CN111551729A - Preparation method of phycoerythrin immunofluorescence probe - Google Patents
Preparation method of phycoerythrin immunofluorescence probe Download PDFInfo
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- CN111551729A CN111551729A CN202010342208.7A CN202010342208A CN111551729A CN 111551729 A CN111551729 A CN 111551729A CN 202010342208 A CN202010342208 A CN 202010342208A CN 111551729 A CN111551729 A CN 111551729A
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Abstract
The invention discloses a preparation method of phycoerythrin immunofluorescence probe, which comprises the steps of marking phycoerythrin on target protein; before the step of labeling the phycoerythrin on the target protein, the method also comprises the step of blocking the free sulfydryl on the target protein or the phycoerythrin. According to the invention, before phycoerythrin is marked and crosslinked on a target protein by adopting a conventional method, the free sulfydryl on the target protein or the phycoerythrin is subjected to sealing treatment, and after the free sulfydryl is sealed, the amine-sulfydryl crosslinking agent can only react with the amino on the sealed target protein or the phycoerythrin and can not react with the sulfydryl on the corresponding protein, so that the amino of the amine-sulfydryl crosslinking agent can be ensured to be completely used for coupling the sulfydryl phycoerythrin or the target protein, the marking efficiency of the phycoerythrin on the target protein is effectively improved, and the obtained phycoerythrin immunofluorescence probe has a higher signal-to-noise ratio during immunodetection.
Description
Technical Field
The invention belongs to the technical field of immunology, and particularly relates to a preparation method of an phycoerythrin immunofluorescence probe.
Background
Phycoerythrin (P-phyerythrin, abbreviated as PE) is a novel fluorescence labeling reagent which is generally used at present and is obtained by separating and purifying red algae. Under the excitation of specific wavelength, phycobiliprotein can emit strong fluorescence, the fluorescence intensity of which is 30-100 times that of fluorescein, and the phycobiliprotein has good light absorption performance and high quantum yield, and has wide excitation and emission range in the visible spectrum region.
Phycoerythrin is used for fluorescence analysis, and has the advantages that the traditional chemical fluorescent dye cannot compare with. Such as: (1) the fluorescent material has a wide absorption spectrum in a wide pH range, and is easy to select a proper excitation wavelength, so that high-efficiency fluorescence emission is obtained, and a specific fluorescence emission peak is generated during excitation; (2) the absorbance and the fluorescence quantum yield are high, the fluorescence intensity is stable, and the sensitivity is high; (3) the fluorescent material has smaller fluorescent background, is not easy to quench, and has longer fluorescent retention period; (4) the water solubility is excellent, the cross-linking and the combination with other molecules are easy, and the non-specific adsorption is less; (5) the pure natural marine organism extract has no toxic or side effect, no radioactivity and safe operation and use.
In the prior art, a PE labeling method is often adopted to combine phycoerythrin with substances such as antibodies, biotin, avidin, immune protein and the like to prepare a fluorescent probe. By detecting the fluorescence emitted by the fluorescent probe, the fluorescent probe can be used for fluorescent microscopic detection, fluorescent immunoassay, bicolor or multicolor fluorescence analysis, cancer cell surface antigen detection, analysis of biomacromolecules such as protein and nucleic acid and the like; can also be used for clinical diagnosis and biological engineering technologies such as immunoassay, fluorescence microscopy, flow cytometry fluorescence measurement and the like.
The conventional PE Labeling (e.g., AnaTag. RTM. R-PE Labeling Kit) is performed by the following steps: (1) thiolating the target protein; (2) activating PE by adopting SMCC; (3) and (3) crosslinking the sulfhydrylation target protein with the activated PE.
The PE labeling method (phycoerythrin immunofluorescence probe labeling method, patent No. 202010087828.0) developed by Xixi organism is carried out by (1) thiolating PE; (2) activating the target protein by adopting SMCC; (3) and (3) crosslinking the sulfhydrylated PE with the activated target protein.
There is also a practice in the prior art (see: Zhaoyijie, Spilan, Suncoan, Onakai, Jaqian, King Yu Ling, Lu lan Jun, Zhang Aihua. fluorescent dye R-phycoerythrin labeled mouse anti-human CD4 monoclonal antibody. microbiological immunological progress [ J ]. 2006, 34 (2): 32-34.) as follows: (1) respectively activating PE and a target protein by adopting SPDP and SMCC; (2) sulfhydrylating the SPDP-activated PE; (3) thiolated PE is cross-linked to SMCC-activated protein of interest.
