CN102313801A - Metabolic labeling method of antibody and application of antibody in fluorescence detection - Google Patents
Metabolic labeling method of antibody and application of antibody in fluorescence detection Download PDFInfo
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- CN102313801A CN102313801A CN201110148435A CN201110148435A CN102313801A CN 102313801 A CN102313801 A CN 102313801A CN 201110148435 A CN201110148435 A CN 201110148435A CN 201110148435 A CN201110148435 A CN 201110148435A CN 102313801 A CN102313801 A CN 102313801A
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Abstract
The invention provides a metabolic labeling method of an antibody and an application of the antibody in fluorescence detection. The method comprises the following steps that: two key compounds containing an azide group and an alkynyl group are respectively synthesized; the compound containing the azide group is added to a cell culture medium, and the azide group is labeled on a sugar chain of IgG through a metabolic pathway of a cell; and a click chemical reaction is carried out on the product obtained in the former step and the compound containing a fluorescence group to realize fluorescence color developing. With the adoption of the method, staining analysis can be performed in the cell level, and the metabolic labeled antibody can be directly used for immune fluorescent staining analysis; moreover, the antibody labeled by using the method can be used with the other existing immune fluorescent technologies synchronously, and the results are not interfered with one another. Compared with the traditional method, the novel antibody labeling method is easy to operate and less in steps, richens the immune fluorescent antibody detecting measures, and has development potential and wide application prospects in the future immune research.
Description
Technical field
The invention belongs to the immunological technique field, relate to the metabolic marker method of antibody and carry out the method for immuning fluorescent dyeing analysis through click chemistry.
Background technology
[0002] click chemistry is the splicing through junior unit, and rapid and reliable is accomplished the chemosynthesis of varied molecule.It stresses the combinatorial chemistry new method that synthesizes the basis with carbon-heteroatom bond (C-X-C) in particular, and reacts advantages of simplicity and high efficiency to obtain molecular diversity by this.Because the click chemistry product is nontoxic, and good stability, be widely used in the outer and body inner analysis of drug screening, drug development, polymerization and other biosome.
Biological coupling technology is a kind of technology of biomacromolecule being modified through micromolecular compound; Use widely, like fluorophore tagged such as protein, nucleic acid, modification, part chelating, labelled with radioisotope etc. in fields such as molecular biology and chemicobiologies.
The immunofluorescence antibody technique is to use special fluorescent marker covalently bound to any a kind of antigen or antibody, and the method that detects through fluorescent microscope.At present, fluorescent markers such as FITC, rhodamine have been widely used in the immunofluorescence analysis technology, in addition, utilize the immune detection system of biotin-avidin system, also are widely used.
Summary of the invention
The objective of the invention is to develop a kind of novel antibody labeling method; This method is compared with classic method possessed processing ease; Advantages such as step is few; To enrich immunology detection system and fluorescence antibody detection means, a kind of new antibody metabolic marker method and the application in fluoroscopic examination thereof are provided.
The present invention at first provides a kind of antibody metabolic marker method, and this method comprises:
Metabolic marker thing when synthesizing as glycoprotein with the non-natural monose that contains azido group; Its sugared structure can replace the normal monose in the NGP; Thereby make and be labeled protein and contain azido group; And then react through the click chemistry between azido group and the azide reaction group, make protein possess the label group that lighting function or coupling can be to be detected.
Described azide reaction group is to form the alkynyl compounds of triazole ring with azido group, and possesses the characteristic of fluorescing or contain the label group.
The described coupling group that possesses the characteristic of fluorescing comprises Cy3, Cy5, FITC, TRITC, PE, DAPI, Texas red, RB200, Indo-1 and quantum dot etc.
Described label group comprises His, GST, FLAG, HA and biotin etc.
The present invention provides a kind of application of antibody in fluorescence labeling and immunofluorescence analysis method of adopting metabolic marker method mark simultaneously.
