The invention relates to the technical field of immunology, in particular to a fluorescent protein and/or coupled protein monoclonal antibody labeling method and a kit thereof.
Background
The monoclonal antibody is an antibody which is generated by secreting and aims at a single antigenic determinant under the condition of in vitro culture by mutually fusing a mouse spleen immune cell and a mouse myeloma cell to form a hybridoma cell by adopting a cell fusion technology. The B lymphocyte can differentiate and proliferate to form specific antibody to the antigen under the stimulation of the antigen. Non-secretory myeloma cells that grow and propagate in vitro can be fused with the B lymphocytes to form hybridoma cells, which are further cloned to have both the ability to produce B lymphocytes with specific antibodies and the ability to indefinitely grow myeloma cells in vitro, and a large amount of high titer, single specific antibodies can be obtained by culturing or injecting the cloned hybridoma cells into animals. This technique is known as monoclonal antibody technique. The monoclonal antibody has the same specificity, and the Ig classes and the subclasses of the monoclonal antibody are the same, because the monoclonal antibody is generated by a cell line formed by the proliferation of a single B lymphocyte (namely, a hybridoma cell with the same gene), the monoclonal antibody has the advantages of high specificity, reproducibility, repeatability, detection sensitivity and the like.
The immunofluorescence technique is also called as fluorescent antibody probe technique, the immunofluorescence technique is that firstly the specific antigen is injected into the body of the experimental animal, the animal generates antibody, the antibody immunoglobulin is separated from the serum of the animal, the fluorescent probe dye is used for marking the antibody to prepare fluorescent marked antibody solution, the fluorescent marked antibody and the specific antigen generate antigen-antibody specific reaction, and the distribution condition of the antibody-antigen complex in the tissue can be observed very sensitively by using the fluorescence microscope technique. The immunofluorescence technique has higher specificity and sensitivity.
The monoclonal antibody immunofluorescence technology is a combination of monoclonal antibody technology and immunofluorescence technology, wherein a fluorescent probe dye which does not affect the immunological characteristics of the monoclonal antibody is combined with the monoclonal antibody, and then the detection is carried out by using methods such as fluorescence microscopy, laser scanning confocal microscopy and the like, namely the monoclonal antibody immunofluorescence technology. Because of its unique advantages, it has wide applications in the diagnosis of influenza virus, detection of pathogenic microorganisms, and the like.
Patent CN111308063A provides a method for labeling phycoerythrin immunofluorescence probe, which adopts amine-sulfhydryl cross-linking agent to activate target protein, simultaneously carries out sulfhydrylation treatment on phycoerythrin, and then cross-links the activated target protein and the sulfhydrylated phycoerythrin, thereby obtaining the immunofluorescence probe. Patent CN111044732A provides a kit for detecting CD45 antigen, which can be used for detecting CD45 antigen after activated fluorescent protein is crosslinked with activated antibody. The traditional label usually adopts SMCC cross-linking agent to couple amino and sulfhydryl groups on antibody and fluorescent protein or other coupling protein, and has the disadvantages that SMCC generates cross-linking products between antibody and antibody, coupling protein/fluorescent protein, the cross-linking efficiency is low, and the signal of the fluorescent labeling protein is poor. There is also a procedure performed in the prior art (Zhaoyije, et al. fluorescent dye R-phycoerythrin labeled mouse anti-human CD4 monoclonal antibody. microbiological immunological progress [ J ] 2006,34(2): 32-34.): SPDP and SMCC are adopted to respectively activate PE and target protein, then the activated PE is sulfhydrylated, and the sulfhydrylated PE is crosslinked with the activated target protein. The method leads to that the signal-to-noise ratio of the yin-yang signal of the finally obtained target protein PE marker is poor and the background signal is strong when fluorescence detection is carried out.
Therefore, it is highly desirable to develop a labeling method with high specificity, simple operation and higher detection efficiency.
Disclosure of Invention
The terms:
unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
PE (R-Phytoerythrin, R-PE): phycoerythrin;
PERCP (Peridinin-Chlorophyl-Protein Complex): a polymethacrylic flavin-chlorophyll-protein complex;
apc (allophycycanin): allophycocyanin;
nem (maleimide): a maleimide;
Sulfo-SMCC (S-SMCC): 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid sulfosuccinimide ester sodium salt;
pbs (phosphate buffered solution): phosphate buffer;
mcab (monoclonal antibody): a monoclonal antibody;
cys (cysteine): (ii) cysteine;
TCEP (Tris (2-carboxythroughout) phosphine): tris (2-carboxyethyl) phosphine;
Sulfo-LC-SPDP (succinimidyl 6- [3(2-pyridyldithio) propioamido ] hexanoate, S-LC-SPDP): succinimide 6- [3(2-pyridyldithio) propionamido ] hexanoate, a heterobifunctional cross-linking agent water-soluble LC-SPDP;
SPDP (succinimidyl 3- (2-pyridyldithio) propionate): succinimide 3- (2-pyridyldithio) propionate;
LC-SPDP (succinimidyl 6- [3 (2-pyridldithio) propioamido ] hexanoate): succinimide 6- [3(2-pyridyldithio) propionamido ] hexanoate, a water-insoluble crosslinking agent;
GMBS (4-Maleimidobutyric acid N-hydroxysuccinimide ester): 4-maleimidobutanoic acid-N-succinimidyl ester;
MBS (3-Maleimidobenzoic acid N-hydroxysuccinimide ester): 3-maleimidobenzoic acid succinimidyl ester;
PDPH (3- (2-pyridyldithio) propionyl hydrate): 3- (2-dithiopyridyl) propionic acid hydrazide.
Aiming at the defects, the invention realizes the directional coupling between the antibody and the fluorescent protein and/or the coupled protein through the sulfhydryl blocking agent, the cross-linking agent and the disulfide bond reducing agent, improves the cross-linking efficiency of the fluorescent protein and/or the coupled protein and the monoclonal antibody mark, further improves the specificity of the fluorescent protein and/or the coupled protein and the monoclonal antibody mark, and enhances the fluorescent signal.
The technical scheme of the application is as follows:
in one aspect, the present invention provides a method for labeling a fluorescent protein and/or a conjugated protein with a monoclonal antibody, comprising the steps of:
(1) carrying out desalination treatment on the fluorescent protein and/or the coupling protein, and then reacting with a sulfhydryl blocking agent;
(2) reacting the amino group of the sulfhydryl-blocked fluorescent protein and/or coupling protein obtained in the step (1) with a cross-linking agent A;
(3) the method comprises the following steps of carrying out a sulfhydrylation reaction on a monoclonal antibody, wherein the sulfhydrylation reaction of the monoclonal antibody comprises a reaction of the monoclonal antibody and a cross-linking agent B and a reduction reaction of the monoclonal antibody-the cross-linking agent B and a disulfide bond reducing agent;
(4) and (3) performing directional coupling reaction on the sulfhydrylation monoclonal antibody obtained in the step (3) and the cross-linked fluorescent protein and/or the coupled protein obtained in the step (2), and after the reaction is completed, performing NEM, Cys and PBS treatment to obtain the fluorescence labeling antibody.
