WO2023143179A1 - Processing method for antigen sample, and use - Google Patents

Processing method for antigen sample, and use Download PDF

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Publication number
WO2023143179A1
WO2023143179A1 PCT/CN2023/072345 CN2023072345W WO2023143179A1 WO 2023143179 A1 WO2023143179 A1 WO 2023143179A1 CN 2023072345 W CN2023072345 W CN 2023072345W WO 2023143179 A1 WO2023143179 A1 WO 2023143179A1
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Prior art keywords
antibody
labeled antibody
glycan
labeled
combined
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PCT/CN2023/072345
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French (fr)
Chinese (zh)
Inventor
李劼
毕昂志
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南京大学
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Publication of WO2023143179A1 publication Critical patent/WO2023143179A1/en

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    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals

Definitions

  • the present application relates to the field of antigen processing, in particular to a method and application for processing antigen samples.
  • IHC/IF immunohistochemistry/immunofluorescence imaging
  • the reported multiple immunohistochemistry/immunofluorescence techniques are mainly divided into the following four categories: (1) Enzyme-based tyramide chemical labeling: such as the Vectra imaging system developed by Akoya Biosciences, which uses microwaves on the same slice to Dissociate the labeled antibody multiple times, and use a narrow-spectrum and detachable fluorescent dye each time to achieve multicolor fluorescence imaging of 7 to 9 colors; (2) Multicolor imaging based on chemically cleavable fluorescent antibodies: such as Guo et al.
  • Multicolor imaging method based on base pairing such as the CODEX technology reported by Nolan et al., using deoxynuclear
  • the nucleotide chain-antibody conjugates image the target, and realize the research on multiple target proteins through the extension of a single base on the primer and the cleavage of the disulfide bond between the base and the fluorescent group by TCEP, which can also reach 40+ Color multiplex fluorescence imaging.
  • the present application provides a method for processing antigen samples.
  • specific site-glycosidic bond enzyme cleavage on labeled antibodies containing glycans, the separation of glycans and antibodies is achieved, and the enzymatic cleavage is Excision of a specific site will not cause damage to the antigen, and can be used for multiple subsequent rounds of multi-channel single-cell analysis, not only for multiple immunohistochemistry/immunofluorescence techniques, but also for protein and nucleic acid sequence amplification in cells Wait for the scene.
  • a method for processing an antigen sample comprising:
  • the labeled antibodies include antibodies and glycans linked to the antibodies, and the glycans contain glycosidic bonds; the antigen samples include multiple sites that can bind to antibodies; ( 2) binding the labeled antibody to the site, or binding the site to the first antibody to obtain a combined first antibody; combining the labeled antibody to the combined first antibody, The bound labeled antibody is obtained; the glycosidic bond in the bound labeled antibody is digested by glycosidase.
  • the above-mentioned processing method is applicable to antibodies labeled by antibody glycan modification, both currently published and unpublished.
  • step (2) several different kinds of labeled antibodies are combined with their corresponding antigens or primary antibodies.
  • the types of the labeled antibodies are 1-10.
  • the treatment method further includes: (3) preparing a labeled antibody that is the same as or different from step (1); binding the labeled antibody to the site in step (1) , or, after the site is combined with a new first antibody, a new combined first antibody is obtained; the new combined first antibody is combined with the labeled antibody in this step to obtain a combined label Antibody; (4) cleavage of the glycosidic bond in the labeled antibody bound in the step (3) by glycosidase.
  • the labeled antibody in the step (3) is different from the labeled antibody in the step (1).
  • the difference between the labeled antibody in the step (3) and the labeled antibody in the step (1) is reflected in the difference of the antibody and the glycan, that is, at least one of the antibody and the glycan is different.
  • the labeled antibody in the step (3) is different from the labeled antibody in the step (1), and is used to bind to different antigens or secondary antibodies, which is beneficial to the comprehensive mapping of subsequent protein maps.
  • the labeled antibody in the step (3) is different from the glycan of the labeled antibody in the step (1), which can increase the diversity of linking structure molecules, and it is easy to select a suitable linking structure according to different scenarios; But in glycans, there are at least two monosaccharides linked by glycosidic bonds.
  • the labeled antibody in the step (3) is the same as the labeled antibody in the step (1).
  • the labeled antibodies are the same, and can be combined with all antigens that can be combined with the labeled antibody, so as to facilitate accurate detection of the content of a certain antigen in the multiple antigen samples.
  • the new first antibody in step (3) is the same or different from the first antibody in step (2).
  • the "new” refers to substances that are not previously used, but whose composition and content may be the same or different; that is, the new first antibody in step (3) and the first antibody in step (2)
  • the composition and content can be the same or different, but the new primary antibody in step (3) is not the primary antibody in step (2).
  • step (2) Since the first antibody in step (2) binds to its corresponding site, even after enzyme digestion, the first antibody still binds to the site, in order to detect other unbound sites, it is necessary to add a new first antibody Bind to its corresponding site.
  • the antigen sample includes multiple antigens, that is, the antigen sample includes at least two antigens.
  • the antigen sample is a mixture of at least two antigens.
  • the antigen sample includes at least one of cells, animal and plant tissues, and human tissues.
  • animal and plant tissues and human tissues include paraffin-embedded tissues, frozen section tissues, etc.
  • the processing method further includes: multiple cycles of step (3) and step (4); the number of multiple cycles is ⁇ 2.
  • the times of combining and enzyme cutting are at least four times.
  • Combining and enzymatic cleavage are a cycle, and the more the number of cycles, the more conducive to the drawing of the protein map.
  • the enzyme cleavage of glycosidic bonds is used, that is, by cleavage of specific sites, which can reduce the damage to the antigen, and the number of cycles can be reduced. Reach 5 or more times.
  • the mass ratio of the labeled antibody to the glycosidase is 1:0.001-1000.
  • the glycosidase includes endoglycosidase and/or exoglycosidase.
  • the glycosidase includes at least one of EndoS, EndoF and Alfc.
  • the glycosidase includes at least two of EndoS, EndoF, and Alfc.
  • the conditions for the enzyme digestion are: the temperature is 0°C-45°C.
  • the glycan is also linked with a labeling structure.
  • the labeling structure is a group for detection or display, and its related signals can be captured by detection equipment for quantitative or qualitative analysis of the corresponding labeled antibody.
  • the labeling structure includes enzymes, fluorescent substances, radionuclides, chemiluminescent agents, quantum dots, biotin or dyes.
  • the dyes include, but are not limited to, Alexa Fluor 488, Alexa Fluor 568, FITC, Cy3 or Cy5.
  • the preparation method of the labeled antibody containing the glycan linked with the labeled structure includes enzymatic reaction and bioorthogonal chemical reaction.
  • the enzymatic reaction includes reacting the glycan and/or monosaccharide linked with the labeled structure with the antibody by a glycosyltransferase to obtain a labeled antibody containing the glycan linked with the labeled structure; or Glycans and/or monosaccharides linked with labeled structures are reacted with antibodies and other glycans and/or monosaccharides to obtain labeled antibodies containing glycans linked with labeled structures.
  • the bioorthogonal chemical reaction includes, but is not limited to: reacting dibenzocyclooctyne or alkyne linked to a labeled structure, an azide combined with a sugar, and an antibody to obtain a A labeled antibody to a glycan; or, react a trans-cyclooctene linked to a labeled structure, a tetrazine or methyltetrazine bound to a sugar, and an antibody to obtain a labeled antibody containing a glycan linked to a labeled structure.
  • said tagging structure and said tagged antibody are linked via said glycan.
  • the labeled antibody is covalently linked to the glycan.
  • the glycans include two or more monosaccharides.
  • the two or more monosaccharides may be in the same glycoform or in different glycoforms.
  • the glycan there are at least two monosaccharides linked by glycosidic bonds.
  • the glycans are N-glycans and/or O-glycans.
  • the N-glycan refers to the sugar linked to Asn through an N atom
  • the O-glycan refers to the sugar linked to Ser/Thr through an O atom
  • the glycans are N-glycans.
  • the monosaccharides include N-acetylglucosamine, mannose, galactose, fucose, N-semi at least one of lactosamines.
  • the polysaccharide is a plurality of monosaccharides connected in any order by covalent or non-covalent bonds.
  • the glycans include natural or non-natural glycans.
  • Natural glycans refer to the configuration and composition of glycans that exist in nature; non-natural glycans refer to artificially synthesized non-naturally occurring glycan configurations or compositions.
  • the polysaccharide is a compound obtained by random arrangement and combination of various monosaccharides.
  • the polysaccharides include monosaccharide-monosaccharide, monosaccharide-monosaccharide-monosaccharide, At least one of monosaccharide-monosaccharide-monosaccharide-monosaccharide.
  • the glycan is linked to at least one labeling structure.
  • the tag structure is linked to any one or more monosaccharides in the glycan.
  • one of said tagging structures is linked to one of said monosaccharides.
  • the glycan is linked to 1-10 marker structures.
  • the glycan is linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 marker structures.
  • the glycan is linked to 1-4 marker structures.
  • the glycan of the present application can be connected with multiple labeling structures, and the labeling structure can be located on any monosaccharide; as shown in Figure 1, the glycosidic bond in the glycan is cleaved by glycosidase to remove the labeling structure .
  • the difference between the labeled antibody containing glycans in the step (3) and the labeled antibody in the step (1) is also reflected in the difference in the labeling structure, that is, the antibody, the glycan or the labeling structure At least one of the different.
  • the other glycan-containing labeled antibody has a different labeling structure from the glycan-containing labeled antibody, which can be used to detect different signals, which is conducive to more accurate and reliable detection results.
  • elution is also performed.
  • the elution is to wash away the cleaved functional groups by the eluent, so as to avoid further damage to the subsequent signal. No. detection is affected.
  • the eluent includes but not limited to DPBS buffer solution, HEPES buffer solution, Tris buffer solution, etc.
  • This application provides a method for processing antigen samples, which uses the specific binding of labeled antibodies containing glycans to antigens or primary antibodies, and then performs specific site-glycosidic bond enzymatic cleavage to realize the glycan-linked labeling structure and Separation of antibodies; after repeated recognition and binding of labeled antibodies containing glycans to antigens and enzymatic cleavage of glycan-linked labeled structures, comprehensive detection of intracellular proteins can be achieved.
  • This application removes glycans or glycans with labeled structures by enzymatic cleavage, which not only can control the cleavage site, but also does not damage other proteins; and can treat multiple proteins at the same time without interfering with each other, greatly improving
  • the speed of in situ processing of intracellular proteins is improved; and due to the mildness and high efficiency of glycosidase, the cutting efficiency is greatly improved, and multiple rounds of processing can be performed, which has a higher structure for in situ processing and detection of intracellular proteins. the accuracy.
  • the treatment method of the present application can not only perform mild cutting in various biochemical and immunochemical scenarios (such as IF, WB), greatly reduce the damage to the antigen, but also perform efficient cutting, and can perform multiple rounds of IF and multiple rounds of WB. detection.
  • Fig. 1 is the schematic diagram of the reaction of the present application to the cleavage of the labeled antibody; wherein, stands for N-acetylglucosamine; represents mannose; represents galactose; represents fucose; Represents N-galactosamine; X represents the labeled structure.
  • Fig. 2 is the schematic diagram of the reaction of the labeled antibodies Ab-1 and Ab-2 prepared by the present application; wherein, stands for N-acetylglucosamine; represents mannose; represents galactose; represents fucose; Represents N-galactosamine; X represents biotin or Cy5.
  • Fig. 3 is the schematic diagram of the reaction of the labeled antibodies Ab-3 and Ab-4 prepared by the present application; wherein, stands for N-acetylglucosamine; represents mannose; represents galactose; represents fucose; Represents N-galactosamine; X represents biotin or Cy5; Represents bioorthogonal reaction conjugation.
  • Fig. 4 is a diagram of the fluorescence results of the mixture after digestion in Example 1 of the present application; the abscissa is the fluorescence intensity, unitless; the ordinate is the relative cell number, the unit is %.
  • Fig. 5 is the residual fluorescence diagram of the mixture of enzymes with different concentrations in Example 2 of the present application after digesting the labeled antibody; Fluorescence rate, in %.
  • Fig. 6 is the residual fluorescence diagram of the mixture of different enzyme types and different labeled antibodies in Example 3 of the present application; the abscissa is from left to right group I, group II (Ab-1), group III (Ab-2) , IV group (Ab-3) and V group (Ab-4), each group is further divided into two groups of 0.5 ⁇ +0.5 ⁇ and 1 ⁇ ; the ordinate is the residual fluorescence rate, the unit is %.
  • Figure 7 is the mass spectrogram of the labeled antibody after digestion with different reaction times in Example 4 of the present application; (a) 0min; (b) 5min; (c) 10min; (d) 20min; the abscissa is the molecular weight, the unit is Da; the ordinate is the strength, the unit is %;
  • Figure 8 is a comparison chart of antigen expression in Example 6 of the present application after different cutting methods at the level of living cells; the abscissa is from left to right: Group A, Group B (DPBS), Group C (50mM TCEP), and Group D (100mM TCEP), E group (200mM TCEP), F group (0.1 ⁇ EndoS+0.1 ⁇ EndoF3), G group (0.5 ⁇ EndoS+0.5 ⁇ EndoF3) and H group (1 ⁇ EndoS+1 ⁇ EndoF3), each group is divided into two groups of Her2 and EGFR ; The vertical axis is the residual fluorescence rate, the unit is %.
