CN111551725B - Method for improving antibody fluorescent dye marking signal - Google Patents
Method for improving antibody fluorescent dye marking signal Download PDFInfo
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- CN111551725B CN111551725B CN202010279571.9A CN202010279571A CN111551725B CN 111551725 B CN111551725 B CN 111551725B CN 202010279571 A CN202010279571 A CN 202010279571A CN 111551725 B CN111551725 B CN 111551725B
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- 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
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- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—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"
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- G—PHYSICS
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- 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
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- G—PHYSICS
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- 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
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Abstract
The invention aims at improving a method for improving a fluorescent dye labeling signal of an antibody, which comprises the following steps: step 1) the sulfhydrylation of an antibody; step 2) labeling the antibody with a fluorescent dye; according to the invention, through a sulfhydrylation amination method, after free amino groups are added on free sulfhydryl groups on proteins or antibodies, small molecular fluorescent labeling is performed, so that the efficiency of fluorescent labeling and a final fluorescent staining signal are improved.
Description
Technical Field
The invention belongs to the field of antibody labeling, and particularly relates to a method for improving a fluorescent dye labeling signal of an antibody.
Background
The traditional labeling reaction is to react succinimidyl ester of small molecular fluorescent dye (FITC, iFluor series dye, alexafluor series dye, etc.) with free amino groups on the protein or antibody to form amide bond covalent coupling, and the dyeing fluorescence effect is limited by the number of free amino groups on the protein or antibody.
Disclosure of Invention
The inventor of the invention adds free amino on free sulfhydryl on protein or antibody by sulfhydryl amination method and then carries out small molecule fluorescent marking, thus improving the efficiency of fluorescent marking and final fluorescent staining signal. Based on this, the object of the present invention is to improve a method for increasing the fluorescent dye labeling signal of an antibody, comprising the steps of:
step 1) the sulfhydrylation of an antibody; step 2) labelling the antibody with a fluorescent dye.
In some embodiments, the thiol group on the antibody is aminated by a haloalkylamine.
In some embodiments, the haloalkylamine is selected from the group consisting of bromoethylamine, chloroethylamine, iodoethylamine; in some embodiments of the invention, the alkylation reaction between bromoethylamine and antibody is carried out as follows:
in some embodiments, the fluorescent dye is a FITC, iFluor-based dye, alexafluor-based dye.
In some embodiments, the step 1) specifically includes: adding sodium bicarbonate solution into the antibody, uniformly mixing, and then adding bromoethylamine solution for reaction, so that bromoethylamine molecules react with free sulfhydryl groups of the antibody;
transferring the solution after the reaction to an ultrafiltration centrifuge tube, adding PBS buffer solution, centrifuging, removing filtrate, adding PBS buffer solution, mixing, centrifuging, and desalting to make the residual amount of unreacted bromoethylamine in the antibody be at least 10% of the total amount of the antibody -3 More than two times; in some embodiments, 100ul of 1m sodium bicarbonate solution is added to 200ug of antibody, and after mixing well, 1.4ul of 50mm bromoethylamine solution is added and reacted overnight at 25 ℃ to allow bromoethylamine molecules to react with free thiols in Ab molecules;
transferring the solution after the reaction to an ultrafiltration centrifuge tube, adding 500uL PBS (containing 0.25mM EDTA) buffer solution for 5min, centrifuging 12000g, removing filtrate, adding 500uL PBS (containing 0.25mM EDTA) buffer solution, mixing, centrifuging, repeating the operation for 5 times, and desalting to give residual unreacted bromoethylamine of at least 10% of total antibody -3 More than two times, as much dilution as possible.