Specifically, the fluorescent protein and/or coupled protein comprises PERCP, PE, PECy5, PECy5.5, PECy7, PerCPCy5.5, APCCy5.5, APCCy7 or APC and the like.
Specifically, in the step (1), the fluorescent protein and/or conjugated protein is desalted so as to replace the fluorescent protein and/or conjugated protein into the PBS buffer solution containing EDTA, so that the interference of free amino groups contained in the fluorescent protein and/or conjugated protein suspension with the reaction is prevented.
The desalting treatment step comprises:
1) shaking and mixing the fluorescent protein and/or the coupling protein suspension uniformly, centrifuging, and removing the supernatant;
2) adding a PBS buffer solution containing EDTA into the precipitate, centrifuging after fully dissolving, and taking the supernatant;
3) transferring the supernatant into an ultrafiltration tube, and performing ultrafiltration treatment on the supernatant by using PBS (containing 0.25mM EDTA) buffer solution to obtain desalted fluorescent protein and/or coupled protein.
Specifically, the sulfhydryl blocking agent in the step (1) comprises an aminoethylation reagent, iodoacetamide, NEM, BMPA or MMTS.
Further specifically, the thiol blocking agent in step (1) is NEM.
Specifically, the molar ratio of the fluorescent protein and/or the conjugated protein to the sulfhydryl blocking agent in the step (1) is 1:3-1: 1000.
Further specifically, the molar ratio of the fluorescent protein and/or the conjugated protein to the thiol blocking agent in the step (1) is 1: 50.
Specifically, the reaction condition of the fluorescent protein and/or the conjugated protein and the sulfhydryl blocking agent in the step (1) is that the reaction is carried out for 1.5h at 25 ℃.
Specifically, the crosslinking agent A in the step (2) comprises S-SMCC, sulfoSMCC, polyethylene glycol SMCC or LC-SMCC.
More specifically, the crosslinking agent A in the step (2) is S-SMCC.
Specifically, the molar ratio of the sulfhydryl-blocked fluorescent protein and/or coupling protein in the step (2) to the cross-linking agent A is 1:5-1: 100.
Further specifically, the molar ratio of the thiol-blocked fluorescent protein and/or conjugated protein to the cross-linking agent A in the step (2) is 1: 20.
Specifically, the reaction condition of the sulfhydryl blocked fluorescent protein and/or coupling protein and the cross-linking agent A in the step (2) is that the reaction is carried out for 1.5h at 25 ℃.
Specifically, the crosslinking agent B in the step (3) comprises S-LC-SPDP, LC-SPDP, PDPH, MBS or GMBS.
More specifically, the crosslinking agent B in the step (3) is S-LC-SPDP.
Specifically, the molar ratio of the monoclonal antibody to the cross-linking agent B in the step (3) is 1:10-1: 50.
Further specifically, the reaction condition of the monoclonal antibody and the cross-linking agent B in the step (3) is that the monoclonal antibody and the cross-linking agent B are reacted for 1h at 25 ℃.
Specifically, the disulfide bond reducing agent in the step (3) comprises dithiothreitol DTT, beta-mercaptoethanol or TCEP.
Further specifically, the disulfide bond reducing agent described in step (3) is TCEP.
Specifically, the reaction of the disulfide bond reducing agent with the monoclonal antibody-crosslinking agent B in the step (3) is carried out at a final concentration of 10 to 50 mM.
Further specifically, the reaction of the disulfide bond reducing agent with the monoclonal antibody-crosslinking agent B described in the step (3) was carried out at a final concentration of 20 mM.
Further specifically, the reaction condition of the monoclonal antibody-crosslinking agent B and the disulfide bond reducing agent in the step (3) is that the reaction is carried out for 1.5h at 25 ℃.
Specifically, the molar ratio of the thiolated monoclonal antibody obtained in step (3) to the crosslinked fluorescent protein and/or conjugated protein obtained in step (2) is 1:0.5-1: 100.
Specifically, the molar ratio of the thiolated monoclonal antibody obtained in step (3) described in step (4) to the crosslinked fluorescent protein and/or conjugated protein obtained in step (2) is 1: 5.
Specifically, the reaction condition of the thiolated monoclonal antibody obtained in the step (3) and the crosslinked fluorescent protein and/or the conjugated protein obtained in the step (2) is 25 ℃ for 3 h.
Specifically, the NEM treatment step in the step (4) is to add 1/10 volumes of NEM after the reaction of the thiolated monoclonal antibody with the cross-linked fluorescent protein and/or the conjugated protein is completed, and react for 1.5h at 25 ℃.
Specifically, the Cys treatment step described in step (4) is performed after the NEM treatment is completed, 1/10 volumes of Cys solution are added, and the reaction is performed overnight at 4 ℃.
Specifically, in the PBS treatment step described in step (4), after Cys treatment is completed, an appropriate amount of PBS (containing 0.1% sodium azide and 0.1% gelatin) buffer is added to make the final concentration of the antibody 0.25 mg/mL.
In another aspect, the invention provides a kit for labeling a fluorescent protein and/or a conjugated protein with a monoclonal antibody, wherein the kit for labeling the fluorescent protein and/or the conjugated protein with the monoclonal antibody comprises the monoclonal antibody, the fluorescent protein and/or the conjugated protein, a crosslinking agent A, a crosslinking agent B, a sulfhydryl blocking agent, a disulfide bond reducing agent, NEM, Cys, PBS (containing 0.25mM EDTA) buffer solution and PBS (containing 0.1% sodium azide and 0.1% gelatin) buffer solution. Wherein the cross-linking agent A is S-SMCC, sulfoSMCC, PEGylated SMCC or LC-SMCC; the cross-linking agent B is S-LC-SPDP, LC-SPDP, PDPH, MBS or GMBS; the sulfhydryl blocking agent is an aminoethylation reagent, iodoacetamide, NEM, BMPA or MMTS; the disulfide bond reducing agent is dithiothreitol DTT, beta-mercaptoethanol or TCEP.
Compared with the prior art, the invention has the advantages that:
(1) according to the invention, a sulfhydryl blocking agent is adopted to treat fluorescent protein and/or coupling protein to block sulfhydryl groups, a cross-linking agent A only reacts with amino groups of the fluorescent protein and/or coupling protein after the sulfhydryl groups are blocked, the prepared fluorescent protein-cross-linking agent A and/or coupling protein-cross-linking agent A directionally reacts with sulfhydryl groups of a monoclonal antibody, the directional coupling of the monoclonal antibody and the fluorescent protein and/or coupling protein is realized, and the reaction specificity of the fluorescent protein and/or coupling protein and the monoclonal antibody is improved.
