CN113884676A - Method for detecting target protein by coupling fluorescent microspheres with antibody - Google Patents
Method for detecting target protein by coupling fluorescent microspheres with antibody Download PDFInfo
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Abstract
The invention discloses a method for detecting target protein by coupling fluorescent microspheres with an antibody. In the invention, antibody labeling and purification are carried out; adding 50 mu L of quantum dots into a 1.5mL microcentrifuge tube, then adding 1 mu of LEDC and 10 mu of Lsulfo-NHS solution, adding phosphate buffer solution to supplement 800 mu L, continuously mixing the solution, and reacting for 20min at room temperature; adding 200 mu L of purified antibody, and oscillating at 37 ℃ for reaction for 2 h; adding 100 mu L of glycine, and sealing unreacted carboxyl on the quantum dots; and centrifuging the product obtained by the coupling reaction at 4 ℃ and 12000r/min for 20min to obtain a precipitate, namely the purified P1 target protein antibody coupled with the fluorescent microspheres, and coupling the fluorescent microspheres with the antibody to detect the target protein in a manner of quantum dot coupled co-product fluorescence efficiency determination, coupled product immunoreactivity determination and quantum dot coupled co-product target protein antigen detection, so that a more rapid and accurate detection manner is achieved, the detection rate is improved, and more convenience is brought to the research of people.
Description
Technical Field
The invention belongs to the technical field of antibody detection, and particularly relates to a method for detecting target protein by coupling fluorescent microspheres with an antibody.
Background
It is easier to judge if the protein has a purification tag and is overexpressed, and the target protein is generally determined by SDS electrophoresis of the different fractions during the purification process. If the tag is not purified, the target protein is detected by SDS electrophoresis or two-dimensional electrophoresis and protein activity assay (if the protein has biological activity, such as an enzyme), or by western blot.
However, the common detection method has low separation rate, long time and high cost, and is not suitable for clinical application.
Disclosure of Invention
The invention aims to: in order to solve the above-mentioned problems, a method for detecting a target protein by coupling fluorescent microspheres to an antibody is provided.
The technical scheme adopted by the invention is as follows: a method for detecting a protein of interest by coupling fluorescent microspheres to an antibody, the method comprising the steps of:
s1, preparing an activation buffer with 50mM pH6.0, and more preferably acetic acid or MESbuffer; suspending the microspheres with an activation buffer to a concentration of 1% w/v;
s2, adding 20mg of EDC into each 1mL of microsphere suspension, incubating for 20 minutes at room temperature, then adding 20mg/mL of EDC again, and continuing to incubate for 20 minutes at room temperature;
s3, centrifuging or ultrafiltering, washing the microspheres twice by using equal volume of coating buffer solution, and finally suspending in the coating buffer solution;
s4, dissolving the antibody to 1mg/mL by using a coating buffer solution, wherein the pH value of the coating buffer solution is 7-9, and the concentration is 50-100 mM;
s5, quickly adding the antibody into the stirred microsphere suspension, continuously stirring, and incubating at room temperature for 2-5 hours;
s6, adding 2.5ul ethanolamine into each 1ml of reaction solution, continuously stirring and incubating for 10 minutes at room temperature;
s7, centrifuging or ultrafiltering, suspending with storage buffer solution, repeating twice, and removing unbound antibody and ethanolamine to obtain fluorescent microsphere antibody;
s8, preparing a target protein polyclonal antibody, namely extracting and purifying P1 recombinant protein, adding an isovolume Freund complete adjuvant immune coupling antibody into the purified recombinant protein, injecting the immune from multiple subcutaneous points, reinforcing the immune from Freund incomplete adjuvant for 3 times, then collecting blood from carotid artery to obtain a coupling antibody for detection, and measuring the titer of the antibody by an ELISA method;
s9 extracting and purifying IgG, crudely extracting immunoglobulin from the collected immune serum by sulfuric acid hinge precipitation method, adding saturated (NH) into the immune serum4)2SO4Making it into 20% solution to remove fibrin; the centrifugal supernatant is sucked up and saturated (NH) is added4)2SO4A solution; making it into 50% solution to remove albumin;