In some embodiments, the step 2) specifically includes: 1. adding 1/10 of 1M sodium bicarbonate buffer to the pre-reaction antibody solution; 2. the specification of the iFluor fluorescent dye is 1mg per tube, the molecular weight is 1000, 100uL of DMSO is added into each tube to be dissolved to a final concentration of 10mM, and the mixture is split into 5uL of each tube and stored at the temperature of minus 20 ℃; 3. adding 1.5uL of fluorescent dye (n (dye): n (protein) =15:1) to the protein solution prepared in the step 2, and slightly shaking to enable the dye to be fully dissolved; the solution is placed in the environment of 25 ℃ in a dark place for reaction for 1h; 4. separating and purifying by an ultrafiltration centrifuge tube; 4.1 charging 0.5mL of 50kd ultrafiltration tube inner tube into a collecting tube, transferring the whole solution into the ultrafiltration tube inner tube, adding 400uL PBS (containing 0.01% NaN) 3 ) Centrifuging 14000g for 10 min; 4.2 pouring the liquid in the collecting tube into the waste liquid tank, adding 500uL PBS (containing 0.01% NaN) into the inner tube 3 ) Centrifuging 14000g for 10min, discarding the waste liquid in the collecting pipe, and repeating the test step for 5-6 times until the color of the waste liquid in the collecting pipe is not displayed as green;
4.3 pouring the waste liquid in the collecting tube, placing the inner tube of the ultrafiltration tube into the collecting tube for 2000g of 2min for centrifugation, sucking the liquid in the collecting tube by using a liquid transfer device, transferring the liquid into a new centrifuge tube, estimating the liquid volume (the liquid volume is about 20-40 uL), and adding a certain amount of PBS (containing 0.01% NaN) 3 ) The final concentration of the antibody is 0.5mg/mL;4.4 rinsing the finished ultrafiltration centrifuge tube with PBS and centrifuging to dry the residual liquid in the tube. 1mL of 20% ethanol is added into a collecting pipe, 500uL of 20% ethanol is added into an inner pipe, a cover is covered, and an ultrafiltration centrifuge tube is placed in a refrigerator at 4 ℃ for storage; 5. the antibody is put in a refrigerator at 4 ℃ for preservation, and information such as the name of the antibody, the name of the fluorescent dye, the concentration of the antibody, the volume of the antibody, the purification date and the like is marked. In other embodiments of the present invention, the step 2) specifically includes: 1. adding 1/10 of 1M sodium bicarbonate buffer to the pre-reaction antibody solution; 2. adding 10uL of FITC fluorescent dye into the protein solution prepared in the step 1, slightly shaking to enable the dye to be fully dissolved and not shaking vigorously, placing the solution in the environment of 25 ℃ in a dark place, and reacting for 1h; 3. separating and purifying by an ultrafiltration centrifuge tube; 4.1, loading the inner tube of a 0.5mL 50kd ultrafiltration centrifuge tube into a collecting tube, transferring all the solution after the reaction into the inner tube of the ultrafiltration centrifuge tube, supplementing 400uL PBS (containing 0.01% NaN 3), and centrifuging 14000g for 10 min; 4.2 pouring the liquid in the collecting pipe into a waste liquid barrel, adding 500uL PBS (containing 0.01% NaN 3) into the inner pipe, centrifuging for 14000g for 10min, discarding the waste liquid in the collecting pipe, and repeating the test step for 5-6 times until the color of the waste liquid in the collecting pipe is not displayed as green; 4.3, pouring the waste liquid in the collecting pipe cleanly, inversely placing the inner pipe of the ultrafiltration pipe into the collecting pipe for 2000g of 2min for centrifugation, sucking the liquid in the collecting pipe by using a liquid transfer device, transferring the liquid into a new centrifuge pipe, estimating the liquid volume (the liquid volume is about 20-40 uL), and adding a certain amount of PBS (containing 0.01% NaN 3) into the liquid to ensure that the final concentration of the antibody is 0.5mg/mL;4.4 flushing the ultrafiltration centrifuge tube with PBS, centrifuging and drying the residual liquid in the tube; 1mL of 20% ethanol is added into a collecting pipe, 500uL of 20% ethanol is added into an inner pipe, a cover is covered, and an ultrafiltration centrifuge tube is placed in a refrigerator at 4 ℃ for storage; 5. placing the antibody in a refrigerator at 4deg.C for preservationAnd labeling information such as antibody names, fluorescent dye names, antibody concentrations, antibody volumes, purification dates and the like.
