CN110229231A - A kind of fluorescent labeled antibody and application - Google Patents

A kind of fluorescent labeled antibody and application Download PDF

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CN110229231A
CN110229231A CN201910644315.2A CN201910644315A CN110229231A CN 110229231 A CN110229231 A CN 110229231A CN 201910644315 A CN201910644315 A CN 201910644315A CN 110229231 A CN110229231 A CN 110229231A
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antibody
sulfonic acid
integer
solution
labeled antibody
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肖义
陈令成
张新富
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Dalian University of Technology
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    • C07—ORGANIC CHEMISTRY
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    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2839—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily
    • C07K16/2845—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily against integrin beta2-subunit-containing molecules, e.g. CD11, CD18
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    • G01N33/531—Production of immunochemical test materials
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    • G01N33/533—Production of labelled immunochemicals with fluorescent label
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Abstract

The present invention relates to a kind of fluorescent labeled antibody and applications, belong to field of fine chemical.This kind of fluorescent labeled antibody is made by being connected with sulfonic acid rhodamine fluorescent chemicals and antibody the removing N- hydroxy maleimide in the buffer solution that Ph is 9 of amino acid pyrrolidinyl ester.Fluorescent chemicals are keyed in antibody molecule by the fluorescent labeled antibody by chemistry, overcome the previous problem for only causing fluorescent dye to be highly soluble in cell liquid with fluorescent dye dip dyeing antibody and leading to tracer effect difference.Meanwhile the sulfonic acid rhodamine that the present invention chooses has excellent water solubility and cell membrane penetration, provides advantage for the mechanism of action of the research antibody in biological cell.The fluorescent labeled antibody can observe antibody mechanism and mechanism in biological tissue or cell by the fluorescence developing of sulfonic acid rhodamine fluorogen, provide a kind of visualization tool in the process for playing the functions such as anti-infective, antitumor, immunological regulation and monitoring for research antibody.

Description

A kind of fluorescent labeled antibody and application
Technical field
The present invention relates to a kind of fluorescent labeled antibody and applications, belong to field of fine chemical.
Background technique
Antibody is most important effector molecule in body immune system, have in conjunction with antigen, conjugated complement, neutralize a toxin, Mediated cell poison promotes the effects of swallowing and pass through the functions such as placenta, playing anti-infective, antitumor, immunological regulation and monitoring.With The development of biotechnology, antibody controlled in alloimmunity repulsion, autoimmune response inhibition, Antiplatelet therapy, cancer Treatment and infectious diseases treatment etc. are all widely used.The appearance of humanized antibody and human antibody is controlling for a variety of diseases Treatment brings new hope, but whether human antibody can solve all problems occurred in the application of mouse antibody clinical, need big Measure the inspection of clinical test.Due to influence antibody immunogenicity factor it is very much, as antigen presentation route, signal transduction system with And individual difference of patient etc., and the humanization of antibody can only solve subproblem, extensive use is also depended on to antibody Effect mechanism and body immune system adjustment mechanism carry out more careful in-depth study.Equally, antibody derivatives can also face The intrinsic problem of immunogenicity, stability, activity, adverse reaction etc. itself.Therefore, research antibody is anti-infective, anti-in performance The mechanism of action when functions such as tumour, immunological regulation and monitoring is of great significance, can be provided for the research and development of antibody drug according to According to, and a series of adverse reactions present in clinical application can be prevented.
The development of Induced Fluorescence Microscopy for the detection and research of large biological molecule provide it is a kind of convenient, fast and Visual tool, most of the fluorescence labeling method currently used for antibody be all by fluorescent chemicals and antibody mixture infection from And it is fluorescently labeled antibody.This method is easy to operate, but the fluorescent chemicals of antibody surface be easy to be rinsed off with And be dissolved into PBS, cause the fluorescence signal of antibody weaker, and background fluorescence interference is stronger, so that the visual research of antibody As a result not ideal enough.
