CN113024665A - Method for labeling antibodies by oligos and antibodies obtained by labeling antibodies - Google Patents
Method for labeling antibodies by oligos and antibodies obtained by labeling antibodies Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/13—Labelling of peptides
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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
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
Abstract
The invention discloses an oligos antibody labeling method and an antibody obtained by labeling, wherein biotinylated oligos is connected with streptavidin, and then the streptavidin is connected with the biotinylated antibody to form an oligos-biotin-streptavidin-biotin-antibody compound. Different oligos labels, forming multiple labeled complexes, can label multiple antibodies simultaneously. The obtained oligos antibody compound has fixed ratio of antibody to oligos molecule, and can be used for corresponding experiments such as double antibody sandwich method.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an oligos antibody labeling method and an antibody obtained by labeling.
Background
At present, an enzyme-linked immunosorbent assay (ELISA) adopts an HRP-labeled antibody, although the detection sensitivity is high, when the HRP catalyzes a TMB substrate reaction, the reaction is easy to reach saturation, so that a high value cannot be continuously increased, the linear range is narrow, and meanwhile, when the same sample is subjected to simultaneous detection of multiple proteins, a large amount of pretreatment is required, and the defects of multiple operation steps, large reagent consumption amount, multiple times of cleaning, low detection flux and the like exist. The colloidal gold immune test strip adopts the nano-gold particles to mark the antibody, the technology is simple and easy to implement, and is suitable for on-site rapid detection, but the detection method is not suitable for quantifying the protein with lower concentration because only semi-quantitative analysis can be carried out, the stability of the detection result is poor, and the detection limit is lower. Latex immunoturbidimetry, which employs latex particles to label antibodies, can perform quantitative detection, but the detection sensitivity is still insufficient, which limits the application.
Disclosure of Invention
In order to solve the defects that detection methods such as ELISA and the like in the current rapid real-time detection of protein are complex, time-consuming, reagent-consuming and low-flux, colloidal gold cannot be quantitatively detected, latex turbidity sensitivity is low and the like, the invention provides an oligos-labeled antibody method, which has the advantages of high flux, high specificity, strong universality, time saving, samples, reagents and the like in the detection process.
A method for labeling an antibody with oligos, comprising the steps of: obtaining biotin-oligos complexes and biotin-antibody complexes; and (3) crosslinking the biotin-oligos complex and the biotin-antibody complex by using streptavidin to obtain an oligos-biotin-streptavidin-biotin-antibody complex.
Further, the molar ratio of the biotin-oligos complex to the streptavidin is (1-4): 1.
further, the preparation of the biotin-oligos complex or the biotin-antibody complex includes: activating biotin to obtain activated biotin; mixing the activated biotin and antibodies or oligos, and carrying out rotary reaction at room temperature to obtain the biotin-antibody complex or the biotin-oligos complex.
Further, the molar ratio of oligos to biotin in the biotin-oligos complex prepared as such is 1:1 to 1: 10.
Further, in the preparation of the biotin-antibody complex, a cross-linking agent is used in combination of EDC and NHS, or EDC and Sulfo-NHS.
Furthermore, the 5 'end or the 3' end of the oligos is modified with an amino group.
Further, the activated biotin is attached to the amino group using a cross-linking agent.
Further, the cross-linking agent comprises beta-cyanoethyl phosphoramidite, tetrazole or nucleoside phosphoramidite.
Further, the length of the base fragment of the oligos is 2-100 bp.
Further, the molar ratio of the oligos mixed with the activated biotin is 1:1 to 1: 20.
Further, the biotin activation comprises the following steps: mixing biotin with EDC and NHS, and carrying out rotary reaction at room temperature for 15-25 minutes to obtain activated biotin; wherein the molar ratio of biotin to EDC is 10: 1, biotin to NHS molar ratio of 10: 1.
in another aspect, the present invention also provides an oligos-labeled antibody, which is an oligos-biotin-streptavidin-biotin-antibody complex labeled by any one of the methods described above.