(2) The monoclonal antibody prepared by the steps not only has sulfydryl formed by reducing the disulfide bond, but also comprises sulfydryl formed by combining and converting the amino of the monoclonal antibody and the crosslinking agent B. Therefore, the number of the fluorescent protein and/or the conjugated protein bound on a single monoclonal antibody is obviously improved, and the intensity of a fluorescent signal is obviously enhanced.
Drawings
FIG. 1 is a scattergram of flow assay of splenocytes from CD4-PE [ GK1.5] and CD8-FITC [53-6.72] co-infected mice (C57) labeled by the monoclonal antibody labeling method of the present invention.
FIG. 2 is a scattergram for flow detection of splenocytes from CD4-PE [ GK1.5] and CD8-FITC [53-6.72] co-infected mice (C57) labeled by the monoclonal antibody labeling method of the present invention (the monoclonal antibody is not cross-linked with Sulfo-LC-SPDP).
FIG. 3 is a dot-blot assay of splenocytes from CD4-PE [ GK1.5] and CD8-FITC [53-6.72] co-infected mice (C57) labeled with a Kit (Saimer fly, Invitrogen, SiteClick R-PE Antibody Labeling Kit).
FIG. 4 is a diagram showing the peaks detected by flow of splenocytes of a mouse (C57) co-infected with CD4-PE [ GK1.5] and CD8-FITC [53-6.72] labeled by the monoclonal antibody labeling method of the present invention.
FIG. 5 is a diagram showing flow detection peaks of splenocytes of a mouse (C57) co-infected with CD4-PE [ GK1.5] and CD8-FITC [53-6.72] labeled by the monoclonal antibody labeling method of the present invention (the monoclonal antibody is not cross-linked with Sulfo-LC-SPDP).
FIG. 6 is a graph showing flow-detected peaks of splenocytes from mice (C57) co-infected with CD4-PE [ GK1.5] and CD8-FITC [53-6.72] labeled with a Kit (Saimerfei, Invitrogen, SiteClick R-PE Antibody Labeling Kit).
FIG. 7 is a dot-blot diagram for flow assay of splenocytes from CD4-PERCP [ GK1.5] and CD3-FITC [145-2C11] co-infected mice (C57) labeled by the monoclonal antibody labeling method of the present invention.
FIG. 8 is a scattergram for flow assay of splenocytes from mice (C57) co-infected with CD4-PERCP [ GK1.5] and CD3-FITC [145-2C11] labeled by the monoclonal antibody labeling method of the present invention (monoclonal antibody not cross-linked with Sulfo-LC-SPDP).
FIG. 9 is a scattergram for flow assay of splenocytes from CD4-PERCP [ GK1.5] and CD3-FITC [145-2C11] co-infected mice (C57) labeled with a Kit (Abcam, PerCP Conjugation Kit-Lightning Kit).
FIG. 10 is a diagram showing flow detection peaks of splenocytes from a mouse (C57) co-infected with CD4-PERCP [ GK1.5] and CD3-FITC [145-2C11] labeled by the monoclonal antibody labeling method of the present invention.
FIG. 11 is a diagram showing flow-detected peaks of splenocytes of a mouse (C57) co-infected with CD4-PERCP [ GK1.5] and CD3-FITC [145-2C11] labeled by the monoclonal antibody labeling method of the present invention (the monoclonal antibody is not cross-linked with Sulfo-LC-SPDP).
FIG. 12 is a diagram showing flow-detected peaks of splenocytes obtained from CD4-PERCP [ GK1.5] and CD3-FITC [145-2C11] co-infected mice (C57) labeled with a Kit (Abcam, PerCP Conjugation Kit-Lighting Kit).
Detailed Description
The present invention will be further illustrated in detail with reference to the following specific examples, which are not intended to limit the present invention but are merely illustrative thereof. The experimental methods used in the following examples are not specifically described, and the materials, reagents and the like used in the following examples are generally commercially available under the usual conditions without specific descriptions.
EXAMPLE 1 preparation of CD4[ GK1.5] -PE (Using the monoclonal antibody labeling method described herein)
Pretreatment of PE suspension
(1) And shaking and uniformly mixing the PE turbid liquid in the reagent tube by using a vortex oscillator.
(2) 0.5mg PE precipitate suspension was placed in a 1.5mL centrifuge tube, centrifuged at 12000g for 5min, and the supernatant was completely aspirated off with a pipette, taking care not to aspirate the precipitate.
(3) Add 100. mu.L of PBS (containing 0.25mM EDTA) buffer to the centrifuge tube to dissolve the PE pellet at the bottom of the centrifuge tube, centrifuge at 12000g for 5min, and absorb the PE supernatant.
(4) A50 Kd ultrafiltration tube was selected, the PE supernatant solution was transferred to the tube, 400. mu.L of PBS (containing 0.25mM EDTA) buffer was added and mixed, 12000g was centrifuged for 5min and the filtrate was removed, 500. mu.L of PBS (containing 0.25mM EDTA) buffer was added and mixed and centrifuged, and this operation was repeated 3 times.
(5) The PE solution after desalting was collected and made to a constant volume of 50. mu.L to obtain a 10mg/mL PE solution.
Capping of the thiol groups of PE
(1) Taking out 50mM NEM from a low-temperature state, placing the NEM in a room-temperature environment, and opening a bottle cap when the temperature of the bottle is balanced to room temperature so as to prevent condensed water in the bottle.
(2) To 50. mu.L PE (10mg/mL) was added 3.5. mu.L of 50mM NEM solution (n)PE:nNEM1:50) and reacted at 25 ℃ for 1.5h to allow binding of the NEM molecule to the free thiol group in the PE molecule and blocking of the free thiol group in the PE molecule.
Crosslinking reaction of PE-NEM with S-SMCC
(1) Taking out S-SMCC (with the solubility of 10mM and mother solution dissolved in sterile pure water) from a low-temperature state, placing the bottle in a room-temperature environment, and opening a bottle cover when the temperature of the bottle is balanced to room temperature so as to prevent condensed water from appearing in the bottle.
(2) 50mg of S-SMCC is dissolved in 11.46mL of sterile water to prepare 10mM mother liquor (the mother liquor is 100 muL/tube and is stored in-20 ℃), one reagent is newly thawed each time when the reagent is used, repeated freezing and thawing are not needed for use, and the residual reagent is discarded after the reagent is used.