The non-natural monose that the key compound that the present invention relates to promptly contains azido group comprises: non-natural monose 1,3,4,6-oxygen acetyl-2-'-deoxy-n-nitrine acetylmannosamine (1,3; 4,6-tetra-O-acetyl-N-azidoacetylmannosamine, Ac4ManNAz), 1,3,4; 6-oxygen acetyl-2-'-deoxy-n-nitrine acetyl sialic acid (1,3,4,6-tetra-O-acetyl-azido sialic acid; Ac4SiaNAz), 1,3,4,6-oxygen acetyl-2-'-deoxy-n-nitrine acetylgalactosamine (1; 3,4,6-tetra-O-acetyl-N-azidoacetylgalactosamine is Ac4GalNAz) with 1; 3,4,6-oxygen acetyl-2-'-deoxy-n-nitrine acetylglucosamine (1,3; 4,6-tetra-O-acetyl-N-azidoacetylglucosamine, Ac4GlcNAz) and detection compound 4-ethinyl-N-ethyl-1, the 8-naphthalimide.
Synthetic non-natural monose joined in the Hybridoma Cell Culture base cultivate; Make it to substitute natural monose, obtain the anti-her2 antibody of the Azide of metabolic marker, the azido group through alkynyl in the copper ion catalysis detection compound and metabolic marker antibody carries out the click chemistry reaction subsequently; Realize fluorescence developing; Its detectability can reach 0.1 μ g, and EC50 is 1 μ g, and carries out the fluorescent dye analysis verification at cellular level.
The present invention has synthesized non-natural monose compd A c4ManNAz, Ac4SiaNAz, Ac4GalNAz, Ac4GlcNAz and detection compound 4-ethinyl-N-ethyl-1 voluntarily; The 8-naphthalimide; Non-natural monose has and the ability that substitutes normal monose; And can specific click chemistry reaction take place with alkynyl, change 4-ethinyl-N-ethyl-1, the electron distributions of 8-naphthalimide.
Carry out metabolic marker with above-mentioned synthetic non-natural monose; Preparation contains azido group ground antibody; Set up the fluorescence antibody analytical approach of using this antibody, verified, confirmed the detection sensitivity and the detectability of this labeling method simultaneously through the immunofluorescence dyeing and the click chemistry method of cell.
The concrete steps of the nitrine labeling method of antibody provided by the invention are following:
The non-natural monose that will contain azido group slowly joins in the cell culture medium, and final concentration is 0.2 mM, join behind the mixing in the hybridoma, and 37 ℃, 5% CO
2Cultivate 72 h, the collecting cell suspension, centrifugal 3 min of 1000 r/min draw supernatant, with the antibody of antibody purification affinity column purifying metabolic marker.
Click chemistry catalytic reaction provided by the invention and fluorophor analytical approach are following:
The non-natural monose of 0.1 ~ 1.0 mmol/L is added to contains 0.5 mmol/L 4-ethinyl-N-ethyl-1,8-naphthalimide, 0.2 mmol/L Tris-triazoleamine, 1 mmol/L CuSO
4And in the PBS solution of 2 mmol/L sodium ascorbates, room temperature reaction 60 min.With 365 nm wavelength excitation reaction product, respectively organize fluorescence intensity with 465 nm emission wavelengths mensuration, draw the concentration dependent typical curve of azido group; Simultaneously, carry out the click chemistry reaction, confirm the Azide labeling effciency of antibody according to the fluorescence intensity of reaction product with the anti-her2 antibody surrogate non-natural monose of 3 μ mol/L metabolic markers.
Beneficial effect of the present invention: the present invention adopts the nitrine labelled antibody to carry out immunofluorescence analysis, and this invention can be used with other existing immunofluorescence techniques simultaneously, and the result does not disturb mutually.The present invention has set up a kind of new immunofluorescence antibody analysis method through a kind of novel antibody labeling technology of exploitation, has enriched immunofluorescence antibody test means, in the immune Research in future, possesses development potentiality and wide application prospect.
Description of drawings
Fig. 1 is the quantitative curve of nitrine;
Fig. 2 is the detection sensitivity of nitrine labelled antibody;
Fig. 3 is the cell dyeing analysis,
A: negative control; B: reaction group; C: positive control;
Fig. 4 is that the triple channel fluorescent dye is analyzed,
A: metabolic marker Her2 antibody staining; The EGFR4 dyeing of B:FITC labelled antibody; C: biotinylated GRP94 dyeing; D: triple channel multiple analysis.