Further, in the oligos-biotin-streptavidin-biotin-antibody complex, the molar ratio of antibody to oligos is (0.3-1): 1.
in yet another aspect, the present invention also provides a capture antibody pairwise linked to the oligos-labeled antibody; for linking the oligo-labeled antibody and adsorbing on an microplate, or for linking the oligo-labeled antibody and magnetic beads.
Further, the step of attaching the oligos-labeled antibody and adsorbing the antibody on an elisa plate comprises the steps of: diluting the capture antibody to 10ug/ml with 0.05M pH9.6 carbonate buffer solution, adding 100ul of the diluted antibody to a 96-well enzyme label plate, standing overnight at 4 ℃, patting the liquid on the plate the next day, adding 300ul of 20mM pH7.8 PBS buffer solution (containing 5% BSA) to seal the 96-well enzyme label plate for 6 hours, patting the liquid on the plate after sealing, drying in a 37-degree oven for 6 hours, putting the enzyme label plate into a self-sealing bag after drying, adding a drying agent, sealing, and storing at 4 ℃.
Further, the linkage of the oligo-labeled antibody and magnetic beads the washed carboxylated magnetic beads were mixed with EDC and NHS, the molar ratio of the number of carboxyl groups on the carboxylated magnetic beads to EDC was 1 to 10, the molar ratio of the number of carboxyl groups on the carboxylated magnetic beads to NHS was 1 to 10, the concentrations of EDC and NHS were 20mg/ml, respectively, and the reaction was performed for 20 minutes at room temperature by rotation; and mixing the dialyzed antibody with activated carboxylated magnetic beads, carrying out a rotation reaction at room temperature for 6 hours, and purifying.
In summary, the technical scheme provided by the invention has the following beneficial effects: high flux, reagent and time saving, simplified complexity of multi-item detection, and ensured detection sensitivity.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 shows the fitting results of the data corresponding to the antibody A of the first embodiment.
FIG. 2 shows the result of fitting the data corresponding to the antibody B of the first embodiment.
FIG. 3 shows the result of fitting data to the first example antibody C.
FIG. 4 shows the fitting results of the data corresponding to the second example antibody A.
FIG. 5 shows the result of fitting data to the second example antibody B.
FIG. 6 shows the fitting results of the data corresponding to the second example antibody C.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
[ first embodiment ] A method for manufacturing a semiconductor device
1) Amino groups were modified at the 5' end of the oligos.
1.1 taking 3 EP tubes of 1.5ml, respectively taking 100ug of oligos-A, oligos-B and oligos-C, and adding the materials into the EP tubes.
1.2 Add 1ml of each of the activating solutions to the 3 EP tubes.
1.3 Add 10ug beta-cyanoethyl phosphoramidite and 10ug tetrazole to the above 3 EP tubes, rotate and react for 3 hours at room temperature.
1.4 after the reaction, purifying, adding the reactant into a chromatographic column for purification, wherein the flow rate of eluent is 5ml/min, obtaining 5' end amino modified oligos, the concentration is 100ug/ml, and storing at 4 ℃ for later use.
2) Biotinylated oligos were prepared.
2.1 weighing 1mg biotin, adding to 1.5ml EP tube containing 1ml activating solution to dissolve.
2.2 0.1mg of EDC and 0.1mg of NHS were weighed out and added to the above activating solution, and the reaction was rotated at room temperature for 20 minutes.
2.3 after the reaction, 0.1ml of each of the reacted liquids was taken out and put into 3 1.5ml of EP tubes, and 1ml of each of the crosslinking liquids was put into 3 1.5ml of EP tubes.
2.4 taking 100ul of each of the above 5' -amino-modified oligos-A, oligos-B and oligos-C, respectively, adding into the above EP tube, and rotating at room temperature for 6 hours.
2.5 after the reaction is finished, purifying, adding the reactant into a chromatographic column for purification, wherein the flow rate of eluent is 5ml/min, and obtaining three biotin-oligos-complexes, which are respectively: biotin-oligos-a complex, biotin-oligos-B complex, biotin-oligos-C complex, stored at 4 degrees for future use.