s10, carrying out antibody labeling and purification; adding 50 mu L of quantum dots into a 1.5mL microcentrifuge tube, then adding 1 mu of LEDC and 10 mu of Lsulfo-NHS solution, adding phosphate buffer solution to supplement 800 mu L, continuously mixing the solution, and reacting for 20min at room temperature; adding 200 mu L of purified antibody, and oscillating at 37 ℃ for reaction for 2 h; adding 100 mu L of glycine, and sealing unreacted carboxyl on the quantum dots; centrifuging the product obtained by the coupling reaction at 4 ℃ at 12000r/min for 20min to obtain a precipitate, namely the purified P1 target protein antibody coupled with the fluorescent microsphere;
s11, measuring the fluorescence efficiency of the quantum dot coupled product: measuring the quantum dot marked antibody by using a fluorescence spectrophotometer, and determining whether the coupling reaction of the antibody and the quantum dot changes the fluorescence efficiency of the quantum dot or not through measuring the fluorescence intensity of a marker;
s12, performing immunoreaction determination of the coupling product, and taking 2 mu L of the target protein whole bacteria antigen; dropping on a nitrocellulose membrane, drying at 4 ℃, and blocking with BSA-PBS 4 ℃ overnight; washing the membrane for 3 times by PBS, drying, and respectively adding a quantum dot coupling-P1 recombinant protein antibody, a quantum dot, a P1 recombinant protein antibody and PBS to react at a position containing a coating target protein antigen; reacting at 37 ℃ for 2h, washing with PBs for 3 times after reaction, and observing the result under an ultraviolet lamp;
s13, detecting a target protein antigen by using a quantum dot coupling product, taking a culture in a logarithmic growth phase, centrifuging for 30min at a speed of 12000r/min, discarding the supernatant, adding 0.01mol/L PBS solution into the precipitate, fully mixing uniformly, centrifuging and washing for 3 times, collecting the precipitate, repeatedly freezing and thawing for 3 times to obtain the target protein thalli antigen, adding 0.01mol/L PBS to prepare a bacterial suspension, taking 5 mu L of bacterial suspension, dropwise adding the 5 mu L bacterial suspension onto a glass slide, naturally drying at room temperature, and fixing by acetone precooled at-20 ℃; mixing the following components in parts by weight: adding 40 diluted quantum drops coupled with P1 recombinant protein polyclonal antibody into the antigen sheet, acting in a 37 ℃ wet box for 45min, washing with PBS for 3 times, naturally drying, adding alkaline buffer glycerol dropwise, adding a cover slip, sealing, and observing under a fluorescence microscope;
s14 respectively detecting Staphylococcus aureus, Escherichia coli, group A streptococcus and Streptococcus pneumoniae antigens according to the steps S11, S12 and S13 with PBS as blank control, and analyzing the test result to finish the whole experiment process
In a preferred embodiment, in step S3, unbound compounds or proteins are removed, and if the particle diameter is greater than 0.2um, the microspheres can be resuspended by centrifugation, then agitated, followed by gentle sonication. The centrifugal force should not be too large, which may result in the microspheres not being easily dispersed. Purification by ultrafiltration or dialysis is also possible. When ultrafiltration is used, the filter pore size is large enough to allow free protein to pass through the filter freely. The buffer used for washing the microspheres is the same as the storage buffer. Any changes in the buffer composition and pH used for washing will cause protein shedding.
In a preferred embodiment, in step S4, the composition of the reaction buffer varies according to the type of protein, and the buffers are acetic acid buffer, phosphate buffer, borate buffer, and MES buffer. Proteins adsorb more readily to the microspheres near the isoelectric point because more hydrophobic sites are exposed on the protein surface near the isoelectric point. In this case, the antibodies are also more compact, and therefore more antibodies are needed for labeling onto the microspheres. However, the final reaction buffer needs to be selected in consideration of the bioactivity of the final antibody microsphere complex, so that several different concentrations of antibody and reaction buffers with different pH values are optimally selected. In the initial experiment, the ionic strength of the reaction buffer is 25-50 mM.