Summary of the invention
To solve problems of the prior art, the present invention provides a kind of fluorescent labeled antibody and application, anti-to study Mechanism of action of the body in biological cell provides a kind of visualization tool.The technical solution adopted by the present invention are as follows: a kind of fluorescence mark Remember that antibody, the general structure of the fluorescent labeled antibody are as follows:
In general formula, R1,
Wherein: the integer of a=0-18, the integer of b=0-18, X-For anion, the anion is BF4 -、Cl-、Br-、 I-、NO3 -、SO4 2-、ClO4 -、CH3COO-、CH3SO3 -Or CF3SO3 -,n1 The integer of=0-18, n2The integer of=0-18；It is describedIt is negatively charged that the positively charged sum of institute is equal to anion institute Lotus sum, R1And R2It can be different groups, R5And R6It can be different groups；
R3,R3And R4It can be different groups；
Wherein: The integer of n=1-10, n3The integer of=1-11, n4The integer of=1-5, n5The integer of=1-5, n6 The integer of=1-11, n7The integer of=1-5, m1The integer of=0-11, n8The integer of=0-5, m2The integer of=0-11, n9=0-11 Integer, n10The integer of=0-5, m3=0-11.The preparation method of a kind of fluorescent labeled antibody, the fluorescent labeled antibody By compound A and antibody, removing N- hydroxy maleimide is made in the buffer solution that Ph is 9.
Wherein, R1、R2、R3、R4, Y, n definition with the definition in general structure.
The application of a kind of fluorescent labeled antibody, the fluorescent labeled antibody for monitoring, tracer antibody and research The mechanism of action of the antibody in every vital movement.
The beneficial effects of the present invention are: a kind of fluorescent labeled antibody and application, this kind of fluorescent labeled antibody is by being connected with ammonia The sulfonic acid rhodamine fluorescent chemicals and antibody of base acid pyrrolidinyl ester remove N- hydroxyl Malaysia acyl in the buffer solution that Ph is 9 Imines is made, and preparation method is simple.Fluorescent chemicals are keyed in antibody molecule by the fluorescent labeled antibody by chemistry, gram It has taken and had only disseminated antibody in the past with fluorescent dye and caused fluorescent dye to be highly soluble in cell liquid and lead to asking for tracer effect difference Topic, meanwhile, the sulfonic acid rhodamine that the present invention chooses has excellent water solubility and cell membrane penetration, is research antibody in biology The mechanism of action in cell provides advantage.The fluorescent labeled antibody can pass through the fluorescence developing of sulfonic acid rhodamine fluorogen Observe antibody mechanism and mechanism in biological tissue or cell, it is anti-infective, antitumor, immune in performance for research antibody It adjusts and provides a kind of visualization tool with the process of the functions such as monitoring.In clinical application for research and development antibody drug and research antibody A series of adverse reactions that may be present provide a kind of new research direction.
Specific embodiment
The present invention is illustrated with following instance but not limited to this, wherein unless otherwise indicated, all numbers and hundred Score is by weight.
Describe specific embodiments of the present invention in detail below in conjunction with technical solution, in the embodiment of the present invention antibody buy in Commercial antibody.
Embodiment 1
Sulfonic acid rhodamine (0.84g, 2mmol) is weighed in reaction flask, 1,2- dichloroethanes is added and is sufficiently stirred, slowly drips Add phosphorus oxychloride, 95 DEG C of back flow reactions, silica gel plate monitors extent of reaction, and after sulfonic acid rhodamine fully reacting, cooling, decompression is steamed It is spare to obtain rhodamine sulfonic acid chloride for solvent out；Amino acid pyrrolidinyl ester (0.74g, 4mmol), triethylamine and acetonitrile are added and reacted Bottle be stirred at room temperature, rhodamine sulfonic acid chloride is dissolved in acetonitrile and is slowly added to reaction solution, react at room temperature, thin-layer chromatography monitoring react into Degree.Product A1Washing extraction, decompression steam solvent, purify through pillar layer separation, and product structure is identified by HRMS.
Embodiment 2
By compound A1It is dissolved in the compound A that 5mM/L is configured in DMSO solvent1Solution, then by 14 μ L 20mg/mL's Mouse anti-human igg4Fc two corresponding anti-solution (article No. A-10651), the compound A of 2 μ L 5mM/L1Solution, 100 μ L pH are 7.40 The NaHCO of PBS and 28 μ L 1M/L3Solution be added reaction flask in mix, be stirred at room temperature 2 hours, product by protein desalination from Stem purify 142 μ L, 2mg/mL B1Solution, freeze or refrigerate it is spare, product structure pass through HRMS identify.