The above PE labeling method has the following disadvantages: because the target protein and phycoerythrin carry both free amino and free sulfydryl, when activated by amine-sulfydryl cross-linking agents such as SMCC, the amine-sulfydryl cross-linking agents can be combined with both amino and sulfydryl, so that when the protein activated by the amine-sulfydryl cross-linking agents is cross-linked with the sulfydryl phycoerythrin, the cross-linking agents which are combined with the sulfydryl on the protein activated by the amine-sulfydryl cross-linking agents are difficult to cross-link with the sulfydryl phycoerythrin, the phycoerythrin combined on the target protein is few, the labeling effect is poor, the positive signal generated by the obtained immunofluorescence probe is weak, the background signal is strong, and the signal-to-noise ratio during detection is low.
Disclosure of Invention
The invention aims to provide a preparation method of a phycoerythrin immunofluorescence probe with high phycoerythrin labeling efficiency.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a preparation method of phycoerythrin immunofluorescence probe comprises the steps of labeling phycoerythrin on target protein; before the step of labeling phycoerythrin onto a target protein, the preparation method of the phycoerythrin immunofluorescence probe further comprises the step of blocking the target protein or free sulfydryl on the phycoerythrin.
In the invention, before the phycoerythrin is marked and crosslinked on the target protein by adopting the conventional method steps, the free sulfydryl on the target protein or the phycoerythrin is sealed, after the free sulfydryl on the target protein or the phycoerythrin is blocked, the subsequent amine-sulfydryl crosslinking agent can only react with the amino on the blocked target protein or the blocked phycoerythrin, without reacting with the sulfydryl on the corresponding protein, thus ensuring that the maleimide group of the amine-sulfydryl cross-linking agent is completely used for coupling the sulfhydrylated phycoerythrin or target protein, thereby effectively improving the cross-linking efficiency of the phycoerythrin and the target protein, improving the marking efficiency of the phycoerythrin to the target protein, when the obtained phycoerythrin immunofluorescence probe is used for immunodetection, a strong positive signal can be obtained, and the signal-to-noise ratio is greatly improved.
Preferably, in the above method for preparing a phycoerythrin immunofluorescent probe, the free thiol group on the target protein is blocked. Compared with the blocking treatment of the free sulfydryl on the phycoerythrin, the phycoerythrin immunofluorescence probe obtained by the blocking treatment of the free sulfydryl on the target protein has stronger positive signals and higher signal-to-noise ratio.
Namely, the preparation method of the phycoerythrin immunofluorescence probe comprises the following steps:
(1) blocking free sulfydryl on the target protein;
(2) activating the sulfhydryl-blocked target protein obtained in the step (1) by adopting an amine-sulfhydryl cross-linking agent;
(3) carrying out sulfhydrylation treatment on phycoerythrin;
(4) and (3) crosslinking the sulfhydrylation phycoerythrin obtained in the step (3) with the activation target protein obtained in the step (2) to obtain the phycoerythrin immunofluorescence probe.
In the preparation method of the phycoerythrin immunofluorescence probe, free sulfydryl on target protein is sealed firstly, and then the activation is carried out by adopting an amine-sulfydryl cross-linking agent, so that the amine-sulfydryl cross-linking agent can only react with amino on the target protein, and all maleimide groups on the amine-sulfydryl cross-linking agent do not participate in the reaction; when the activated target protein is crosslinked with the sulfhydrylated phycoerythrin, all maleimide groups of the amine-sulfhydryl crosslinking agent on the activated target protein are used for reacting with sulfhydryls on the sulfhydrylated phycoerythrin, so that more phycoerythrin can be combined on the target protein, the phycoerythrin labeling efficiency is higher, and the obtained phycoerythrin immunofluorescence probe can also obtain stronger positive signals when in immunoassay, so that the signal to noise ratio is further improved.
In the preparation method of the phycoerythrin immunofluorescence probe, phycoerythrin is not required to be activated in advance, sulfhydrylation treatment is directly carried out on the phycoerythrin, and then the phycoerythrin is crosslinked with a target protein which is activated in advance, so that the operation is simple and convenient, and the signal-to-noise ratio of the phycoerythrin immunofluorescence probe in use can be further improved.
In the prior art, a plurality of methods for blocking free sulfydryl on protein exist. The method which is simple and convenient to operate is selected, namely, in the step (1) of the preparation method of the phycoerythrin immunofluorescence probe, the sulfhydryl blocking agent is adopted to block the target protein.
Preferably, in the above method for preparing a phycoerythrin immunofluorescent probe, the blocking treatment comprises: mixing the target protein and the sulfhydryl blocking agent uniformly according to the molar ratio of 1:10-1:100, and reacting for 1-2 h at room temperature.