3) Preparation of streptavidin-biotin-oligos complexes
3.1 Take 3 1.5ml EP tubes and add 50ul of each of the three biotin-oligos complexes to 1.5ml EP tubes.
3.2 and then adding 50ul of streptavidin respectively, and shaking to react for 30 minutes at room temperature. The molar ratio of oligos to streptavidin was 1: 2.
3.3 after the reaction is finished, adding the reactant into a chromatographic column for purification, collecting and obtaining a streptavidin-biotin-oligos-A compound, a streptavidin-biotin-oligos-B compound and a streptavidin-biotin-oligos-C compound at the eluent flow rate of 5ml/min, and storing for later use at 4 degrees.
4) Preparation of biotinylated antibodies
4.1 weighing 1mg biotin, adding to 1.5ml EP tube containing 1ml activating solution to dissolve.
4.2 0.1mg of EDC and 0.1mg of NHS were weighed out and added to the above activating solution, and the reaction was rotated at room temperature for 20 minutes.
4.3 after the reaction, 0.1ml of each of the reacted liquids was taken out and put into 3 1.5ml of EP tubes, and 1ml of each of the crosslinking liquids was put into 3 1.5ml of EP tubes.
4.4 the above-mentioned 1.5ml EP tube was filled with 1mg of antibody A, 1mg of antibody B and 1mg of antibody C, respectively, and the reaction was carried out for 6 hours at room temperature by rotation.
4.5 after the reaction, the liquid was taken out separately and added into dialysis bags with 8000-year 14000 pore size for overnight dialysis, 1L of dialysate was added every 1 hour for 12 times in total.
4.6 after dialysis, three biotin-antibody complexes were obtained, respectively: and 4, storing the biotin-antibody A complex, the biotin-antibody B complex and the biotin-antibody C complex at 4 degrees for later use.
5) Preparation of oligo-biotin-streptavidin-biotin-antibody complexes
5.1 adding 50ul biotin-antibody A complex into a 1.5ml EP tube; then adding streptavidin-biotin-oligos-A compound, and shaking to react for 30 minutes at room temperature. Wherein the molar ratio of the biotin-antibody A complex to the streptavidin-biotin-oligos-A complex is 2: 1.
5.2 after the reaction is finished, adding the reactant into a chromatographic column for purification, and collecting the obtained oligos-A-biotin-streptavidin-biotin-antibody A complex, wherein the flow rate of eluent is 5 ml/min. And 4 degrees are stored for later use.
5.3 Using the procedures of steps 5.1 and 5.2, oligos-B-biotin-streptavidin-biotin-antibody B complexes and oligos-C-biotin-streptavidin-biotin-antibody C complexes were obtained.
6) Elisa method for detecting marking effect
6.1 the three oligo-biotin-streptavidin-biotin-antibody complexes obtained in step 5 were mixed together in equal proportion and coated on a 96-well microplate with each well coated with 900ng of 300-.
6.2 the coated ELISA plates were blocked with 2% BSA in a blocking solution at 37 ℃ for 5 hours.
6.3 after the sealing is finished, patting the mixture dry, and drying the mixture for 5 hours in a 37-DEG oven.
6.4 drying, taking out, putting into a self-sealing bag, adding a drying agent, sealing at 4 ℃ and storing for later use.
6.5 the recombinant proteins corresponding to the antibody A, the antibody B and the antibody C are respectively prepared into: 0ng/ml, 2.4ng/ml, 9.8ng/ml, 39.1ng/ml, 156.3ng/ml, 625ng/ml, 2500ng/ml, 10000 ng/ml.
6.6 100ul of the recombinant protein prepared above was taken, added to the above enzyme-labeled plate, and reacted at 37 ℃ for 1 hour.
6.7 after the reaction is complete, the plate is washed 5 times with wash solution and then patted dry.