  • Fig. 9 is an image of cells before and after enzyme cleavage in Example 7 of the present application; (a) before cleavage; (b) after cleavage.
  • Fig. 10 is the staining diagram of paraffin sections before and after enzyme cleavage in Example 8 of the present application; (a) before cleavage; (b) after cleavage.
  • Fig. 11 is the enzyme cleavage diagram of Example 5 of the present application; (a) protein gel characterization diagram before and after cleavage; (b) cleavage efficiency diagram for antibody-horseradish peroxidase (Ab-HRP); abscissa is control group and glycosidase group; the ordinate is the cutting effect, and the unit is %.
  • Ab-HRP antibody-horseradish peroxidase
  • abscissa is control group and glycosidase group
  • the ordinate is the cutting effect, and the unit is %.
  • Figure 12 is a comparison chart of antigen retention after different cleavage methods at the fixed cell level in Example 6 of the present application; (A) antigen retention after cleavage of HER2; (B) antigen retention after cleavage of EGFR; abscissa is the number of treatments, from left to right: treatment once, treatment three times and treatment five times; the vertical axis is the amount of antigen preservation, no unit.
  • Figure 13 is a comparison chart of the relative antigen retention of HER2 after different cutting methods on the surface of the imprinted membrane in Example 6 of the present application; (A) the antigen retention after cutting HER2 once; (B) the antigen after cutting EGFR three times Preservation amount; the vertical axis is the antigen preservation rate, no unit.
  • Protein A resin purification add NMab protein A (NMab Protein A) in an amount of 100 ⁇ L/1mg (antibody to be purified), add it to an affinity chromatography column, centrifuge at a centrifugal force of 1000g for 1min, and use 400 ⁇ L phosphate buffer solution (DPBS buffer solution) washed 3 times. Add antibody, mix and incubate for 1h. Centrifuge for 1 min with a centrifugal force of 1000 g, and wash 3 times with DPBS buffer solution.
  • NMab Protein A NMab Protein A
  • DPBS buffer solution phosphate buffer solution
  • concentration value in the following is the concentration of the substance in the mixture of the current step.
  • Trastuzumab (3 ⁇ ), MnCl 2 (5mM), MgCl 2 (10mM), Tris-HCl buffer solution (10mM, pH 7.4), uridine diphosphate galactose (UDP-Galactose , 5mM), ⁇ -1,4-galactosyltransferase ( ⁇ -1,4-galactosyltransferase 1, ⁇ 4GalT1, 0.4 ⁇ ), fucosyltransferase (Fucosyltransferase, FT, 1 ⁇ ) and guanosine diphosphate-rock Cocose-biotin (Guanosine diphosphate fucose-biotin, GDP-Fuc-Biotin, 2mM) was subjected to a one-pot reaction at 30°C and 300rpm in a metal bath for 40h. The protein A resin was used for affinity purification, and the purified product was concentrated and desalted into DPBS buffer solution to obtain the labeled antibody
  • Trastuzumab (3 ⁇ ), MnCl 2 (5mM), MgCl 2 (10mM), Tris-HCl buffer solution (10mM, pH 7.4), uridine diphosphate galactose (UDP-Galactose , 5mM), ⁇ -1,4-galactosyltransferase ( ⁇ -1,4-galactosyltransferase 1, ⁇ 4GalT1, 0.4 ⁇ ), fucosyltransferase (Fucosyltransferase, FT, 1 ⁇ ) and guanosine diphosphate-rock Cocose-fluorescent dyes (Guanosine diphosphate fucose-Cy5, GDP-Fuc-Cy5, 2mM) were subjected to a one-pot reaction at 30°C and 300rpm in a metal bath for 40h. After affinity purification with protein A resin, the purified product was concentrated and desalted into DPBS buffer solution to obtain the labeled antibody Ab-2.
  • the preparation methods of labeled antibodies Ab-1 and Ab-2 are obtained through enzymatic reactions, specifically, through glycosyltransferases, reacting monosaccharides linked with labeled structures with antibodies and other monosaccharides, as shown in Figure 2 shown.
  • UDP-GalNAz Uridine diphosphate-acetylated-N-azidoacetylgalactosamine
  • ⁇ -1,4-galactosyltransferase ⁇ -1,4-galactosyltransferase 1, ⁇ -4GalT1, 0.5 ⁇
  • UDP-GalNAz uridine diphosphate-acetylated-N-azidoacetylgalactosamine
  • ⁇ -1,4-galactosyltransferase ⁇ -1,4-galactosyltransferase 1, ⁇ -4GalT1, 0.5 ⁇
  • the preparation methods of labeled antibodies Ab-3 and Ab-4 are both through click chemistry, specifically, the click chemistry includes: dibenzocyclooctyne linked to the label structure, alkyne azide combined with sugar, and Antibodies were reacted, as shown in Figure 3.
  • the protein A resin was used for affinity purification, and the
  • UDP-GalNAz uridine diphosphate-acetylated-N-azidoacetylgalactosamine
  • ⁇ -1,4-galactosyltransferase ⁇ -1,4-galactosyltransferase 1, ⁇ -4GalT1, 0.5 ⁇
  • Adherent cultured SK-OV-3 cells (human ovarian cancer cells, used to express HER2) were digested with trypsin, resuspended with FACS buffer solution after centrifugation, and centrifuged and washed three times. After being resuspended with FACS buffer solution, the same amount was divided into 96-well plates and marked as blank group, control group and experimental group respectively, with ten wells in each group. Among them, the blank group was not treated; both the control group and the experimental group were added with labeled antibody Ab-1 (the final concentration of labeled antibody Ab-1 in each group was 10 ⁇ g/mL), and incubated on ice for 30 minutes.
  • Adherent cultured SK-OV-3 cells (human ovarian cancer cells, used to express HER2) were digested with trypsin, resuspended with FACS buffer solution after centrifugation, and centrifuged and washed three times. After resuspending with FACS buffer solution, the same amount was added to a 96-well plate, marked as control group and experimental group respectively, with ten wells in each group. Among them, both the control group and the experimental group were added with labeled antibody Ab-1 (10 ⁇ g/mL), and incubated on ice for 30 min. After centrifugation, resuspend with FACS buffer solution, and repeat centrifugation and washing 3 times.
  • EndoS the final concentration of EndoS was 0.1 ⁇ , 0.1 ⁇ group
  • EndoS the final concentration of EndoS was 0.5 ⁇ , 0.5 ⁇ group
  • EndoS The final concentration of EndoS was 1 ⁇ , 1 ⁇ group
  • EndoS can enzymatically cut the labeled antibody and reduce the degree of fluorescence of the labeled antibody, but increasing the concentration of EndoS found that the proportion of residual fluorescence did not decrease significantly, indicating that the increase in the concentration of a single enzyme did not promote enzyme cleavage effect.
  • Adherent cultured SK-OV-3 cells (human ovarian cancer cells, used to express HER2) were digested with trypsin, resuspended with FACS buffer solution after centrifugation, and centrifuged and washed three times. After being resuspended with FACS buffer solution, the same amount was added to a 96-well plate and marked as two groups of control group and eight groups of experimental group, with ten wells in each group. Among them, labeled antibodies Ab-1, Ab-2, Ab-3, and Ab-4 were added to the control group and the experimental group respectively (the final concentration of each labeled antibody was 10 ⁇ g/mL), and incubated on ice for 30 minutes.
  • the antibody-horseradish peroxidase (Ab-HRP) conjugate based on sugar chain modification was cleaved by this method and characterized by protein glue.
  • the results are shown in (A) of Figure 11.
  • coupled small molecules such as biotin, fluorescent dyes
  • efficient cleavage of large molecules such as HRP here.
  • the SK-OV-3 cells were respinned with RIPA lysate and then ultrasonically crushed to extract the whole protein, run on the gel and transfer to the membrane, incubate the cleavable antibody, and then perform enzyme digestion or not, the result is shown in (B) of Figure 11
  • the method of the present application compared with the control group (DPBS), the method of the present application also has high cutting efficiency to the antibody conjugate on the surface of the imprinted membrane, which makes the method of the present application not only applicable to multiple rounds of immunofluorescence, but also applicable to multiple rounds of immunofluorescence. Potential for blotting experiments.
  • Adherent cultured SK-OV-3 cells (human ovarian cancer cells, used to express HER2) were digested with trypsin, resuspended with FACS buffer solution after centrifugation, and centrifuged and washed three times. After being resuspended with FACS buffer solution, the same amount was added to a 96-well plate, and eight groups were marked as the control group and the experimental group, with ten wells in each group. Among them, labeled antibody Ab-1 (10 ⁇ g/mL) was added to the control group and the experimental group respectively, and incubated on ice for 30 min. After centrifugation, resuspend with FACS buffer solution, and repeat centrifugation and washing 3 times.
  • the expression of the antigens Her2 and EGFR was detected for the mixture after 5 times of cleavage by the above enzymes, and the detection results are shown in FIG. 8 .
  • glycosidase method was used on the western blot membrane to compare with the traditional stripping solution.
  • the stripping solution was processed as follows: SK-OV-3 cells (human ovarian cancer cells, expressing HER2) were respinned with RIPA lysate, then ultrasonically crushed to extract the whole protein, and the blotted membrane was processed after gel transfer.
  • the conditions were: 1 ⁇ TBST, 0.5 ⁇ EndoS+0.5 ⁇ EndoF3 (the final concentration of EndoS was 0.5 ⁇ , the final concentration of EndoF3 was 0.5 ⁇ ; 0.5 ⁇ EndoS+0.5 ⁇ EndoF3 group) and stripping buffer (stripping buffer) at room temperature for 30 minutes, in After 1 and 3 treatments, the antibodies (the primary antibody is HER2/ErbB2(29D8) Rabbit mAb, the secondary antibody is anti-Rabbit IgG-HRP) were incubated to detect the antigen Her2, and the results are shown in Figure 13. (TBST group is the control group in WB experiments, TBST is often used in blot membrane washing buffer, which is considered low antigen damage)
  • glycosidase cleavage treatment sample method proposed in this patent has verified that this method is compared with other cutting methods at the level of living cells (Figure 8), the level of fixed cells ( Figure 12) and the surface of imprinted membranes (Figure 13). or elution reagents have the advantage of being milder, especially after multiple rounds of repeated elutions, this method exhibits more complete retention of antigens (eg HER2, EGFR).
  • antigens eg HER2, EGFR
  • NCI-N87 cells human gastric cancer cells, HER2+, GFP-
  • MDA-MB-231 cells human breast cancer cells, HER2-, GFP+
  • HER2/ErbB2 (29D8) Rabbit mAb (volume ratio is 1:100 (the volume of the solution in the culture dish)) and incubate on ice for 1 h, wash with DPBS buffer solution for 5 min, repeat 3 times; add the modified secondary antibody Anti Rabbit IgG-Glycan-Cy5 (final concentration 10 ⁇ g/mL) was incubated at room temperature in the dark for 1 h, washed with DPBS buffer solution for 5 min, and repeated 3 times; DAPI (4',6-diamidino-2-phenyl indole) at room temperature in the dark for 10 min, washed with DPBS buffer solution for 5 min, Repeated 3 times; imaging was performed with an inverted fluorescence microscope, and images were processed and channel merged with ImageJ.
  • HER2/ErbB2 (29D8) Rabbit mAb (volume ratio is 1:100 (the volume of the solution in the culture dish)) and incubate on ice for 1 h, wash with DP
  • this method can achieve effective and specific cleavage.
  • green is MDA-MB-231 cells (expressing GFP), which do not highly express HER2 antigen and thus only appear green, while NCI-N87 cells highly expressing HER2 antigen (not expressing GFP ) only shows red but not green.
  • FIG. 9( b ) after combined enzyme cleavage, the red fluorescence signal disappeared, while the green fluorescence remained. This shows that this method only selectively cuts specific imaging, while not affecting other antigens.
  • the NCI-N87 paraffin tissue sections were dewaxed in an oven at 65°C for 45 minutes, and then placed in xylene-gradient ethanol solutions for complete dewaxing.
  • the slices were microwaved in antigen retrieval solution (EDTA-Tris, pH 8.0) to expose the antigens. After natural cooling, they were washed with DPBS buffer solution for 5 min, and repeated 3 times.
  • EDTA-Tris antigen retrieval solution
  • BSA bovine serum albumin
  • this method can also achieve effective and specific cutting for fluorescence imaging of tissue sections.
  • the red fluorescent signal is the imaging result of NCI-N87 paraffin tissue section.
  • the red fluorescent signal almost disappeared after adding combined restriction enzymes. This shows that this method can also specifically achieve fluorescent cutting at the level of paraffin tissue sections.
  • the monomers used in the experimental examples can be purchased or obtained through simple preparation, and the preparation process is also in the prior art, so no detailed description is given in the specification.