Preferably, the sealing treatment comprises: mixing the target protein and the sulfhydryl blocking agent uniformly according to the molar ratio of 1:40-1:60, and reacting for 1-2 h at room temperature.
Unless otherwise specified, the room temperature in the present invention means a room temperature condition of 25 ℃ or higher.
In the above method for preparing phycoerythrin immunofluorescent probe, the thiol blocking agent comprises at least one of N-ethylmaleimide and iodoacetamide (IAAm).
Preferably, the thiol blocking agent is N-ethylmaleimide (NEM). NEM can block the free thiol group mildly under neutral conditions.
As a further preferred aspect, the sealing treatment comprises: and uniformly mixing the target protein and N-ethylmaleimide according to the molar ratio of 1:50, and reacting at room temperature for 1.5 h.
Preferably, after the blocking treatment is completed, desalting treatment is performed on the blocking treatment reaction solution to remove the unreacted thiol blocking agent remaining in the blocking treatment reaction solution, so as to ensure that the finally obtained thiol-blocked target protein has high purity.
In the step (2) of the preparation method of phycoerythrin immunofluorescence probe, the activating treatment includes: and (2) uniformly mixing the sulfhydryl-blocked target protein obtained in the step (1) with an amine-sulfhydryl crosslinking agent in a molar ratio of 1:10-1:100, and reacting at room temperature for 1-2 h. When the free thiol groups on the target protein are blocked, the amine-thiol crosslinking agent can only be attached to the amino groups of the target protein.
Preferably, the activation treatment comprises: and (2) uniformly mixing the sulfhydryl-blocked target protein obtained in the step (1) with an amine-sulfhydryl crosslinking agent in a molar ratio of 1:40-1:60, and reacting at room temperature for 1-2 h.
In the above method for preparing phycoerythrin immunofluorescent probe, the amine-mercapto crosslinking agent comprises succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (Sulfo-SMCC), N-hydroxysuccinimide 3- (2-pyridinedimercapto) propionate (SPDP), or N-succinimidyl 6- (3-maleimidopropionamido) hexanoate (SMPH).
Preferably, the activation treatment comprises: and (2) uniformly mixing the sulfhydryl-blocked target protein obtained in the step (1) and SMCC in a molar ratio of 1:20, and reacting at room temperature for 1.5 h.
Similarly, preferably, the activation reaction solution is also desalted after the activation treatment to remove the unreacted amine-mercapto crosslinking agent remaining in the activation reaction solution.
In the step (3) of the preparation method of phycoerythrin immunofluorescence probe, the sulfhydrylation treatment comprises: mixing phycoerythrin and 2-iminosulfane hydrochloride (trail's regent) in a molar ratio of 1:10-1:50, and reacting at room temperature in the dark for 1-2 h.
Preferably, the sulfhydrylation treatment comprises: mixing phycoerythrin and 2-iminosulfane hydrochloride in a molar ratio of 1:10-1:20, and reacting at room temperature in the dark for 1.5 h.
As a further preferred, the thiolation treatment includes: mixing phycoerythrin and 2-iminosulfane hydrochloride in a molar ratio of 1:10, and reacting at room temperature in the dark for 1.5 h.
Similarly, preferably, before the sulfhydrylation treatment, the phycoerythrin needs to be desalted to prevent the interference of free amino groups contained in the phycoerythrin with the sulfhydrylation reaction; after the sulfhydrylation treatment, the reaction solution is also desalted to remove the unreacted 2-iminosulfane hydrochloride remaining in the activated reaction solution.
In the step (4) of the preparation method of the phycoerythrin immunofluorescence probe, the sulfhydrylated phycoerythrin obtained in the step (3) and the activated target protein obtained in the step (2) are mixed uniformly according to the molar ratio of 1:1-30:1, and then are placed at room temperature to react for 2-4 hours in a dark place.
Preferably, in the step (4), the sulfhydrylated phycoerythrin obtained in the step (3) and the activated target protein obtained in the step (2) are mixed uniformly according to the mol ratio of 1:1-10:1, and then are placed at room temperature and are protected from light for reaction for 2-4 hours.
More preferably, in the step (4), the thiolated phycoerythrin obtained in the step (3) and the activated target protein obtained in the step (2) are mixed uniformly in a molar ratio of 5:1, and then the mixture is left to react at room temperature for 3 hours in the absence of light.
