CN111551725A - Method for improving antibody fluorescent dye labeling signal - Google Patents

Method for improving antibody fluorescent dye labeling signal Download PDF

Info

Publication number
CN111551725A
CN111551725A CN202010279571.9A CN202010279571A CN111551725A CN 111551725 A CN111551725 A CN 111551725A CN 202010279571 A CN202010279571 A CN 202010279571A CN 111551725 A CN111551725 A CN 111551725A
Authority
CN
China
Prior art keywords
antibody
tube
solution
bromoethylamine
pbs
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN202010279571.9A
Other languages
Chinese (zh)
Other versions
CN111551725B (en
Inventor
张洋
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zhejiang Zhengxi Biotechnology Co ltd
Original Assignee
Zhejiang Zhengxi Biomedical Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Zhejiang Zhengxi Biomedical Co ltd filed Critical Zhejiang Zhengxi Biomedical Co ltd
Priority to CN202010279571.9A priority Critical patent/CN111551725B/en
Publication of CN111551725A publication Critical patent/CN111551725A/en
Application granted granted Critical
Publication of CN111551725B publication Critical patent/CN111551725B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
    • Y02A40/81—Aquaculture, e.g. of fish

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Physics & Mathematics (AREA)
  • Biomedical Technology (AREA)
  • General Physics & Mathematics (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Optics & Photonics (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention aims to provide a method for improving a fluorescent dye labeling signal of an antibody, which comprises the following steps: step 1) aminating the sulfhydryl group of the antibody; step 2) labeling the antibody with a fluorescent dye; according to the invention, free amino is added to free sulfhydryl on protein or antibody by a sulfydryl amination method, and then micromolecule fluorescent labeling is carried out, so that the fluorescent labeling efficiency and the final fluorescent staining signal are improved.