Embodiment
The fluorescence labeling of Anti-HP15
1 mg NHS-FITC or NHS-rhodamine are dissolved among the 100 μ L DMSO, draw 4.7 μ L NHS-FITC subsequently or 3.5 μ L NHS-rhodamines dropwise add 1 mL Anti-HP15 antibody-solutions (0.1 mol/L, pH=9.0 Na
2CO
3-NaHCO
3, 1 mg/mL IgG) in, after stirring 1 h under the room temperature, centrifugal 10 min of 5000 r/min draw supernatant and in PBS (pH=7.2,0.02 mol/L Na
2HPO
4-NaH
2PO
4, 0.15 mol/L NaCl) in fully dialysis, remove unreacted NHS-FITC and NHS-rhodamine, the Anti-HP15 antibody of acquisition FITC or rhodamine mark is used for positive control.
Embodiment 2
Synthesizing of key compound (the non-natural monose that contains azido group)
The synthetic method of crucial compound is following: select to carry out Azide and acetylation, the non-natural monose that the acetylizad specificity of synthetic respectively preparation is modified like precursor monose such as N-acetylated mannan sugar, N-acetyl sialic acid, N-acetyl galactose and N-acetyl glucosamines through optimizing glycosyl protection strategy; To 4 bromo-1, add ethamine in the 8-naphthalimide, product is collected in the reaction back in ethanolic solution; Add TMS alkynes; In the tetrahydrofuran system,, obtain 4-ethinyl-N-ethyl-1, the 8-naphthalimide through tetrakis triphenylphosphine palladium and cuprous iodide catalytic reaction.
The metabolic marker of anti-her2 antibody
In the cell culture medium that the non-natural monose that embodiment 2 is synthesized slowly join, final concentration is 0.2 mM, join behind the mixing in the hybridoma, and 37 ℃, 5% CO
2Cultivate 72 h, the collecting cell suspension, centrifugal 3 min of 1000 r/min draw supernatant, with the antibody of antibody purification affinity column purifying metabolic marker.
Embodiment 4
The nitrine labelled amount of metabolic marker antibody is analyzed
With 1,3,4 of variable concentrations, 6-oxygen acetyl-2-'-deoxy-n-nitrine acetylglucosamine is added to and contains 0.5 mmol/L4-ethinyl-N-ethyl-1,8-naphthalimide, 0.2 mmol/L Tris-triazoleamine, 1 mmol/L CuSO
4And in the PBS solution of 2 mmol/L sodium ascorbates, behind room temperature 60 min, in 365 nm exciting lights measure down 465 nm down respectively organize fluorescence intensity, the drafting concentration dependent typical curve of azido group (Fig. 1); Simultaneously; With 3 μ mol/L metabolic marker antibody surrogates 1; 3,4,6-oxygen acetyl-2-'-deoxy-n-nitrine acetylglucosamine carries out the click chemistry reaction; Confirm to convert the Azide labeling effciency of antibody and learn that antibody labeling efficient is 3 azido groups of the about mark of each molecular antibody according to the fluorescence intensity of reaction product.
Embodiment 5
The sensitivity analysis of nitrine labelled antibody
Adopt enzyme linked immunosorbent assay analysis method to measure the sensitivity of metabolic marker detection of antibodies.100 μ L membranous antigens, the 5 μ g that in 96 hole ELISA Plates, add ultrasonication; 37 ° of C adopt 4 ° of C sealings of 1% BSA to spend the night after encapsulating 2 h; The antibody, the antibody of FITC mark and the antibody (10,1,0.1,0.01,0.001 and 0.001 μ g/mL) of rhodamine mark that add the identical metabolic marker of the concentration of 100 μ L serial dilutions subsequently, room temperature reaction 3 h.Fully after the washing, in nitrine labeled reactant group, add and contain 0.5 mmol/L 4-ethinyl-N-ethyl-1,8-naphthalimide, 0.2 mmol/L Tris-triazoleamine, 1 mmol/L CuSO
4And the PBS solution of 2 mmol/L sodium ascorbates, after the washing, measure the fluorescence intensity in every hole under the 365 nm exciting lights, emission wavelength is 465 nm; The antibody of the antibody of FITC mark and rhodamine mark excites and measures respectively the fluorescence intensity under 518 nm and 590 nm with respective excitation wavelength (488 nm and 570 nm).The EC of metabolic marker antibody
50Value is more lower slightly than control group; But at the same order of magnitude; Its detection limit is about 0.1 μ g; Be that the sensitivity of metabolic marker detection of antibodies is almost consistent with the detection sensitivity of existing commercial labelled reagent NHS-FITC and NHS-rhodamine, detectability is consistent, can be applied to (Fig. 2) in the immunofluorescence analysis as a kind of effective compensation process.