Compared with the prior art, the invention has the beneficial effects that:
in the preparation method of the phycoerythrin immunofluorescence probe, before the phycoerythrin is marked and cross-linked to the target protein by adopting the conventional method steps, the free sulfydryl on the target protein or the phycoerythrin is firstly sealed, after the free sulfydryl on the target protein or the phycoerythrin is sealed, the subsequent amine-sulfydryl cross-linking agent can only react with the amino on the sealed target protein or the sealed phycoerythrin and can not react with the sulfydryl on the corresponding protein, thus ensuring that all the amino of the amine-sulfydryl cross-linking agent is used for coupling the sulfydryl phycoerythrin or the target protein, effectively improving the cross-linking efficiency of the phycoerythrin and the target protein, improving the marking efficiency of the phycoerythrin on the target protein, and obtaining the phycoerythrin immunofluorescence probe can also obtain stronger positive signals when the obtained phycoerythrin immunofluorescence probe is used for immunodetection, the signal-to-noise ratio is greatly improved.
Drawings
FIG. 1 is a dot-blot analysis of the phycoerythrin immunofluorescence probe NEM-CD62L-PE prepared in example 1 co-stained mouse splenocytes with CD3-ifluor 488;
FIG. 2 is a flow detection scattergram of mouse splenocytes co-stained with phycoerythrin immunofluorescence probe CD62L-PE and CD3-ifluor488 prepared in comparative example 1;
FIG. 3 is a histogram of the results of detecting the phycoerythrin fluorescence signal intensity of mouse splenocytes stained with the phycoerythrin immunofluorescence probe NEM-CD62L-PE prepared in example 1;
FIG. 4 is a histogram of the results of detecting the phycoerythrin fluorescence signal intensity of mouse splenocytes stained with the phycoerythrin immunofluorescence probe CD62L-PE prepared in comparative example 1;
FIG. 5 is a graph of the overlay alignment of FIGS. 3 and 4;
FIG. 6 is a flow-through detection scattergram of phycoerythrin immunofluorescence probe NEM-CD4-PE prepared in example 2 and CD3-ifluor488 co-stained mouse splenocytes;
FIG. 7 is a flow-through detection scattergram of mouse splenocytes co-stained with phycoerythrin immunofluorescence probe CD4-PE and CD3-ifluor488 prepared in comparative example 2;
FIG. 8 is a histogram of the results of detecting the phycoerythrin fluorescence signal intensity of mouse splenocytes stained with the phycoerythrin immunofluorescence probe NEM-CD4-PE prepared in example 2;
FIG. 9 is a histogram of results of detecting phycoerythrin fluorescence signal intensity of mouse splenocytes stained with the phycoerythrin immunofluorescence probe CD4-PE prepared in comparative example 2;
FIG. 10 is a graph of the overlay alignment of FIGS. 8 and 9;
FIG. 11 is a dot-plot for flow detection of phycoerythrin immunofluorescence probe NEM-CD8-PE prepared in example 3 co-staining mouse splenocytes with CD 19-FITC;
FIG. 12 is a flow detection scattergram of phycoerythrin immunofluorescence probe CD8-PE and CD19-FITC co-stained mouse splenocytes prepared in comparative example 3;
FIG. 13 is a graph showing the results of measuring the phycoerythrin fluorescence signal intensity of mouse splenocytes stained with the phycoerythrin immunofluorescence probe NEM-CD8-PE prepared in example 3;
FIG. 14 is a graph showing the results of detecting the phycoerythrin fluorescence signal intensity of mouse splenocytes stained with the phycoerythrin immunofluorescence probe CD8-PE prepared in comparative example 3;
fig. 15 is a graph of the overlay alignment of fig. 13 and 14.
Detailed Description
The technical solution of the present invention will be further described in detail with reference to the accompanying drawings and the detailed description.