Description

Method for improving antibody fluorescent dye labeling signal
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
According to the traditional labeling reaction, succinimide ester of micromolecule fluorescent dye (FITC, iFluor series dye, alexafluor series dye and the like) reacts with free amino on the protein or the antibody to form amido bond covalent coupling, and the dyeing fluorescent effect is limited by the number of the free amino on the protein or the antibody.
Disclosure of Invention
The inventor adds free amino groups on free sulfydryl on protein or antibody by sulfydryl amination method and then carries out small molecule fluorescent labeling, thereby improving the efficiency of fluorescent labeling and the final fluorescent staining signal. Based on this, the present invention aims to provide a method for increasing the signal of a fluorescent dye label of an antibody, comprising the steps of:
step 1) aminating the sulfhydryl group of the antibody; step 2) labeling the antibody with a fluorescent dye.
In some embodiments, the thiol group on the antibody is aminated with a haloalkylamine.
In some embodiments, the haloalkylamine is selected from bromoethylamine, chloroethylamine, iodoethylamine; in some embodiments of the invention, bromoethylamine is used to carry out an alkylation reaction with an antibody, as follows:
Figure BDA0002446049020000011
in some embodiments, the fluorescent dye is a FITC, iFluor-based dye, an alexafluor-based dye.
In some embodiments, said step 1) comprises in particular: adding a sodium bicarbonate solution into the antibody, uniformly mixing, and then adding a bromoethylamine solution for reaction to ensure that bromoethylamine molecules react with free sulfydryl of the antibody;
transferring the solution after the reaction into an ultrafiltration centrifugal tube, adding PBS buffer solution, centrifuging, removing filtrate, adding PBS buffer solution, mixing, centrifuging, desalting to make the residual amount of unreacted bromoethylamine in the antibody at least 10 of the total amount of the antibody-3Doubling the weight; in some embodiments, 200ug of antibody was added with 100uL of 1M sodium bicarbonate solution, mixed well and then added with 1.4uL of 50mM bromoethylamine solution and reacted overnight at 25 ℃ to allow the bromoethylamine molecules to react with the free thiol groups in the Ab molecules;
transferring the solution after the reaction to an ultrafiltration centrifugal tube, adding 500uL of PBS (containing 0.25mM EDTA) buffer solution for 5min, centrifuging at 12000g, removing the filtrate, adding 500uL of PBS (containing 0.25mM EDTA) buffer solution, mixing, centrifuging, repeating the operation for 5 times, desalting to obtain the residual content of unreacted bromoethylamine in the antibody of at least 10% of the total amount of the antibody-3More than twice, and diluting as much as possible.
In some embodiments, the step 2) specifically includes: 1. adding 1/10 of 1M sodium bicarbonate buffer to the pre-reacted antibody solution; 2. the specification of the iFluor fluorescent dye is 1mg per tube, the molecular weight is 1000, 100uLDMSO is added into each tube, the final concentration is 10mM, and the mixture is prepared byIt is packaged into 5uL per tube, and stored at-20 deg.C; 3. adding 1.5uL of fluorescent dye into the protein solution prepared in the step 2 (n (dye): n (protein) ═ 15:1), and slightly shaking to fully dissolve the dye; placing the solution in a dark environment at 25 ℃ for reaction for 1 h; 4. separating and purifying by an ultrafiltration centrifugal tube; 4.1 put 0.5mL 50kd ultrafiltration centrifuge tube inner tube into a collection tube, transfer all the reacted solution into the ultrafiltration centrifuge tube inner tube, and supplement 400uL PBS (containing 0.01% NaN)3)14000g for 10 min; 4.2 pour the liquid in the collection tube into the waste liquid bucket, add 500uL PBS (containing 0.01% NaN) into the inner tube3)14000g for 10min, discarding the waste liquid in the collecting tube, repeating the test step for 5-6 times until the color of the waste liquid in the collecting tube is not green;
4.3 the waste liquid in the collecting tube is completely poured, the tube in the ultrafiltration tube is placed upside down in the collecting tube for 2000g 2min for centrifugation, the liquid in the collecting tube is sucked out by a pipette and transferred into a new centrifugal tube, the liquid volume is estimated (the liquid volume is about 20-40 uL), and a certain amount of PBS (containing 0.01 percent NaN) is added into the liquid volume3) The final concentration of the antibody was set to 0.5 mg/mL; 4.4 rinsing the used ultrafiltration centrifuge tube with PBS and centrifuging to spin-dry the residual liquid in the tube. Adding 1mL of 20% ethanol into the collection tube, adding 500uL of 20% ethanol into the inner tube, covering the inner tube with a cover, and placing the ultrafiltration centrifugal tube in a refrigerator at 4 ℃ for storage; 5. and (3) storing the antibody in a refrigerator at 4 ℃, and labeling the information such as the name of the antibody, the name of a fluorescent dye, the concentration of the antibody, the volume of the antibody, the purification date and the like. In other specific embodiments of the present invention, the step 2) specifically includes: 1. adding 1/10 of 1M sodium bicarbonate buffer to the pre-reacted antibody solution; 2. adding the protein solution prepared in the step 1 into 10uL FITC fluorescent dye, slightly shaking to fully dissolve the dye, not shaking violently, placing the solution in an environment at 25 ℃ in a dark place, and reacting for 1 h; 3. separating and purifying by an ultrafiltration centrifugal tube; 4.1, putting 0.5mL of 50kd inner tube of an ultrafiltration centrifugal tube into a collecting tube, transferring all the solution after the reaction into the inner tube of the ultrafiltration centrifugal tube, supplementing 400uL of PBS (containing 0.01 percent of NaN3), and centrifuging for 14000g for 10 min; 4.2 pour the liquid in the collection tube into the waste liquid barrel, add 500uL into the inner tubePBS (containing 0.01% NaN3), centrifuging at 14000g for 10min, discarding the waste liquid in the collecting tube, and repeating the test step for 5-6 times until the color of the waste liquid in the collecting tube is not green; 4.3, completely pouring the waste liquid in the collecting tube, inverting the tube in the ultrafiltration tube, putting the tube in the collecting tube for 2000g and 2min for centrifugation, sucking out the liquid in the collecting tube by using a pipette, transferring the liquid into a new centrifugal tube, estimating the volume of the liquid (the volume of the liquid is about 20-40 uL), and supplementing a certain amount of PBS (containing 0.01 percent NaN3) into the new centrifugal tube to ensure that the final concentration of the antibody is 0.5 mg/mL; 4.4 washing the used ultrafiltration centrifugal tube by PBS, and centrifugally drying the residual liquid in the tube; adding 1mL of 20% ethanol into the collection tube, adding 500uL of 20% ethanol into the inner tube, covering the inner tube with a cover, and placing the ultrafiltration centrifugal tube in a refrigerator at 4 ℃ for storage; 5. and (3) storing the antibody in a refrigerator at 4 ℃, and labeling the information such as the name of the antibody, the name of a fluorescent dye, the concentration of the antibody, the volume of the antibody, the purification date and the like.