Embodiment 6
Immunofluorescence analysis
MCF-7 SK-BR-3 was cultivated 3 days in the RPMI that contains 10% FBS 1640 nutrient culture media, then with 1.25 * 10
5Cell transfer overnight incubation in the laser co-focusing culture plate; Subsequently with fixing 30 min of 4% paraformaldehyde room temperature; Fully after the washing sealing; Add 1 mg/mL metabolic marker antibody, add behind room temperature reaction 3 h and contain 0.5 mmol/L 4-ethinyl-N-ethyl-1,8-naphthalimide, 0.2 mmol/L Tris-triazoleamine, 1 mmol/L CuSO
4And the reaction of the PBS solution catalyzing of 2 mmol/L sodium ascorbates, observe under 365 nm through laser confocal microscope.Simultaneously, adopt the corresponding FITC mark two anti-cell fluorescences that carry out of identical unlabelled antibody and (embodiment 1 preparation) to dye, under 488 nm, analyze as positive control; Only add catalytic reaction solution and do not add an anti-cell and observe down in 365 nm as negative control group.When adopting the metabolic marker antibody staining to analyze, can observe blue-fluorescence (Fig. 3 B) under 365 nm significantly, and in not adding the negative control of antibody, almost not have fluorescence signal (Fig. 3 A); In positive control; Adopt the two anti-immuning fluorescent dyeing analysis that carry out of FITC mark; And fluorescence location and use nitrine labelled antibody coloration result consistent (Fig. 3 C); Be the method developed of the present invention no matter in detection sensitivity, still all suitable on the accuracy of testing result with existent method, can be widely used in the immunofluorescence check and analysis.
Embodiment 7
Complex staining is analyzed
MCF-7 SK-BR-3 is cultivated 3 d with the RPMI 1640 that contains 10% FBS, then, with 1.25 * 10
5Cell transfer is in the laser co-focusing double dish; Overnight incubation; The paraformaldehyde of adding 4% is fixed after the PBS washing, fully adds metabolic marker antibody, the anti-EGFR4 antibody of rabbit and biotinylated anti-GRP94 antibody simultaneously after the washing sealing, behind room temperature reaction 1 h; In double dish, add 0.5 mmol/L 4-ethinyl-N-ethyl-1,8-naphthalimide, 0.2 mmol/L Tris-triazoleamine, 1 mmol/L CuSO
4And the PBS solution of 2 mmol/L sodium ascorbates; Room temperature reaction 1 h; Fully after the washing, in double dish, add FITC mark goat anti-rabbit igg and strepadivin-CY3, behind room temperature 1 h; Fully washing is observed the fluorescence signal under 365 nm, 488 nm and the 550nm exciting light respectively with laser confocal microscope.When excitation wavelength is 365 nm, can observe tangible blue-fluorescence signal (Fig. 4 A) in the interior survey of cell membrane, i.e. 4-ethinyl-N-ethyl-1, the 8-naphthalimide produces autofluorescence after changing electron distributions; Under 488 nm exciting lights, can observe tangible FITC green fluorescence signal (Fig. 4 B) in the cell membrane outside; Under 550 nm exciting lights, can observe tangible Cy3 red fluorescence signal (Fig. 4 C).Fluorescence signal stack result such as Fig. 4 D show that 3 kinds of fluorescence signals can effectively merge, and signal do not disturb mutually, and promptly the fluorescence antibody analytical approach behind the metabolic marker can and be used with existing conventional method.