Abstract

Provided in the present application are a processing method for an antigen sample, and the use. The processing method comprises: (1) preparing a labeled antibody and an antigen sample, the labeled antibody comprising an antibody and a glycan bound to the antibody, the glycan containing a glycosidic bond, and the antigen sample comprising a plurality of sites capable of binding to the antibody; (2) binding the labeled antibody to the sites, or binding the sites to a first antibody so as to obtain a bound first antibody; and binding the labeled antibody to the bound first antibody so as to obtain a bound labeled antibody; and by means of glycoside hydrolases, performing enzyme digestion on the glycosidic bond in the bound labeled antibody. For a labeled antibody containing a glycan, enzyme digestion of a specific site, namely a glycosidic bond, is performed, so as to separate the glycan and the antibody, and enzyme digestion, which cuts a specific site, will not damage the antigen, and can be used for multiple rounds of subsequent multi-channel fluorescence imaging (or other detection methods), thereby implementing hypermulticolor pathological analysis.

Description

一种对抗原样本的处理方法和应用A method and application for processing antigen samples 技术领域technical field
本申请涉及抗原处理领域，特别涉及一种对抗原样本的处理方法和应用。The present application relates to the field of antigen processing, in particular to a method and application for processing antigen samples.
背景技术Background technique
传统的免疫组织化学/免疫荧光成像(IHC/IF)是组织病理学领域常用的一种诊断技术，其基于成像的方式将病理学信息(如生物标志物的表达量等)转换为荧光检测信号(如荧光强度等)，实现病理特征可视化的目标，以利于研究开发新的疾病治疗靶标。但传统技术的弊端在于其只允许在每个组织切片上实现一种标志物的标记，这使我们错过了从有限的样本中获得更多诊断信息的机会。基于此不足，多重免疫组织化学/免疫荧光(mIHC/mIF)技术应运而生，其突破点在于允许在单个组织切片上同时检测多个标记物。Traditional immunohistochemistry/immunofluorescence imaging (IHC/IF) is a commonly used diagnostic technique in the field of histopathology, which converts pathological information (such as the expression of biomarkers, etc.) into fluorescent detection signals based on imaging (such as fluorescence intensity, etc.), to achieve the goal of visualizing pathological features, so as to facilitate the research and development of new disease treatment targets. But the disadvantage of traditional technology is that it only allows one marker to be marked on each tissue section, which makes us miss the opportunity to obtain more diagnostic information from limited samples. Based on this deficiency, the technique of multiplex immunohistochemistry/immunofluorescence (mIHC/mIF) emerged as the times require, and its breakthrough lies in allowing simultaneous detection of multiple markers on a single tissue section.
已经被报道的多重免疫组织化学/免疫荧光技术主要分为以下四类：(1)基于酶催化的酪酰胺化学标记：如Akoya Biosciences公司研发的Vectra成像***，其通过在同一张切片上使用微波多次解离标记后的抗体，同时每次使用窄光谱且可拆分的荧光染料，可以实现7～9色的多色荧光成像；(2)基于化学可切割荧光抗体的多色成像：如Guo等报道使用含叠氮的连接臂偶联荧光基团与抗体，在每轮成像之后使用还原剂三(2-羧乙基)膦(TCEP)对连接臂实现化学切割，可以达到12色荧光成像；(3)基于非特异性抗体洗脱的多轮染色：如Pelkmans等开发的4i平台，其通过使用主要成分为盐酸胍(GC)和三(2-羧乙基)膦(TCEP)的洗脱剂，破坏抗原-抗体间非共价相互作用进行洗脱，可以达到40+色多重荧光成像；(4)基于碱基配对的多色成像方法：如Nolan等报道的CODEX技术，使用脱氧核苷酸链-抗体偶联物对靶标进行成像，通过引物上单个碱基的延伸及TCEP对碱基-荧光基团间二硫键的切割来实现对多重目标蛋白的研究，同样可以达到40+色多重荧光成像。The reported multiple immunohistochemistry/immunofluorescence techniques are mainly divided into the following four categories: (1) Enzyme-based tyramide chemical labeling: such as the Vectra imaging system developed by Akoya Biosciences, which uses microwaves on the same slice to Dissociate the labeled antibody multiple times, and use a narrow-spectrum and detachable fluorescent dye each time to achieve multicolor fluorescence imaging of 7 to 9 colors; (2) Multicolor imaging based on chemically cleavable fluorescent antibodies: such as Guo et al. reported that an azide-containing linker was used to couple the fluorophore to the antibody, and after each round of imaging, the reducing agent tris(2-carboxyethyl)phosphine (TCEP) was used to chemically cut the linker to achieve 12-color fluorescence. Imaging; (3) Multiple rounds of staining based on non-specific antibody elution: such as the 4i platform developed by Pelkmans et al. Remove the agent, destroy the non-covalent interaction between antigen and antibody for elution, and can achieve 40+ color multiple fluorescence imaging; (4) Multicolor imaging method based on base pairing: such as the CODEX technology reported by Nolan et al., using deoxynuclear The nucleotide chain-antibody conjugates image the target, and realize the research on multiple target proteins through the extension of a single base on the primer and the cleavage of the disulfide bond between the base and the fluorescent group by TCEP, which can also reach 40+ Color multiplex fluorescence imaging.
以上的四类技术均使用到微波或化学还原剂等对抗原不温和的切割试剂，在多色成像过程中存在多轮成像过程中抗原失真的问题，进而限制多轮荧光成像的循环上限，这限制了多色荧光成像的通量上限。基于此，开发对抗原温和的且能够进行有效切割的物质和方法具有较大的实际意义和进步，也是抗原领域急需解决的关键问题之一。The above four types of technologies all use microwave or chemical reducing agent and other cutting reagents that are not mild to the antigen. In the process of multi-color imaging, there is the problem of antigen distortion during multiple rounds of imaging, which in turn limits the upper limit of the cycle of multiple rounds of fluorescence imaging. An upper limit to the throughput of multicolor fluorescence imaging. Based on this, the development of substances and methods that are gentle on antigens and capable of effectively cutting antigens has great practical significance and progress, and is also one of the key issues in the field of antigens that needs to be solved urgently.
发明内容Contents of the invention
针对上述问题，本申请提供了一种对抗原样本的处理方法，通过对含有聚糖的标记抗体，进行特定位点-糖苷键的酶切，实现聚糖与抗体的分离，并且该酶切是特定位点的切除，不会对抗原造成损伤，可用于后续多轮的多通道单细胞分析，不仅可以用于多重免疫组织化学/免疫荧光技术，还可用于包括细胞中蛋白、核酸序列扩增等场景。 In response to the above problems, the present application provides a method for processing antigen samples. By performing specific site-glycosidic bond enzyme cleavage on labeled antibodies containing glycans, the separation of glycans and antibodies is achieved, and the enzymatic cleavage is Excision of a specific site will not cause damage to the antigen, and can be used for multiple subsequent rounds of multi-channel single-cell analysis, not only for multiple immunohistochemistry/immunofluorescence techniques, but also for protein and nucleic acid sequence amplification in cells Wait for the scene.
根据本申请的一方面，提供了一种对抗原样本的处理方法，所述处理方法包括：According to one aspect of the present application, a method for processing an antigen sample is provided, the processing method comprising:
(1)准备标记抗体和抗原样本；所述标记抗体包含有抗体和与该抗体连接的聚糖，该聚糖中含有糖苷键；所述抗原样本包括多个可与抗体结合的位点；(2)将所述标记抗体与所述位点结合，或，所述位点与第一抗体结合，得到结合后的第一抗体；将所述标记抗体与所述结合后的第一抗体结合，得到结合后的标记抗体；通过糖苷酶对所述结合后的标记抗体中的糖苷键进行酶切。(1) Prepare labeled antibodies and antigen samples; the labeled antibodies include antibodies and glycans linked to the antibodies, and the glycans contain glycosidic bonds; the antigen samples include multiple sites that can bind to antibodies; ( 2) binding the labeled antibody to the site, or binding the site to the first antibody to obtain a combined first antibody; combining the labeled antibody to the combined first antibody, The bound labeled antibody is obtained; the glycosidic bond in the bound labeled antibody is digested by glycosidase.
上述处理方法适用目前公开和非公开的通过抗体聚糖修饰进行标记的抗体。The above-mentioned processing method is applicable to antibodies labeled by antibody glycan modification, both currently published and unpublished.
可选地，在所述步骤(2)中，将若干个不同种类的标记抗体与其相对应的抗原或第一抗体结合。Optionally, in the step (2), several different kinds of labeled antibodies are combined with their corresponding antigens or primary antibodies.
可选地，在所述步骤(2)中，所述标记抗体的种类为1～10种。Optionally, in the step (2), the types of the labeled antibodies are 1-10.
可选地，在步骤(2)之后，所述处理方法还包括：(3)准备与步骤(1)相同或不同的标记抗体；将该标记抗体与步骤(1)中的所述位点结合，或，所述位点与新的第一抗体结合后，得到新的结合后的第一抗体；将该新的结合后的第一抗体与本步骤中的标记抗体结合，得到结合后的标记抗体；(4)通过糖苷酶对所述步骤(3)中结合后的所述标记抗体中的糖苷键进行酶切。Optionally, after step (2), the treatment method further includes: (3) preparing a labeled antibody that is the same as or different from step (1); binding the labeled antibody to the site in step (1) , or, after the site is combined with a new first antibody, a new combined first antibody is obtained; the new combined first antibody is combined with the labeled antibody in this step to obtain a combined label Antibody; (4) cleavage of the glycosidic bond in the labeled antibody bound in the step (3) by glycosidase.
可选地，所述步骤(3)中的标记抗体与所述步骤(1)中的标记抗体不同。Optionally, the labeled antibody in the step (3) is different from the labeled antibody in the step (1).
可选地，所述步骤(3)中的标记抗体与所述步骤(1)中的标记抗体的不同体现在抗体、聚糖的不同，即，抗体、聚糖中的至少一个不同。Optionally, the difference between the labeled antibody in the step (3) and the labeled antibody in the step (1) is reflected in the difference of the antibody and the glycan, that is, at least one of the antibody and the glycan is different.
可选地，所述步骤(3)中的标记抗体与所述步骤(1)中的标记抗体的抗体不同，用于结合不同的抗原或二抗，有利于后续蛋白质图谱的全面绘制。Optionally, the labeled antibody in the step (3) is different from the labeled antibody in the step (1), and is used to bind to different antigens or secondary antibodies, which is beneficial to the comprehensive mapping of subsequent protein maps.
可选地，所述步骤(3)中的标记抗体与所述步骤(1)中的标记抗体的聚糖不同，可以增加连接结构分子的多样性，易于根据不同的场景选择合适的连接结构；但是在聚糖中，至少存在通过糖苷键连接的两个单糖。Optionally, the labeled antibody in the step (3) is different from the glycan of the labeled antibody in the step (1), which can increase the diversity of linking structure molecules, and it is easy to select a suitable linking structure according to different scenarios; But in glycans, there are at least two monosaccharides linked by glycosidic bonds.
可选地，所述步骤(3)中的标记抗体与所述步骤(1)中的标记抗体相同。Optionally, the labeled antibody in the step (3) is the same as the labeled antibody in the step (1).
标记抗体相同，可以同所有能与该标记抗体的抗原进行结合，便于准确检测该多种抗原样本中某一抗原的含量。The labeled antibodies are the same, and can be combined with all antigens that can be combined with the labeled antibody, so as to facilitate accurate detection of the content of a certain antigen in the multiple antigen samples.
可选地，所述步骤(3)中新的第一抗体与步骤(2)中的第一抗体相同或不相同。Optionally, the new first antibody in step (3) is the same or different from the first antibody in step (2).
可选地，所述“新”指不是前述已经使用过的物质，但是组成、含量可以相同或不同的物质；即步骤(3)中新的第一抗体与步骤(2)中的第一抗体组成、含量可以相同或不同，但步骤(3)中新的第一抗体不是步骤(2)中的第一抗体。Optionally, the "new" refers to substances that are not previously used, but whose composition and content may be the same or different; that is, the new first antibody in step (3) and the first antibody in step (2) The composition and content can be the same or different, but the new primary antibody in step (3) is not the primary antibody in step (2).
由于步骤(2)中的第一抗体与其相对应的位点结合后，即使经过酶切，第一抗体仍与位点结合，为了检测其他未结合的位点，需要再加入新的第一抗体与其相对应的位点结合。Since the first antibody in step (2) binds to its corresponding site, even after enzyme digestion, the first antibody still binds to the site, in order to detect other unbound sites, it is necessary to add a new first antibody Bind to its corresponding site.
可选地，所述抗原样本包含多种抗原，即所述抗原样本至少包括两种抗原。Optionally, the antigen sample includes multiple antigens, that is, the antigen sample includes at least two antigens.
可选地，所述抗原样本为至少两种抗原的混合状态。Optionally, the antigen sample is a mixture of at least two antigens.
可选地，所述抗原样本包括细胞、动植物组织、人类组织中的至少一种。 Optionally, the antigen sample includes at least one of cells, animal and plant tissues, and human tissues.
可选地，所述动植物组织、人类组织包括石蜡包埋组织，冰冻切片组织等Optionally, the animal and plant tissues and human tissues include paraffin-embedded tissues, frozen section tissues, etc.
可选地，所述处理方法还包括：将步骤(3)和步骤(4)多次循环；所述多次循环的次数为≥2。Optionally, the processing method further includes: multiple cycles of step (3) and step (4); the number of multiple cycles is ≥2.