In a preferred embodiment, in step S5, the protein solution is added to the microsphere suspension rapidly under rapid stirring. Small volumes, swirling mixing can occur. When large, the stirring is vigorous in a flask or beaker and the protein solution is added quickly into the middle of the vortex.
In a preferred embodiment, in step S6, ethanolamine is added to react with excess carboxyl groups present after the addition of the protein.
In a preferred embodiment, in step S7, unbound compounds or proteins are removed, and if the particle diameter is greater than 0.2um, the microspheres can be resuspended by centrifugation, then agitated, followed by gentle sonication. The centrifugal force should not be too large, which may result in the microspheres not being easily dispersed. Purification by ultrafiltration or dialysis is also possible. When ultrafiltration is used, the filter pore size is large enough to allow free protein to pass through the filter freely. The buffer used for washing the microspheres is the same as the storage buffer. Any changes in the buffer composition and pH used for washing will cause protein shedding.
In a preferred embodiment, in step S9, the albumin is removed and then saturated (NH) solution is added4)2SO4And (3) solution. And (3) making the solution into 33% solution to remove the globulin, and dissolving the precipitate in PBS to obtain the crude Mp polyclonal antibody. Loading Na0H treated DEAE-SephadexA-50 column, balancing to I with o.01mol/LPBS, adding ammonium sulfate to crude immunoglobulin after H7.4, eluting with eluent, collecting by tube, and measuring the concentration of purified antibody with ultraviolet spectrophotometer.
In a preferred embodiment, in the step S11, the excitation light used in the test is 488nm, and the emission spectrum of 605nm is scanned.
In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:
1. according to the invention, the fluorescent microsphere is coupled with the antibody to detect the target protein in a mode of quantum dot pair co-product fluorescence efficiency measurement, coupled product immunoreactivity measurement and quantum dot pair co-product target protein antigen detection, so that a more rapid and accurate detection mode is achieved, the detection rate is improved, and more convenience is brought to the research of people.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are some embodiments of the present invention, but not all 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.
Example (b):
a method for detecting a protein of interest by coupling fluorescent microspheres to an antibody, the method comprising the steps of:
s1, preparing an activation buffer with 50mM pH6.0, and more preferably acetic acid or MESbuffer; suspending the microspheres with an activation buffer to a concentration of 1% w/v;
s2, adding 20mg of EDC into each 1mL of microsphere suspension, incubating for 20 minutes at room temperature, then adding 20mg/mL of EDC again, and continuing to incubate for 20 minutes at room temperature;
s3, centrifuging or ultrafiltering, washing the microspheres twice by using equal volume of coating buffer solution, and finally suspending in the coating buffer solution; in step S3, unbound compounds or proteins are removed, and if the particle diameter is greater than 0.2um, the microspheres may be resuspended by centrifugation, then agitated, followed by gentle sonication. The centrifugal force should not be too large, which may result in the microspheres not being easily dispersed. Purification by ultrafiltration or dialysis is also possible. When ultrafiltration is used, the filter pore size is large enough to allow free protein to pass through the filter freely. The buffer used for washing the microspheres is the same as the storage buffer. Any buffer composition and pH change of the buffer used for washing will cause protein shedding;
s4, dissolving the antibody to 1mg/mL by using a coating buffer solution with the pH of 7 and the concentration of 50 mM; in step S4, the composition of the reaction buffer varies according to the type of protein, and commonly used buffers include acetic acid buffer, phosphate buffer, borate buffer, and MES buffer. Proteins adsorb more readily to the microspheres near the isoelectric point because more hydrophobic sites are exposed on the protein surface near the isoelectric point. In this case, the antibodies are also more compact, and therefore more antibodies are needed for labeling onto the microspheres. However, the final reaction buffer needs to be selected in consideration of the bioactivity of the final antibody microsphere complex, so that several different concentrations of antibody and reaction buffers with different pH values are optimally selected. Starting the experiment, the ionic strength of the reaction buffer is 25 mM;
s5, rapidly adding the antibody into the stirred microsphere suspension, continuously stirring, and incubating for 2 hours at room temperature; in step S5, the protein solution is rapidly added to the microsphere suspension under rapid stirring. Small volumes, swirling mixing can occur. When the volume is large, the stirring is violent in a flask or a beaker, and the protein solution is quickly added into the middle of the vortex;
s6, adding 2.5ul ethanolamine into each 1ml of reaction solution, continuously stirring and incubating for 10 minutes at room temperature; in step S6, ethanolamine is added to react with the excessive carboxyl groups after protein addition;