Another object of the present invention is to provide a method for increasing the signal of a fluorescent dye for protein labeling, which is characterized in that the method comprises the following steps:
step 1) the sulfhydrylation of a protein; step 2) labelling the protein with a fluorescent dye.
In some preferred embodiments, the thiol groups on the protein are aminated by means of a haloalkylamine; preferably, the haloalkylamine is selected from the group consisting of bromoethylamine, chloroethylamine, iodoethylamine.
It is a further object of the present invention to provide a thiol-amino derivative of an antibody or a thiol-amino derivative of a protein obtainable by the method as described above.
Still another object of the present invention is to apply the above method to the industrial production of antibodies, proteins, raw materials reagents and raw materials.
According to the invention, through the amination reaction of the sulfhydryl of the antibody or the protein, the efficiency of fluorescent marking and the final fluorescent staining signal are obviously improved. Through amination reaction of sulfhydryl groups of the antibody or protein, binding sites of the antibody or protein and small molecule dyes such as FITC, iFluor, alexa fluor and the like (succinimidyl ester of the small molecule dye reacts with free amino groups on the protein or antibody to form amide bond covalent coupling) are increased, so that the number of fluorescent dyes marked on a single antibody or protein is increased, and fluorescent marking efficiency and final fluorescent staining signals are improved.
Drawings
FIG. 1 is a graph showing the signal of iFluor 488-labeled CD4 antibody of comparative example 1 of the present invention;
FIG. 2 is a graph showing the signal of iFluor 488-labeled CD4 antibody in example 1 of the present invention;
FIG. 3 is a chart showing FITC-labeled signals of CD4 antibodies in comparative example 2 of the present invention;
FIG. 4 is a chart showing FITC-labeled signals of CD4 antibodies in example 2 of the present invention.
Detailed Description
The term "Ab" as used herein means: an antibody;
"SMCC" means: n-hydroxysuccinimide ester of 4- (N-maleimidomethyl) cyclohexanecarboxylic acid
"FITC" means: fluorescein isothiocyanate;
"iFluor" means: the iFluor series of highly water-soluble fluorescent dyes produced by AAT Bioquest corporation;
reagents used in the following embodiments:
1. anti-mouse CD4 monoclonal antibody [ GK1.5] at a concentration of 1-5 mg/mL;
2.1M sodium bicarbonate (molecular weight: 84.01):
84mg of sodium bicarbonate solid was weighed and dissolved in 1mL of deionized water; sodium bicarbonate cannot be stored for a long time.
3.10% sodium azide (NaN 3) solution:
2g of sodium azide solid was weighed and dissolved in 20mL deionized water. When weighing sodium azide, the toxicity of the sodium azide needs to be paid attention to, and the direct contact of the sodium azide by hands is avoided, so that the skin contact is avoided.
Pbs (0.01% sodium azide) buffer:
500uL of 10% sodium azide solution was added to 500mL of PBS.
FITC dye liquor preparation:
accurately weighing 5mg of FITC powder, dissolving the powder into 0.5mL of DMSO, uniformly mixing the powder and preparing 10mg/mL of FITC dye solution. Split charging according to the amount of 10 mu L/branch; and freezing at-20deg.C in dark place.
Preparation of ifluor fluorescent dye:
the iFluor fluorescent dye has a specification of 1mg per tube and a molecular weight of 1000, 100uL of DMSO per tube is added to a final concentration of 10mM, and the mixture is split into 5uL per tube and stored at-20deg.C.
Example 1 protocol for iFluor fluorescent labelling
1. Antibody pre-reaction with bromoethylamine
Step 1) taking out 50mM bromoethylamine from a low temperature state, placing the bromoethylamine in a room temperature environment, and opening a bottle cap when the temperature of the bottle is balanced to room temperature so as to avoid condensed water in the bottle.