Example 1
In this example, a method for preparing an immunofluorescent phycoerythrin probe, using an anti-mouse CD62L monoclonal antibody [ MEL-14] (hereinafter referred to as CD62L antibody) as a target protein, is described, the method comprising the steps of:
(1) blocking free sulfydryl on the target protein;
the method specifically comprises the following steps:
(1.1) 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 the room temperature so as to prevent condensed water from appearing in the bottle;
(1.2) to 200. mu.g of CD62L antibody, 1.4. mu.l of 50mM NEM solution (n (Ab): N (NEM) ≈ 1: 50) was added, mixed well, and then reacted at room temperature of 25 ℃ or higher for 1.5 hours;
in this step, NEM molecules bind to the free thiol group in the CD62L antibody molecule, blocking the free thiol group in the CD62L antibody molecule;
(1.3) transferring the reaction solution obtained in the step (1.2) into an ultrafiltration centrifugal tube, adding 500. mu.l of PBS (containing 0.25mM EDTA) buffer solution, centrifuging at 12000g for 5 min, removing the filtrate, adding 500. mu.l of PBS (containing 0.25mM EDTA) buffer solution, mixing uniformly, centrifuging, and repeating the operation for 5 times;
the step removes the residual NEM which does not participate in the reaction solution as much as possible by desalting treatment to ensure that the residual NEM in the reaction solution obtained after desalting is at least 10 of the total amount of the antibody-3Doubling the weight;
(1.4) collecting the desalted sulfhydryl-blocked antibody solution, and fixing the volume of the sulfhydryl-blocked antibody solution to 40 mul (5 mg/ml);
(2) activating the sulfhydryl-blocked target protein obtained in the step (1) by adopting an amine-sulfhydryl cross-linking agent;
the method specifically comprises the following steps:
(2.1) taking out the SMCC from a low-temperature state, placing the SMCC in a room-temperature environment, and opening a bottle cover when the temperature of the bottle is balanced to the room temperature so as to prevent condensed water from appearing in the bottle;
(2.2) weighing a proper amount of SMCC, dissolving in a proper amount of DMSO, and preparing into mother liquor with the concentration of 10 mg/ml; subpackaging the mother liquor with 5 μ l/tube, and storing at-20 deg.C; one reagent is melted again when the reagent is used every time, repeated freezing and thawing use is not needed, and the residual reagent is discarded after use;
(2.3) adding 0.8. mu.l (10 mg/ml) of SMCC solution (n (Ab): n (SMCC) ≈ 1: 20) to 40. mu.l (5 mg/ml) of thiol-blocked antibody solution, and reacting at 25 ℃ or higher for 1.5 hours;
in the step, succinyl ester in the SMCC molecule reacts and is combined with primary amino in a sulfhydryl-blocked antibody molecule;
(2.4) transferring the reaction solution obtained in the step (2.3) into an ultrafiltration centrifugal tube, adding 500. mu.l of PBS (containing 0.25mM EDTA) buffer solution, centrifuging at 12000g for 5 min, removing the filtrate, adding 500. mu.l of PBS (containing 0.25mM EDTA) buffer solution, mixing uniformly, centrifuging, and repeating the operation for 5 times;
the step removes the SMCC residue which does not participate in the reaction liquid as much as possible through desalting treatment, and ensures that the SMCC residue in the reaction liquid obtained after desalting is at least 10 of the total amount of the antibody-3Doubling the weight;
(2.5) collecting the desalted activated antibody solution, and fixing the volume of the activated antibody solution to 80 mu l (2 mg/ml);
(3) carrying out sulfhydrylation treatment on phycoerythrin;
the method specifically comprises the following steps:
(3.1) pre-treating phycoerythrin suspension;
the method comprises the following steps:
(a) uniformly shaking phycoerythrin suspension (with the concentration of 5 mg/ml) in a reagent tube by using a vortex oscillator, then taking out 0.5g (about 100 mu l) of the suspension, placing the suspension in a 1.5ml centrifugal tube, centrifuging the suspension for 5 min at 12000g, completely sucking away supernatant by using a pipette, and taking care not to suck away phycoerythrin precipitate;
(b) adding 100 μ l PBS (containing 0.25mM EDTA) buffer solution into a centrifuge tube, dissolving phycoerythrin precipitate at the bottom of the centrifuge tube, centrifuging at 12000g for 5 min, and collecting phycoerythrin supernatant;
(c) placing phycoerythrin supernatant in 50kd ultrafiltration centrifuge tube, adding 400 μ l PBS (containing 0.25mM EDTA) buffer solution, mixing, centrifuging at 12000g for 5 min, and removing filtrate; adding 500 μ l PBS (containing 0.25mM EDTA) buffer solution, mixing, centrifuging, repeating the operation for 3 times, collecting desalted phycoerythrin solution, and diluting to 50 μ l to obtain phycoerythrin solution with concentration of 10 mg/ml;
(3.2) weighing a proper amount of trail's regent, dissolving in a proper amount of deionized water, and preparing into 2mg/ml (14 mM) mother liquor; subpackaging the mother liquor at 5 μ l/tube, storing at-20 deg.C, thawing one new tube each time without repeated freeze thawing, discarding the rest reagent
(3.3) adding 2.3 mul of traut's solution (n (PE): n (traut's) ≈ 1: 10) into 50 mul of phycoerythrin solution (10 mg/ml), mixing, and reacting at above 25 deg.C in the dark for 1.5 h;
(3.4) after completion of the reaction, the reaction solution was transferred to an ultrafiltration centrifuge tube, 500. mu.l of PBS (containing 0.25mM EDTA) buffer was added thereto, centrifugation was carried out for 5 min at 12000g, the filtrate was removed, and 500. mu.l of PBS (containing 0.25mM EDTA) buffer was added thereto and the mixture was centrifuged; this operation was repeated 5 times;
(3.5) collecting the desalted sulfhydrylated phycoerythrin solution, detecting the concentration of the phycoerythrin, and fixing the volume of the sulfhydrylated phycoerythrin solution to 40 mul (the concentration is 10 mg/ml);
(4) crosslinking the thiolated phycoerythrin obtained in the step (3) with the activated target protein obtained in the step (2) to obtain the phycoerythrin immunofluorescence probe NEM-CD62L-PE of the embodiment;
the method specifically comprises the following steps:
(4.1) uniformly mixing 5 μ l (2 mg/ml) of the activated antibody solution obtained in the step (2) with 5 μ l (10 mg/ml) of the thiolated phycoerythrin solution obtained in the step (3) (n (Ab): n (PE) = 1: 5), and then placing the mixture at the temperature of more than 25 ℃ for reaction in a dark place for 3 hours;
(4.2) after completion of the reaction, 30. mu.l of a PBS buffer containing 0.25mM EDTA was added to the reaction system so that the final concentration of phycoerythrin-labeled CD62L antibody (i.e., NEM-CD 62L-PE) was 0.25mg/ml, and after completion of the operation, the reaction mixture was left to stand at 4 ℃ for a long period of time.