Another object of the present invention is to provide a method for increasing a signal of a fluorescent dye label of a protein, which comprises the steps of:
step 1) carrying out sulfydryl amination on protein; step 2) labeling the protein with a fluorescent dye.
In some preferred embodiments, the thiol group on the protein is aminated with a haloalkylamine; preferably, the haloalkylamine is selected from bromoethylamine, chloroethylamine, iodoethylamine.
It is still another object of the present invention to provide a mercaptoaminated derivative of an antibody or a mercaptoaminated derivative of a protein obtained by the method as described above.
The invention also aims to apply the method to the industrial production of antibodies, proteins, raw material reagents and raw material medicaments.
According to the invention, through carrying out amination reaction on the sulfhydryl group of the antibody or the protein, the efficiency of fluorescent labeling and the final fluorescent staining signal are obviously improved. Through carrying out amination reaction on sulfydryl of the antibody or protein, the binding sites of the antibody or protein and small molecular dyes such as FITC, iFluor and alexa Fluor are increased (succinimide ester of the small molecular dyes reacts with free amino on the protein or the antibody to form amido bond covalent coupling), so that the number of fluorescent dyes marked on a single antibody or protein is increased, and the fluorescent marking efficiency and the final fluorescent staining signal are improved.
Drawings
FIG. 1 is a graph showing the signal of iFluor488 labeling of the CD4 antibody in comparative example 1;
FIG. 2 is a signal diagram of iFluor488 labeling of the CD4 antibody in example 1 of the present invention;
FIG. 3 is a FITC labeling signal diagram for the CD4 antibody in comparative example 2 of the present invention;
FIG. 4 is a FITC labeling signal diagram for the CD4 antibody in example 2 of the present invention.
Detailed Description
The term "Ab" as used herein means: an antibody;
"SMCC" means: 4- (N-Maleimidomethyl) cyclohexanecarboxylic acid N-hydroxysuccinimide ester
"FITC" refers to: fluorescein isothiocyanate;
"iFluor" means: iFluor series highly water-soluble fluorescent dyes manufactured by AAT Bioquest corporation;
reagents used in the following embodiments:
1. an anti-mouse CD4 monoclonal antibody [ GK1.5] with the concentration of 1-5 mg/mL;
2.1M sodium bicarbonate (molecular weight: 84.01):
weighing 84mg of sodium bicarbonate solid, and dissolving in 1mL of deionized water; sodium bicarbonate is not storable for a long time.
3.10% sodium azide (NaN3) solution:
2g of sodium azide solid was weighed and dissolved in 20mL of deionized water. When sodium azide is weighed, the sodium azide needs to be toxic and does not need to be directly contacted by hands, so that the contact with the skin is avoided.
PBS (0.01% sodium azide) buffer:
to 500mL of PBS was added 500uL of 10% sodium azide solution.
Preparing a FITC dye solution:
5mg of FITC powder is accurately weighed, dissolved in 0.5mL of DMSO, and mixed uniformly to prepare 10mg/mL of FITC dye solution. Subpackaging according to 10 μ L/branch; and (4) freezing and storing at minus 20 ℃ in a dark place.
Preparation of iFluor fluorescent dye:
the specification of the iFluor fluorescent dye is 1mg per tube, the molecular weight is 1000, 100uL DMSO is added into each tube to dissolve the dye until the final concentration is 10mM, the dye is subpackaged into 5uL per tube, and the dye is stored at the temperature of-20 ℃.
Example 1 iFluor fluorescent labeling protocol
First, pre-reaction of antibody 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 cover when the temperature of the bottle is balanced to the room temperature so as to prevent condensed water from appearing in the bottle.
Step 2) 100uL of 1M sodium bicarbonate solution was added to 200ug of antibody, mixed well and then 1.4uL of 50mM bromoethylamine solution (n (ab): n (SMCC) 1: 50) (if the total amount of the selected antibody is increased, the amount of bromoethylamine added is correspondingly increased), and the bromoethylamine molecule reacts with the free thiol in the Ab molecule at 25 ℃ overnight.
Step 3) transferring the solution after the reaction to an ultrafiltration centrifugal tube, adding 500uL PBS (containing 0.25mM EDTA) buffer solution for 5min, centrifuging 12000g, removing the filtrate, adding 500uL PBS (containing 0.25mM EDTA) buffer solution, mixing, centrifuging, repeating the operation for 5 times, desalting to ensure that the residual content of the unreacted bromoethylamine in the antibody is at least 10 of the total amount of the antibody-3More than twice, and diluting as much as possible.
And 4) collecting the desalted high-concentration pre-reaction antibody protein solution, measuring the volume of the collected solution by using a liquid transfer device, and fixing the volume of the antibody solution to be 100uL (2 mg/mL).
Second, antibody and fluorescent dye labeling
Step 1) to the pre-reacted antibody solution was added 1/10 of 1M sodium bicarbonate buffer.
And step 2) the specification of the iFluor fluorescent dye is 1mg per tube, the molecular weight is 1000, 100uL DMSO is added into each tube to dissolve the dye until the final concentration is 10mM, the dye is subpackaged into 5uL per tube, and the dye is placed at the temperature of minus 20 ℃ for storage.
Step 3) add 1.5uL of fluorescent dye to the protein solution prepared in step 2 (n (dye): n (protein) ═ 15:1), the dye was dissolved sufficiently with gentle shaking and vigorous shaking was not allowed. The solution is placed in an environment at 25 ℃ in the dark and reacted for 1 h.