6.8 the diluted three oligos-biotin-streptavidin-biotin-antibody complexes (stock solution was diluted 1000-fold with marker diluent) were added to 100ul and reacted at 37 ℃ for 30 minutes.
6.9 after the reaction is complete, the plate is washed 5 times with wash solution, then patted dry and the reconstituted solution is added.
6.10 the oligonucleotides labeled on the various antibodies were detected using a gene sequencer. The signal intensity of the oligos is proportional to the content of the target protein in the sample, and the content of the target protein in the sample is reflected by the measured signal intensity of the oligos.
7) Results of the experiment
The detection results of the recombinant protein corresponding to the antibody A are shown in Table 1.
TABLE 1
Referring to fig. 1, Logistic four parameter fit was used: r2=0.99967。
The detection results of the recombinant protein corresponding to the antibody B are shown in Table 2:
TABLE 2
Referring to fig. 2, Logistic four parameter fit was used: r2=0.99983。
The detection results of the recombinant protein corresponding to the antibody C are shown in table 3:
TABLE 3
Referring to fig. 3, Logistic four parameter fit was used: r2=0.99994。
8) Buffer solution and raw material for experiment
8.1 activation solution-20 mM pH6.0 MES buffer: 4.265g MES and 800ml ultrapure water were taken, the pH was adjusted to 6.0 with NaOH, and the volume was adjusted to 1000 ml.
8.2 Cross-linker-50 mM pH6.5 MES buffer: 10.6625g MES and 800ml ultrapure water were taken, the pH was adjusted to 6.5 with NaOH, and the volume was adjusted to 1000 ml.
8.3 dialysate-PBS buffer pH 7.8: collecting 0.55g KH2PO4、6.5g Na2HPO4·12H2O, 8g NaCl and 800ml ultrapure water, the pH was adjusted to 7.8 with HCl, and the volume was adjusted to 1000 ml.
8.4 blocking solution-PBS buffer at pH7.6 (2% BSA): collecting 0.55g KH2PO4、6.5g Na2HPO4·12H2O, 8g NaCl, 1g KCl, 20g BSA, 800ml ultrapure water, adjusting the pH to 7.5 with HCl, and then making the volume to 1000 ml.
8.5 eluentPBST buffer at pH7.5 (0.1% Tween-20): collecting 0.55g KH2PO4、6.5g Na2HPO4·12H2O, 8g NaCl, 1g KCl, 5g BSA, 1ml Tween-20 and 800ml ultrapure water, adjusting the pH value to 7.5 by using HCl, and then fixing the volume to 1000 ml.
8.6 marker dilutions-PBS buffer pH7.3 (5% BSA): collecting 0.55g KH2PO4、6.5g Na2HPO4·12H2O, 8g NaCl, 1g KCl, 50g BSA, 1ml Tween-20 and 800ml ultrapure water, adjusting the pH value to 7.5 by using HCl, and then fixing the volume to 1000 ml.
[ second embodiment ]
1) Three oligos-labeled antibodies were prepared, namely oligos-a-biotin-streptavidin-biotin-antibody a complex, oligos-B-biotin-streptavidin-biotin-antibody B complex, oligos-C-biotin-streptavidin-biotin-antibody C complex.
See the first example for the preparation method except that amino groups are modified at the 3' ends of the oligos.
2) Method for crosslinking antibody by magnetic beads
2.1 mixing the above three oligo labeled antibodies in equal proportion, cross-linking with carboxylated magnetic beads, linking the amino group of the antibody with the carboxyl group of the magnetic beads, and linking 0.1-1mg antibody per 1mg magnetic beads.
2.2 washing magnetic beads: and (3) absorbing 1mg of magnetic beads by a pipette gun, adding the magnetic beads into a 2ml centrifuge tube, putting the centrifuge tube on a magnetic frame, carrying out magnetic adsorption for 2 minutes, absorbing the supernatant by the pipette gun, adding 1ml of activating solution, taking out the centrifuge tube, shaking up and down for redissolving, putting the centrifuge tube on the magnetic frame, cleaning the magnetic beads for 5 times in total according to the method, and adding 1ml of the activating solution for redissolving after the last cleaning.