Embodiment 3
Sulfonic acid rhodamine (0.96g, 2mmol) is weighed in reaction flask, 1,2- dichloroethanes is added and is sufficiently stirred, slowly drips Add phosphorus oxychloride, 95 DEG C of back flow reactions, silica gel plate monitors extent of reaction, and after sulfonic acid rhodamine fully reacting, cooling, decompression is steamed It is spare to obtain rhodamine sulfonic acid chloride for solvent out；Amino acid pyrrolidinyl ester (0.86g, 4mmol), triethylamine and acetonitrile are added and reacted Bottle be stirred at room temperature, rhodamine sulfonic acid chloride is dissolved in acetonitrile and is slowly added to reaction solution, react at room temperature, thin-layer chromatography monitoring react into Degree.Product A2Washing extraction, decompression steam solvent, purify through pillar layer separation, and product structure is identified by HRMS.
Embodiment 4
By compound A2It is dissolved in the compound A that 5mM/L is configured in DMSO solvent2Solution, then by 14 μ L 20mg/mL's Mouse anti-human igg Fab two corresponding anti-solution (article No. SA1-19255), the compound A of 2 μ L 5mM/L2Solution, 100 μ L pH are 7.40 PBS and 28 μ L 1M/L NaHCO3Solution is added in reaction flask and mixes, and is stirred at room temperature 2 hours, and product passes through protein desalination Centrifugal column purify 142 μ L, 2mg/mL B2Solution, freeze or refrigerate it is spare, product structure pass through HRMS identify.
Embodiment 5
Synthetic method is the same as embodiment 3.
Embodiment 6
Synthetic method is the same as embodiment 4.
Embodiment 7
Synthetic method is the same as embodiment 3.
Embodiment 8
Synthetic method is the same as embodiment 4.
Embodiment 9
Sulfonic acid rhodamine (1.26g, 2mmol) is weighed in reaction flask, 1,2- dichloroethanes is added and is sufficiently stirred, slowly drips Add phosphorus oxychloride, 95 DEG C of back flow reactions, silica gel plate monitors extent of reaction, and after sulfonic acid rhodamine fully reacting, cooling, decompression is steamed It is spare to obtain rhodamine sulfonic acid chloride for solvent out；Amino acid pyrrolidinyl ester (0.91g, 4mmol), triethylamine and acetonitrile are added and reacted Bottle be stirred at room temperature, rhodamine sulfonic acid chloride is dissolved in acetonitrile and is slowly added to reaction solution, react at room temperature, thin-layer chromatography monitoring react into Degree.Product A5Washing extraction, decompression steam solvent, purify through pillar layer separation, and product structure is identified by HRMS.
Embodiment 10
By compound A5It is dissolved in the compound A that 5mM/L is configured in DMSO solvent5Solution, then by 14 μ L 20mg/mL's Mouse anti-human igg (H+L) two corresponding anti-solution (article No. MA5-14728), the compound A of 2 μ L 5mM/L5Solution, 100 μ L pH are The NaHCO of 7.40 PBS and 28 μ L 1M/L3Solution is added in reaction flask and mixes, and is stirred at room temperature 2 hours, product passes through protein Desalination centrifugal column purify 142 μ L, 2mg/mL B5Solution, freeze or refrigerate it is spare, product structure pass through HRMS identify.
Embodiment 11
Sulfonic acid rhodamine (0.96g, 2mmol) is weighed in reaction flask, 1,2- dichloroethanes is added and is sufficiently stirred, slowly drips Add phosphorus oxychloride, 95 DEG C of back flow reactions, silica gel plate monitors extent of reaction, and after sulfonic acid rhodamine fully reacting, cooling, decompression is steamed It is spare to obtain rhodamine sulfonic acid chloride for solvent out；Amino acid pyrrolidinyl ester (0.92g, 4mmol), triethylamine and acetonitrile are added and reacted Bottle be stirred at room temperature, rhodamine sulfonic acid chloride is dissolved in acetonitrile and is slowly added to reaction solution, react at room temperature, thin-layer chromatography monitoring react into Degree.Product A6Washing extraction, decompression steam solvent, purify through pillar layer separation, and product structure is identified by HRMS, product structure It is identified by HRMS.