Step 4) separating and purifying the ultrafiltration centrifugal tube (the pipette tip does not contact the filter membrane of the inner tube of the ultrafiltration tube in the whole operation process so as not to puncture the filter membrane)
Step 4.1) putting 0.5mL of 50kd inner tube of an ultrafiltration centrifugal tube into a collecting tube, transferring all the solution after reaction into the inner tube of the ultrafiltration centrifugal tube, and supplementing 400uL of PBS (containing 0.01% NaN)3)14000g for 10 min.
And 4.2) pouring the liquid in the collecting tube into a waste liquid barrel, adding 500uL PBS (containing 0.01 percent NaN3) into the inner tube, centrifuging for 14000g for 10min, discarding the waste liquid in the collecting tube, and repeating the test step for 5-6 times until the color of the waste liquid in the collecting tube is not green.
Step 4.3) emptying the waste liquid in the collecting tube, inverting the tube in the ultrafiltration tube, putting the tube in the collecting tube for 2000g 2min for centrifugation, sucking out the liquid in the collecting tube by a pipette, transferring the liquid into a new centrifugal 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 antibody concentration was set to 0.5 mg/mL.
And 4.4) washing the used ultrafiltration centrifugal tube by PBS and centrifugally drying 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, the lid was closed, and the ultrafiltration tube was placed in a refrigerator at 4 ℃ for storage.
And 5) storing the antibody in a refrigerator at 4 ℃, and labeling information such as antibody name, fluorescent dye name, antibody concentration, antibody volume, purification date and the like.
Example 2 FITC fluorescent labeling protocol
First, pre-reaction of antibody 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 cover when the temperature of the bottle is balanced to the room temperature so as to prevent condensed water from appearing in the bottle.
Step 2) 100uL of 1M sodium bicarbonate solution was added to 200ug of antibody, mixed well and then 1.4uL of 50mM bromoethylamine solution (n (ab): n (bromoethylamine) ≈ 1: 50) (if the total amount of the selected antibody is increased, the amount of bromoethylamine added is correspondingly increased), and the bromoethylamine molecule reacts with the free thiol in the Ab molecule at 25 ℃ overnight.
Step 3) transferring the solution after the reaction to an ultrafiltration centrifugal tube, adding 500uL PBS (containing 0.25mM EDTA) buffer solution for 5min, centrifuging 12000g, removing the filtrate, adding 500uL PBS (containing 0.25mM EDTA) buffer solution, mixing, centrifuging, repeating the operation for 5 times, desalting to ensure that the residual content of the unreacted bromoethylamine in the antibody is at least 10 of the total amount of the antibody-3More than twice, and diluting as much as possible.
And 4) collecting the desalted high-concentration pre-reaction antibody protein solution, measuring the volume of the collected solution by using a liquid transfer device, and fixing the volume of the antibody solution to be 100uL (2 mg/mL).
Second, antibody and FITC fluorescent dye label
Step 1) to the pre-reacted antibody solution was added 1/10 of 1M sodium bicarbonate buffer.
And 2) adding the protein solution prepared in the step 1 into 10uL FITC fluorescent dye, and slightly shaking to fully dissolve the dye, wherein the dye cannot be violently shaken. The solution is placed in an environment at 25 ℃ in the dark and reacted for 1 h.
Step 3) separating and purifying the ultrafiltration centrifugal tube (the pipette tip does not contact the filter membrane of the inner tube of the ultrafiltration tube in the whole operation process so as not to puncture the filter membrane)
Step 4.1) 0.5mL of 50kd inner tube of an ultrafiltration centrifugal tube is put into a collection tube, the solution after the reaction is completely transferred into the inner tube of the ultrafiltration centrifugal tube, 400uL of PBS (containing 0.01% NaN3) is added, and 14000g of solution is centrifuged for 10 min.
And 4.2) pouring the liquid in the collecting tube into a waste liquid barrel, adding 500uL PBS (containing 0.01 percent NaN3) into the inner tube, centrifuging for 14000g for 10min, discarding the waste liquid in the collecting tube, and repeating the test step for 5-6 times until the color of the waste liquid in the collecting tube is not green.
And 4.3) completely pouring the waste liquid in the collecting tube, inverting the tube in the ultrafiltration tube, putting the tube in the collecting tube for 2000g 2min for centrifugation, sucking out the liquid in the collecting tube by using a pipette, transferring the liquid into a new centrifugal tube, estimating the volume of the liquid (the volume of the liquid is about 20-40 uL), and supplementing a certain amount of PBS (containing 0.01% NaN3) into the new centrifugal tube to ensure that the final concentration of the antibody is 0.5 mg/mL.
And 4.4) washing the used ultrafiltration centrifugal tube by PBS and centrifugally drying 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, the lid was closed, and the ultrafiltration tube was placed in a refrigerator at 4 ℃ for storage.
And 5) storing the antibody in a refrigerator at 4 ℃, and labeling information such as antibody name, fluorescent dye name, antibody concentration, antibody volume, purification date and the like.
Comparative example 1 iFluor fluorescent labeling protocol
Step 1): A1M sodium bicarbonate solution is prepared in advance, and the sodium bicarbonate solution can be stored in a refrigerator at 4 ℃ for 2 weeks and is easily decomposed after a long time.
Step 2) protein concentration was diluted to 2mg/mL, approximately 0.01mM, with PBS (containing 0.01% sodium azide) buffer, and 1/10 of 1M sodium bicarbonate buffer was added to the protein solution.
Take the CD4(GK1.5 clone) antibody as an example: and 8, 8 months and 14 days in 2018, wherein the protein concentration is 2.5mg/mL, the antibody purity is 91%, and the antibody concentration is 2.275 mg/mL.
88uL of 2.5mg/mL antibody solution was taken, 12uL of PBS (containing 0.01% sodium azide) buffer was added, the solution was diluted to 100uL of 1mg/mL antibody solution, and 10uL of sodium bicarbonate solution was added to the solution and mixed well.
Step 3): the specification of the iFluor fluorescent dye is 1mg per tube, the molecular weight is 1000, 100uL DMSO is added into each tube to dissolve the dye until the final concentration is 10mM, the dye is subpackaged into 5uL per tube, and the dye is stored at the temperature of-20 ℃.
