CN113777300A - Detection kit for quantitatively detecting new coronavirus antibody in human blood and detection method thereof - Google Patents
Detection kit for quantitatively detecting new coronavirus antibody in human blood and detection method thereof Download PDFInfo
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Abstract
The invention discloses a detection kit for quantitatively detecting a new coronavirus antibody in human blood and a detection method thereof, wherein the detection kit comprises an immunochromatography reagent strip, the immunochromatography reagent strip comprises a bottom lining, an NC (numerical control) membrane, a first sample pad, a second sample pad, a release pad and absorbent paper, the first sample pad and the absorbent paper are respectively laminated at the left end and the right end of the NC membrane, and a T line and a C line are arranged on the NC membrane; the second sample pad is superposed on the first sample pad, the release pad is superposed on the first sample pad, the RBD antibody and the rabbit IgG which are fluorescently labeled by the quantum dots are mixed and then sprayed on the release pad, the T line is coated with the recombinant RBD antigen, and the C line is coated with the goat anti-rabbit secondary antibody GAR. According to the invention, the detection sensitivity is improved through the quantum dot fluorescent label, the blood sample is firstly dripped on the first sample pad, and then the chromatography propelling liquid is dripped on the second sample pad, so that the positive antibody in the blood sample can be combined with the recombinant RBD antigen coated on the T line of the NC membrane before the RBD antibody of the quantum dot fluorescent label, and the accuracy of the detection result is greatly improved.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a detection kit for quantitatively detecting a new coronavirus antibody in human blood and a detection method thereof.
Background
The novel coronavirus pneumonia is pneumonia caused by novel coronavirus (COVID-19), and some severe patients can have respiratory distress syndrome or sepsis shock and even die. Patients infected with the novel coronavirus (COVID-19) have symptoms of fever, hypodynamia, dry cough, gradual dyspnea and the like in the early stage. Has strong infectivity. Can cause major public health safety incidents.
At present, a plurality of countries are inoculated with the novel coronavirus vaccine in a large scale in the global scope, and the detection of the antibody generation condition after the vaccine inoculation has great significance for preventing the novel coronavirus infection of people, and the vaccine should be reseeded for the people who are injected with the vaccine and can not generate the antibody, thereby being further beneficial to preventing the infection. However, the conventional test reagent strip is generally structured as shown in fig. 1, and it usually has only one sample application hole on the front side of the release liner, and the release liner is sprayed with a fluorescent-labeled antibody, and the T-line of the NC membrane is coated with a corresponding antigen, when a blood sample is added for chromatographic test, the fluorescent-labeled antibody is easy to contact the antigen on the T-line in advance under the pushing action of chromatography, in this case, even if the blood sample contains a positive antibody, a faint fluorescent strip is easy to be displayed on the T-line, and the test shows a false negative result, which makes the test accuracy not high.
Disclosure of Invention
In view of the above technical problems in the prior art, the present invention aims to provide a detection kit for quantitatively detecting a new coronavirus antibody in human blood and a detection method thereof.
The detection kit for quantitatively detecting the new coronavirus antibody in the human blood is characterized by comprising an immunochromatography reagent strip, wherein the immunochromatography reagent strip comprises a bottom lining, an NC (numerical control) membrane, a first sample pad, a second sample pad, a release pad and absorbent paper; the sample pad II, the release pad, the sample pad I, the NC film and the absorbent paper are sequentially overlapped and assembled on the bottom liner from left to right, the sample pad I and the absorbent paper are respectively overlapped at the left end and the right end of the NC film, a detection area is formed on the surface of the NC film, and a detection line T line close to the sample pad I and a control line C line close to the absorbent paper are arranged on the NC film; the right end of the release pad is laminated on the left end of the first sample pad, and the second sample pad is laminated on the left end of the release pad;
coupling rabbit IgG to a carboxyl quantum dot microsphere to obtain QM-rabbit IgG; coupling the RBD antibody to a carboxyl quantum dot microsphere to obtain a QM-RBD antibody;
and mixing the QM-RBD antibody and the QM-rabbit IgG, spraying the mixture on a release pad of an immunochromatography reagent strip, coating a recombinant RBD antigen on an NC membrane of a T line area, and coating a goat anti-rabbit secondary antibody GAR on an NC membrane of a C line area.
The method for quantitatively detecting the new coronavirus antibody in the human blood by using the detection kit is characterized by comprising the following operation steps of:
1) making a standard curve: preparing a series of standard working solutions with different concentrations by using a pH buffer solution by using a monoclonal antibody of the recombinant new coronavirus RBD as a standard substance, wherein a blank reference solution is the pH buffer solution; respectively spotting the immunochromatography reagent strips of the detection kit, dripping standard working solution or blank control solution onto a sample pad I of the immunochromatography reagent strip, then dripping pH buffer solution onto a sample pad II to serve as chromatography pushing solution, measuring a fluorescence value of a T line by using a dry fluorescence chromatography analyzer after chromatography for a period of time, drawing a standard curve by taking an average fluorescence value as a vertical coordinate and taking the concentration of a standard substance as a horizontal coordinate, and performing linear regression to obtain a linear equation;
2) detecting a blood sample: dripping a blood sample on a sample pad I of the immunochromatography reagent strip, then dripping a pH buffer solution on a sample pad II to serve as a chromatography pushing solution, measuring a T-line fluorescence value by using a dry fluorescence chromatography analyzer after chromatography is carried out for a period of time, and substituting the measured T-line fluorescence value into the linear equation in the step 1), so that the content of the new coronavirus antibody in the blood sample can be calculated.