即整个处理方法中，结合和酶切的次数至少为四次。结合和酶切为一个循环，循环次数越多越有利于蛋白质图谱的绘制，本申请由于使用对糖苷键的酶切，即通过对特定位点进行切割，可以减少对抗原的损伤，循环次数可达到5次或以上。That is, in the whole treatment method, the times of combining and enzyme cutting are at least four times. Combining and enzymatic cleavage are a cycle, and the more the number of cycles, the more conducive to the drawing of the protein map. In this application, the enzyme cleavage of glycosidic bonds is used, that is, by cleavage of specific sites, which can reduce the damage to the antigen, and the number of cycles can be reduced. Reach 5 or more times.
可选地，所述标记抗体和所述糖苷酶的质量比为1:0.001～1000。Optionally, the mass ratio of the labeled antibody to the glycosidase is 1:0.001-1000.
可选地，所述糖苷酶包括内切糖苷酶和/或外切糖苷酶。Optionally, the glycosidase includes endoglycosidase and/or exoglycosidase.
可选地，所述糖苷酶包括EndoS、EndoF、Alfc中的至少一种。Optionally, the glycosidase includes at least one of EndoS, EndoF and Alfc.
可选地，所述糖苷酶包括EndoS、EndoF、Alfc中的至少两种。Optionally, the glycosidase includes at least two of EndoS, EndoF, and Alfc.
可选地，所述酶切的条件为：温度为0℃～45℃。Optionally, the conditions for the enzyme digestion are: the temperature is 0°C-45°C.
可选地，所述聚糖还连接有标记结构。Optionally, the glycan is also linked with a labeling structure.
可选地，所述标记结构为用于检测或显示的基团，能够被检测设备捕捉其相关信号，用于相应标记抗体的定量或定性的分析。Optionally, the labeling structure is a group for detection or display, and its related signals can be captured by detection equipment for quantitative or qualitative analysis of the corresponding labeled antibody.
可选地，所述标记结构包括酶、荧光物质、放射性核素、化学发光剂、量子点、生物素或染料。Optionally, the labeling structure includes enzymes, fluorescent substances, radionuclides, chemiluminescent agents, quantum dots, biotin or dyes.
可选地，所述染料包括但不限于Alexa Fluor 488、Alexa Fluor 568、FITC、Cy3或Cy5。Optionally, the dyes include, but are not limited to, Alexa Fluor 488, Alexa Fluor 568, FITC, Cy3 or Cy5.
可选地，含连接有标记结构的聚糖的标记抗体，其制备方法包括酶促反应、生物正交化学反应。Optionally, the preparation method of the labeled antibody containing the glycan linked with the labeled structure includes enzymatic reaction and bioorthogonal chemical reaction.
可选地，所述酶促反应包括通过糖基转移酶，将连接有标记结构的聚糖和/或单糖、与抗体反应，得到含连接有标记结构的聚糖的标记抗体；或通过糖基转移酶，将连接有标记结构的聚糖和/或单糖与抗体、其他聚糖和/单糖反应，得到含连接有标记结构的聚糖的标记抗体。Optionally, the enzymatic reaction includes reacting the glycan and/or monosaccharide linked with the labeled structure with the antibody by a glycosyltransferase to obtain a labeled antibody containing the glycan linked with the labeled structure; or Glycans and/or monosaccharides linked with labeled structures are reacted with antibodies and other glycans and/or monosaccharides to obtain labeled antibodies containing glycans linked with labeled structures.
可选地，所述生物正交化学反应包括但不限于：将与标记结构连接的二苯并环辛炔或炔、与糖结合的叠氮化物以及抗体进行反应，得到含连接有标记结构的聚糖的标记抗体；或，将与标记结构连接的反式环辛烯、与糖结合的四嗪或甲基四嗪化物以及抗体进行反应，得到含连接有标记结构的聚糖的标记抗体。Optionally, the bioorthogonal chemical reaction includes, but is not limited to: reacting dibenzocyclooctyne or alkyne linked to a labeled structure, an azide combined with a sugar, and an antibody to obtain a A labeled antibody to a glycan; or, react a trans-cyclooctene linked to a labeled structure, a tetrazine or methyltetrazine bound to a sugar, and an antibody to obtain a labeled antibody containing a glycan linked to a labeled structure.
可选地，所述标记结构和所述标记抗体通过所述聚糖连接。Optionally, said tagging structure and said tagged antibody are linked via said glycan.
可选地，所述标记抗体与所述聚糖共价连接。Optionally, the labeled antibody is covalently linked to the glycan.
可选地，所述聚糖包括两个或以上的单糖。Optionally, the glycans include two or more monosaccharides.
可选地，所述两个或以上的单糖可以为相同糖型或不同糖型。Optionally, the two or more monosaccharides may be in the same glycoform or in different glycoforms.
可选地，在所述聚糖中，至少存在两个通过糖苷键连接的单糖。Optionally, in the glycan, there are at least two monosaccharides linked by glycosidic bonds.
可选地，所述聚糖为N-聚糖和/或O-聚糖。Optionally, the glycans are N-glycans and/or O-glycans.
可选地，所述N-聚糖是指糖通过一个N原子连接在Asn；所述O聚糖是指糖通过O原子连接在Ser/Thr。Optionally, the N-glycan refers to the sugar linked to Asn through an N atom; the O-glycan refers to the sugar linked to Ser/Thr through an O atom.
可选地，所述聚糖为N-聚糖。Optionally, the glycans are N-glycans.
可选地，所述单糖包括N-乙酰葡萄糖胺、甘露糖、半乳糖、岩藻糖、N-半 乳糖胺中的至少一种。Optionally, the monosaccharides include N-acetylglucosamine, mannose, galactose, fucose, N-semi at least one of lactosamines.
可选地，所述聚糖为多个单糖通过共价键或非共价键以任意顺序连接。Optionally, the polysaccharide is a plurality of monosaccharides connected in any order by covalent or non-covalent bonds.
可选地，所述聚糖包括天然型聚糖或非天然性聚糖。Optionally, the glycans include natural or non-natural glycans.
天然型聚糖是指自然界中存在的聚糖的构型和组成；非天然性聚糖是指人工合成出的非自然存在的聚糖构型或组成。Natural glycans refer to the configuration and composition of glycans that exist in nature; non-natural glycans refer to artificially synthesized non-naturally occurring glycan configurations or compositions.
可选地，所述聚糖为多种单糖随机排列组合得到的化合物。Optionally, the polysaccharide is a compound obtained by random arrangement and combination of various monosaccharides.
可选地，所述聚糖包括单糖-单糖、单糖-单糖-单糖、 单糖-单糖-单糖-单糖中的至少一种。Optionally, the polysaccharides include monosaccharide-monosaccharide, monosaccharide-monosaccharide-monosaccharide, At least one of monosaccharide-monosaccharide-monosaccharide-monosaccharide.
可选地，所述聚糖连接至少一个标记结构。Optionally, the glycan is linked to at least one labeling structure.
可选地，所述标记结构与所述聚糖中的任意一个或多个单糖连接。Optionally, the tag structure is linked to any one or more monosaccharides in the glycan.
可选地，一个所述标记结构与一个所述单糖连接。Optionally, one of said tagging structures is linked to one of said monosaccharides.
可选地，所述聚糖与1～10个标记结构连接。Optionally, the glycan is linked to 1-10 marker structures.
可选地，所述聚糖与1个、2个、3个、4个、5个、6个、7个、8个、9个、或10个标记结构连接。Optionally, the glycan is linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 marker structures.
可选地，所述聚糖与1～4个标记结构连接。Optionally, the glycan is linked to 1-4 marker structures.
可选地，本申请的聚糖中可与多个标记结构连接，标记结构可位于任意一个单糖上；如图1所示，通过糖苷酶对聚糖中的糖苷键进行切割，去除标记结构。Optionally, the glycan of the present application can be connected with multiple labeling structures, and the labeling structure can be located on any monosaccharide; as shown in Figure 1, the glycosidic bond in the glycan is cleaved by glycosidase to remove the labeling structure .
可选地，所述步骤(3)中的其他含有聚糖的标记抗体与所述步骤(1)中的标记抗体的不同还体现在标记结构的不同，即，抗体、聚糖或标记结构中的至少一个不同。Optionally, the difference between the labeled antibody containing glycans in the step (3) and the labeled antibody in the step (1) is also reflected in the difference in the labeling structure, that is, the antibody, the glycan or the labeling structure At least one of the different.
可选地，所述其他含有聚糖的标记抗体的抗体和所述含有聚糖的标记抗体的标记结构不同，可用于检测不同的信号，有利于检测结果更加精准、可靠。Optionally, the other glycan-containing labeled antibody has a different labeling structure from the glycan-containing labeled antibody, which can be used to detect different signals, which is conducive to more accurate and reliable detection results.
可选地，在所述酶切后，还进行洗脱。Optionally, after the digestion, elution is also performed.
洗脱是通过洗脱剂对于切割下来的功能性基团进行洗去，避免对于后续信 号检测造成影响。The elution is to wash away the cleaved functional groups by the eluent, so as to avoid further damage to the subsequent signal. No. detection is affected.
可选地，所述洗脱剂包括但不限于DPBS缓冲溶液、HEPES缓冲溶液、Tris缓冲溶液等Optionally, the eluent includes but not limited to DPBS buffer solution, HEPES buffer solution, Tris buffer solution, etc.
与现有技术相比，本申请具有以下有益效果：Compared with the prior art, the present application has the following beneficial effects:
本申请提供一种对抗原样本的处理方法，采用含有聚糖的标记抗体与抗原或第一抗体的特异性结合，之后进行特定位点-糖苷键的酶切，实现聚糖连接的标记结构与抗体的分离；经过含有聚糖的标记抗体对抗原的多次识别、结合和对聚糖链接的标记结构的酶切，可以实现对细胞内的蛋白质的全面检测。本申请通过酶切去除聚糖或带有标记结构的聚糖，不仅可以对切割位点进行控制，而且还对于其他蛋白没有损伤；并且能够同时对于多种蛋白质进行处理，互相不干扰，大大提高了对细胞内蛋白质原位处理的速度；且由于糖苷酶的温和型以及高效性，大大提高了切割效率，通过又能够进行多轮处理，对于细胞内蛋白质原位处理和检测的结构有着更高的准确度。This application provides a method for processing antigen samples, which uses the specific binding of labeled antibodies containing glycans to antigens or primary antibodies, and then performs specific site-glycosidic bond enzymatic cleavage to realize the glycan-linked labeling structure and Separation of antibodies; after repeated recognition and binding of labeled antibodies containing glycans to antigens and enzymatic cleavage of glycan-linked labeled structures, comprehensive detection of intracellular proteins can be achieved. This application removes glycans or glycans with labeled structures by enzymatic cleavage, which not only can control the cleavage site, but also does not damage other proteins; and can treat multiple proteins at the same time without interfering with each other, greatly improving The speed of in situ processing of intracellular proteins is improved; and due to the mildness and high efficiency of glycosidase, the cutting efficiency is greatly improved, and multiple rounds of processing can be performed, which has a higher structure for in situ processing and detection of intracellular proteins. the accuracy.
本申请的处理方法不仅能够在多种生化学和免疫化学场景(如IF、WB)进行温和切割，大大降低对于抗原的损伤，还能够进行高效切割，并且能够进行多轮IF和多轮WB的检测。The treatment method of the present application can not only perform mild cutting in various biochemical and immunochemical scenarios (such as IF, WB), greatly reduce the damage to the antigen, but also perform efficient cutting, and can perform multiple rounds of IF and multiple rounds of WB. detection.
附图说明Description of drawings
图1是本申请对标记抗体切割的反应原理图；其中，代表N-乙酰葡萄糖胺；代表甘露糖；代表半乳糖；代表岩藻糖；代表N-半乳糖胺；X代表标记结构。Fig. 1 is the schematic diagram of the reaction of the present application to the cleavage of the labeled antibody; wherein, stands for N-acetylglucosamine; represents mannose; represents galactose; represents fucose; Represents N-galactosamine; X represents the labeled structure.
图2是本申请制备得到的标记抗体Ab-1和Ab-2的反应原理图；其中，代表N-乙酰葡萄糖胺；代表甘露糖；代表半乳糖；代表岩藻糖；代表N-半乳糖胺；X代表生物素或Cy5。Fig. 2 is the schematic diagram of the reaction of the labeled antibodies Ab-1 and Ab-2 prepared by the present application; wherein, stands for N-acetylglucosamine; represents mannose; represents galactose; represents fucose; Represents N-galactosamine; X represents biotin or Cy5.
图3是本申请制备得到的标记抗体Ab-3和Ab-4的反应原理图；其中，代表N-乙酰葡萄糖胺；代表甘露糖；代表半乳糖；代表岩藻糖；代表N-半乳糖胺；X代表生物素或Cy5；代表生物正交反应结合。Fig. 3 is the schematic diagram of the reaction of the labeled antibodies Ab-3 and Ab-4 prepared by the present application; wherein, stands for N-acetylglucosamine; represents mannose; represents galactose; represents fucose; Represents N-galactosamine; X represents biotin or Cy5; Represents bioorthogonal reaction conjugation.
图4是本申请实施例1酶切后混合物的荧光结果图；横坐标为荧光强度，无单位；纵坐标为相对细胞数量，单位为％。Fig. 4 is a diagram of the fluorescence results of the mixture after digestion in Example 1 of the present application; the abscissa is the fluorescence intensity, unitless; the ordinate is the relative cell number, the unit is %.