Step 4): to the protein solution prepared in step 2, 1.5uL of fluorescent dye (n (dye): n (protein) ═ 15:1) was added, and the dye was dissolved sufficiently with gentle shaking and was not shaken vigorously. The solution was placed in a room temperature environment in the dark, and gently shaken every 10min for reaction for 1 h.
Step 5): the square clamping piece blocking the bottom of the purification column at the pipe orifice is rotated and screwed off forcibly, the white round gasket is placed at the bottom of the purification column (two gaskets should be placed, the thinner gasket is placed at the lowest part, and the other gasket is placed on the thinner gasket), and the gasket can be pressed firmly by a pipette tip or tweezers. Firstly, adding 2mL of filler suspension into a purification column tube, placing the purification column tube in a centrifuge, centrifuging for 3min at 1100g, removing the filler preservation solution in the tube (the purification column tube can be placed on a 1.5mL centrifuge tube with a cover removed and a tube bottom cut off for centrifuging), 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 is about 1.5mL, and the tube is finally centrifuged once for 3min at 1100g to remove the residual filler preserving fluid in the filler. (this step may be performed starting near the end of the reaction of the protein with the fluorescent dye, and if it is too early, it may dry out easily inside the packing, which may result in a smaller volume of labeled protein being collected and remaining in the seasoning; the column may be placed in a 50mL centrifuge tube during centrifugation.)
And 6) after the reaction of the protein and the fluorescent dye is finished, 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, 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 tube in a refrigerator at 4 ℃ for storage, and marking information such as antibody name, fluorescent dye name, antibody concentration, antibody volume, purification date and the like on the collecting tube.
Comparative example 2 monoclonal antibody FITC labeling Standard operating procedure
Step 1) taking 0.1mL of monoclonal antibody, adding 10 mu L of 1M sodium bicarbonate solution, and uniformly mixing to prepare a monoclonal antibody reaction solution.
And 2) taking 10uL FITC dye solution, adding the FITC dye solution into the monoclonal antibody reaction solution, and uniformly mixing.
And 3) reacting at room temperature in a dark place for 1h, and uniformly mixing every 10 min.
Step 4) separating and purifying the ultrafiltration centrifugal tube (the pipette tip does not contact the filter membrane of the inner tube of the ultrafiltration tube in the whole operation process so as not to puncture the filter membrane)
Step 4.1) 0.5mL of 50kd inner tube of the ultrafiltration centrifugal tube is put into a collection tube, the solution after the reaction is completely transferred into the inner tube of the ultrafiltration centrifugal tube, 400uL of PBS (containing 0.01% of NaN3) is added, and 10000g of the solution is centrifuged for 5 min.
And 4.2) pouring the liquid in the collecting tube into a waste liquid barrel, adding 500uL PBS (containing 0.01% NaN3) into the inner tube for 14000g for 10min for centrifugation, discarding the waste liquid in the collecting tube, and repeating the test step for 5-6 times until the color of the waste liquid in the collecting tube is not green.
And 4.3) completely pouring the waste liquid in the collecting tube, inverting the tube in the ultrafiltration tube, putting the tube in the collecting tube for 2000g 2min for centrifugation, sucking out the liquid in the collecting tube by using a pipette, transferring the liquid into a new centrifugal tube, estimating the volume of the liquid (the volume of the liquid is about 20-40 uL), and supplementing a certain amount of PBS (containing 0.01% NaN3) into the new centrifugal tube to ensure that the final concentration of the antibody is 1 mg/mL.
And 4.4) washing the used ultrafiltration centrifugal tube by PBS and centrifugally drying 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, the lid was closed, and the ultrafiltration tube was placed in a refrigerator at 4 ℃ for storage.
Step 5) storing the labeled product at 4 ℃ in a dark place.
Test example antibody Positive fluorescent Signal test
Antibody positive fluorescence signal test method: the fluorescently labeled antibodies (bromoethylamine-treated CD4-iFluor488, bromoethylamine-treated CD4-FITC, untreated CD4-iFluor488, and untreated CD4-FITC) were co-stained with CD3-PE for 1million mouse splenocytes (0.25 ug of labeled CD4 antibody and 0.125ug of CD3-PE antibody were added to each reaction system) at the same concentration, and the fluorescence signals were detected by a flow cytometer after 30 minutes of staining.
Anti-mouse CD4 monoclonal antibody [ GK1.5] is used as a target protein, after the pre-treatment by bromoethylamine, iFluor488 labeling is carried out on a CD4 antibody (figure 2), figure 1 shows that the iFluor labeled CD4 antibody is directly carried out without the treatment of the bromoethylamine, and the positive fluorescence signal of the iFluor labeled CD4 antibody after the treatment of the bromoethylamine is stronger (comparing the signal values at the upper right corner of figures 1 and 2, the experiment represents at least 3 groups of repetition, at least 2 samples are tested in each group, and CD3-PE and labeled CD4-iFluor488 are used for co-staining mouse splenocytes in the experiment.
An anti-mouse CD4 monoclonal antibody [ GK1.5] is taken as a target protein, after the pre-treatment by bromoethylamine, FITC labeling is carried out on a CD4 antibody (figure 4), figure I is a CD4 antibody directly subjected to FITC labeling without being treated by the bromoethylamine, and after the treatment by the bromoethylamine, a positive fluorescence signal of the FITC-labeled CD4 antibody is stronger (comparing signal values at the upper right corner of figures 3 and 4, experiments represent at least 3 repeated groups, at least 2 samples are tested in each group, and CD3-PE and labeled CD4-FITC are used for co-staining mouse splenocytes in the experiments.
Table 1:
Figure BDA0002446049020000071
as can be known from table 1 above, by comparing the positive signal values marked by CD4, we can find that the positive signal value marked by CD4 can be significantly improved by performing an amination reaction on the thiol group of the antibody, and the efficiency and signal of the antibody fluorescence labeling are improved.