s7, centrifuging or ultrafiltering, suspending with storage buffer solution, repeating twice, and removing unbound antibody and ethanolamine to obtain fluorescent microsphere antibody; in step S7, unbound compounds or proteins are removed, and if the particle diameter is greater than 0.2um, the microspheres may be resuspended by centrifugation, then agitated, followed by gentle sonication. The centrifugal force should not be too large, which may result in the microspheres not being easily dispersed. Purification by ultrafiltration or dialysis is also possible. When ultrafiltration is used, the filter pore size is large enough to allow free protein to pass through the filter freely. The buffer used for washing the microspheres is the same as the storage buffer. Any buffer composition and pH change of the buffer used for washing will cause protein shedding;
s8, preparing a target protein polyclonal antibody, namely extracting and purifying P1 recombinant protein, adding an isovolume Freund complete adjuvant immune coupling antibody into the purified recombinant protein, injecting the immune from multiple subcutaneous points, reinforcing the immune from Freund incomplete adjuvant for 3 times, then collecting blood from carotid artery to obtain a coupling antibody for detection, and measuring the titer of the antibody by an ELISA method;
s9 extraction of IgGPurifying, subjecting the immune serum to crude extraction by sulfuric acid-hinge precipitation, adding saturated (NH) into the immune serum4)2SO4Making it into 20% solution to remove fibrin; the centrifugal supernatant is sucked up and saturated (NH) is added4)2SO4A solution; making it into 50% solution to remove albumin; in step S9, albumin is removed and then saturation (NH) is added4)2SO4And (3) solution. And (3) making the solution into 33% solution to remove the globulin, and dissolving the precipitate in PBS to obtain the crude Mp polyclonal antibody. Loading DEAE-SephadexA-50 treated by Na0H into a column, balancing to I with o.01mol/LPBS, adding ammonium sulfate to crude extract immunoglobulin after H7.4, eluting with eluent, collecting by tube, and measuring the concentration of purified antibody with ultraviolet spectrophotometer;
s10, carrying out antibody labeling and purification; adding 50 mu L of quantum dots into a 1.5mL microcentrifuge tube, then adding 1 mu of LEDC and 10 mu of Lsulfo-NHS solution, adding phosphate buffer solution to supplement 800 mu L, continuously mixing the solution, and reacting for 20min at room temperature; adding 200 mu L of purified antibody, and oscillating at 37 ℃ for reaction for 2 h; adding 100 mu L of glycine, and sealing unreacted carboxyl on the quantum dots; centrifuging the product obtained by the coupling reaction at 4 ℃ at 12000r/min for 20min to obtain a precipitate, namely the purified P1 target protein antibody coupled with the fluorescent microsphere;
s11, measuring the fluorescence efficiency of the quantum dot coupled product: measuring the quantum dot marked antibody by using a fluorescence spectrophotometer, and determining whether the coupling reaction of the antibody and the quantum dot changes the fluorescence efficiency of the quantum dot or not through measuring the fluorescence intensity of a marker; in step S11, the excitation light used in the test is 488nm, and the emission spectrum of 605nm is scanned;
s12, performing immunoreaction determination of the coupling product, and taking 2 mu L of the target protein whole bacteria antigen; dropping on a nitrocellulose membrane, drying at 4 ℃, and blocking with BSA-PBS 4 ℃ overnight; washing the membrane for 3 times by PBS, drying, and respectively adding a quantum dot coupling-P1 recombinant protein antibody, a quantum dot, a P1 recombinant protein antibody and PBS to react at a position containing a coating target protein antigen; reacting at 37 ℃ for 2h, washing with PBs for 3 times after reaction, and observing the result under an ultraviolet lamp;
s13, detecting a target protein antigen by using a quantum dot coupling product, taking a culture in a logarithmic growth phase, centrifuging for 30min at a speed of 12000r/min, discarding the supernatant, adding 0.01mol/L PBS solution into the precipitate, fully mixing uniformly, centrifuging and washing for 3 times, collecting the precipitate, repeatedly freezing and thawing for 3 times to obtain the target protein thalli antigen, adding 0.01mol/L PBS to prepare a bacterial suspension, taking 5 mu L of bacterial suspension, dropwise adding the 5 mu L bacterial suspension onto a glass slide, naturally drying at room temperature, and fixing by acetone precooled at-20 ℃; mixing the following components in parts by weight: adding 40 diluted quantum drops coupled with P1 recombinant protein polyclonal antibody into the antigen sheet, acting in a 37 ℃ wet box for 45min, washing with PBS for 3 times, naturally drying, adding alkaline buffer glycerol dropwise, adding a cover slip, sealing, and observing under a fluorescence microscope;
and S14, respectively detecting the staphylococcus aureus, the escherichia coli, the streptococcus pneumoniae and the streptococcus pneumoniae antigens according to the operations of the steps S11, S12 and S13 by using PBS as a blank control, and finally analyzing the test result so as to finish the whole experimental process.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.