Step 2) 100uL of 1M sodium bicarbonate solution was added to 200ug of antibody, and after mixing well, 1.4uL of 50mM bromoethylamine solution (n (Ab): n (SMCC) ≡1): 50 (if the total amount of the selected antibody is increased, the adding amount of the bromoethylamine can be correspondingly increased), and the reaction is carried out overnight at 25 ℃ so that the bromoethylamine molecules react with free sulfhydryl groups in Ab molecules.
Step 3) transferring the solution after the reaction to an ultrafiltration centrifuge tube, adding 500uL PBS (containing 0.25mM EDTA) buffer solution for 5min, centrifuging 12000g, removing filtrate, adding 500uL PBS (containing 0.25mM EDTA) buffer solution, mixing, centrifuging, repeating the operation for 5 times, and desalting to make the residual amount of unreacted bromoethylamine in the antibody be at least 10% of the total amount of the antibody -3 More than two times, as much dilution as possible.
Step 4) collecting the desalted high-concentration pre-reacted antibody protein solution, and taking the volume of the collected solution by a pipette, wherein the volume of the antibody solution is fixed to 100uL (2 mg/mL).
2. Antibody and fluorescent dye labeling
Step 1) 1/10 of 1M sodium bicarbonate buffer was added to the pre-reacted antibody solution.
Step 2) iFluor fluorochromes were 1mg per tube, 1000 molecular weight, 100uL DMSO per tube was added to a final concentration of 10mM, and the mixture was split into 5uL per tube and stored at-20 ℃.
Step 3) 1.5uL of fluorescent dye was added to the protein solution prepared in step 2 (n (dye): n (protein) =15:1), gently shaking to dissolve the dye sufficiently, and not shaking vigorously. The solution was placed in the environment at 25℃in the dark and reacted for 1h.
Step 4) separation and purification of the ultrafiltration centrifuge tube (the gun head of the liquid transfer device does not need to contact the filter membrane of the inner tube of the ultrafiltration tube in the whole operation process so as to avoid puncturing the filter membrane)
Step 4.1) charging the inner tube of a 0.5mL 50kd ultrafiltration centrifuge tube into a collecting tube, transferring the whole solution after the reaction into the inner tube of the ultrafiltration centrifuge tube, and supplementing 400uL PBS (containing 0.01% NaN) 3 ) Centrifuge 14000g for 10 min.
Step 4.2) pouring the liquid in the collecting pipe into a waste liquid barrel, adding 500uL PBS (containing 0.01% NaN 3) into the inner pipe, centrifuging for 14000g for 10min, discarding the waste liquid in the collecting pipe, and repeating the test step 5-6 times until the color of the waste liquid in the collecting pipe is not displayed as green.
Step 4.3) pouring the waste liquid in the clean collecting pipe, inversely placing the inner pipe of the ultrafiltration pipe into the collecting pipe for 2000g of 2min for centrifugation, sucking the liquid in the collecting pipe by using a liquid transfer device, transferring the liquid into a new centrifuge pipe, estimating the liquid volume (the liquid volume is about 20-40 uL), and adding a certain amount of PBS (containing 0.01% NaN) into the liquid 3 ) The final concentration of antibody was set to 0.5mg/mL.
Step 4.4) rinsing the finished ultrafiltration centrifuge tube with PBS, and centrifuging to dry the residual liquid in the tube. 1mL of 20% ethanol was added to the collection tube, 500uL of 20% ethanol was added to the inner tube and the cap was closed, and the ultrafiltration centrifuge tube was stored in a refrigerator at 4 ℃.
And 5) placing the antibody in a refrigerator at 4 ℃ for preservation, and marking information such as antibody names, fluorescent dye names, antibody concentration, antibody volume, purification date and the like.
EXAMPLE 2 FITC fluorescent labelling protocol
1. Antibody pre-reaction with bromoethylamine
Step 1) taking out 50mM bromoethylamine from a low temperature state, placing the bromoethylamine in a room temperature environment, and opening a bottle cap when the temperature of the bottle is balanced to room temperature so as to avoid condensed water in the bottle.