(3) To 50. mu.L of the solution of 10mg/mL S-SMCC (n) was added 6.69. mu.L of 10mg/mL S-SMCC per 50. mu.L of the E-NEM (10mg/mL) solutionPE-NEM:nS-SMCC1:20) and reacting at 25 ℃ for 1.5h to combine the succinyl ester in the S-SMCC molecule with the primary amino group in the PE molecule.
(4) The solution after completion of the reaction was transferred to an ultrafiltration centrifuge tube, 500. mu.L of PBS (containing 0.25mM EDTA) buffer was added thereto, 12000g was centrifuged for 5min and the filtrate was removed,adding 500 μ L PBS (containing 0.25mM EDTA) buffer, mixing, centrifuging, repeating the above operation 5 times, desalting to obtain 10% unreacted S-SMCC residue in protein-3The dilution was as large as possible.
(5) The desalted high concentration crosslinked protein solution is collected, and the volume of the collected solution is measured by a pipette to 40. mu.L (10 mg/mL).
4. Monoclonal antibody (McAb) sulfhydrylation reaction
(1) 2mg of Sulfo-LC-SPDP was dissolved in 200. mu.L of deionized water to prepare a 20mM solution. Mu.g McAb (2mg/mL) was added with 1.5. mu. L S-LC-SPDP, reacted at 25 ℃ for 1h, the solution after completion of the reaction was transferred to an ultrafiltration centrifuge tube, 500. mu.L of LPBS (containing 0.25mM EDTA) buffer was added, centrifugation was performed for 5min at 12000g and the filtrate was removed, 500. mu.L of PBS (containing 0.25mM EDTA) buffer was added, and the operation was repeated 5 times by mixing and centrifugation. The volume of the antibody was made 50. mu.L (2 mg/mL).
(2) An appropriate amount of TCEP was weighed, 1g TCEP was dissolved in 6mL deionized water to dissolve TCEP, pH2.5, pH was adjusted to 7.0 with 10N NaOH, volume was adjusted to 7mL to obtain 0.5M TCEP (stock solution 100. mu.L/tube stored at-20 ℃ C.). When the reagent is used, one reagent is melted again every time, repeated freeze thawing is not needed, and the residual reagent is discarded after use.
(3) To 50. mu.L of the antibody, 2. mu.L of TCEP was added to a final concentration of about 20mM, and the reaction was carried out at 25 ℃ for 1.5 hours in the absence of light.
(4) The solution after completion of the reaction was transferred to an ultrafiltration centrifuge tube, and 500. mu.L of PBS (containing 0.25mM EDTA) buffer was added, and then, 12000g was centrifuged for 5min to remove the filtrate, and 500. mu.L of PBS (containing 0.25mM EDTA) buffer was added, and the mixture was centrifuged by mixing and repeating the operation 5 times.
(5) Collecting the desalted high-concentration sulfhydrylation monoclonal antibody solution, measuring the volume of the collected solution by using a pipette, estimating the antibody concentration by using 80% protein recovery rate, and fixing the volume of the antibody protein solution to 40 mu L (2mg/mL) to obtain the McAb-SH intermediate.
Crosslinking reaction of PE with antibody
The PE cross-linked protein and the sulfhydrylation monoclonal antibody solution obtained in the step 3 and the step 4 are mixed according to the molar ratio nMcAb-SH:nNEM-PE-S-SMCCThe mixture was mixed at a ratio of 1:5, 40. mu.L (2mg/mL) of the thiolated monoclonal antibody was added to 40. mu.L (10mg/mL) of S-SMCC-crosslinked NEM-PE, the mixture was reacted at 25 ℃ for 3 hours in the dark, 1/10 volumes (8. mu.L) of NEM (50mM) were added to the reaction system, the reaction was carried out at 25 ℃ for 1.5 hours, 8. mu.L of 1M Cys solution was added after the reaction, the mixture was mixed well, and the mixture was left to stand at 4 ℃ for overnight reaction. After the reaction is finished, adding a proper amount of PBS (containing 0.1% of sodium azide and 0.1% of gelatin) solution into the reaction system, fixing the volume to 320 mu L, enabling the final concentration of the antibody to be 0.25mg/mL, and after the operation is finished, placing the antibody at 4 ℃ for long-term storage.
When the flow detection is carried out, the antibody solution after the cross-linking reaction is diluted according to the ratio of 1:100 and then is detected, wherein the detection reaction system is as follows: 100 mu L of flow buffer (PBS + 0.5% FBS + 0.2% EDTA), 2.5 mu g/mL of CD4-PE [ GK1.5] and 5 mu g/mL of CD8-FITC [53-6.72] and 10^6cells of mouse (C57); the reaction conditions are as follows: carrying out water bath at 25 ℃ and light-proof reaction for 15min, washing for 3 times by using 1mL of flow buffer solution, and fixing the volume to 500 mu L; the door setting method comprises the following steps: lymph +.
As shown in FIG. 1, when splenocytes of mice (C57) were co-infected with CD4-PE [ GK1.5] and CD8-FITC [53-6.72] prepared in this example, significant cell clustering appeared, and the negative cell population, CD4-CD 8-lymphocyte (Q4), CD4+ lymphocyte (Q3), and CD8+ lymphocyte (Q1), were found to be 69.1%, 21.0%, and 9.73%, respectively, in normal ratios.
Comparative example 1 preparation of CD4[ GK1.5] -PE (labeling with the monoclonal antibody described in the present invention, but without crosslinking with Sulfo-LC-SPDP)
Pretreatment of PE suspension
(1) And shaking and uniformly mixing the PE turbid liquid in the reagent tube by using a vortex oscillator.
(2) 0.5mg PE precipitate suspension was placed in a 1.5mL centrifuge tube, centrifuged at 12000g for 5min, and the supernatant was completely aspirated off with a pipette, taking care not to aspirate the precipitate.
(3) Add 100. mu.L of PBS (containing 0.25mM EDTA) buffer to the centrifuge tube to dissolve the PE pellet at the bottom of the centrifuge tube, centrifuge at 12000g for 5min, and absorb the PE supernatant.
(4) A50 Kd ultrafiltration tube was selected, the PE supernatant solution was transferred to the tube, 400. mu.L of PBS (containing 0.25mM EDTA) buffer was added and mixed, 12000g was centrifuged for 5min and the filtrate was removed, 500. mu.L of PBS (containing 0.25mM EDTA) buffer was added and mixed and centrifuged, and this operation was repeated 3 times.
(5) The PE solution after desalting was collected and made to a constant volume of 50. mu.L to obtain a 10mg/mL PE solution.
Capping of the thiol groups of PE
(1) Taking out 50mM NEM from a low-temperature state, placing the NEM in a room-temperature environment, and opening a bottle cap when the temperature of the bottle is balanced to room temperature so as to prevent condensed water in the bottle.