Embodiment 12
By compound A6It is dissolved in the compound A that 5mM/L is configured in DMSO solvent6Solution, then by 14 μ L 20mg/mL's Mouse anti-human igg Fab two corresponding anti-solution (article No. SA1-19255), the compound A of 2 μ L 5mM/L6Solution, 100 μ L pH are 7.40 PBS and 28 μ L 1M/L NaHCO3Solution is added in reaction flask and mixes, and is stirred at room temperature 2 hours, and product passes through protein desalination Centrifugal column purify 142 μ L, 2mg/mL B6Solution, freeze or refrigerate it is spare, product structure pass through HRMS identify.
Embodiment 13
Synthetic method is the same as embodiment 11.
Embodiment 14
Synthetic method is the same as embodiment 12.
Embodiment 15
Synthetic method is the same as embodiment 11.
Embodiment 16
Synthetic method is the same as embodiment 12.
Embodiment 17
Sulfonic acid rhodamine (0.90g, 2mmol) is weighed in reaction flask, 1,2- dichloroethanes is added and is sufficiently stirred, slowly drips Add phosphorus oxychloride, 95 DEG C of back flow reactions, silica gel plate monitors extent of reaction, and after sulfonic acid rhodamine fully reacting, cooling, decompression is steamed It is spare to obtain rhodamine sulfonic acid chloride for solvent out；Amino acid pyrrolidinyl ester (0.92g, 4mmol), triethylamine and acetonitrile are added and reacted Bottle be stirred at room temperature, rhodamine sulfonic acid chloride is dissolved in acetonitrile and is slowly added to reaction solution, react at room temperature, thin-layer chromatography monitoring react into Degree.Product A9Washing extraction, decompression steam solvent, purify through pillar layer separation, and product structure is identified by HRMS.
Embodiment 18
By compound A9It is dissolved in the compound A that 5mM/L is configured in DMSO solvent9Solution, then by 14 μ L 20mg/mL's Rabbit anti-mouse igg (H+L) two corresponding anti-solution (article No. A27022), the compound A of 2 μ L 5mM/L9Solution, 100 μ L pH are 7.40 The NaHCO of PBS and 28 μ L 1M/L3Solution be added reaction flask in mix, be stirred at room temperature 2 hours, product by protein desalination from Stem purify 142 μ L, 2mg/mL B9Solution, freeze or refrigerate it is spare, product structure pass through HRMS identify.
Embodiment 19
Synthetic method is the same as embodiment 17.
Embodiment 20
Synthetic method is the same as embodiment 18.
Embodiment 21
Synthetic method is the same as embodiment 17.
Embodiment 22
Synthetic method is the same as embodiment 18.
Embodiment 23
Sulfonic acid rhodamine (1.2g, 2mmol) is weighed in reaction flask, 1,2- dichloroethanes is added and is sufficiently stirred, slowly drips Add phosphorus oxychloride, 95 DEG C of back flow reactions, silica gel plate monitors extent of reaction, and after sulfonic acid rhodamine fully reacting, cooling, decompression is steamed It is spare to obtain rhodamine sulfonic acid chloride for solvent out；Amino acid pyrrolidinyl ester (0.9g, 4mmol), triethylamine and acetonitrile are added and reacted Bottle be stirred at room temperature, rhodamine sulfonic acid chloride is dissolved in acetonitrile and is slowly added to reaction solution, react at room temperature, thin-layer chromatography monitoring react into Degree.Product A12Washing extraction, decompression steam solvent, purify through pillar layer separation, and product structure is identified by HRMS.
Embodiment 24
By compound A12It is dissolved in the compound A that 5mM/L is configured in DMSO solvent12Solution, then by 14 μ L 20mg/mL Rabbit anti-human igg1Two corresponding anti-solution (article No. SA5-10202), the compound A of 2 μ L 5mM/L12Solution, 100 μ L pH are 7.40 PBS and 28 μ L 1M/L NaHCO3Solution is added in reaction flask and mixes, and is stirred at room temperature 2 hours, and product passes through protein desalination Centrifugal column purify 142 μ L, 2mg/mL B12Solution, freeze or refrigerate it is spare, product structure pass through HRMS identify.
Embodiment 25
Synthetic method is the same as embodiment 23.
Embodiment 26
Synthetic method is the same as embodiment 24.