Step 2) 100uL of 1M sodium bicarbonate solution was added to 200ug of antibody, and after mixing well, 1.4uL of 50mM bromoethylamine solution (n (Ab): n (bromoethylamine) ≡1:50 (if the total amount of the selected antibody is increased, the adding amount of the bromoethylamine can be correspondingly increased), and the reaction is carried out overnight at 25 ℃ so that the bromoethylamine molecules react with free sulfhydryl groups in Ab molecules.
Step 3) transferring the solution after the reaction to an ultrafiltration centrifuge tube, adding 500uL PBS (containing 0.25mM EDTA) buffer solution for 5min, centrifuging 12000g, removing filtrate, adding 500uL PBS (containing 0.25mM EDTA) buffer solution, mixing, centrifuging, repeating the operation for 5 times, and desalting to make the residual amount of unreacted bromoethylamine in the antibody be at least 10% of the total amount of the antibody -3 More than two times, as much dilution as possible.
Step 4) collecting the desalted high-concentration pre-reacted antibody protein solution, and taking the volume of the collected solution by a pipette, wherein the volume of the antibody solution is fixed to 100uL (2 mg/mL).
2. Antibody and FITC fluorescent dye label
Step 1) 1/10 of 1M sodium bicarbonate buffer was added to the pre-reacted antibody solution.
Step 2) to the protein solution prepared in step 1, 10uL of FITC fluorescent dye was added, and the dye was sufficiently dissolved by gentle shaking, and could not be shaken vigorously. The solution was placed in the environment at 25℃in the dark and reacted for 1h.
Step 3) separation and purification of the ultrafiltration centrifuge tube (the gun head of the liquid transfer device does not need to contact the filter membrane of the inner tube of the ultrafiltration tube in the whole operation process so as to avoid puncturing the filter membrane)
Step 4.1) 0.5mL of 50kd ultrafiltration tube inner tube was placed in a collection tube, and the whole solution after completion of the reaction was transferred into the ultrafiltration tube inner tube, 400uL of PBS (containing 0.01% NaN 3) was added, and 14000g was centrifuged for 10 min.
Step 4.2) pouring the liquid in the collecting pipe into a waste liquid barrel, adding 500uL PBS (containing 0.01% NaN 3) into the inner pipe, centrifuging for 14000g for 10min, discarding the waste liquid in the collecting pipe, and repeating the test step 5-6 times until the color of the waste liquid in the collecting pipe is not displayed as green.
Step 4.3) pouring the waste liquid in the clean collecting pipe, inversely placing the inner pipe of the ultrafiltration pipe into the collecting pipe for 2000g of 2min for centrifugation, sucking the liquid in the collecting pipe by using a liquid transfer device, transferring the liquid into a new centrifugal pipe, estimating the liquid volume (the liquid volume is about 20-40 uL), and adding a certain amount of PBS (containing 0.01% NaN 3) into the liquid to ensure that the final concentration of the antibody is 0.5mg/mL.
Step 4.4) rinsing the finished ultrafiltration centrifuge tube with PBS, and centrifuging to dry the residual liquid in the tube. 1mL of 20% ethanol was added to the collection tube, 500uL of 20% ethanol was added to the inner tube and the cap was closed, and the ultrafiltration centrifuge tube was stored in a refrigerator at 4 ℃.
And 5) placing the antibody in a refrigerator at 4 ℃ for preservation, and marking information such as antibody names, fluorescent dye names, antibody concentration, antibody volume, purification date and the like.
Comparative example 1 protocol for iFluor fluorescent labelling
Step 1): 1M sodium bicarbonate solution is prepared in advance, and the sodium bicarbonate solution can be stored in a refrigerator at 4 ℃ for 2 weeks, so that the sodium bicarbonate solution is easy to decompose after long time.