图5是本申请实施例2不同浓度的酶对标记抗体酶切后混合物的残存荧光图；横坐标从左到右依次为对照组、0.1λ组、0.5λ组和1λ组；纵坐标为残存荧光率，单位为％。Fig. 5 is the residual fluorescence diagram of the mixture of enzymes with different concentrations in Example 2 of the present application after digesting the labeled antibody; Fluorescence rate, in %.
图6是本申请实施例3不同酶种类和不同标记抗体酶切后混合物的残存荧光图；横坐标从左到右依次为I组、II组(Ab-1)、III组(Ab-2)、IV组(Ab-3)和V组(Ab-4)，每组又分为0.5λ+0.5λ和1λ两组；纵坐标为残存荧光率，单位为％。Fig. 6 is the residual fluorescence diagram of the mixture of different enzyme types and different labeled antibodies in Example 3 of the present application; the abscissa is from left to right group I, group II (Ab-1), group III (Ab-2) , IV group (Ab-3) and V group (Ab-4), each group is further divided into two groups of 0.5λ+0.5λ and 1λ; the ordinate is the residual fluorescence rate, the unit is %.
图7是本申请实施例4不同反应时间酶切后标记抗体的质谱谱图；(a) 0min；(b)5min；(c)10min；(d)20min；横坐标为分子量，单位为Da；纵坐标为强度，单位为％；Figure 7 is the mass spectrogram of the labeled antibody after digestion with different reaction times in Example 4 of the present application; (a) 0min; (b) 5min; (c) 10min; (d) 20min; the abscissa is the molecular weight, the unit is Da; the ordinate is the strength, the unit is %;
图8是本申请实施例6在活细胞层面不同切割方式处理后抗原表达量的对比图；横坐标从左到右依次为A组、B组(DPBS)、C组(50mM TCEP)、D组(100mM TCEP)、E组(200mM TCEP)、F组(0.1λEndoS+0.1λEndoF3)、G组(0.5λEndoS+0.5λEndoF3)和H组(1λEndoS+1λEndoF3)，每组又分为Her2和EGFR两组；纵坐标为残存荧光率，单位为％。Figure 8 is a comparison chart of antigen expression in Example 6 of the present application after different cutting methods at the level of living cells; the abscissa is from left to right: Group A, Group B (DPBS), Group C (50mM TCEP), and Group D (100mM TCEP), E group (200mM TCEP), F group (0.1λEndoS+0.1λEndoF3), G group (0.5λEndoS+0.5λEndoF3) and H group (1λEndoS+1λEndoF3), each group is divided into two groups of Her2 and EGFR ; The vertical axis is the residual fluorescence rate, the unit is %.
图9是本申请实施例7酶切割前和酶切割后的细胞成像图；(a)切割前；(b)切割后。Fig. 9 is an image of cells before and after enzyme cleavage in Example 7 of the present application; (a) before cleavage; (b) after cleavage.
图10是本申请实施例8酶切割前和酶切割后的石蜡切片染色图；(a)切割前；(b)切割后。Fig. 10 is the staining diagram of paraffin sections before and after enzyme cleavage in Example 8 of the present application; (a) before cleavage; (b) after cleavage.
图11是本申请实施例5的酶切图；(a)切割前后的蛋白胶表征图；(b)对抗体-辣根过氧化物酶(Ab-HRP)的切割效率图；横坐标为对照组和糖苷酶组；纵坐标为切割效果，单位为％。Fig. 11 is the enzyme cleavage diagram of Example 5 of the present application; (a) protein gel characterization diagram before and after cleavage; (b) cleavage efficiency diagram for antibody-horseradish peroxidase (Ab-HRP); abscissa is control group and glycosidase group; the ordinate is the cutting effect, and the unit is %.
图12是本申请实施例6在固定细胞层面不同切割方式处理后抗原保存量的对比图；(A)对HER2切割后的抗原保存量；(B)对EGFR切割后的抗原保存量；横坐标为处理次数，从左到右依次为处理一次、处理三次和处理五次；纵坐标为抗原保存量，无单位。Figure 12 is a comparison chart of antigen retention after different cleavage methods at the fixed cell level in Example 6 of the present application; (A) antigen retention after cleavage of HER2; (B) antigen retention after cleavage of EGFR; abscissa is the number of treatments, from left to right: treatment once, treatment three times and treatment five times; the vertical axis is the amount of antigen preservation, no unit.
图13是本申请实施例6在印迹膜表面不同切割方式处理后HER2的相对抗原保存量的对比图；(A)对HER2切割一次后的抗原保存量；(B)对EGFR切割三次后的抗原保存量；纵坐标为抗原保存率，无单位。Figure 13 is a comparison chart of the relative antigen retention of HER2 after different cutting methods on the surface of the imprinted membrane in Example 6 of the present application; (A) the antigen retention after cutting HER2 once; (B) the antigen after cutting EGFR three times Preservation amount; the vertical axis is the antigen preservation rate, no unit.
具体实施方式Detailed ways
下面结合附图对本申请的具体实施方式进行描述。The specific implementation manner of the present application will be described below in conjunction with the accompanying drawings.
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的试剂、材料和操作步骤均为相应领域内广泛使用的试剂、材料和常规步骤。In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the reagents, materials and operation steps used herein are all reagents, materials and routine procedures widely used in the corresponding fields.
需要说明的是，以下实施例中所展现的应用方式仅为针对材料的优选应用方式，下面描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。另外，如果没有明确说明，在下面的实施例中所采用的所有试剂均为市场上可以购得的，或者可以按照本文或已知的方法合成的，对于没有列出的反应条件，也均为本领域技术人员容易获得的。下列实施例中未注明具体条件的操作方法，通常按照常规条件，或按照制造厂商所建议的条件。It should be noted that the application modes shown in the following examples are only preferred application modes for materials, and the examples described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. In addition, if not clearly stated, all reagents used in the following examples are commercially available, or can be synthesized according to this article or known methods, and for the reaction conditions not listed, they are all readily available to those skilled in the art. The operating methods not indicated in the following examples are generally in accordance with conventional conditions, or in accordance with the conditions suggested by the manufacturer.
Protein A树脂纯化：将NMab蛋白A(NMab Protein A)，加入量为100μL/1mg(待纯化抗体)，加入到亲和层析柱中，以1000g的离心力进行离心1min，用400μL磷酸盐缓冲溶液(DPBS缓冲溶液)洗涤3次。加入抗体，混匀共孵1h。以1000g的离心力进行离心1min，用DPBS缓冲溶液洗涤3次。加入甘氨酸(200mM)，加入量为35μL/100μg(待纯化抗体)混匀共孵10 min，以1000g的离心力进行离心1min，立即向洗脱液中加入Tris溶液(1M)，加入量为7μL/100μg(待纯化抗体)。Protein A resin purification: add NMab protein A (NMab Protein A) in an amount of 100μL/1mg (antibody to be purified), add it to an affinity chromatography column, centrifuge at a centrifugal force of 1000g for 1min, and use 400μL phosphate buffer solution (DPBS buffer solution) washed 3 times. Add antibody, mix and incubate for 1h. Centrifuge for 1 min with a centrifugal force of 1000 g, and wash 3 times with DPBS buffer solution. Add glycine (200mM) in an amount of 35μL/100μg (antibody to be purified), mix and incubate for 10 Centrifuge for 1 min at a centrifugal force of 1000 g, and immediately add Tris solution (1 M) to the eluate in an amount of 7 μL/100 μg (antibody to be purified).
原料及其来源
Raw materials and their sources
GDP-Fuc-Cy5:
GDP-Fuc-Cy5:
GDP-Fuc-Biotin:
GDP-Fuc-Biotin:
DBCO-FITC: DBCO-FITC:
DBCO-Biotin:
DBCO-Biotin:
下列中各浓度数值均为该物质在当前步骤的混合物中的浓度。Each concentration value in the following is the concentration of the substance in the mixture of the current step.
制备含有聚糖和标记结构的标记抗体Preparation of labeled antibodies containing glycans and tagged structures
(1)标记抗体Ab-1(1) Labeled antibody Ab-1
将曲妥珠单抗(3λ)、MnCl2(5mM)、MgCl2(10mM)、Tris-HCl缓冲溶液(10mM，pH＝7.4)、尿苷二磷酸-半乳糖(Uridine diphosphate galactose，UDP-Galactose，5mM)、β-1,4半乳糖基转移酶(β-1,4-galactosyltransferase 1，β4GalT1，0.4λ)、岩藻糖基转移酶(Fucosyltransferase，FT，1λ)和鸟苷二磷酸-岩藻糖-生物素(Guanosine diphosphate fucose-biotin，GDP-Fuc-Biotin，2mM)进行一锅法反应,在30℃、300rpm的金属浴中反应40h。用蛋白A树脂亲和纯化，将纯化后的产品进行浓缩并脱盐到DPBS缓冲溶液中，得到标记抗体Ab-1。(由岩唐公司提供)Trastuzumab (3λ), MnCl 2 (5mM), MgCl 2 (10mM), Tris-HCl buffer solution (10mM, pH=7.4), uridine diphosphate galactose (UDP-Galactose , 5mM), β-1,4-galactosyltransferase (β-1,4-galactosyltransferase 1, β4GalT1, 0.4λ), fucosyltransferase (Fucosyltransferase, FT, 1λ) and guanosine diphosphate-rock Cocose-biotin (Guanosine diphosphate fucose-biotin, GDP-Fuc-Biotin, 2mM) was subjected to a one-pot reaction at 30°C and 300rpm in a metal bath for 40h. The protein A resin was used for affinity purification, and the purified product was concentrated and desalted into DPBS buffer solution to obtain the labeled antibody Ab-1. (Provided by Yan Tang Company)
(2)标记抗体Ab-2(2) Labeled antibody Ab-2
将曲妥珠单抗(3λ)、MnCl2(5mM)、MgCl2(10mM)、Tris-HCl缓冲溶液(10mM，pH＝7.4)、尿苷二磷酸-半乳糖(Uridine diphosphate galactose，UDP-Galactose，5mM)、β-1,4半乳糖基转移酶(β-1,4-galactosyltransferase 1，β4GalT1，0.4λ)、岩藻糖基转移酶(Fucosyltransferase，FT，1λ)和鸟苷二磷酸-岩藻糖-荧光染料(Guanosine diphosphate fucose-Cy5，GDP-Fuc-Cy5，2mM)进行一锅法反应,在30℃、300rpm的金属浴中反应40h。用蛋白A树脂亲和纯化，将纯化后的产品进行浓缩并脱盐到DPBS缓冲溶液中，得到标记抗体Ab-2。(由岩唐公司提供)Trastuzumab (3λ), MnCl 2 (5mM), MgCl 2 (10mM), Tris-HCl buffer solution (10mM, pH=7.4), uridine diphosphate galactose (UDP-Galactose , 5mM), β-1,4-galactosyltransferase (β-1,4-galactosyltransferase 1, β4GalT1, 0.4λ), fucosyltransferase (Fucosyltransferase, FT, 1λ) and guanosine diphosphate-rock Cocose-fluorescent dyes (Guanosine diphosphate fucose-Cy5, GDP-Fuc-Cy5, 2mM) were subjected to a one-pot reaction at 30°C and 300rpm in a metal bath for 40h. After affinity purification with protein A resin, the purified product was concentrated and desalted into DPBS buffer solution to obtain the labeled antibody Ab-2. (Provided by Yan Tang Company)
其中，标记抗体Ab-1和Ab-2的制备方法均是通过酶促反应，具体为通过糖基转移酶，将连接有标记结构的单糖与抗体、其他单糖反应得到，具体如图2所示。Among them, the preparation methods of labeled antibodies Ab-1 and Ab-2 are obtained through enzymatic reactions, specifically, through glycosyltransferases, reacting monosaccharides linked with labeled structures with antibodies and other monosaccharides, as shown in Figure 2 shown.
(3)标记抗体Ab-3(3) Labeled antibody Ab-3
将曲妥珠单抗(3λ)、MnCl2(10mM)、Tris-HCl缓冲溶液(50mM,pH＝7.4)、 尿苷二磷酸-乙酰化-N-叠氮乙酰半乳糖胺(UDP-GalNAz，5mM)和β-1,4半乳糖基转移酶(β-1,4-galactosyltransferase 1，β-4GalT1，0.5λ)进行一锅法反应,在30℃、300rpm的金属浴中反应24h。用蛋白A树脂亲和纯化后脱盐到DPBS缓冲溶液中，加入20当量的二苯并环辛炔-生物素(DBCO-Biotin)，30℃反应过夜，脱盐并到浓缩DPBS缓冲溶液中，得到标记抗体Ab-3。Trastuzumab (3λ), MnCl 2 (10mM), Tris-HCl buffer solution (50mM, pH=7.4), Uridine diphosphate-acetylated-N-azidoacetylgalactosamine (UDP-GalNAz, 5mM) and β-1,4-galactosyltransferase (β-1,4-galactosyltransferase 1, β-4GalT1, 0.5λ ) for a one-pot reaction at 30°C, 300rpm in a metal bath for 24h. After affinity purification with protein A resin, desalt into DPBS buffer solution, add 20 equivalents of dibenzocyclooctyne-biotin (DBCO-Biotin), react overnight at 30°C, desalt and put into concentrated DPBS buffer solution to obtain the label Antibody Ab-3.