Embodiment 27
Sulfonic acid rhodamine (1.31g, 2mmol) is weighed in reaction flask, 1,2- dichloroethanes is added and is sufficiently stirred, slowly drips Add phosphorus oxychloride, 95 DEG C of back flow reactions, silica gel plate monitors extent of reaction, and after sulfonic acid rhodamine fully reacting, cooling, decompression is steamed It is spare to obtain rhodamine sulfonic acid chloride for solvent out；Amino acid pyrrolidinyl ester (0.9g, 4mmol), triethylamine and acetonitrile are added and reacted Bottle be stirred at room temperature, rhodamine sulfonic acid chloride is dissolved in acetonitrile and is slowly added to reaction solution, react at room temperature, thin-layer chromatography monitoring react into Degree.Product A14Washing extraction, decompression steam solvent, purify through pillar layer separation, and product structure is identified by HRMS.
Embodiment 28
By compound A14It is dissolved in the compound A that 5mM/L is configured in DMSO solvent14Solution, then by 14 μ L 20mg/mL Mouse anti-human igg (H+L) two corresponding anti-solution (article No. MA5-14728), the compound A of 2 μ L 5mM/L14Solution, 100 μ L pH are The NaHCO of 7.40 PBS and 28 μ L 1M/L3Solution is added in reaction flask and mixes, and is stirred at room temperature 2 hours, product passes through protein Desalination centrifugal column purify 142 μ L, 2mg/mL B14Solution, freeze or refrigerate it is spare, product structure pass through HRMS identify.
Embodiment 29
Synthetic method is the same as embodiment 27.
Embodiment 30
Synthetic method is the same as embodiment 28.
Embodiment 31
Sulfonic acid rhodamine (1.0g, 2mmol) is weighed in reaction flask, 1,2- dichloroethanes is added and is sufficiently stirred, slowly drips Add phosphorus oxychloride, 95 DEG C of back flow reactions, silica gel plate monitors extent of reaction, and after sulfonic acid rhodamine fully reacting, cooling, decompression is steamed It is spare to obtain rhodamine sulfonic acid chloride for solvent out；Amino acid pyrrolidinyl ester (1.08g, 4mmol), triethylamine and acetonitrile are added and reacted Bottle be stirred at room temperature, rhodamine sulfonic acid chloride is dissolved in acetonitrile and is slowly added to reaction solution, react at room temperature, thin-layer chromatography monitoring react into Degree.Product A16Washing extraction, decompression steam solvent, purify through pillar layer separation, and product structure is identified by HRMS.
Embodiment 32
By compound A16It is dissolved in the compound A that 5mM/L is configured in DMSO solvent16Solution, then by 14 μ L 20mg/mL ZO-1 Anti-TNF-α liquid solution (article No. 61-7300), the compound A of 2 μ L 5mM/L16Solution, 100 μ L pH are 7.40 The NaHCO of PBS and 28 μ L 1M/L3Solution be added reaction flask in mix, be stirred at room temperature 2 hours, product by protein desalination from Stem purify 142 μ L, 2mg/mL B16Solution, freeze or refrigerate it is spare, product structure pass through HRMS identify.
Embodiment 33
Synthetic method is the same as embodiment 31.
Embodiment 34
Synthetic method is the same as embodiment 32.
Embodiment 35
Sulfonic acid rhodamine (1.48g, 2mmol) is weighed in reaction flask, 1,2- dichloroethanes is added and is sufficiently stirred, slowly drips Add phosphorus oxychloride, 95 DEG C of back flow reactions, silica gel plate monitors extent of reaction, and after sulfonic acid rhodamine fully reacting, cooling, decompression is steamed It is spare to obtain rhodamine sulfonic acid chloride for solvent out；Amino acid pyrrolidinyl ester (1.08g, 4mmol), triethylamine and acetonitrile are added and reacted Bottle be stirred at room temperature, rhodamine sulfonic acid chloride is dissolved in acetonitrile and is slowly added to reaction solution, react at room temperature, thin-layer chromatography monitoring react into Degree.Product A18Washing extraction, decompression steam solvent, purify through pillar layer separation, and product structure is identified by HRMS.