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 (8)
1. A method for detecting a target protein by coupling fluorescent microspheres with an antibody is characterized in that: the method for detecting the target protein by coupling the fluorescent microspheres with the antibody comprises the following steps:
s1, preparing an activation buffer with the pH value of 50mM being 6.0, and suspending the microspheres by using the activation buffer to enable the concentration to be 1% w/v;
s2, adding 20mg of EDC into each 1mL of microsphere suspension, incubating for 20 minutes at room temperature, then adding 20mg/mL of EDC again, and continuing to incubate for 20 minutes at room temperature;
s3, centrifuging or ultrafiltering, washing the microspheres twice by using equal volume of coating buffer solution, and finally suspending in the coating buffer solution;
s4, dissolving the antibody to 1mg/mL by using a coating buffer solution, wherein the pH value of the coating buffer solution is 7-9, and the concentration is 50-100 mM;
s5, quickly adding the antibody into the stirred microsphere suspension, continuously stirring, and incubating at room temperature for 2-5 hours;
s6, adding 2.5ul ethanolamine into each 1ml of reaction solution, continuously stirring and incubating for 10 minutes at room temperature;
s7, centrifuging or ultrafiltering, suspending with storage buffer solution, repeating twice, and removing unbound antibody and ethanolamine to obtain fluorescent microsphere antibody;
s8, preparing a target protein polyclonal antibody, namely extracting and purifying P1 recombinant protein, adding an isovolume Freund complete adjuvant immune coupling antibody into the purified recombinant protein, injecting the immune from multiple subcutaneous points, reinforcing the immune from Freund incomplete adjuvant for 3 times, then collecting blood from carotid artery to obtain a coupling antibody for detection, and measuring the titer of the antibody by an ELISA method;
s9 extracting and purifying IgG, crudely extracting immunoglobulin from the collected immune serum by sulfuric acid hinge precipitation method, adding saturated (NH) into the immune serum4)2SO4Making it into 20% solution to remove fibrin; adding the supernatant after centrifugationSaturation (NH)4)2SO4A solution; making it into 50% solution to remove albumin;
s10, carrying out antibody labeling and purification; adding 50 mu L of quantum dots into a 1.5mL microcentrifuge tube, then adding 1 mu of LEDC and 10 mu of Lsulfo-NHS solution, adding phosphate buffer solution to supplement 800 mu L, and reacting for 20min at room temperature; adding 200 mu L of purified antibody, and oscillating at 37 ℃ for reaction for 2 h; adding 100 mu L of glycine, and sealing unreacted carboxyl on the quantum dots; centrifuging the product obtained by the coupling reaction at 4 ℃ at 12000r/min for 20min to obtain a precipitate, namely the purified P1 target protein antibody coupled with the fluorescent microsphere;
s11, measuring the fluorescence efficiency of the quantum dot coupled product: measuring the quantum dot marked antibody by using a fluorescence spectrophotometer, and determining whether the coupling reaction of the antibody and the quantum dot changes the fluorescence efficiency of the quantum dot or not through measuring the fluorescence intensity of a marker;
s12, performing immunoreaction determination of the coupling product, and taking 2 mu L of the target protein whole bacteria antigen; dropping on a nitrocellulose membrane, drying at 4 ℃, and blocking with BSA-PBS 4 ℃ overnight; washing the membrane for 3 times by PBS, drying, and respectively adding a quantum dot coupling-P1 recombinant protein antibody, a quantum dot, a P1 recombinant protein antibody and PBS to react at a position containing a coating target protein antigen; reacting at 37 ℃ for 2h, washing with PBs for 3 times after reaction, and observing the result under an ultraviolet lamp;