(4)标记抗体Ab-4(4) Labeled antibody Ab-4
将曲妥珠单抗(3λ)、MnCl2(10mM)、Tris-HCl缓冲溶液(50mM,pH＝7.4)、尿苷二磷酸-乙酰化-N-叠氮乙酰半乳糖胺(UDP-GalNAz，5mM)和β-1,4半乳糖基转移酶(β-1,4-galactosyltransferase 1，β-4GalT1，0.5λ)进行一锅法反应,在30℃、300rpm的金属浴中反应24h。用蛋白A树脂亲和纯化后脱盐到DPBS缓冲溶液中，加入20当量的二苯并环辛炔-染料(DBCO-FITC)，30℃反应过夜，脱盐并到浓缩DPBS缓冲溶液中，得到标记抗体Ab-4。Trastuzumab (3λ), MnCl 2 (10mM), Tris-HCl buffer solution (50mM, pH=7.4), uridine diphosphate-acetylated-N-azidoacetylgalactosamine (UDP-GalNAz, 5mM) and β-1,4-galactosyltransferase (β-1,4-galactosyltransferase 1, β-4GalT1, 0.5λ) for a one-pot reaction at 30°C, 300rpm in a metal bath for 24h. After affinity purification with protein A resin, desalt into DPBS buffer solution, add 20 equivalents of dibenzocyclooctyne-dye (DBCO-FITC), react overnight at 30°C, desalt and put into concentrated DPBS buffer solution to obtain labeled antibody Ab-4.
其中，标记抗体Ab-3和Ab-4的制备方法均是通过点击化学，具体为所述点击化学包括：将与标记结构连接的二苯并环辛炔、与糖结合的炔叠氮化物以及抗体进行反应得到，具体如图3所示。Wherein, the preparation methods of labeled antibodies Ab-3 and Ab-4 are both through click chemistry, specifically, the click chemistry includes: dibenzocyclooctyne linked to the label structure, alkyne azide combined with sugar, and Antibodies were reacted, as shown in Figure 3.
(5)标记抗体Ab-2(5) Labeled antibody Ab-2
将二抗anti Human IgG或二抗anti Rabbit IgG(3λ)、MnCl2(5mM)、MgCl2(10mM)、Tris-HCl缓冲溶液(10mM，pH＝7.4)、尿苷二磷酸-半乳糖(Uridine diphosphate galactose，UDP-Galactose，5mM)、β-1,4半乳糖基转移酶(β-1,4-galactosyltransferase 1，β4GalT1，0.4λ)、岩藻糖基转移酶(Fucosyltransferase，FT，1λ)和鸟苷二磷酸-岩藻糖-荧光染料(Guanosine diphosphate fucose-Cy5，GDP-Fuc-Cy5，2mM)进行一锅法反应,在30℃、300rpm的金属浴中反应40h。用蛋白A树脂亲和纯化，将纯化后的产品进行浓缩并脱盐到DPBS缓冲溶液中，得到二抗anti human IgG-Glycan-Cy5或二抗anti Rabbit IgG-Glycan-Cy5。The secondary antibody anti Human IgG or anti Rabbit IgG (3λ), MnCl 2 (5mM), MgCl 2 (10mM), Tris-HCl buffer solution (10mM, pH=7.4), uridine diphosphate-galactose (Uridine diphosphate galactose, UDP-Galactose, 5mM), β-1,4-galactosyltransferase (β-1,4-galactosyltransferase 1, β4GalT1, 0.4λ), fucosyltransferase (Fucosyltransferase, FT, 1λ) and Guanosine diphosphate-fucose-fluorescent dye (Guanosine diphosphate fucose-Cy5, GDP-Fuc-Cy5, 2mM) was subjected to a one-pot reaction in a metal bath at 30°C and 300rpm for 40h. The protein A resin was used for affinity purification, and the purified product was concentrated and desalted into DPBS buffer solution to obtain the secondary antibody anti human IgG-Glycan-Cy5 or secondary antibody anti Rabbit IgG-Glycan-Cy5.
(6)标记抗体Ab-HRP(6) Labeled antibody Ab-HRP
将二抗anti Rabbit IgG(3λ)、MnCl2(10mM)、Tris-HCl缓冲溶液(50mM,pH＝7.4)、尿苷二磷酸-乙酰化-N-叠氮乙酰半乳糖胺(UDP-GalNAz，5mM)和β-1,4半乳糖基转移酶(β-1,4-galactosyltransferase 1，β-4GalT1，0.5λ)进行一锅法反应,在30℃、300rpm的金属浴中反应24h。用蛋白A树脂亲和纯化后脱盐到DPBS缓冲溶液中，加入20当量的二苯并环辛炔-染料(DBCO-HRP)，30℃反应过夜，脱盐并到浓缩DPBS缓冲溶液中，得到标记抗体Ab-HRP(抗体-辣根过氧化物酶复合物)。The secondary antibody anti Rabbit IgG (3λ), MnCl 2 (10mM), Tris-HCl buffer solution (50mM, pH=7.4), uridine diphosphate-acetylated-N-azidoacetylgalactosamine (UDP-GalNAz, 5mM) and β-1,4-galactosyltransferase (β-1,4-galactosyltransferase 1, β-4GalT1, 0.5λ) for a one-pot reaction at 30°C, 300rpm in a metal bath for 24h. After affinity purification with protein A resin, desalt into DPBS buffer solution, add 20 equivalents of dibenzocyclooctyne-dye (DBCO-HRP), react overnight at 30°C, desalt and put into concentrated DPBS buffer solution to obtain labeled antibody Ab-HRP (antibody-horseradish peroxidase complex).
实施例1Example 1
用胰酶消化贴壁培养的SK-OV-3细胞(人卵巢癌细胞，用于表达HER2)，离心后用FACS缓冲溶液重悬，离心洗涤重复3次。用FACS缓冲溶液重悬后等量分组加入到96孔板中，分别标记为空白组、对照组和实验组4组，每组十孔。其中，空白组不加处理；对照组和实验组均加入标记抗体Ab-1(标记抗体Ab-1在各组中的终浓度为10μg/mL)，冰上孵育30min。离心后用FACS缓冲 溶液重悬，离心洗涤重复3次。用FACS缓冲溶液重悬后，对照组不加处理；实验组分别加入EndoS(0.1λ，EndoS组)、EndoF3(0.1λ，EndoF3组)、EndoM(0.1λ，EndoM组)和PNGaseF(0.1λ，PNGaseF组)，在37℃孵育30min，离心后用FACS缓冲溶液重悬，离心洗涤重复3次。(全程需在避光下进行，以免荧光基团发生淬灭)1λ＝1mg/mLAdherent cultured SK-OV-3 cells (human ovarian cancer cells, used to express HER2) were digested with trypsin, resuspended with FACS buffer solution after centrifugation, and centrifuged and washed three times. After being resuspended with FACS buffer solution, the same amount was divided into 96-well plates and marked as blank group, control group and experimental group respectively, with ten wells in each group. Among them, the blank group was not treated; both the control group and the experimental group were added with labeled antibody Ab-1 (the final concentration of labeled antibody Ab-1 in each group was 10 μg/mL), and incubated on ice for 30 minutes. FACS buffer after centrifugation The solution was resuspended, and centrifuged and washed three times. After resuspended with FACS buffer solution, the control group was not treated; the experimental group was added with EndoS (0.1λ, EndoS group), EndoF3 (0.1λ, EndoF3 group), EndoM (0.1λ, EndoM group) and PNGaseF (0.1λ, PNGaseF group), incubated at 37°C for 30 min, resuspended with FACS buffer solution after centrifugation, and repeated centrifugation and washing 3 times. (The whole process needs to be carried out in the dark to avoid quenching of the fluorescent group) 1λ=1mg/mL
对上述酶切割后的混合物进行流式细胞术检测，检测结果如图4所示。Flow cytometry detection was performed on the mixture after the enzyme cleavage, and the detection results are shown in FIG. 4 .
如图4所示，流式结果表明，EndoM和PNGaseF在37℃，30min内无法对修饰后的抗体进行切割；而EndoS和EndoF3能发生一定切割，其荧光残存率在30％以上。As shown in Figure 4, the results of flow cytometry showed that EndoM and PNGaseF could not cleave the modified antibody within 30 minutes at 37°C; while EndoS and EndoF3 could cleave to a certain extent, and their fluorescence residual rate was above 30%.
实施例2Example 2
用胰酶消化贴壁培养的SK-OV-3细胞(人卵巢癌细胞，用于表达HER2)，离心后用FACS缓冲溶液重悬，离心洗涤重复3次。用FACS缓冲溶液重悬后等量分组加入到96孔板中，分别标记为对照组和实验组3组，每组十孔。其中，对照组和实验组均加入标记抗体Ab-1(10μg/mL)，冰上孵育30min。离心后用FACS缓冲溶液重悬，离心洗涤重复3次。用FACS缓冲溶液重悬后，对照组不加处理；实验组分别加入EndoS(EndoS的终浓度为0.1λ，0.1λ组)、EndoS(EndoS的终浓度为0.5λ，0.5λ组)和EndoS(EndoS的终浓度为1λ，1λ组)，在37℃孵育30min，离心后用FACS缓冲溶液重悬，离心洗涤重复3次。(全程需在避光下进行，以免荧光基团发生淬灭)Adherent cultured SK-OV-3 cells (human ovarian cancer cells, used to express HER2) were digested with trypsin, resuspended with FACS buffer solution after centrifugation, and centrifuged and washed three times. After resuspending with FACS buffer solution, the same amount was added to a 96-well plate, marked as control group and experimental group respectively, with ten wells in each group. Among them, both the control group and the experimental group were added with labeled antibody Ab-1 (10 μg/mL), and incubated on ice for 30 min. After centrifugation, resuspend with FACS buffer solution, and repeat centrifugation and washing 3 times. After resuspension with FACS buffer solution, the control group was not treated; the experimental group was added EndoS (the final concentration of EndoS was 0.1λ, 0.1λ group), EndoS (the final concentration of EndoS was 0.5λ, 0.5λ group) and EndoS ( The final concentration of EndoS was 1λ, 1λ group), incubated at 37°C for 30min, resuspended with FACS buffer solution after centrifugation, and repeated centrifugation and washing 3 times. (The whole process needs to be carried out in the dark to avoid quenching of fluorescent groups)
对上述酶切割后的混合物进行流式细胞术检测，检测结果如图5所示。Flow cytometry detection was performed on the mixture after the enzyme cleavage, and the detection results are shown in FIG. 5 .
通过图5可知，EndoS可以对标记抗体进行酶切割，减少标记抗体的荧光程度，但是提高EndoS的浓度，发现荧光残存的比例并没有明显降低，表明单种酶浓度的提高，对于酶切割没有促进作用。It can be seen from Figure 5 that EndoS can enzymatically cut the labeled antibody and reduce the degree of fluorescence of the labeled antibody, but increasing the concentration of EndoS found that the proportion of residual fluorescence did not decrease significantly, indicating that the increase in the concentration of a single enzyme did not promote enzyme cleavage effect.
实施例3Example 3
用胰酶消化贴壁培养的SK-OV-3细胞(人卵巢癌细胞，用于表达HER2)，离心后用FACS缓冲溶液重悬，离心洗涤重复3次。用FACS缓冲溶液重悬后等量分组加入到96孔板中，分别标记为对照组两组和实验组八组，每组十孔。其中，对照组和实验组分别加入标记抗体Ab-1、Ab-2、Ab-3、Ab-4(各标记抗体的终浓度均为10μg/mL)，冰上孵育30min。离心后用FACS缓冲溶液重悬，离心洗涤重复3次。用FACS缓冲溶液重悬后，对照组不加处理；实验组分别加入0.5λEndoS+0.5λEndoF3(EndoS的终浓度为0.5λ，EndoF3的终浓度为0.5λ；0.5λ+0.5λ+Ab-1组、0.5λ+0.5λ+Ab-2组、0.5λ+0.5λ+Ab-3组、0.5λ+0.5λ+Ab-4组)和1λEndoS(EndoS的终浓度为1λ，1λ+Ab-1组、1λ+Ab-2组、1λ+Ab-3组、1λ+Ab-4组)，在37℃孵育30min，离心后用FACS缓冲溶液重悬，离心洗涤重复3次。(全程需在避光下进行，以免荧光基团发生淬灭)Adherent cultured SK-OV-3 cells (human ovarian cancer cells, used to express HER2) were digested with trypsin, resuspended with FACS buffer solution after centrifugation, and centrifuged and washed three times. After being resuspended with FACS buffer solution, the same amount was added to a 96-well plate and marked as two groups of control group and eight groups of experimental group, with ten wells in each group. Among them, labeled antibodies Ab-1, Ab-2, Ab-3, and Ab-4 were added to the control group and the experimental group respectively (the final concentration of each labeled antibody was 10 μg/mL), and incubated on ice for 30 minutes. After centrifugation, resuspend with FACS buffer solution, and repeat centrifugation and washing 3 times. After resuspending with FACS buffer solution, the control group was not treated; the experimental group was added with 0.5λEndoS+0.5λEndoF3 (the final concentration of EndoS was 0.5λ, and the final concentration of EndoF3 was 0.5λ; 0.5λ+0.5λ+Ab-1 group , 0.5λ+0.5λ+Ab-2 group, 0.5λ+0.5λ+Ab-3 group, 0.5λ+0.5λ+Ab-4 group) and 1λEndoS (the final concentration of EndoS is 1λ, 1λ+Ab-1 group , 1λ+Ab-2 group, 1λ+Ab-3 group, 1λ+Ab-4 group), incubated at 37°C for 30min, resuspended with FACS buffer solution after centrifugation, and repeated centrifugation and washing 3 times. (The whole process needs to be carried out in the dark to avoid quenching of fluorescent groups)
对上述酶切割后的混合物进行流式细胞术检测，检测结果如图6所示。Flow cytometry detection was performed on the mixture after the enzyme cleavage, and the detection results are shown in FIG. 6 .