2.3 weigh 0.3mg EDC and 0.3mg NHS, add to 1ml magnetic beads after reconstitution, and rotate the reaction for 30 minutes at room temperature.
2.4 after the reaction, washing the magnetic beads for 5 times by using a washing liquid, wherein the washing mode is the same as the above, and after the last washing, adding 1ml of a cross-linking liquid for redissolving to obtain the activated magnetic beads.
2.5 washing antibody: adding 0.5mg of the mixed antibody into an ultrafiltration centrifugal tube, supplementing the volume to 200ul with an activating solution, centrifuging for 10min at 8000r/min, supplementing the volume to 200ul with the activating solution after the centrifugation is finished, repeating the operation, and cleaning for 5 times.
2.6 after washing, the antibody in the ultrafiltration tube is taken out, added into the activated magnetic beads and rotated overnight at room temperature for reaction.
2.7 washing antibody-magnetic bead complexes: and after the reaction is finished, placing the centrifuge tube on a magnetic frame, cleaning for 5 times by using a cleaning solution in the same way, after the cleaning is finished, adding 1ml of complex solution, and placing the antibody-magnetic bead complex in a 4-degree refrigerator for later use.
3) Detecting marking effect
3.1 diluting the magnetic beads with the cross-linked antibodies by 50 times with a re-solution, sucking 100ul of the diluted antibody-magnetic bead compound by a pipette gun, and adding the diluted antibody-magnetic bead compound into an enzyme label plate.
3.2 the recombinant proteins corresponding to the antibody A, the antibody B and the antibody C are respectively prepared into: 0ng/ml, 2.4ng/ml, 9.8ng/ml, 39.1ng/ml, 156.3ng/ml, 625ng/ml, 2500ng/ml, 10000 ng/ml. 100ul of the recombinant protein prepared above was added to the above enzyme-labeled plate, and reacted at 37 ℃ for 30 min.
3.3 after the reaction, the ELISA plate is placed on the magnetic plate, and is adsorbed for 2 minutes, and the supernatant is absorbed by a pipette. Adding 300ul of cleaning solution into each hole, taking out the ELISA plate, placing the ELISA plate on a shaking instrument, shaking for 1 minute, placing the ELISA plate on a magnetic plate, and washing the plate for 5 times in total according to the above cleaning method.
3.4 after completion of washing, 100ul of the above diluted three oligo-labeled antibodies (stock solution was diluted 1000-fold with a diluent for a label) was added and reacted at 37 ℃ for 15 minutes.
3.5 after the reaction, the ELISA plate was placed on a magnetic plate, the plate was washed 5 times with a washing solution, and the PBS redissolution was added.
3.6 the oligonucleotides labeled on the various antibodies were detected using a gene sequencer. The signal intensity of the oligos is proportional to the content of the target protein in the sample, and the content of the target protein in the sample is reflected by the measured signal intensity of the oligos.
4) Results of the experiment
4.1 the results of the detection of the recombinant protein corresponding to antibody A are shown in Table 4:
TABLE 4
Referring to fig. 4, Logistic four parameter fit was used: r2=0.9978。
4.2 the results of the detection of the recombinant protein corresponding to antibody B are shown in Table 5:
TABLE 5
Referring to fig. 5, Logistic four parameter fit was used: r2=0.99387。
4.3 the results of the detection of the recombinant protein corresponding to antibody C are shown in Table 6:
TABLE 6
Referring to fig. 6, Logistic four parameter fit was used: r2=0.99902。
5) Buffer solution and raw material for experiment
5.1 reconstituted solution-PBS buffer pH7.8 (containing 0.5% BSA): collecting 0.55g KH2PO4、6.5g Na2HPO4·12H2O, 8g NaCl, 5g BSA, 0.5ml Proclin 300, 800ml ultrapure water, adjusting the pH to 7.8 with HCl, and then making the volume to 1000 ml.