Example 2
A phycoerythrin immunofluorescence probe NEM-CD4-PE was prepared in the same manner as in example 1, using an anti-mouse CD4 monoclonal antibody [ GK1.5] as a target protein.
Example 3
Phycoerythrin immunofluorescence probe NEM-CD8-PE was prepared in the same manner as in example 1, using anti-mouse CD8 monoclonal antibody [53-6.7] as the target protein.
Comparative example 1
This comparative example describes a labeling method of an immunofluorescent probe for phycoerythrin, using an anti-mouse CD62L monoclonal antibody [ MEL-14] as a target protein, the labeling method comprising the steps of:
(1) activating the CD62L antibody with an amine-sulfhydryl crosslinker;
the method specifically comprises the following steps:
(1-1) taking out the SMCC from a low-temperature state, placing the SMCC in a room-temperature environment, and opening a bottle cover when the temperature of the bottle is balanced to the room temperature so as to prevent condensed water from appearing in the bottle;
(1-2) weighing a proper amount of SMCC, dissolving in a proper amount of DMSO, and preparing into a mother solution of 10 mg/ml; subpackaging the mother liquor at 5 μ l/tube, storing at-20 deg.C, thawing one new tube each time without repeated freeze thawing, and discarding the residual reagent after use.
(1-3) preparing a CD62L antibody solution with the concentration of 4mg/mL, adding 1.6 mu l of a 10mg/mL SMCC solution into every 50 mu l of CD62L antibody solution, uniformly mixing, and reacting at the room temperature of more than 25 ℃ for 1.5 h to enable succinyl ester in SMCC molecules to react and combine with primary amino groups in CD62L molecules;
(1-4) after completion of the reaction, transferring the reaction solution into an ultrafiltration centrifuge tube, adding 500. mu.l of PBS (containing 0.25mM EDTA) buffer solution, centrifuging for 5 min at 12000g, removing the filtrate, adding 500. mu.l of PBS (containing 0.25mM EDTA) buffer solution, mixing and centrifuging; repeating the above operation 1-5 times to reduce the amount of unreacted SMCC in the reaction solution to at least 10 times of the total amount of antibody-3More than twice, diluting as much as possible;
(1-5) collecting the desalted high concentration activated antibody protein solution, measuring the CD62L antibody concentration, and adjusting the volume of the activated CD62L antibody solution to 2 mg/ml.
(2) Carrying out sulfhydrylation treatment on phycoerythrin to obtain sulfhydrylated phycoerythrin;
the method specifically comprises the following steps:
(2-1) carrying out desalination treatment on phycoerythrin;
the method specifically comprises the following steps:
(2-1-1) uniformly shaking phycoerythrin suspension (with the concentration of 5 mg/mL) in the reagent tube by using a vortex oscillator, then taking out 0.5g (about 100 mu l) of the suspension to be placed in a 1.5mL centrifugal tube, centrifuging for 5 min at 12000g, completely sucking away supernatant by using a pipette, and taking care not to suck away phycoerythrin precipitate;
(2-1-2) adding 100 μ l of PBS (containing 0.25mM EDTA) buffer solution into a centrifugal tube to fully dissolve phycoerythrin precipitate at the bottom of the centrifugal tube, centrifuging at 12000g for 5 min, and sucking phycoerythrin supernatant;
(2-1-3) placing phycoerythrin supernatant in a 50kd ultrafiltration centrifugal tube, adding 400 μ l PBS (containing 0.25mM EDTA) buffer solution, mixing, centrifuging at 12000g for 5 min, and removing filtrate; then adding 500. mu.l PBS (containing 0.25mM EDTA) buffer solution, mixing well, centrifuging, repeating the operation for 5 times, collecting desalted phycoerythrin solution, and fixing the volume to 50. mu.l to obtain phycoerythrin solution with concentration of 10 mg/mL.