Step 2) the protein concentration was diluted to 2mg/mL with PBS (containing 0.01% sodium azide) buffer, approximately 0.01mM, and then 1/10 of 1M sodium bicarbonate buffer was added to the protein solution.
Take the CD4 (GK 1.5 clone) antibody as an example: purification was performed on day 14 of 8.2018, with a protein concentration of 2.5mg/mL, an antibody purity of 91% and an antibody concentration of 2.275mg/mL.
88uL of 2.5mg/mL of antibody solution was taken, 12uL of PBS (containing 0.01% sodium azide) buffer was added, diluted to 100uL of 1mg/mL of antibody solution, and 10uL of sodium bicarbonate solution was added to the solution and mixed well.
Step 3): the iFluor fluorescent dye has a specification of 1mg per tube and a molecular weight of 1000, 100uL of DMSO per tube is added to a final concentration of 10mM, and the mixture is split into 5uL per tube and stored at-20deg.C.
Step 4): 1.5uL of fluorescent dye (n (dye): n (protein) =15:1) was added to the protein solution prepared in step 2, and the dye was sufficiently dissolved by gentle shaking, and could not be vigorously shaken. The solution was placed in a room temperature environment in the dark, gently shaken every 10min, and reacted for 1h.
Step 5): the square card blocked at the pipe orifice at the bottom of the purification column is forcibly rotated and screwed off, a white round gasket is placed at the bottom of the purification column (two gaskets should be placed, a thinner gasket is placed at the lowest part, and the other gasket is placed on the thinner gasket), and the gasket can be lightly pressed by a pipette head or forceps. Firstly, adding 2mL of filler suspension into a purification column, placing the purification column into a centrifuge, centrifuging for 3min at 1100g, removing filler preservation solution in the tube (the purification column can be placed on a 1.5mL centrifuge tube with a cover removed and a tube bottom cut for centrifugation), then adding 0.5-1mL of filler suspension into the tube, centrifuging for 3min at 1100g, and removing the filler preservation solution. The thickness of the filler in the tube should be about 1.5mL, and finally the tube is centrifuged once at 1100g for 3min to remove the remaining filler preservation solution in the filler. ( This step may begin at the end of the reaction of the protein with the fluorochrome and if prepared too early, the interior of the filling may be easily dried, which may result in less volume of the final collected tagged protein remaining in the seasoning. The purification column may be placed in a 50mL centrifuge tube during centrifugation. )
And 6) after the reaction of the protein and the fluorescent dye is completed, adding the mixed solution of the protein and the fluorescent dye into a purification column, placing a collecting pipe at the bottom of the purification column pipe, centrifuging for 5min at 1100g, and enabling the marked protein to exist in the collecting pipe at the bottom.
And 7) placing the antibody in the collecting pipe in a refrigerator at 4 ℃ for storage, and marking information such as the name of the antibody, the name of the fluorescent dye, the concentration of the antibody, the volume of the antibody, the purification date and the like on the collecting pipe.
Comparative example 2 monoclonal antibody FITC labelling Standard procedure
Step 1) taking 0.1mL of monoclonal antibody, adding 10 mu L of 1M sodium bicarbonate solution, and uniformly mixing to prepare monoclonal antibody reaction solution.
Step 2) taking 10uL of FITC dye solution, adding the 10uL of FITC dye solution into the monoclonal antibody reaction solution, and uniformly mixing.
And 3) carrying out a light-shielding reaction for 1h at room temperature, and uniformly mixing every 10 min.
Step 4) separation and purification of the ultrafiltration centrifuge tube (the gun head of the liquid transfer device does not need to contact the filter membrane of the inner tube of the ultrafiltration tube in the whole operation process so as to avoid puncturing the filter membrane)
Step 4.1) 0.5mL of 50kd ultrafiltration tube inner tube was placed in a collection tube, and the whole solution after completion of the reaction was transferred into the ultrafiltration tube inner tube, 400uL of PBS (containing 0.01% NaN 3) was added, and 10000g was centrifuged for 5 min.