5.2 sample dilutions-pH 7.4 PBS buffer: collecting 0.55g KH2PO4、6.5g Na2HPO4·12H2O, 8g NaCl, 1g KCl, 0.5ml Proclin 300 and 800ml ultrapure water, adjusting the pH to 7.4 by using HCl, and then fixing the volume to 1000 ml.
5.3 preparing a cleaning solution: PBS buffer at pH 7.3: collecting 0.55g KH2PO4、6.5g Na2HPO4·12H2O、8g NaCl, 1ml of Tween-20, 0.5ml of Proclin 300 and 800ml of ultrapure water, adjusting the pH value to 7.3 by using HCl, and then fixing the volume to 1000 ml.
The method for labeling the antibodies by the oligos is not limited to the combination of the 3 different oligos, but also is not limited to the connection mode of the capture antibody and other substances, and in practical application, more than ten oligos and even hundreds of oligos can be combined, and more than ten and more or even hundreds of target proteins can be detected at one time only by adding one sample. Compared with the traditional detection methods of Elisa, colloidal gold, latex immunoturbidimetry and the like, the method has the greatest advantages of high flux, reagent and time saving, simplification of the complexity of multi-item detection and guarantee of detection sensitivity.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (10)
1. A method for labeling an antibody with oligos, comprising the steps of:
obtaining biotin-oligos complexes and biotin-antibody complexes;
and (3) crosslinking the biotin-oligos complex and the biotin-antibody complex by using streptavidin to obtain an oligos-biotin-streptavidin-biotin-antibody complex.
2. The method according to claim 1, wherein the molar ratio of the biotin-oligos complex to the streptavidin is (1-4): 1.
3. the method according to claim 1, wherein the preparation of the biotin-oligos complex or the biotin-antibody complex comprises:
activating biotin to obtain activated biotin;
mixing the activated biotin and antibodies or oligos, and carrying out rotary reaction at room temperature to obtain the biotin-antibody complex or the biotin-oligos complex.
4. The method according to claim 3, wherein the 5 'end or the 3' end of the oligos is modified with an amino group; linking the activated biotin to the amino group using a cross-linking agent; the cross-linking agent comprises beta-cyanoethyl phosphoramidite, tetrazole or nucleoside phosphoramidite.
5. The method according to claim 3, wherein the oligonucleotides have a base fragment length of 2-100 bp.
6. The method according to claim 3, wherein the molar ratio of the oligos to the activated biotin is 1 (1-20).
7. The method of claim 3, wherein the activating biotin comprises the steps of:
mixing biotin with EDC and NHS, and carrying out rotary reaction at room temperature for 15-25 minutes to obtain activated biotin; wherein the molar ratio of biotin to EDC is 10: 1, biotin to NHS molar ratio of 10: 1.
8. an oligos-labeled antibody, wherein the oligos-labeled antibody is an oligos-biotin-streptavidin-biotin-antibody complex labeled by the method of any one of claims 1 to 7.
9. The oligos-labeled antibody of claim 8, wherein the mole ratio of antibody to oligos in the oligos-biotin-streptavidin-biotin-antibody complex is (0.3-1): 1.
10. a capture antibody conjugated to the oligos-labeled antibody of claim 8 or 9; for linking the oligo-labeled antibody and adsorbing on an microplate, or for linking the oligo-labeled antibody and magnetic beads.
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| CN112005115A (en) * | 2018-02-12 | 2020-11-27 | 10X基因组学有限公司 | Methods of characterizing multiple analytes from a single cell or cell population |
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| CN106574301A (en) * | 2014-06-23 | 2017-04-19 | 斯坦福大学托管董事会 | On-slide staining by primer extension |
| CN112005115A (en) * | 2018-02-12 | 2020-11-27 | 10X基因组学有限公司 | Methods of characterizing multiple analytes from a single cell or cell population |
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