(2-2) carrying out sulfhydrylation treatment on the desalted phycoerythrin;
the method specifically comprises the following steps:
(2-2-1) weighing a proper amount of trail's regent, dissolving in a proper amount of deionized water, and preparing into 2mg/ml (14 mM) mother liquor; subpackaging the mother liquor at 5 μ l/tube, storing at-20 deg.C, thawing one new tube each time without repeated freeze thawing, and discarding the rest reagents after use.
(2-2-2) adding 4.6. mu.l of traut's solution into each 50. mu.l of phycoerythrin solution (10 mg/mL), uniformly mixing, placing at the room temperature of more than 25 ℃, and reacting for 1.5 h in a dark place;
(2-2-3) after completion of the reaction, transferring the reaction solution into an ultrafiltration centrifuge tube, adding 500. mu.l of PBS (containing 0.25mM EDTA) buffer solution, centrifuging at 12000g for 5 min, removing the filtrate, adding 500. mu.l of PBS (containing 0.25mM EDTA) buffer solution, mixing well, and centrifuging; this operation was repeated 5 times;
(2-2-4) collecting the desalted sulfhydrylated phycoerythrin solution, detecting the concentration of the phycoerythrin, and fixing the volume of the sulfhydrylated phycoerythrin solution to a constant volume to make the concentration of the sulfhydrylated phycoerythrin solution be 10 mg/ml.
(3) Crosslinking the sulfhydrylation phycoerythrin with the activated target protein to obtain the phycoerythrin immunofluorescence probe CD62L-PE of the comparative example;
the method specifically comprises the following steps:
uniformly mixing the activated CD62L antibody solution obtained in the step (1) and the sulfhydrylated phycoerythrin solution obtained in the step (2) according to a molar ratio of 1:3, namely adding 3 mu l (10 mg/ml) of sulfhydrylated phycoerythrin solution into 5 mu l (2 mg/ml) of activated CD62L antibody solution, then placing the mixture at a temperature of over 25 ℃ for reaction in a dark place for 3 hours, adding 28 mu l of PBS (containing 0.25mM EDTA) buffer solution into a reaction system after the reaction is finished, enabling the final concentration of the phycoerythrin-labeled CD62L antibody (namely CD 62L-PE) to be 0.25mg/ml, and placing the reaction solution at 4 ℃ for long-term storage after the operation is finished.
Comparative example 2
The phycoerythrin immunofluorescence probe CD4-PE was prepared by the same method as comparative example 1, using anti-mouse CD4 monoclonal antibody [ GK1.5] as the target protein.
Comparative example 3
The phycoerythrin immunofluorescence probe CD8-PE was prepared in the same manner as in comparative example 1, using an anti-mouse CD8 monoclonal antibody [53-6.7] as the target protein.
Taking phycoerythrin immunofluorescence probes NEM-CD62L-PE and CD62L-PE, co-staining 1million mouse splenocytes with CD3-iFluor488 according to the same concentration (0.25 mu g of marked iFluor marked antibody and 0.125 mu g of marked PE antibody are added into each reaction system), detecting fluorescence signals through a flow cytometer after staining for 15 min, and repeatedly detecting at least three groups, wherein each group comprises at least two samples; the detection results are shown in fig. 1, 2, 3, 4 and 5.
As shown in fig. 1 and 2, mouse splenocytes stained with NEM-CD62L-PE had stronger positive fluorescence signals in the R6 region (28845 vs 20178) compared to CD 62L-PE. As shown in FIGS. 3, 4 and 5, spleen cells of mice stained with NEM-CD62L-PE had higher values of PE fluorescence signal than CD62L-PE, which is consistent with the results of the assays of FIGS. 1 and 2.
Similarly, phycoerythrin immunofluorescence probes NEM-CD4-PE and CD4-PE are taken to carry out co-staining on splenocytes of the 1million mice with CD3-iFluor488 according to the same concentration (0.25 mu g of labeled iFluor labeled antibody and 0.125 mu g of labeled PE labeled antibody are added into each reaction system), fluorescent signals are detected by a flow cytometer after staining for 15 min, at least three groups are repeatedly detected, and at least two samples in each group are detected; the detection results are shown in fig. 6, 7, 8, 9 and 10.