Step 4.2) pouring the liquid in the collecting pipe into a waste liquid barrel, adding 500uL PBS (containing 0.01% NaN 3) into the inner pipe, centrifuging for 14000g for 10min, discarding the waste liquid in the collecting pipe, and repeating the test step for 5-6 times until the color of the waste liquid in the collecting pipe is not displayed as green.
Step 4.3) pouring the waste liquid in the clean collecting pipe, inversely placing the inner pipe of the ultrafiltration pipe into the collecting pipe for 2000g of 2min for centrifugation, sucking the liquid in the collecting pipe by using a liquid transfer device, transferring the liquid into a new centrifugal pipe, estimating the liquid volume (the liquid volume is about 20-40 uL), and adding a certain amount of PBS (containing 0.01% NaN 3) into the liquid to ensure that the final concentration of the antibody is 1mg/mL.
Step 4.4) rinsing the finished ultrafiltration centrifuge tube with PBS, and centrifuging to dry the residual liquid in the tube. 1mL of 20% ethanol was added to the collection tube, 500uL of 20% ethanol was added to the inner tube and the cap was closed, and the ultrafiltration centrifuge tube was stored in a refrigerator at 4 ℃.
Step 5) storing the marked product at 4 ℃ in a dark place.
Test example antibody-positive fluorescent Signal test
Antibody positive fluorescent signal test method: the fluorescence-labeled antibodies (bromoethylamine-treated CD4-iFluor488, bromoethylamine-treated CD4-FITC, untreated CD4-iFluor488, untreated CD 4-FITC) were co-stained with CD3-PE on 1million mouse spleen cells at the same concentration (0.25 ug of labeled CD4 antibody and 0.125ug of CD3-PE antibody were added to each reaction system) and after 30 minutes staining, fluorescence signals were detected by flow cytometry.
With anti-mouse CD4 monoclonal antibody [ GK1.5] as target protein, after pretreatment with bromoethylamine, the CD4 antibody was iflur 488-labeled (fig. 2), fig. 1 shows that direct iflur-labeled CD4 antibody was not bromoethylamine-treated, and that the iflur-labeled CD4 antibody positive fluorescent signal was stronger after bromoethylamine-treatment (compare fig. 1 with upper right-hand signal values of fig. 2, experiments representing at least 3 replicates of each group, at least 2 samples of each group, and mice spleen cells were co-stained with CD3-PE and labeled CD 4-iflur 488 in the experiments.
FITC-labeled CD4 antibodies were directly labeled with anti-mouse CD4 monoclonal antibody [ GK1.5] as target protein after pretreatment with bromoethylamine (FIG. 4), and FITC-labeled CD4 antibodies were directly labeled with non-bromoethylamine (FIG. 4), and FITC-labeled CD4 antibodies were positive with stronger fluorescent signals after treatment with bromoethylamine (see FIG. 3 versus upper right corner signal values of FIG. 4, experiments representing at least 3 replicates, at least 2 samples per set of experiments, and mouse spleen cells were co-stained with CD3-PE and labeled CD4-FITC in the experiments).
Table 1:
from the above table 1, it can be known that, by comparing the CD 4-labeled positive signal values, we can find that by performing an amination reaction on the thiol group of the antibody, the CD 4-labeled positive signal value can be significantly and greatly improved, and the efficiency and signal of fluorescent labeling of the antibody can be improved.
Claims (10)
1. A method for increasing the signal of an antibody fluorescent dye, comprising the steps of:
step 1) amination of sulfhydryl groups of an antibody, wherein sulfhydryl groups on the antibody undergo an amination reaction through haloalkylamine; the haloalkylamine is selected from bromoethylamine, chloroethylamine and iodoethylamine;
step 2) labelling the antibody with a fluorescent dye.
2. The method according to claim 1, wherein the step 1) specifically includes: adding sodium bicarbonate solution into the antibody, uniformly mixing, and then adding bromoethylamine solution for reaction, so that bromoethylamine molecules react with free sulfhydryl groups of the antibody; transferring the solution after the reaction to an ultrafiltration centrifuge tube, adding PBS buffer solution, centrifuging, removing filtrate, adding PBS buffer solution, mixing, centrifuging, and desalting to make the residual amount of unreacted bromoethylamine in the antibody be at least 10% of the total amount of the antibody -3 More than two times.