(2) To 50. mu.L PE (10mg/mL) was added 3.5. mu.L of 50mM NEM solution (n)PE:nNEM1:50) and reacted at 25 ℃ for 1.5h to allow binding of the NEM molecule to the free thiol group in the PE molecule and blocking of the free thiol group in the PE molecule.
Crosslinking reaction of PE-NEM with S-SMCC
(1) Taking out S-SMCC (with the solubility of 10mM and mother solution dissolved in sterile pure water) from a low-temperature state, placing the bottle in a room-temperature environment, and opening a bottle cover when the temperature of the bottle is balanced to room temperature so as to prevent condensed water from appearing in the bottle.
(2) 50mg of S-SMCC is dissolved in 11.46mL of sterile water to prepare 10mM mother liquor (the mother liquor is 100 muL/tube and is stored in-20 ℃), one reagent is newly thawed each time when the reagent is used, repeated freezing and thawing are not needed for use, and the residual reagent is discarded after the reagent is used.
(3) To 50. mu.L of the solution of 10mg/mL S-SMCC (n) was added 6.69. mu.L of 10mg/mL S-SMCC per 50. mu.L of the E-NEM (10mg/mL) solutionPE-NEM:nS-SMCC1:20) and reacting at 25 ℃ for 1.5h to combine the succinyl ester in the S-SMCC molecule with the primary amino group in the PE molecule.
(4) Transferring the solution after reaction to ultrafiltration centrifuge tube, adding 500 μ L PBS (containing 0.25mM EDTA) buffer solution, centrifuging for 5min at 12000g, removing filtrate, adding 500 μ L PBS (containing 0.25mM EDTA) buffer solution, mixing, centrifuging, repeating the operation for 5 times, desalting to obtain unreacted S-SMCC residue in protein of 10% of total amount of protein-3The dilution was as large as possible.
(5) The desalted high concentration crosslinked protein solution is collected, and the volume of the collected solution is measured by a pipette to 40. mu.L (10 mg/mL).
4. Monoclonal antibody (McAb) sulfhydrylation reaction
(1) An appropriate amount of TCEP was weighed, 1g TCEP was dissolved in 6mL deionized water to dissolve TCEP, pH2.5, pH was adjusted to 7.0 with 10N NaOH, volume was adjusted to 7mL to obtain 0.5M TCEP (stock solution 100. mu.L/tube stored at-20 ℃ C.). When the reagent is used, one reagent is melted again every time, repeated freeze thawing is not needed, and the residual reagent is discarded after use.
(2) mu.L of TCEP was added to 50. mu.L of the antibody (2mg/mL) at a final concentration of about 20mM, and reacted at 25 ℃ for 1.5h in the absence of light at room temperature.
(3) The solution after completion of the reaction was transferred to an ultrafiltration centrifuge tube, and 500. mu.L of PBS (containing 0.25mM EDTA) buffer was added, and then, 12000g was centrifuged for 5min to remove the filtrate, and 500. mu.L of PBS (containing 0.25mM EDTA) buffer was added, and the mixture was centrifuged by mixing and repeating the operation 5 times.
(4) Collecting the desalted high-concentration sulfhydrylation monoclonal antibody solution, measuring the volume of the collected solution by using a pipette, estimating the antibody concentration by using 80% protein recovery rate, and fixing the volume of the antibody protein solution to 40 mu L (2mg/mL) to obtain the McAb-SH intermediate.
Crosslinking reaction of PE with antibody
The PE cross-linked protein and the sulfhydrylation monoclonal antibody solution obtained in the step 3 and the step 4 are mixed according to the molar ratio nMcAb-SH:nNEM-PE-S-SMCCThe mixture was mixed at a ratio of 1:5, 40. mu.L (2mg/mL) of the thiolated monoclonal antibody was added to 40. mu.L (10mg/mL) of S-SMCC-crosslinked NEM-PE, the mixture was reacted at 25 ℃ for 3 hours in the dark, 1/10 volumes (8. mu.L) of NEM (50mM) were added to the reaction system, the reaction was carried out at 25 ℃ for 1.5 hours, 8. mu.L of 1M Cys solution was added after the reaction, the mixture was mixed well, and the mixture was left to stand at 4 ℃ for overnight reaction. After the reaction is finished, adding a proper amount of PBS (containing 0.1% of sodium azide and 0.1% of gelatin) solution into the reaction system, fixing the volume to 320 mu L, enabling the final concentration of the antibody to be 0.25mg/mL, and after the operation is finished, placing the antibody at 4 ℃ for long-term storage.
When the flow detection is carried out, the antibody solution after the cross-linking reaction is diluted according to the ratio of 1:100 and then is detected, wherein the detection reaction system is as follows: 100 mu L of flow buffer (PBS + 0.5% FBS + 0.2% EDTA), 2.5 mu g/mL of CD4-PE [ GK1.5] and 5 mu g/mL of CD8-FITC [53-6.72] and 10^6cells of mouse (C57); the reaction conditions are as follows: carrying out water bath at 25 ℃ and light-proof reaction for 15min, washing for 3 times by using 1mL of flow buffer solution, and fixing the volume to 500 mu L; the door setting method comprises the following steps: lymph +.
As shown in FIG. 2, when splenocytes of mice (C57) were co-infected with CD4-PE [ GK1.5] and CD8-FITC [53-6.72] prepared in this comparative example, significant cell clustering occurred, and the negative cell population, CD4-CD 8-lymphocyte (Q4), CD4+ lymphocyte (Q3), and CD8+ lymphocyte (Q1), were found to be present in a proportion of 67.2%, 22.1%, and 10.7%, respectively, in a proportion of normal.
Comparative example 2
The Labeling method of the comparative example was performed using a Kit (Sammerfei, Invitrogen, SiteClick R-PE Antibody Labeling Kit), and after Labeling, flow detection was performed using the same method as in example 1, and the detection results are shown in FIG. 3.
As is clear from FIG. 3, when spleen cells of mice (C57) were co-infected with CD4-PE [ GK1.5] and CD8-FITC [53-6.72] prepared in this comparative example, a significant cell population was observed, and the negative cell population was 68.4% in the region of CD4-CD 8-lymphocytes (Q4), 19.2% in the region of CD4+ lymphocytes (Q3), and 12.3% in the region of CD8+ lymphocytes (Q1), which were normal.
The results of the flow assays of example 1 and comparative examples 1-2 were further compared and are shown in FIGS. 4-6.
As can be seen from FIGS. 4 to 6, the fluorescence signal value (110442) of the Labeling method of the present invention with Sulfo-LC-SPDP cross-linking was significantly higher than the signal value (66250) without Sulfo-LC-SPDP cross-linking, and the signal value of the Labeling method of the present invention was significantly higher than the signal value (55184) of the Labeling method with a Kit (Sammer fly, Invitrogen, SiteClick R-PE Antibody Labeling Kit).