As shown in FIGS. 6 and 7, spleen cells from mice stained with NEM-CD4-PE had a stronger positive fluorescence signal in the Q2 (upper right panel) region than CD4-PE (53560 vs 40325).
As shown in FIGS. 8, 9 and 10, spleen cells of mice stained with NEM-CD4-PE had higher values of PE fluorescence signal than CD4-PE, which is consistent with the results of the assays of FIGS. 6 and 7.
Similarly, phycoerythrin immunofluorescence probes NEM-CD8-PE and CD8-PE are taken to carry out co-staining on spleen cells of a 1million mouse with CD19-FITC according to the same concentration (0.25 mu g of labeled FITC labeled antibody and 0.125 mu g of labeled PE antibody are added into each reaction system), fluorescent signals are detected by a flow cytometer after staining for 15 min, at least three groups are repeatedly detected, and at least two samples in each group are repeatedly detected; the detection results are shown in fig. 11, 12, 13, 14 and 15.
As shown in FIGS. 11 and 12, spleen cells from mice stained with NEM-CD8-PE had a stronger positive fluorescence signal in the Q4 (lower left panel) region (19417 vs 14075) than CD 8-PE.
As shown in FIGS. 13, 14 and 15, spleen cells of mice stained with NEM-CD8-PE had higher values of PE fluorescence signal than CD8-PE, which is consistent with the results of the assays of FIGS. 11 and 12.
Claims (10)
1. A preparation method of phycoerythrin immunofluorescence probe comprises the steps of labeling phycoerythrin on target protein; the preparation method is characterized in that before the step of marking the phycoerythrin on the target protein, the preparation method of the phycoerythrin immunofluorescence probe further comprises the step of blocking the target protein or free sulfydryl on the phycoerythrin.
2. The method for preparing phycoerythrin immunofluorescence probe according to claim 1, comprising the steps of:
(1) blocking free sulfydryl on the target protein;
(2) activating the sulfhydryl-blocked target protein obtained in the step (1) by adopting an amine-sulfhydryl cross-linking agent;
(3) carrying out sulfhydrylation treatment on phycoerythrin;
(4) and (3) crosslinking the sulfhydrylation phycoerythrin obtained in the step (3) with the activation target protein obtained in the step (2) to obtain the phycoerythrin immunofluorescence probe.
3. The method for preparing phycoerythrin immunofluorescence probe according to claim 2, wherein in step (1), the target protein is blocked with a thiol blocking agent.
4. The method for preparing phycoerythrin immunofluorescence probe according to claim 3, wherein the blocking treatment comprises: mixing the target protein and the sulfhydryl blocking agent uniformly according to the molar ratio of 1:10-1:100, and reacting for 1-2 h at room temperature.
5. The method of claim 3, wherein the thiol blocking agent comprises at least one of N-ethylmaleimide and iodoacetamide.
6. The method for preparing phycoerythrin immunofluorescence probe according to claim 5, wherein the thiol blocking agent is N-ethylmaleimide.
7. The method for preparing phycoerythrin immunofluorescent probe according to any one of claims 1 to 6, wherein in the step (2), the activation treatment comprises: and (2) uniformly mixing the sulfhydryl-blocked target protein obtained in the step (1) with an amine-sulfhydryl crosslinking agent in a molar ratio of 1:10-1:100, and reacting at room temperature for 1-2 h.
8. The method of claim 2, wherein the amine-mercapto crosslinker comprises succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate, sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate, N-hydroxysuccinimide 3- (2-pyridinedimercapto) propionate or N-succinimidyl 6- (3-maleimidopropionamido) hexanoate.
9. The method for preparing phycoerythrin immunofluorescent probe according to any one of claims 1 to 6, wherein in the step (3), the thiolation treatment comprises: mixing phycoerythrin and 2-iminosulfane hydrochloride in a molar ratio of 1:10-1:50, and reacting at room temperature in the dark for 1-2 h.
10. The method for preparing phycoerythrin immunofluorescence probe according to any one of claims 1 to 6, wherein in step (4), the thiolated phycoerythrin obtained in step (3) and the activated target protein obtained in step (2) are mixed uniformly in a molar ratio of 1:1-30:1, and then placed at room temperature in the dark for reaction for 2-4 hours.
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| CN114377154A (en) * | 2021-12-08 | 2022-04-22 | 中国科学院上海高等研究院 | X-ray fluorescence and fluorescence dual-mode imaging probe based on synchrotron radiation light source, preparation method and application |
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| WO2021217768A1 (en) | 2021-11-04 |
| CN111551729B (en) | 2021-02-09 |
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