3. The method of claim 1, wherein the fluorescent dye is FITC, iFluor-based dye, alexafluor-based dye.
4. The method of claim 3, wherein 100uL of 1m sodium bicarbonate solution is added to the 200ug antibody, and after mixing, 1.4uL of 50mm bromoethylamine solution is added and reacted overnight at 25 ℃ to react the bromoethylamine molecules with free sulfhydryl groups in the Ab molecules; after the completion of the reaction, the solution was transferred to an ultrafiltration centrifuge tube, and 500. Mu.L of PBS buffer containing 0.25mM EDTA was added thereto for 5min, and 12000 and g were separatedRemoving the filtrate, adding 500uL PBS buffer containing 0.25mM EDTA, mixing, centrifuging, repeating the operation for 5 times, desalting to give residual amount of unreacted bromoethylamine in the antibody of at least 10% of the total amount of the antibody -3 More than two times, as much dilution as possible.
5. The method of claim 3, wherein,
the step 2) specifically comprises the following steps:
1. adding 1/10 of 1M sodium bicarbonate buffer to the pre-reaction antibody solution;
2. adding fluorescent dye into the antibody solution prepared in the step 1, and slightly shaking to enable the dye to be fully dissolved; the solution is placed in the environment of 25 ℃ in a dark place for reaction for 1h;
4. separating and purifying by an ultrafiltration centrifuge tube;
4.1, filling an inner tube of a 0.5mL 50kd ultrafiltration centrifuge tube into a collecting tube, completely transferring the solution after the reaction into the inner tube of the ultrafiltration centrifuge tube, and supplementing 400uL with 0.01 percent NaN 3 Is centrifuged for 14000g and 10 min;
4.2 pouring the liquid in the collecting tube into a waste liquid barrel, adding 500uL of NaN 0.01% into the inner tube 3 Centrifuging 14000g for 10min, discarding the waste liquid in the collecting pipe, and repeating the test step for 5-6 times until the color of the waste liquid in the collecting pipe is not displayed as green;
4.3, pouring the waste liquid in the clean collecting pipe, inversely placing the inner pipe of the ultrafiltration pipe into the collecting pipe for centrifugation for 2000g 2min, sucking the liquid in the collecting pipe by using a liquid transfer device, transferring the liquid into a new centrifuge pipe, estimating the volume of the liquid, and supplementing a certain amount of NaN containing 0.01 percent 3 To a final antibody concentration of 0.5mg/mL;
4.4, flushing the ultrafiltration centrifuge tube which is completed by using PBS, and centrifuging and drying residual liquid in the tube; 1mL of 20% ethanol is added into a collecting tube, 500uL of 20% ethanol is added into an inner tube, a cover is covered, and an ultrafiltration centrifuge tube is placed in a refrigerator at 4 ℃ for storage;
5. the antibodies were stored in a refrigerator at 4 ℃ and labeled with the antibody name, fluorescent dye name, antibody concentration, antibody volume and purification date information.
6. An antibody thiol-aminated derivative obtained by the method according to any one of claims 1 to 5.
7. The method according to any one of claims 1 to 5, wherein the method is used for the industrial production of antibodies and drug substances.
8. A method for increasing the signal of a protein fluorescent dye, the method comprising the steps of:
step 1) amination of sulfhydryl groups of a protein, wherein the sulfhydryl groups on the protein undergo an amination reaction through haloalkylamine; the haloalkylamine is selected from bromoethylamine, chloroethylamine and iodoethylamine;
step 2) labelling the protein with a fluorescent dye.
9. A thiol-amino derivative of a protein obtainable by the process of claim 8.
10. The use of the method of claim 8 in the industrial production of proteins and pharmaceutical substances.
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