Embodiment 36
By compound A18It is dissolved in the compound A that 5mM/L is configured in DMSO solvent18Solution, then by 14 μ L 20mg/mL CD11b monoclonal antibody solution (article No. 14-0112-82), the compound A of 2 μ L 5mM/L18Solution, 100 μ L pH are The NaHCO of 7.40 PBS and 28 μ L 1M/L3Solution is added in reaction flask and mixes, and is stirred at room temperature 2 hours, product passes through protein Desalination centrifugal column purify 142 μ L, 2mg/mL B18Solution, freeze or refrigerate it is spare, product structure pass through HRMS identify.
Embodiment 37
Synthetic method is the same as embodiment 35.
Embodiment 38
Synthetic method is the same as embodiment 36.
Embodiment 39
Sulfonic acid rhodamine (1.55g, 2mmol) is weighed in reaction flask, 1,2- dichloroethanes is added and is sufficiently stirred, slowly drips Add phosphorus oxychloride, 95 DEG C of back flow reactions, silica gel plate monitors extent of reaction, and after sulfonic acid rhodamine fully reacting, cooling, decompression is steamed It is spare to obtain rhodamine sulfonic acid chloride for solvent out；Amino acid pyrrolidinyl ester (1.08g, 4mmol), triethylamine and acetonitrile are added and reacted Bottle be stirred at room temperature, rhodamine sulfonic acid chloride is dissolved in acetonitrile and is slowly added to reaction solution, react at room temperature, thin-layer chromatography monitoring react into Degree.Product A20Washing extraction, decompression steam solvent, purify through pillar layer separation, and product structure is identified by HRMS.
Embodiment 40
By compound A20It is dissolved in the compound A that 5mM/L is configured in DMSO solvent20Solution, then by 14 μ L 20mg/mL Mouse anti-human igg (H+L) two corresponding anti-solution (article No. MA5-14728), the compound A of 2 μ L 5mM/L20Solution, 100 μ L pH are The NaHCO of 7.40 PBS and 28 μ L 1M/L3Solution is added in reaction flask and mixes, and is stirred at room temperature 2 hours, product passes through protein Desalination centrifugal column purify 142 μ L, 2mg/mL B20Solution, freeze or refrigerate it is spare, product structure pass through HRMS identify.
Embodiment 41
Sulfonic acid rhodamine (1.30g, 2mmol) is weighed in reaction flask, 1,2- dichloroethanes is added and is sufficiently stirred, slowly drips Add phosphorus oxychloride, 95 DEG C of back flow reactions, silica gel plate monitors extent of reaction, and after sulfonic acid rhodamine fully reacting, cooling, decompression is steamed It is spare to obtain rhodamine sulfonic acid chloride for solvent out；Amino acid pyrrolidinyl ester (1.08g, 4mmol), triethylamine and acetonitrile are added and reacted Bottle be stirred at room temperature, rhodamine sulfonic acid chloride is dissolved in acetonitrile and is slowly added to reaction solution, react at room temperature, thin-layer chromatography monitoring react into Degree.Product A21Washing extraction, decompression steam solvent, purify through pillar layer separation, and product structure is identified by HRMS.
Embodiment 42
By compound A21It is dissolved in the compound A that 5mM/L is configured in DMSO solvent21Solution, then by 14 μ L 20mg/mL Mouse anti-human igg (H+L) two corresponding anti-solution (article No. MA5-14728), the compound A of 2 μ L 5mM/L21Solution, 100 μ L pH are The NaHCO of 7.40 PBS and 28 μ L 1M/L3Solution is added in reaction flask and mixes, and is stirred at room temperature 2 hours, product passes through protein Desalination centrifugal column purify 142 μ L, 2mg/mL B21Solution, freeze or refrigerate it is spare, product structure pass through HRMS identify.
Embodiment 43
Sulfonic acid rhodamine (1.56g, 2mmol) is weighed in reaction flask, 1,2- dichloroethanes is added and is sufficiently stirred, slowly drips Add phosphorus oxychloride, 95 DEG C of back flow reactions, silica gel plate monitors extent of reaction, and after sulfonic acid rhodamine fully reacting, cooling, decompression is steamed It is spare to obtain rhodamine sulfonic acid chloride for solvent out；Amino acid pyrrolidinyl ester (1.08g, 4mmol), triethylamine and acetonitrile are added and reacted Bottle be stirred at room temperature, rhodamine sulfonic acid chloride is dissolved in acetonitrile and is slowly added to reaction solution, react at room temperature, thin-layer chromatography monitoring react into Degree.Product A22Washing extraction, decompression steam solvent, purify through pillar layer separation, and product structure is identified by HRMS.