Claims (10)

1. A method for increasing the signal of a fluorescent dye label on an antibody, comprising the steps of:
step 1) aminating the sulfhydryl group of the antibody; step 2) labeling the antibody with a fluorescent dye.
2. The method of claim 1, wherein the thiol group on the antibody is aminated with a haloalkylamine.
3. The method of claim 1, wherein the haloalkylamine is selected from the group consisting of bromoethylamine, chloroethylamine, iodoethylamine.
4. The method according to claim 1, wherein the step 1) specifically comprises: adding a sodium bicarbonate solution into the antibody, uniformly mixing, and then adding a bromoethylamine solution for reaction to ensure that bromoethylamine molecules react with free sulfydryl of the antibody;
transferring the solution after the reaction into an ultrafiltration centrifugal tube, adding PBS buffer solution, centrifuging, removing filtrate, adding PBS buffer solution, mixing, centrifuging, desalting to make the residual amount of unreacted bromoethylamine in the antibody at least 10 of the total amount of the antibody-3More than twice.
5. The method of claim 1, wherein the fluorescent dye is FITC, iFluor-based dye, alexafluor-based dye.
6. The method of claim 4, wherein 100uL of 1M sodium bicarbonate solution is added to 200ug of antibody, mixed well and then 1.4uL of 50mM bromoethylamine solution is added and reacted overnight at 25 ℃ to allow the bromoethylamine molecules to react with the free thiol groups in the Ab molecules;
transferring the solution after the reaction to an ultrafiltration centrifugal tube, adding 500uL of PBS (containing 0.25mM EDTA) buffer solution for 5min, centrifuging at 12000g, removing the filtrate, adding 500uL of PBS (containing 0.25mM EDTA) buffer solution, mixing, centrifuging, repeating the operation for 5 times, desalting to obtain the residual content of unreacted bromoethylamine in the antibody of at least 10% of the total amount of the antibody-3More than twice, and diluting as much as possible.
7. The method of claim 5,
the step 2) specifically comprises the following steps:
1. adding 1/10 of 1M sodium bicarbonate buffer to the pre-reacted antibody solution;
2. adding a fluorescent dye into the antibody solution prepared in the step 1, and slightly shaking to fully dissolve the dye; placing the solution in a dark environment at 25 ℃ for reaction for 1 h;
4. separating and purifying by an ultrafiltration centrifugal tube;
4.1, filling 0.5mL of 50kd inner tube of the ultrafiltration centrifugal tube into a collecting tube, transferring all the solution after the reaction into the inner tube of the ultrafiltration centrifugal tube, and supplementing 400uL of PBS (containing 0.01% NaN)3)14000g for 10 min;
4.2, pouring the liquid in the collecting tube into a waste liquid barrel, adding 500uL PBS (containing 0.01 percent NaN3) into the inner tube, centrifuging for 14000g for 10min, discarding the waste liquid in the collecting tube, and repeating the test step for 5-6 times until the color of the waste liquid in the collecting tube is not green;
4.3, the waste liquid in the collecting tube is completely poured, the tube in the ultrafiltration tube is placed in the collecting tube upside down for 2000g 2min for centrifugation, the liquid in the collecting tube is sucked out by a liquid transfer machine and transferred to a new centrifugal tube, the volume of the liquid is estimated, and a certain amount of PBS (containing 0.01 percent NaN) is added into the liquid3) The final concentration of the antibody was set to 0.5 mg/mL;
4.4, washing the used ultrafiltration centrifugal tube by PBS, and centrifugally drying the residual liquid in the tube; adding 1mL of 20% ethanol into the collection tube, adding 500uL of 20% ethanol into the inner tube, covering the inner tube with a cover, and placing the ultrafiltration centrifugal tube in a refrigerator at 4 ℃ for storage;
5. and (3) storing the antibody in a refrigerator at 4 ℃, and labeling the information such as the name of the antibody, the name of a fluorescent dye, the concentration of the antibody, the volume of the antibody, the purification date and the like.
8. A method for increasing a signal labeled with a protein fluorescent dye, the method comprising the steps of:
step 1) carrying out sulfydryl amination on protein; step 2) labeling the protein with a fluorescent dye.
Preferably, the thiol group on the protein is aminated by a haloalkylamine; preferably, the haloalkylamine is selected from bromoethylamine, chloroethylamine, iodoethylamine.
9. An antibody mercaptoaminated derivative or a protein mercaptoaminated derivative obtained by the method of any one of claims 1 to 8.
10. The method of claims 1-8, for use in the industrial production of antibodies, proteins, raw materials reagents and pharmaceutical products.
CN202010279571.9A 2020-04-10 2020-04-10 Method for improving antibody fluorescent dye marking signal Active CN111551725B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202010279571.9A CN111551725B (en) 2020-04-10 2020-04-10 Method for improving antibody fluorescent dye marking signal