Embodiment 8
Synthetic and the chromogenic assay of fluorescent reagent
Respectively fluorophors such as Cy3, Cy5, FITC, TRITC, PE, DAPI, Texas red, RB200, Indo-1, quantum dot are carried out chemical modification, make it to contain alkynyl group.Carry out the click chemistry reaction according to embodiment 6 subsequently, make the azido group reaction on alkynyl and the antibody, form stable triazole ring, and then make sample to be detected possess lighting function, be convenient to detect.
Embodiment 9
Synthetic and the chromogenic assay of fluorescent reagent
To contain label groups such as His, GST, FLAG, HA, biotin respectively and carry out chemical modification, make it to contain alkynyl group.Carry out the click chemistry reaction according to embodiment 6 subsequently, make the azido group reaction on alkynyl and the antibody, form stable triazole ring, add the corresponding affinity ligand of various labels subsequently, and then make sample to be detected possess lighting function, be convenient to detect.
Claims (7)
1. the metabolic marker method of an antibody is characterized in that:
Metabolic marker thing when synthesizing as glycoprotein with the non-natural monose that contains azido group; Replace the normal monose in the NGP; Thereby make and be labeled protein and contain azido group; And then react through the click chemistry between azido group and the azide reaction group, make protein possess the label group that lighting function or coupling can be to be detected.
2. the metabolic marker method of antibody according to claim 1 is characterized in that the concrete steps of this method are following:
The non-natural monose that will contain azido group slowly joins in the cell culture medium, and final concentration is 0.2 mM, join behind the mixing in the hybridoma, and 37 ℃, 5% CO
2Cultivate 72 h, the collecting cell suspension, centrifugal 3 min of 1000 r/min draw supernatant, with the antibody of antibody purification affinity column purifying metabolic marker.
3. method according to claim 1 and 2 is characterized in that the described non-natural monose that contains azido group comprises 1,3,4; 6-oxygen acetyl-2-'-deoxy-n-nitrine acetylmannosamine, 1,3,4,6-oxygen acetyl-2-'-deoxy-n-nitrine acetyl sialic acid, 1; 3,4,6-oxygen acetyl-2-'-deoxy-n-nitrine acetylgalactosamine and 1; 3,4,6-oxygen acetyl-2-'-deoxy-n-nitrine acetylglucosamine.
4. method according to claim 1 and 2 is characterized in that described azide reaction group is to form the alkynyl compounds of triazole ring with azido group, and self possesses the coupling group of the characteristic of fluorescing or contain the label group.
5. method according to claim 4 is characterized in that the described coupling group that possesses the characteristic of fluorescing comprises Cy3, Cy5, FITC, TRITC, PE, DAPI, Texas red, RB200, Indo-1 and quantum dot.
6. method according to claim 4 is characterized in that described label group comprises His, GST, FLAG, HA and biotin.
7. the application of metabolic marker antibody in the fluoroscopic examination analytical approach of the said method preparation of claim 1.