s13, detecting a target protein antigen by using a quantum dot coupling product, taking a culture in a logarithmic growth phase, centrifuging for 30min at a speed of 12000r/min, discarding the supernatant, adding 0.01mol/L PBS solution into the precipitate, fully mixing uniformly, centrifuging and washing for 3 times, collecting the precipitate, repeatedly freezing and thawing for 3 times to obtain the target protein thalli antigen, adding 0.01mol/L PBS to prepare a bacterial suspension, taking 5 mu L of bacterial suspension, dropwise adding the 5 mu L bacterial suspension onto a glass slide, naturally drying at room temperature, and fixing by acetone precooled at-20 ℃; mixing the following components in parts by weight: adding 40 diluted quantum drops coupled with P1 recombinant protein polyclonal antibody into the antigen sheet, acting in a 37 ℃ wet box for 45min, washing with PBS for 3 times, naturally drying, adding alkaline buffer glycerol dropwise, adding a cover slip, sealing, and observing under a fluorescence microscope;
and S14, respectively detecting the staphylococcus aureus, the escherichia coli, the streptococcus pneumoniae and the streptococcus pneumoniae antigens according to the operations of the steps S11, S12 and S13 by using PBS as a blank control, and finally analyzing the test result so as to finish the whole experimental process.
2. The method of claim 1, wherein the protein of interest is detected by coupling fluorescent microspheres to antibodies, wherein the fluorescent microspheres comprise: in the step S3, unbound compounds or proteins are removed, and if the particle diameter is larger than 0.2um, the microspheres may be resuspended by centrifugation, and then stirred, followed by mild ultrasonic dispersion.
3. The method of claim 1, wherein the protein of interest is detected by coupling fluorescent microspheres to antibodies, wherein the fluorescent microspheres comprise: in step S4, the composition of the reaction buffer varies according to the type of protein, and the buffers are acetic acid buffer, phosphate buffer, borate buffer, and MES buffer.
4. The method of claim 1, wherein the protein of interest is detected by coupling fluorescent microspheres to antibodies, wherein the fluorescent microspheres comprise: in the step S5, the protein solution is rapidly added into the microsphere suspension under rapid stirring; small volumes may provide vortex mixing; when large, the stirring is vigorous in a flask or beaker and the protein solution is added quickly into the middle of the vortex.
5. The method of claim 1, wherein the protein of interest is detected by coupling fluorescent microspheres to antibodies, wherein the fluorescent microspheres comprise: in step S6, ethanolamine is added to react with the excess carboxyl groups after the protein addition.
6. The method of claim 1, wherein the protein of interest is detected by coupling fluorescent microspheres to antibodies, wherein the fluorescent microspheres comprise: in the step S7, unbound compounds or proteins are removed, and if the particle diameter is larger than 0.2um, the microspheres may be resuspended by centrifugation, and then stirred, followed by mild ultrasonic dispersion.
7. The method of claim 1, wherein the protein of interest is detected by coupling fluorescent microspheres to antibodies, wherein the fluorescent microspheres comprise: in step S9, the albumin is removed and then saturated (NH) solution is added4)2SO4A solution; and (3) making the solution into 33% solution to remove the globulin, and dissolving the precipitate in PBS to obtain the crude Mp polyclonal antibody.
8. The method of claim 1, wherein the protein of interest is detected by coupling fluorescent microspheres to antibodies, wherein the fluorescent microspheres comprise: in step S11, the excitation light used in the test was 488nm, and the emission spectrum was scanned at 605 nm.
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