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202010279571.9A CN111551725B (en) 2020-04-10 2020-04-10 Method for improving antibody fluorescent dye marking signal

Publications (2)

Publication Number Publication Date
CN111551725A true CN111551725A (en) 2020-08-18
CN111551725B CN111551725B (en) 2023-07-28

Family

ID=72002294

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202010279571.9A Active CN111551725B (en) 2020-04-10 2020-04-10 Method for improving antibody fluorescent dye marking signal

Country Status (1)

Country Link
CN (1) CN111551725B (en)

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5877310A (en) * 1997-04-25 1999-03-02 Carnegie Mellon University Glycoconjugated fluorescent labeling reagents
US20030148399A1 (en) * 2000-03-24 2003-08-07 Ryoichi Nemori Film for assaying protease activity
WO2008027075A2 (en) * 2006-02-16 2008-03-06 The Scripps Research Institute Sulfhydryl-reactive, water soluble dyes
CN101900723A (en) * 2009-05-27 2010-12-01 中国科学技术大学 Application of nano-gold directly bonded with luminol in immunoassay
CN202614678U (en) * 2011-05-18 2012-12-19 东南大学 Detection micro needle capable of strengthening Raman and fluorescence signals
CN103323607A (en) * 2013-06-25 2013-09-25 青岛科技大学 Method for simultaneously measuring two-component plant hormones
CN104459147A (en) * 2013-09-18 2015-03-25 深圳迈瑞生物医疗电子股份有限公司 Biomarker preserving liquid, biomarker reagent and method
CN107235866A (en) * 2017-06-13 2017-10-10 重庆医科大学 A kind of fluorescence probe, preparation method and applications for being used to detect sulfenic acids protein
US20180275134A1 (en) * 2015-01-22 2018-09-27 Konica Minolta, Inc. Biological Substance Quantitation Method, Pathological Diagnosis Support System, And Recording Medium Storing Computer Readable Program