通过图6可知，无论是单酶还是混合酶，对于不同的标记抗体，都具有较 为明显的酶切割效果；针对同种标记抗体，总体来讲，混合酶的酶切割效果优于单酶的酶切割效果。It can be seen from Figure 6 that no matter it is a single enzyme or a mixed enzyme, for different labeled antibodies, it has a relatively high It is an obvious enzyme cleavage effect; for the same type of labeled antibody, generally speaking, the enzyme cleavage effect of the mixed enzyme is better than that of the single enzyme.
实施例4Example 4
对试验组(0.5λ+0.5λ+Ab-1组)酶切割后的混合物进行质谱表征，检测结果如图7所示(+4代表连接四个生物素；+3代表连接三个生物素；+2代表连接两个生物素；+1代表连接一个生物素)。Mass spectrometry was performed on the mixture of the test group (0.5λ+0.5λ+Ab-1 group) after enzymatic cleavage, and the detection results are shown in Figure 7 (+4 represents the connection of four biotins; +3 represents the connection of three biotins; +2 for linking two biotins; +1 for linking one biotin).
通过图7可知，连接有四个生物素的峰值(+4)在5min后逐渐降低，直至20min时，几乎完全消失；而连接有两个生物素的峰值(+2)在0-5min逐渐增多，是由于连接有三个生物素转化而成；并且连接有两个生物素的峰值(+2)在5-10min的增多是由于+4转化而成；在10-20min，连接有两个生物素的峰值(+2)逐渐降低，直至几乎与基线持平。该图表明，混合酶对于标记抗体进行酶切割，且切割较为完全。It can be seen from Figure 7 that the peaks (+4) connected with four biotins gradually decreased after 5 minutes, and almost completely disappeared at 20 minutes; while the peaks (+2) connected with two biotins gradually increased at 0-5 minutes , is due to the conversion of three biotins; and the increase of the peak (+2) with two biotins at 5-10min is due to the conversion of +4; at 10-20min, there are two biotins connected The peak value of (+2) decreases gradually until it is almost equal to the baseline. The figure shows that the mixed enzymes cleaved the labeled antibody, and the cleavage was relatively complete.
实施例5Example 5
对基于糖链修饰的抗体-辣根过氧化物酶(Ab-HRP)偶联物用本方法切割，通过蛋白胶表征，结果如图11的(A)图所示，本酶切方法除了能对偶联小分子(如生物素、荧光染料)进行有效切割，对于大分子(如此处的HRP)也能有效切割。The antibody-horseradish peroxidase (Ab-HRP) conjugate based on sugar chain modification was cleaved by this method and characterized by protein glue. The results are shown in (A) of Figure 11. In addition to being able to Efficient cleavage of coupled small molecules (such as biotin, fluorescent dyes), and efficient cleavage of large molecules (such as HRP here).
进一步，将SK-OV-3细胞用RIPA裂解液重旋后超声破碎提取全蛋白，跑胶转膜后孵育可切割抗体，再进行酶切与否的处理，结果如图11的(B)图所示，相对于对照组(DPBS)，本申请的方法对印迹膜表面的抗体偶联物也有高切割效率，这使得本申请的方法除了应用于多轮免疫荧光，同样具有应用于多轮免疫印迹实验的潜力。Further, the SK-OV-3 cells were respinned with RIPA lysate and then ultrasonically crushed to extract the whole protein, run on the gel and transfer to the membrane, incubate the cleavable antibody, and then perform enzyme digestion or not, the result is shown in (B) of Figure 11 As shown, compared with the control group (DPBS), the method of the present application also has high cutting efficiency to the antibody conjugate on the surface of the imprinted membrane, which makes the method of the present application not only applicable to multiple rounds of immunofluorescence, but also applicable to multiple rounds of immunofluorescence. Potential for blotting experiments.
实施例6Example 6
用胰酶消化贴壁培养的SK-OV-3细胞(人卵巢癌细胞，用于表达HER2)，离心后用FACS缓冲溶液重悬，离心洗涤重复3次。用FACS缓冲溶液重悬后等量分组加入到96孔板中，分别标记为对照组和实验组八组，每组十孔。其中，对照组和实验组分别加入标记抗体Ab-1(10μg/mL)，冰上孵育30min。离心后用FACS缓冲溶液重悬，离心洗涤重复3次。用FACS缓冲溶液重悬后，对照组不加处理；实验组分别加相同体积的DPBS(DPBS组)、TCEP(TCEP的终浓度为50mM，50mM TCEP组)、TCEP(TCEP的终浓度为100mM，100mM TCEP组)、TCEP(TCEP的终浓度为200mM，200mM TCEP组)、0.1λEndoS+0.1λEndoF3(EndoS的终浓度为0.1λ，EndoF3的终浓度为0.1λ；0.1λEndoS+0.1λEndoF3组)、0.5λEndoS+0.5λEndoF3(EndoS的终浓度为0.5λ，EndoF3的终浓度为0.5λ；0.5λEndoS+0.5λEndoF3组)和1λEndoS+1λEndoF3(EndoS的终浓度为1λ，EndoF3的终浓度为1λ；1λEndoS+1λEndoF3组)，在37℃孵育30min，离心后用FACS缓冲溶液重悬，离心洗涤重复3次；在实验组再次加入相应物质(DPBS、50mM TCEP、100mM TCEP、200mM TCEP、0.1λEndoS+0.1λEndoF3、0.5λEndoS+0.5λEndoF3和1λ EndoS+1λEndoF3)，进行孵育后洗涤，共循环5次。(全程需在避光下进行，以免荧光基团发生淬灭；三(2-羧乙基)膦：TCEP)Adherent cultured SK-OV-3 cells (human ovarian cancer cells, used to express HER2) were digested with trypsin, resuspended with FACS buffer solution after centrifugation, and centrifuged and washed three times. After being resuspended with FACS buffer solution, the same amount was added to a 96-well plate, and eight groups were marked as the control group and the experimental group, with ten wells in each group. Among them, labeled antibody Ab-1 (10 μg/mL) was added to the control group and the experimental group respectively, and incubated on ice for 30 min. After centrifugation, resuspend with FACS buffer solution, and repeat centrifugation and washing 3 times. After resuspending with FACS buffer solution, the control group was not treated; the experimental group was added with the same volume of DPBS (DPBS group), TCEP (the final concentration of TCEP was 50mM, 50mM TCEP group), TCEP (the final concentration of TCEP was 100mM, 100mM TCEP group), TCEP (the final concentration of TCEP is 200mM, 200mM TCEP group), 0.1λEndoS+0.1λEndoF3 (the final concentration of EndoS is 0.1λ, the final concentration of EndoF3 is 0.1λ; 0.1λEndoS+0.1λEndoF3 group), 0.5 λEndoS+0.5λEndoF3 (the final concentration of EndoS is 0.5λ, the final concentration of EndoF3 is 0.5λ; 0.5λEndoS+0.5λEndoF3 group) and 1λEndoS+1λEndoF3 (the final concentration of EndoS is 1λ, the final concentration of EndoF3 is 1λ; 1λEndoS+1λEndoF3 group), incubated at 37°C for 30min, centrifuged, resuspended with FACS buffer solution, and washed by centrifugation for 3 times; in the experimental group, the corresponding substances (DPBS, 50mM TCEP, 100mM TCEP, 200mM TCEP, 0.1λEndoS+0.1λEndoF3, 0.5 λEndoS+0.5λEndoF3 and 1λ EndoS+1λEndoF3), wash after incubation, and cycle 5 times in total. (The whole process needs to be carried out in the dark to avoid quenching of the fluorescent group; tris(2-carboxyethyl)phosphine: TCEP)
对上述酶切割5次后的混合物，检测抗原Her2和EGFR的表达，检测结果如图8所示。The expression of the antigens Her2 and EGFR was detected for the mixture after 5 times of cleavage by the above enzymes, and the detection results are shown in FIG. 8 .
同样地，使用多聚甲醛对细胞进行固定之后，按照上述条件处理1次、3次和5次后检测抗原Her2和EGFR的表达，检测结果如图12所示。Similarly, after the cells were fixed with paraformaldehyde, the expressions of the antigens Her2 and EGFR were detected after treatment according to the above conditions for 1 time, 3 times and 5 times, and the detection results are shown in FIG. 12 .
通过图8和图12可知，对于活细胞和固定细胞而言，DPBS组中的Her2和EGFR几乎没有影响；但是TCEP对抗原的损伤非常大，无论是低浓度还是高浓度在五次循环后，EGFR和Her2都严重损坏，无法用于后续多轮检测；而本申请的混合酶对于EGFR和EGFR损伤较小，与DPBS组相近，表明了本申请的酶切割在保证高效切割的情况下，还能对于抗原有着较小的损伤，具有较大的抗原温和性。It can be seen from Figure 8 and Figure 12 that for living cells and fixed cells, Her2 and EGFR in the DPBS group have almost no effect; but TCEP has a very large damage to the antigen, whether it is low concentration or high concentration After five cycles, Both EGFR and Her2 are severely damaged and cannot be used for subsequent rounds of detection; while the mixed enzyme of the present application has less damage to EGFR and EGFR, which is similar to the DPBS group, indicating that the enzyme cleavage of the present application can also cleavage while ensuring high-efficiency cleavage. It can have less damage to the antigen and has greater antigen mildness.
除此之外，在免疫印迹膜上使用上述糖苷酶的方法处理，与传统的剥离液进行对比。In addition, the above-mentioned glycosidase method was used on the western blot membrane to compare with the traditional stripping solution.
剥离液的处理方式为：将SK-OV-3细胞(人卵巢癌细胞，表达HER2)用RIPA裂解液重旋后超声破碎提取全蛋白，跑胶转膜后对印迹膜进行处理。条件分别为：1×TBST、0.5λEndoS+0.5λEndoF3(EndoS的终浓度为0.5λ，EndoF3的终浓度为0.5λ；0.5λEndoS+0.5λEndoF3组)和剥离液(stripping buffer)室温处理30分钟，在处理1次和3次后，孵育抗体(一抗为HER2/ErbB2(29D8)Rabbit mAb，二抗为anti-Rabbit IgG-HRP)对抗原Her2进行检测，结果如图13所示。(TBST组为WB实验中对照组，TBST常用于印迹膜洗涤缓冲液，被认为低抗原损伤)The stripping solution was processed as follows: SK-OV-3 cells (human ovarian cancer cells, expressing HER2) were respinned with RIPA lysate, then ultrasonically crushed to extract the whole protein, and the blotted membrane was processed after gel transfer. The conditions were: 1×TBST, 0.5λEndoS+0.5λEndoF3 (the final concentration of EndoS was 0.5λ, the final concentration of EndoF3 was 0.5λ; 0.5λEndoS+0.5λEndoF3 group) and stripping buffer (stripping buffer) at room temperature for 30 minutes, in After 1 and 3 treatments, the antibodies (the primary antibody is HER2/ErbB2(29D8) Rabbit mAb, the secondary antibody is anti-Rabbit IgG-HRP) were incubated to detect the antigen Her2, and the results are shown in Figure 13. (TBST group is the control group in WB experiments, TBST is often used in blot membrane washing buffer, which is considered low antigen damage)
通过图13可知，在处理一次后，使用剥离液的方式仅为对照组的75％；在处理三次后，与对照组相比，仅剩25％；本申请糖苷酶的酶切，在一次处理后，与对照组几乎无差别；在处理三次后，仍保留75％的抗原。表明，本申请地酶切割方法对于印迹膜层面的抗原同样较传统的商业化剥离液具有较大的抗原温和性。It can be seen from Figure 13 that after one treatment, the method of using stripping solution is only 75% of that of the control group; after three treatments, compared with the control group, only 25% remains; After treatment, there was almost no difference from the control group; after three treatments, 75% of the antigen remained. It shows that the enzymatic cleavage method of the present application is also more mild to the antigen than the traditional commercial stripping solution for the antigen on the imprinted membrane layer.
通过上述可知，本专利提出的糖苷酶切处理样本方法，在活细胞层面(图8)、固定细胞层面(图12)及印迹膜表面(图13)验证了本方法相较于其他的切割方法或洗脱试剂都具有更加温和的优势，尤其是在多轮重复洗脱之后，该方法展现出来对抗原(如HER2、EGFR)具有更完整的保留。From the above, it can be seen that the glycosidase cleavage treatment sample method proposed in this patent has verified that this method is compared with other cutting methods at the level of living cells (Figure 8), the level of fixed cells (Figure 12) and the surface of imprinted membranes (Figure 13). or elution reagents have the advantage of being milder, especially after multiple rounds of repeated elutions, this method exhibits more complete retention of antigens (eg HER2, EGFR).