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| CN103454268A (en) * | 2013-04-19 | 2013-12-18 | 南京工业大学 | Revertose quantitative detection method based on click reaction |
| CN103604791A (en) * | 2013-11-29 | 2014-02-26 | 广西师范大学 | Fluorescence spectroscopic method for measuring azides ions |
| CN103755778A (en) * | 2014-02-24 | 2014-04-30 | 北京大学 | Immunofluorescent labeling method for protein in living cell |
| CN104749369A (en) * | 2013-12-31 | 2015-07-01 | 中国科学院深圳先进技术研究院 | Fluorescent labeling method for living organisms having cell membrane structures |
| CN107810273A (en) * | 2015-04-23 | 2018-03-16 | 西纳福克斯股份有限公司 | With for or derived from β (1,4) N acetylgalactosamine transferases glycosyl transferase modified glucoprotein method |
| CN108169474A (en) * | 2018-01-12 | 2018-06-15 | 中国科学院成都生物研究所 | A kind of novel cell fixative |
| CN108728397A (en) * | 2017-04-17 | 2018-11-02 | 中国科学院微生物研究所 | The method that the foreign protein that insect protein or insect expression system are expressed is marked |
| CN108865918A (en) * | 2017-05-09 | 2018-11-23 | 中国科学院微生物研究所 | A method of the gram-positive bacterium of label survival and its cell wall |
| CN109897881A (en) * | 2019-03-21 | 2019-06-18 | 中国科学院深圳先进技术研究院 | The metabolism modification of non-togavirus labeling method orthogonal with biology in situ and application |
| CN112082976A (en) * | 2019-06-14 | 2020-12-15 | 天津方得生物科技有限公司 | In-vitro drug sensitivity detection method based on drug probe and tissue slice |
| CN113897412A (en) * | 2021-09-23 | 2022-01-07 | 中国科学院城市环境研究所 | Screening method of drug-resistant microorganisms by fluorescence method |
| US11634448B2 (en) | 2016-06-15 | 2023-04-25 | The General Hospital Corporation | Metabolic labeling and molecular enhancement of biological materials using bioorthogonal reactions |
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| CN103454268B (en) * | 2013-04-19 | 2016-02-24 | 南京工业大学 | A kind of reducing sugar quantitative detecting method based on click-reaction |
| CN103454268A (en) * | 2013-04-19 | 2013-12-18 | 南京工业大学 | Revertose quantitative detection method based on click reaction |
| CN103604791A (en) * | 2013-11-29 | 2014-02-26 | 广西师范大学 | Fluorescence spectroscopic method for measuring azides ions |
| CN103604791B (en) * | 2013-11-29 | 2016-01-20 | 广西师范大学 | A kind of fluorescence spectrum method measuring azides ion |
| CN104749369A (en) * | 2013-12-31 | 2015-07-01 | 中国科学院深圳先进技术研究院 | Fluorescent labeling method for living organisms having cell membrane structures |
| CN103755778A (en) * | 2014-02-24 | 2014-04-30 | 北京大学 | Immunofluorescent labeling method for protein in living cell |
| CN107810273B (en) * | 2015-04-23 | 2022-09-27 | 西纳福克斯股份有限公司 | Method for modifying glycoproteins with a glycosyltransferase that is or is derived from a beta- (1,4) -N-acetylgalactosamine transferase |
| CN107810273A (en) * | 2015-04-23 | 2018-03-16 | 西纳福克斯股份有限公司 | With for or derived from β (1,4) N acetylgalactosamine transferases glycosyl transferase modified glucoprotein method |
| US11634448B2 (en) | 2016-06-15 | 2023-04-25 | The General Hospital Corporation | Metabolic labeling and molecular enhancement of biological materials using bioorthogonal reactions |
| CN108728397A (en) * | 2017-04-17 | 2018-11-02 | 中国科学院微生物研究所 | The method that the foreign protein that insect protein or insect expression system are expressed is marked |
| CN108728397B (en) * | 2017-04-17 | 2022-05-20 | 中国科学院微生物研究所 | Method for marking insect protein or foreign protein expressed by insect expression system |
| CN108865918B (en) * | 2017-05-09 | 2022-01-07 | 中国科学院微生物研究所 | Method for marking viable gram-positive bacteria and cell walls thereof |
| CN108865918A (en) * | 2017-05-09 | 2018-11-23 | 中国科学院微生物研究所 | A method of the gram-positive bacterium of label survival and its cell wall |
| CN108169474B (en) * | 2018-01-12 | 2020-09-25 | 中国科学院成都生物研究所 | Novel cell fixing agent |
| CN108169474A (en) * | 2018-01-12 | 2018-06-15 | 中国科学院成都生物研究所 | A kind of novel cell fixative |
| CN109897881A (en) * | 2019-03-21 | 2019-06-18 | 中国科学院深圳先进技术研究院 | The metabolism modification of non-togavirus labeling method orthogonal with biology in situ and application |
| CN112082976A (en) * | 2019-06-14 | 2020-12-15 | 天津方得生物科技有限公司 | In-vitro drug sensitivity detection method based on drug probe and tissue slice |
| CN113897412A (en) * | 2021-09-23 | 2022-01-07 | 中国科学院城市环境研究所 | Screening method of drug-resistant microorganisms by fluorescence method |
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Application publication date: 20120111 |