Example 2 preparation of CD4[ GK1.5] -PERCP (Using the monoclonal antibody labeling method described herein)
PERCP thiol blocking reaction
(1) Taking out 50mM NEM from a low-temperature state, placing the NEM in a room-temperature environment, and opening a bottle cap when the temperature of the bottle is balanced to room temperature so as to prevent condensed water in the bottle.
(2) To 25. mu.L of PERCP (10mg/mL) was added 7.5. mu.L of 50mM NEM solution (n)PERCP:nNEM1:50) and reacted at 25 ℃ for 1.5h to allow the NEM molecule to bind to and block the free thiol group of the PERCP molecule.
Crosslinking reaction of PERCP-NEM with S-SMCC
(1) Taking out S-SMCC (with the solubility of 10mM and mother solution dissolved in sterile pure water) from a low-temperature state, placing the bottle in a room-temperature environment, and opening a bottle cover when the temperature of the bottle is balanced to room temperature so as to prevent condensed water from appearing in the bottle.
(2) 50mg of S-SMCC is dissolved in 11.46mL of sterile water to prepare 10mM mother liquor (the mother liquor is 100 muL/tube and is stored in-20 ℃), one reagent is newly thawed each time when the reagent is used, repeated freezing and thawing are not needed for use, and the residual reagent is discarded after the reagent is used.
(3) To 32.5. mu.L of PERCP-NEM (10mg/mL) solution was added 13.75. mu.L of 10mg/mL S-SMCC solution (nPERCP-NEM:nS-SMCC1:20) and reacting at 25 ℃ for 1.5h to combine the succinyl ester in the S-SMCC molecule with the primary amino group in the PERCP molecule.
(4) Transferring the solution after reaction to ultrafiltration centrifuge tube, adding 500 μ L PBS (containing 0.25mM EDTA) buffer solution, centrifuging for 5min at 12000g, removing filtrate, adding 500 μ L PBS (containing 0.25mM EDTA) buffer solution, mixing, centrifuging, repeating the operation for 5 times, desalting to obtain unreacted S-SMCC residue in protein of 10% of total amount of protein-3The dilution was as large as possible.
(5) The desalted high concentration crosslinked protein solution was collected, and the volume of the collected solution was measured by pipette to 32. mu.L (7.2mg/mL) of the protein solution volume.
3. Monoclonal antibody (McAb) sulfhydrylation reaction
(1) 2mg of Sulfo-LC-SPDP was dissolved in 200. mu.L of deionized water to prepare a 20mM solution. Mu.g McAb (2mg/mL) was added with 1.5. mu. L S-LC-SPDP, reacted at 25 ℃ for 1h, the solution after completion of the reaction was transferred to an ultrafiltration centrifuge tube, 500. mu.L of PBS (containing 0.25mM EDTA) buffer was added, centrifugation was performed for 5min at 12000g, the filtrate was removed, 500. mu.L of PBS (containing 0.25mM EDTA) buffer was added, and the mixture was centrifuged uniformly, and this operation was repeated 5 times. The volume of the antibody was made 50. mu.L (2 mg/mL).
(2) An appropriate amount of TCEP was weighed, 1g TCEP was dissolved in 6mL deionized water to dissolve TCEP, pH2.5, pH was adjusted to 7.0 with 10N NaOH, volume was adjusted to 7mL to obtain 0.5M TCEP (stock solution 100. mu.L/tube stored at-20 ℃ C.). When the reagent is used, one reagent is melted again every time, repeated freeze thawing is not needed, and the residual reagent is discarded after use.
(3) mu.L of TCEP was added to 50. mu.L of the antibody at a final concentration of about 20mM, and reacted at 25 ℃ for 1.5h in the dark.
(4) The solution after completion of the reaction was transferred to an ultrafiltration centrifuge tube, and 500. mu.L of PBS (containing 0.25mM EDTA) buffer was added, and then, 12000g was centrifuged for 5min to remove the filtrate, and 500. mu.L of PBS (containing 0.25mM EDTA) buffer was added, and the mixture was centrifuged by mixing and repeating the operation 5 times.
(5) Collecting the desalted high-concentration sulfhydrylation monoclonal antibody solution, measuring the volume of the collected solution by using a pipette, estimating the antibody concentration by using 80% protein recovery rate, and fixing the volume of the antibody protein solution to 40 mu L (2mg/mL) to obtain the McAb-SH intermediate.
Crosslinking reaction of PERCP with antibody
The PERCP cross-linked protein and the thiolated monoclonal antibody solution obtained in the step 3 and the step 4 are mixed according to a molar ratio nMcAb-SH:nNEM-PERCP-S-SMCCThe mixture was mixed at a ratio of 1:5, 32. mu.L (7.2mg/mL) of S-SMCC-crosslinked NEM-PERCP was added to 40. mu.L (2mg/mL) of the thiolated monoclonal antibody, the resulting mixture was reacted at 25 ℃ for 3 hours while keeping out of the light, 1/10 volumes (8. mu.L) of NEM (50mM) were added to the reaction system, the reaction was carried out at 25 ℃ for 1.5 hours, and after the reaction, 8. mu.L of 1M Cys solution was added and mixed well, followed by allowing the mixture to stand at 4 ℃ for overnight reaction. After the reaction is finished, adding a proper amount of PBS (containing 0.1% of sodium azide and 0.1% of gelatin) solution into the reaction system, fixing the volume to 320 mu L, enabling the final concentration of the antibody to be 0.25mg/mL, and after the operation is finished, placing the antibody at 4 ℃ for long-term storage.
When the flow detection is carried out, the antibody solution after the cross-linking reaction is diluted according to the ratio of 1:100 and then is detected, wherein the detection reaction system is as follows: 100 mu L of flow buffer (PBS + 0.5% FBS + 0.2% EDTA), 2.5 mu g/mL of CD4-PERCP [ GK1.5] and 5 mu g/mL of CD3-FITC [145-2C11] and 10^6cells of mouse (C57); the reaction conditions are as follows: carrying out water bath at 25 ℃ and light-proof reaction for 15min, washing for 3 times by using 1mL of flow buffer solution, and fixing the volume to 500 mu L; the door setting method comprises the following steps: lymph +.
As shown in FIG. 7, when splenocytes of mice (C57) were co-infected with CD4-PERCP [ GK1.5] and CD3-FITC [145-2C11] prepared in this example, distinct cell groups appeared, and the negative cell group, CD4-CD 3-lymphocyte (Q4) region, CD4+ lymphocyte (Q2) region, and CD3+ lymphocyte (Q2+ Q3) region were 68.5%, 16.8%, and 30.6%, respectively, were normal.