Embodiment 44
By compound A22It is dissolved in the compound A that 5mM/L is configured in DMSO solvent22Solution, then by 14 μ L 20mg/mL Mouse anti-human igg (H+L) two corresponding anti-solution (article No. MA5-14728), the compound A of 2 μ L 5mM/L22Solution, 100 μ L pH are The NaHCO of 7.40 PBS and 28 μ L 1M/L3Solution is added in reaction flask and mixes, and is stirred at room temperature 2 hours, product passes through protein Desalination centrifugal column purify 142 μ L, 2mg/mL B22Solution, freeze or refrigerate it is spare, product structure pass through HRMS identify.

Claims (3)

1. a kind of fluorescent labeled antibody, which is characterized in that the general structure of the fluorescent labeled antibody is as follows:
In general formula, R1, R2= Wherein: a= The integer of 0-18, the integer of b=0-18, X-For anion, the anion is BF4 -、Cl-、Br-、I-、NO3 -、SO4 2-、ClO4 -、 CH3COO-、CH3SO3 -Or CF3SO3 -, R5,R6=The integer of e=0-18, n1=0- 18 integer, n2The integer of=0-18；
It is describedThe positively charged sum of institute is equal to the negatively charged sum of anion institute, R1And R2It can be different groups, R5And R6It can be different groups；
R3, R4=R3And R4It can be different groups；
Y=
Wherein: Y1=Y2= The integer of n=1-10, n3The integer of=1-11, n4The integer of=1-5, n5The integer of=1-5, n6 The integer of=1-11, n7The integer of=1-5, m1The integer of=0-11, n8The integer of=0-5, m2The integer of=0-11, n9=0-11 Integer, n10The integer of=0-5, m3=0-11.
2. the preparation method of one kind fluorescent labeled antibody according to claim 1, it is characterised in that: the fluorescent labeled antibody By compound A and antibody, removing N- hydroxy maleimide is made in the buffer solution that Ph is 9.
Wherein, R1、R2、R3、R4, n definition with the definition in general structure.
3. the application of one kind fluorescent labeled antibody according to claim 1, it is characterised in that: the fluorescent labeled antibody is used for The mechanism of action of monitoring, tracer antibody and research antibody in every vital movement.
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CN115636843A (en) * 2022-09-27 2023-01-24 大连理工大学 Multifunctional fluorescent dye and application thereof

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US5798276A (en) * 1995-06-07 1998-08-25 Molecular Probes, Inc. Reactive derivatives of sulforhodamine 101 with enhanced hydrolytic stability
CN101526534A (en) * 2009-03-10 2009-09-09 沈鹤柏 Fluorescence immune chromatography test paper and preparing method and application thereof
CN102012425A (en) * 2010-12-29 2011-04-13 南开大学 New immuno-fluorenscence labelling method
JP2016027340A (en) * 2011-09-09 2016-02-18 コニカミノルタ株式会社 Fluorescent labelling compound for detecting biological substance
CN109456341A (en) * 2018-10-19 2019-03-12 大连理工大学 Sulfonamide rhodamine compound and preparation method and application of the one kind with alkynyl or the derivative site of azido

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FR2295426A2 (en) * 1974-12-20 1976-07-16 Block Engineering ANTIBODIES MARKS AND THEIR MARKING PROCESS
US5798276A (en) * 1995-06-07 1998-08-25 Molecular Probes, Inc. Reactive derivatives of sulforhodamine 101 with enhanced hydrolytic stability
CN101526534A (en) * 2009-03-10 2009-09-09 沈鹤柏 Fluorescence immune chromatography test paper and preparing method and application thereof
CN102012425A (en) * 2010-12-29 2011-04-13 南开大学 New immuno-fluorenscence labelling method
JP2016027340A (en) * 2011-09-09 2016-02-18 コニカミノルタ株式会社 Fluorescent labelling compound for detecting biological substance
CN109456341A (en) * 2018-10-19 2019-03-12 大连理工大学 Sulfonamide rhodamine compound and preparation method and application of the one kind with alkynyl or the derivative site of azido

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115636843A (en) * 2022-09-27 2023-01-24 大连理工大学 Multifunctional fluorescent dye and application thereof

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Application publication date: 20190913