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5877310A (en) * 1997-04-25 1999-03-02 Carnegie Mellon University Glycoconjugated fluorescent labeling reagents
US20030148399A1 (en) * 2000-03-24 2003-08-07 Ryoichi Nemori Film for assaying protease activity
WO2008027075A2 (en) * 2006-02-16 2008-03-06 The Scripps Research Institute Sulfhydryl-reactive, water soluble dyes
CN101900723A (en) * 2009-05-27 2010-12-01 中国科学技术大学 Application of nano-gold directly bonded with luminol in immunoassay
CN202614678U (en) * 2011-05-18 2012-12-19 东南大学 Detection micro needle capable of strengthening Raman and fluorescence signals
CN103323607A (en) * 2013-06-25 2013-09-25 青岛科技大学 Method for simultaneously measuring two-component plant hormones
CN104459147A (en) * 2013-09-18 2015-03-25 深圳迈瑞生物医疗电子股份有限公司 Biomarker preserving liquid, biomarker reagent and method
US20180275134A1 (en) * 2015-01-22 2018-09-27 Konica Minolta, Inc. Biological Substance Quantitation Method, Pathological Diagnosis Support System, And Recording Medium Storing Computer Readable Program
CN107235866A (en) * 2017-06-13 2017-10-10 重庆医科大学 A kind of fluorescence probe, preparation method and applications for being used to detect sulfenic acids protein

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
李响;李向丽;谭贵良;吴世嘉;王周平;: "磁分离结合CdTe发光量子点标记黄曲霉毒素B1免疫检测新方法", 食品与生物技术学报, no. 03, pages 40 - 46 *

Also Published As

Publication number Publication date
CN111551725B (en) 2023-07-28

Similar Documents

Publication Publication Date Title
CN102575244B (en) Dissociation method and dissociation agent for avidin and biotin
JPH08278239A (en) Method for bonding biological material
CN102323409B (en) Virus infection detection method
CA2483945A1 (en) Method for simultaneously detecting an antigen and an antibody of an infectious microorganism
CN111551725A (en) Method for improving antibody fluorescent dye labeling signal
CN102245637A (en) Antibody against n-terminal region of beta-chain of hemoglobin
Perlmann et al. Soluble antigens in microsomes and other cell-fractions of rat liver
CN114085842B (en) Swine-derived monoclonal genetic engineering antibody of sai Ka virus, and preparation method and application thereof
CN107855115B (en) Affinity chromatographic column, preparation method and application
JP2000002707A (en) Diagnosis of feline immunodeficiency virus infection using env/gag polypeptide marker
CN109374886B (en) Infectious bovine rhinotracheitis virus antibody detection kit and application thereof
CN113373025B (en) Coating composition, device and method suitable for capturing rare cells
CN108828239B (en) Biosensing analysis method for detecting estrogen binding activity of water sample
CN106596876B (en) A kind of accurate method for qualitative and quantitative detection of oil-adjuvant vaccine
EP0078734B1 (en) Procedure for immunological determinations using two antibodies, the second antibody being purified by affinity chromatography, and supports therefor
CN109541219A (en) A kind of immune blotting detection method of Ancient Silk Textile
EP0665893B1 (en) A method and an apparatus for detecting cells
CN104730233A (en) Serum diluent in ELISA detection
CN101290315B (en) Clenbuterol ELISA method and reagent kit and method for making same
CH629894A5 (en) Process for separating the free fractions from the bound fractions in an immunoassay procedure
JPS60500348A (en) Peptide and protein sequence determination device
CN109738630A (en) A kind of excretion body Iem Methods of highly sensitive magnetic bead sorting association colloid gold label
US20040068101A1 (en) Methods for selectively staining water soluble protein using reactive dye and its applications thereof
CN108484725A (en) A kind of polypeptide for distinguishing the wild malicious antibody of detection PCV2 and vaccine antibody and its application in ELISA detection kit
CN113567206B (en) Membrane protein based on metal organic framework nano material and membrane-associated protein extraction method

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
TA01 Transfer of patent application right
TA01 Transfer of patent application right

Effective date of registration: 20230630

Address after: 313200 Floor 4, Building 3, No. 926, Changhong East Street, Kangqian Street, Deqing County, Huzhou, Zhejiang Province

Applicant after: Huzhou Zhengxi Medical Laboratory Co.,Ltd.

Address before: No. 926, Changhong East Street, Fuxi Street, Deqing County, Huzhou, Zhejiang Province, 313299

Applicant before: ZHEJIANG ZHENGXI BIOMEDICAL Co.,Ltd.

GR01 Patent grant
GR01 Patent grant
TA01 Transfer of patent application right
TA01 Transfer of patent application right

Effective date of registration: 20230726

Address after: 313299 Building 1, Building 3, No. 926, Changhong East Street, Fuxi Street, Deqing County, Huzhou, Zhejiang Province (Mount Mogan National High tech Zone) (self declared)

Applicant after: Zhejiang Zhengxi Biotechnology Co.,Ltd.

Address before: 313200 Floor 4, Building 3, No. 926, Changhong East Street, Kangqian Street, Deqing County, Huzhou, Zhejiang Province

Applicant before: Huzhou Zhengxi Medical Laboratory Co.,Ltd.