Comparative example 3 preparation of CD4[ GK1.5] -PERCP (labeling with the monoclonal antibody described in the present invention, but the monoclonal antibody was not crosslinked with Sulfo-LC-SPDP)
PERCP thiol blocking reaction
(1) Taking out 50mM NEM from a low-temperature state, placing the NEM in a room-temperature environment, and opening a bottle cap when the temperature of the bottle is balanced to room temperature so as to prevent condensed water in the bottle.
(2) To 25. mu.L of PERCP (10mg/mL) was added 7.5. mu.L of 50mM NEM solution (n)PERCP:nNEM1:50) and reacted at 25 ℃ for 1.5h to allow the NEM molecule to bind to and block the free thiol group of the PERCP molecule.
Crosslinking reaction of PERCP-NEM with S-SMCC
(1) Taking out S-SMCC (with the solubility of 10mM and mother solution dissolved in sterile pure water) from a low-temperature state, placing the bottle in a room-temperature environment, and opening a bottle cover when the temperature of the bottle is balanced to room temperature so as to prevent condensed water from appearing in the bottle.
(2) 50mg of S-SMCC is dissolved in 11.46mL of sterile water to prepare 10mM mother liquor (the mother liquor is 100 muL/tube and is stored in-20 ℃), one reagent is newly thawed each time when the reagent is used, repeated freezing and thawing are not needed for use, and the residual reagent is discarded after the reagent is used.
(3) To 32.5. mu.L of PERCP-NEM (10mg/mL) solution was added 13.75. mu.L of 10mg/mL S-SMCC solution (nPERCP-NEM:nS-SMCC1:20) at 25 ℃ for 1.5h to make S-SMCC molecules in the solutionThe succinyl ester reacts with the primary amino group of the PERCP molecule to bind.
(4) Transferring the solution after reaction to ultrafiltration centrifuge tube, adding 500 μ L PBS (containing 0.25mM EDTA) buffer solution, centrifuging for 5min at 12000g, removing filtrate, adding 500 μ L PBS (containing 0.25mM EDTA) buffer solution, mixing, centrifuging, repeating the operation for 5 times, desalting to obtain unreacted S-SMCC residue in protein of 10% of total amount of protein-3The dilution was as large as possible.
(5) The desalted high concentration crosslinked protein solution was collected, and the volume of the collected solution was measured by pipette to 32. mu.L (7.2mg/mL) of the protein solution volume.
3. Monoclonal antibody (McAb) sulfhydrylation reaction
(1) An appropriate amount of TCEP was weighed, 1g TCEP was dissolved in 6mL deionized water to dissolve TCEP, pH2.5, pH was adjusted to 7.0 with 10N NaOH, volume was adjusted to 7mL to obtain 0.5M TCEP (stock solution 100. mu.L/tube stored at-20 ℃ C.). When the reagent is used, one reagent is melted again every time, repeated freeze thawing is not needed, and the residual reagent is discarded after use.
(2) mu.L of TCEP was added to 50. mu.L of the antibody (2mg/mL) at a final concentration of about 20mM, and reacted at 25 ℃ for 1.5h in the absence of light at room temperature.
(3) The solution after completion of the reaction was transferred to an ultrafiltration centrifuge tube, and 500. mu.L of PBS (containing 0.25mM EDTA) buffer was added, and then, 12000g was centrifuged for 5min to remove the filtrate, and 500. mu.L of PBS (containing 0.25mM EDTA) buffer was added, and the mixture was centrifuged by mixing and repeating the operation 5 times.
(4) Collecting the desalted high-concentration sulfhydrylation monoclonal antibody solution, measuring the volume of the collected solution by using a pipette, estimating the antibody concentration by using 80% protein recovery rate, and fixing the volume of the antibody protein solution to 40 mu L (2mg/mL) to obtain the McAb-SH intermediate.
Crosslinking reaction of PERCP with antibody
The PERCP cross-linked protein and the thiolated monoclonal antibody solution obtained in the step 3 and the step 4 are mixed according to a molar ratio nMcAb-SH:nNEM-PERCP-S-SMCCMixing evenly according to the proportion of 1:5, adding 32 mu L (7.2mg/mL) S-SMCC cross-linking NEM-PERCP into 40 mu L (2mg/mL) of thiolated monoclonal antibody, reacting for 3h at 25 ℃ in a dark place, and then adding 1/10 bodies into the reaction systemAfter reaction for 1.5h at 25 ℃ with a volume (8. mu.L) of NEM (50mM), 8. mu.L of 1M Cys solution was added and mixed well, and then left to react overnight at 4 ℃. After the reaction is finished, adding a proper amount of PBS (containing 0.1% of sodium azide and 0.1% of gelatin) solution into the reaction system, fixing the volume to 320 mu L, enabling the final concentration of the antibody to be 0.25mg/mL, and after the operation is finished, placing the antibody at 4 ℃ for long-term storage.
When the flow detection is carried out, the antibody solution after the cross-linking reaction is diluted according to the ratio of 1:100 and then is detected, wherein the detection reaction system is as follows: 100 mu L of flow buffer (PBS + 0.5% FBS + 0.2% EDTA), 2.5 mu g/mL of CD4-PERCP [ GK1.5] and 5 mu g/mL of CD3-FITC [145-2C11] and 10^6cells of mouse (C57); the reaction conditions are as follows: carrying out water bath at 25 ℃ and light-proof reaction for 15min, washing for 3 times by using 1mL of flow buffer solution, and fixing the volume to 500 mu L; the door setting method comprises the following steps: lymph +.
As shown in FIG. 8, when splenocytes of mice (C57) were co-infected with CD4-PERCP [ GK1.5] and CD3-FITC [145-2C11] prepared in this comparative example, distinct cell groups were observed, and the negative cell group, CD4-CD 3-lymphocyte (Q4) region, CD4+ lymphocyte (Q2) region, and CD3+ lymphocyte (Q2+ Q3) region were found to be 70.8%, 16.9%, and 28.5%, respectively, normal.
Comparative example 4
The labeling method of this comparative example was performed using a Kit (Abcam, PerCP Conjugation Kit-Lighting Kit), and after labeling, flow assay was performed in the same manner as in example 1, and the assay results are shown in FIG. 3.
As is clear from FIG. 9, when splenocytes of mice (C57) were co-infected with CD4-PERCP [ GK1.5] and CD3-FITC [145-2C11] prepared in this comparative example, distinct cell groups were observed, and the negative cell group, CD4-CD 3-lymphocyte (Q4) region, CD4+ lymphocyte (Q2) region, and CD3+ lymphocyte (Q2+ Q3) region were found to be 69.3%, 17.5%, and 30.3%, respectively, normal.
The results of the flow assays of example 2 and comparative examples 3-4 were further compared and are shown in FIGS. 10-12.