实施例7Example 7
将NCI-N87细胞(人胃癌细胞，HER2+，GFP-)与MDA-MB-231细胞(人乳腺癌细胞，HER2-，GFP+)共同培养在培养皿里，加入4％多聚甲醛在室温下固定30min。加入一抗HER2/ErbB2(29D8)Rabbit mAb(体积比按1:100(培养皿中溶液的体积))在冰上孵育1h，用DPBS缓冲溶液洗5min，重复3次；加入修饰后的二抗anti Rabbit IgG-Glycan-Cy5(终浓度为10μg/mL)在室温下避光孵育1h，用DPBS缓冲溶液洗5min，重复3次；加入DAPI(4',6-二脒基-2-苯基吲哚)在室温下避光孵育10min，用DPBS缓冲溶液洗5min， 重复3次；用倒置荧光显微镜进行成像，并用imageJ对图像进行处理和通道合并。立即加入含0.5λEndoS+0.5λEndoF3(EndoS的终浓度为0.5λ，EndoF3的终浓度为0.5λ)的DPBS缓冲溶液在37℃下孵育30min，用DPBS缓冲溶液洗5min，重复3次。再次用倒置荧光显微镜进行成像及图像处理，结果如图9所示。(全程需在避光下进行，以免荧光基团发生淬灭)NCI-N87 cells (human gastric cancer cells, HER2+, GFP-) and MDA-MB-231 cells (human breast cancer cells, HER2-, GFP+) were co-cultured in a culture dish, and fixed by adding 4% paraformaldehyde at room temperature 30min. Add the primary antibody HER2/ErbB2 (29D8) Rabbit mAb (volume ratio is 1:100 (the volume of the solution in the culture dish)) and incubate on ice for 1 h, wash with DPBS buffer solution for 5 min, repeat 3 times; add the modified secondary antibody Anti Rabbit IgG-Glycan-Cy5 (final concentration 10 μg/mL) was incubated at room temperature in the dark for 1 h, washed with DPBS buffer solution for 5 min, and repeated 3 times; DAPI (4',6-diamidino-2-phenyl indole) at room temperature in the dark for 10 min, washed with DPBS buffer solution for 5 min, Repeated 3 times; imaging was performed with an inverted fluorescence microscope, and images were processed and channel merged with ImageJ. Immediately add DPBS buffer solution containing 0.5λEndoS+0.5λEndoF3 (the final concentration of EndoS is 0.5λ, the final concentration of EndoF3 is 0.5λ) and incubate at 37°C for 30min, wash with DPBS buffer solution for 5min, repeat 3 times. Imaging and image processing were performed again with an inverted fluorescence microscope, and the results are shown in Figure 9. (The whole process needs to be carried out in the dark to avoid quenching of fluorescent groups)
通过图9可知对于细胞表面的荧光成像，本方法能够实现有效的特异性切割。如图9(a)所示，绿色是MDA-MB-231细胞(表达GFP)，这类细胞并不高表达HER2抗原因而只呈现绿色，而高表达HER2抗原的NCI-N87细胞(不表达GFP)只成像显示红色而不呈现绿色。如图9(b)所示，在组合酶切之后，红色的荧光信号消失，而绿色荧光保留。这说明本方法只对特异性的成像选择性切割，同时不影响其他抗原。It can be seen from FIG. 9 that for the fluorescence imaging of the cell surface, this method can achieve effective and specific cleavage. As shown in Figure 9(a), green is MDA-MB-231 cells (expressing GFP), which do not highly express HER2 antigen and thus only appear green, while NCI-N87 cells highly expressing HER2 antigen (not expressing GFP ) only shows red but not green. As shown in FIG. 9( b ), after combined enzyme cleavage, the red fluorescence signal disappeared, while the green fluorescence remained. This shows that this method only selectively cuts specific imaging, while not affecting other antigens.
实施例8Example 8
将NCI-N87石蜡组织切片置于65℃烘箱中脱蜡45min，再将切片置于二甲苯-梯度乙醇溶液里进行完全脱蜡。将切片置于抗原修复液(EDTA-Tris,pH8.0)中微波处理以暴露抗原，自然冷却后，用DPBS缓冲溶液洗5min，重复3次。加入3％BSA(牛血清白蛋白)封闭30min，轻轻甩干封闭液后加入一抗Herceptin(終浓度10ug/ml，曲妥珠单抗)于4℃孵育过夜，用DPBS缓冲溶液洗5min，重复3次；加入修饰后的二抗anti human IgG-Glycan-Cy5(终浓度为10μg/mL)在室温下避光孵育1h，用DPBS缓冲溶液洗5min，重复3次；加入DAPI在室温下避光孵育10min，用DPBS缓冲溶液洗5min，重复3次；用倒置荧光显微镜进行成像，并用软件imageJ对图像进行处理和通道合并，结果如图10所示。The NCI-N87 paraffin tissue sections were dewaxed in an oven at 65°C for 45 minutes, and then placed in xylene-gradient ethanol solutions for complete dewaxing. The slices were microwaved in antigen retrieval solution (EDTA-Tris, pH 8.0) to expose the antigens. After natural cooling, they were washed with DPBS buffer solution for 5 min, and repeated 3 times. Add 3% BSA (bovine serum albumin) to block for 30 min, dry the blocking solution gently, add primary antibody Herceptin (final concentration 10ug/ml, trastuzumab) and incubate overnight at 4°C, wash with DPBS buffer solution for 5 min, Repeat 3 times; add the modified secondary antibody anti human IgG-Glycan-Cy5 (final concentration is 10 μg/mL) and incubate at room temperature in the dark for 1 h, wash with DPBS buffer solution for 5 min, repeat 3 times; add DAPI at room temperature in the dark Incubate with light for 10 min, wash with DPBS buffer solution for 5 min, and repeat 3 times; use an inverted fluorescence microscope to image, and use the software imageJ to process the image and merge the channels. The results are shown in Figure 10.
之后立即加入含0.5λEndoS+0.5λEndoF3(EndoS的终浓度为0.5λ，EndoF3的终浓度为0.5λ)的DPBS缓冲溶液在37℃下孵育30min，用DPBS缓冲溶液洗5min，重复3次。再次用倒置荧光显微镜进行成像及图像处理，结果如图10所示。(全程需在避光下进行，以免荧光基团发生淬灭)Immediately after that, add DPBS buffer solution containing 0.5λEndoS+0.5λEndoF3 (final concentration of EndoS is 0.5λ, final concentration of EndoF3 is 0.5λ), incubate at 37°C for 30min, wash with DPBS buffer solution for 5min, repeat 3 times. Imaging and image processing were performed again with an inverted fluorescence microscope, and the results are shown in Figure 10. (The whole process needs to be carried out in the dark to avoid quenching of fluorescent groups)
通过图10可知，对于组织切片的荧光成像，本方法也能够实现有效的特异性切割。如图10(a)所示，红色的荧光信号是NCI-N87石蜡组织切片的成像结果。如图10(b)所示，在加入组合酶切之后，红色荧光信号几乎消失。这说明本方法在石蜡组织切片水平上也能特异性地实现荧光切割。It can be seen from FIG. 10 that this method can also achieve effective and specific cutting for fluorescence imaging of tissue sections. As shown in Figure 10(a), the red fluorescent signal is the imaging result of NCI-N87 paraffin tissue section. As shown in Fig. 10(b), the red fluorescent signal almost disappeared after adding combined restriction enzymes. This shows that this method can also specifically achieve fluorescent cutting at the level of paraffin tissue sections.
在本发明中，实验例中只选择了部分结构作为代表来说明本申请的制备方法和效果等，其他没有列举的结构均具有相似的效果。In the present invention, only some structures are selected as representatives in the experimental examples to illustrate the preparation method and effects of the present application, and other structures not listed have similar effects.
实验例中所用到的单体均可以购买得到或经过简单制备得到，制备工艺也均为现有技术，故在说明书中未进行详细描述。The monomers used in the experimental examples can be purchased or obtained through simple preparation, and the preparation process is also in the prior art, so no detailed description is given in the specification.
还需要说明的是，本申请人对该系列结构做了大量的试验，有时候为了更好的与现有体系进行对比，存在同一个结构和体系，做了不止一次试验的情况，因此，不同次的试验可能会存在一定的误差。 It should also be noted that the applicant has done a lot of tests on this series of structures. Sometimes, in order to better compare with the existing system, there are cases where the same structure and system have been tested more than once. Therefore, different There may be some errors in the experiment.
最后应说明的是：以上所述的各实施例仅用于说明本发明的技术方案，而非对其限制；尽管参照前述实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述实施例所记载的技术方案进行修改，或者对其中部分或全部技术特征进行等同替换；而这些修改或替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。Finally, it should be noted that: the above-described embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand : It is still possible to modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements to some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the various embodiments of the present invention range.
上面结合附图对本申请优选的具体实施方式和实施例作了详细说明，但是本申请并不限于上述实施方式，在本领域技术人员所具备的知识范围内，还可以在不脱离本申请构思的前提下做出各种变化。 The preferred specific implementations and examples of the present application have been described in detail above in conjunction with the accompanying drawings, but the present application is not limited to the above-mentioned implementations, and within the scope of knowledge of those skilled in the art, it is also possible to Various changes are made.

Claims (10)

  1. 一种对抗原样本的处理方法，其特征在于，包括：A method for processing an antigen sample, comprising:
    (1)准备标记抗体和抗原样本；(1) Prepare labeled antibody and antigen samples;
    所述标记抗体包含有抗体和与该抗体连接的聚糖，该聚糖中含有糖苷键；The labeled antibody comprises an antibody and a glycan linked to the antibody, and the glycan contains a glycosidic bond;
    所述抗原样本包括多个可与抗体结合的位点；The antigen sample includes multiple sites that can be combined with antibodies;
    (2)将所述标记抗体与所述位点结合，或，(2) binding said labeled antibody to said site, or,
    所述位点与第一抗体结合，得到结合后的第一抗体；将所述标记抗体与所述结合后的第一抗体结合，得到结合后的标记抗体；The site is combined with the first antibody to obtain the combined first antibody; the labeled antibody is combined with the combined first antibody to obtain the combined labeled antibody;
    通过糖苷酶对所述结合后的标记抗体中的糖苷键进行酶切。The glycosidic bond in the bound labeled antibody is digested by glycosidase.
  2. 根据权利要求1的处理方法，其特征在于，在步骤(2)之后，还包括：The processing method according to claim 1, characterized in that, after step (2), further comprising:
    (3)：准备与步骤(1)相同或不同的标记抗体；(3): Prepare the same or different labeled antibody as in step (1);
    将该标记抗体与步骤(1)中的所述位点结合，或，binding the labeled antibody to the site in step (1), or,
    所述位点与新的第一抗体结合后，得到新的结合后的第一抗体；将该新的结合后的第一抗体与本步骤中的标记抗体结合，得到结合后的标记抗体；After the site is combined with the new first antibody, a new combined first antibody is obtained; the new combined first antibody is combined with the labeled antibody in this step to obtain a combined labeled antibody;
    (4)：通过糖苷酶对所述步骤(3)中结合后的所述标记抗体中的糖苷键进行酶切。(4): cleavage of the glycosidic bond in the labeled antibody bound in the step (3) by glycosidase.
  3. 根据权利要求2所述的处理方法，其特征在于，所述抗原样本中包含多种抗原；The processing method according to claim 2, wherein the antigen sample comprises multiple antigens;
    将步骤(3)和步骤(4)多次循环；Repeat step (3) and step (4);
    所述多次循环的次数为≥2。The number of said multiple cycles is ≥2.
  4. 根据权利要求1所述的处理方法，其特征在于，所述糖苷酶包括内切糖苷酶和/或外切糖苷酶。The treatment method according to claim 1, wherein the glycosidase comprises an endoglycosidase and/or an exoglycosidase.
  5. 根据权利要求1所述的处理方法，其特征在于，所述糖苷酶包括EndoS、EndoF、Alfc中的至少一种。The treatment method according to claim 1, wherein the glycosidase comprises at least one of EndoS, EndoF, and Alfc.
  6. 根据权利要求1所述的处理方法，其特征在于，所述糖苷酶包括EndoS、EndoF、Alfc中的至少两种。The treatment method according to claim 1, wherein the glycosidase comprises at least two of EndoS, EndoF, and Alfc.
  7. 根据权利要求1或2所述的处理方法，其特征在于，步骤(2)和步骤(4)中，所述酶切的条件为：温度为0℃～45℃。The treatment method according to claim 1 or 2, characterized in that, in step (2) and step (4), the conditions for the enzyme digestion are: the temperature is 0°C-45°C.
  8. 根据权利要求1所述的处理方法，其特征在于，所述聚糖还连接有标记结构；The processing method according to claim 1, wherein the glycan is also connected with a labeling structure;
    所述标记结构和所述标记抗体通过所述聚糖连接；said tagging structure and said tagged antibody are linked via said glycan;
  9. 根据权利要求8所述的处理方法，其特征在于，所述聚糖连接至少一个标记结构。The treatment method according to claim 8, characterized in that the glycan is linked to at least one labeling structure.
  10. 根据权利要求1所述的处理方法，其特征在于，在所述聚糖中，至少存在两个通过糖苷键连接的单糖。 The processing method according to claim 1, characterized in that, in the polysaccharide, there are at least two monosaccharides linked by glycosidic bonds.
PCT/CN2023/072345 2022-01-28 2023-01-16 Processing method for antigen sample, and use WO2023143179A1 (en)

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WO1996031776A1 (en) * 1995-04-06 1996-10-10 Miltenyi Bioteh, Inc. Multiparameter cell separation using releasable colloidal magnetic particles
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