As can be seen from FIGS. 10 to 12, the fluorescence signal value of PERCP crosslinked with Sulfo-LC-SPDP (9025) is significantly higher than that of the labeled protein without Sulfo-LC-SPDP (6655), and the signal value of the labeled protein with the labeling method of the present invention is significantly higher than that of the labeled protein with Kit (Abcam, PerCP Conjugation Kit-Lighting Kit) (4228).
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (11)

1. A method for labeling a fluorescent protein and/or a conjugated protein with a monoclonal antibody, comprising the steps of:
(1) carrying out desalination treatment on the fluorescent protein and/or the coupled protein, and then reacting with a sulfhydryl blocking agent, wherein the sulfhydryl blocking agent is NEM;
(2) reacting the amino group of the sulfhydryl-blocked fluorescent protein and/or coupling protein obtained in the step (1) with a cross-linking agent A, wherein the cross-linking agent A is S-SMCC;
(3) the method comprises the following steps of carrying out a sulfhydrylation reaction on a monoclonal antibody, wherein the sulfhydrylation reaction of the monoclonal antibody comprises a reaction of the monoclonal antibody and a cross-linking agent B and a reduction reaction of the monoclonal antibody-cross-linking agent B and a disulfide bond reducing agent, the cross-linking agent B is S-LC-SPDP, and the disulfide bond reducing agent is TCEP;
(4) and (3) performing directional coupling reaction on the sulfhydrylation monoclonal antibody obtained in the step (3) and the cross-linked fluorescent protein and/or the coupled protein obtained in the step (2), and after the reaction is completed, performing NEM, Cys and PBS treatment to obtain the fluorescence labeling antibody.
2. The method of claim 1, wherein the fluorescent protein and/or conjugated protein comprises PERCP, PE, PECy5, PECy5.5, PECy7, PerCPCy5.5, APCCy5.5, APCCy7 or APC.
3. The method for labeling a fluorescent protein and/or conjugated protein with a monoclonal antibody according to claim 1, wherein the desalting treatment in step (1) comprises the steps of:
1) shaking and mixing the fluorescent protein and/or the coupling protein suspension uniformly, centrifuging, and removing the supernatant;
2) adding a PBS buffer solution containing EDTA into the precipitate, centrifuging after fully dissolving, and taking the supernatant;
3) and transferring the supernatant into an ultrafiltration tube, and performing ultrafiltration treatment on the supernatant by using PBS (phosphate buffer solution) containing 0.25mM EDTA to obtain the desalted fluorescent protein and/or coupled protein.
4. The method for labeling a fluorescent protein and/or conjugated protein with a monoclonal antibody according to claim 1, wherein the molar ratio of the fluorescent protein and/or conjugated protein to the thiol blocking agent in step (1) is 1:3 to 1: 1000; the reaction condition of the fluorescent protein and/or the conjugated protein and the sulfhydryl blocking agent is that the reaction lasts for 1.5h at 25 ℃.
5. The method for labeling a fluorescent protein and/or conjugated protein with a monoclonal antibody according to claim 1, wherein the molar ratio of the thiol-blocked fluorescent protein and/or conjugated protein to the crosslinking agent A in step (2) is 1:5 to 1: 100; the reaction condition of the sulfhydryl blocked fluorescent protein and/or coupling protein and the cross-linking agent A is that the reaction lasts for 1.5h at 25 ℃.
6. The method for labeling a fluorescent protein and/or conjugated protein with a monoclonal antibody according to claim 1, wherein the molar ratio of the monoclonal antibody to the crosslinking agent B in the step (3) is 1:10 to 1: 50; the reaction condition of the monoclonal antibody and the cross-linking agent B is that the monoclonal antibody reacts for 1h at 25 ℃.
7. The method for labeling a fluorescent protein and/or conjugated protein with a monoclonal antibody according to claim 1, wherein the disulfide bond reducing agent and the monoclonal antibody-crosslinking agent B in the step (3) are reacted at a final concentration of 10 to 50 mM; the reaction condition of the monoclonal antibody-cross-linking agent B and the disulfide bond reducing agent is that the reaction lasts for 1.5h at 25 ℃.
8. The method for labeling a fluorescent protein and/or conjugated protein with a monoclonal antibody according to claim 1, wherein the molar ratio of the thiolated monoclonal antibody obtained in step (3) to the crosslinked fluorescent protein and/or conjugated protein obtained in step (2) in step (4) is 1:0.5 to 1: 100; and (3) reacting the sulfhydrylation monoclonal antibody obtained in the step (4) with the cross-linked fluorescent protein and/or the coupling protein obtained in the step (2) for 3 hours at 25 ℃.
9. The method for labeling a fluorescent protein and/or conjugated protein with a monoclonal antibody according to any one of claims 1 to 8,
the molar ratio of the fluorescent protein and/or the coupling protein to the sulfhydryl blocking agent in the step (1) is 1: 50;
the mol ratio of the sulfhydryl blocked fluorescent protein and/or coupling protein in the step (2) to the cross-linking agent A is 1: 20;
the final reaction concentration of the disulfide bond reducing agent and the monoclonal antibody-cross-linking agent B in the step (3) is 20 mM;
the mol ratio of the sulfhydrylation monoclonal antibody obtained in the step (3) in the step (4) to the cross-linked fluorescent protein and/or the coupling protein obtained in the step (2) is 1: 5.
10. The method for labeling a fluorescent protein and/or conjugated protein with a monoclonal antibody according to claim 1,
the NEM treatment step in the step (4) is that after the reaction of the sulfhydrylation monoclonal antibody and the cross-linked fluorescent protein and/or the coupling protein is completed, 1/10 volumes of NEM are added, and the reaction is carried out for 1.5h at 25 ℃;
the Cys treatment step in the step (4) is that after the NEM treatment is completed, 1/10 volumes of Cys solution is added, and the reaction is carried out overnight at 4 ℃;
in the PBS treatment step described in step (4), after Cys treatment was completed, an appropriate amount of PBS buffer containing 0.1% sodium azide and 0.1% gelatin was added to give a final antibody concentration of 0.25 mg/mL.
11. A kit for labeling a fluorescent protein and/or a conjugated protein with a monoclonal antibody is characterized by comprising the monoclonal antibody, the fluorescent protein and/or the conjugated protein, a cross-linking agent A, a cross-linking agent B, a sulfhydryl blocking agent, a disulfide bond reducing agent, NEM, Cys, a PBS buffer solution containing 0.25mM EDTA, a PBS buffer solution containing 0.1% of sodium azide and 0.1% of gelatin, wherein the cross-linking agent A is S-SMCC; the cross-linking agent B is S-LC-SPDP; the sulfhydryl blocking agent is NEM; the disulfide bond reducing agent is TCEP.
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