The method for quantitatively detecting the new coronavirus antibody in the human blood is characterized in that in the step 1) or the step 2), the pH buffer solution is PBS buffer solution with pH =7.4 or MES buffer solution with pH = 6.
The method for quantitatively detecting the new coronavirus antibody in the human blood is characterized in that in the step 1), 20-40 ul, preferably 30ul of standard working solution or blank control solution is dripped into a first sample pad of an immunochromatography reagent strip; the volume of the pH buffer solution dropped on the second sample pad is 80-100 ul, preferably 90 ul; and (3) measuring the T-line fluorescence value by using a dry fluorescence chromatographic analyzer after the chromatography time is 12-20 min, preferably 15 min.
The method for quantitatively detecting the new coronavirus antibody in the human blood is characterized in that in the step 2), the sample volume of a blood sample is 20-40 ul, and preferably 30 ul; the volume of the pH buffer solution dropped on the second sample pad is 80-100 ul, preferably 90 ul; and (3) measuring the T-line fluorescence value by using a dry fluorescence chromatographic analyzer after the chromatography time is 12-20 min, preferably 15 min.
The method for quantitatively detecting the new coronavirus antibody in the human blood is characterized in that in the step 2), the blood sample is whole blood, plasma or serum; when the blood sample is whole blood or plasma, the blood sample is diluted to 3 to 5 times the original volume with a pH buffer solution and then used for measurement.
The detection kit for quantitatively detecting the new coronavirus antibody in the human blood is characterized in that the carboxyl quantum dot microspheres are quantum dot fluorescent microspheres with carboxyl functional groups on the surfaces, and the preparation process of the QM-rabbit IgG or QM-RBD antibody is as follows: and activating by using an EDC/NHS cross-linking agent, covalently cross-linking the rabbit IgG or RBD antibody to the quantum dot fluorescent microsphere, and blocking redundant spark groups by using BSA and glycine to obtain the quantum dot fluorescence-labeled rabbit IgG or RBD antibody.
The detection kit for quantitatively detecting the new coronavirus antibody in the human blood is characterized in that the specific preparation steps of the QM-rabbit IgG or QM-RBD antibody are as follows:
adjusting an S1 microsphere buffer system: mixing and diluting MES buffer solution with the concentration of 20mmol/L, pH =6 and carboxyl quantum dot microsphere solution with the concentration of 1 mu mol/L according to the volume ratio of 1:1 to obtain a microsphere buffer system;
s2 microsphere activation: quickly and uniformly mixing EDC-HCl solution with the concentration of 20mg/ml and NHS solution with the concentration of 20mg/ml according to the volume ratio of 1:1, reacting for 15min at 37 ℃, centrifuging for 20min at 8000g, discarding the supernatant, and resuspending the precipitate by using a microsphere buffer system prepared in the step S1;
s3 antibody-coupled microspheres: step S2, immediately adding rabbit IgG or RBD antibody after microsphere activation, rapidly mixing uniformly, and reacting for 1h at 37 ℃;
s4 wash and block: adding 20 mu L of sealing agent, mixing uniformly, reacting for 0.5h at 37 ℃, centrifuging for 15min at 8000g, discarding the supernatant, and suspending and dispersing the precipitate by using buffer solution to finish the coupling.
Compared with the prior art, the invention has the following beneficial effects:
1. according to the invention, the detection sensitivity is improved through the quantum dot fluorescent marker, the left end and the right end of the release pad of the immunochromatography reagent strip are respectively overlapped with the second sample pad and the first sample pad, the blood sample is firstly dripped on the first sample pad, so that the blood sample firstly chromatographs a certain region, then the PBS buffer solution is immediately dripped on the second sample pad to serve as a chromatography pushing solution, and the positive antibody in the blood sample can be combined with the recombinant RBD antigen coated on the T line of the NC membrane before the RBD antibody of the quantum dot fluorescent marker, so that the detection accuracy is improved.
2. The detection principle of the invention is based on the principle of a competition method, and the positive antibody in the blood sample and the RBD antibody which is sprayed on the release pad and is fluorescently marked by the quantum dots compete for being combined with the recombinant RBD antigen coated on the T line of the NC membrane. When the improved reagent strip is used for detecting an antibody negative blood sample, the binding effect of the quantum dot fluorescence-labeled RBD antibody and the recombinant RBD antigen is strongest, and the T-line fluorescence value is highest; when the amount of the positive antibody in the blood sample is proper, under the competitive action of the positive antibody in the blood sample, the binding action of the RBD antibody of the quantum dot fluorescent marker and the recombinant RBD antigen is weakened, the T-line fluorescence value is weakened to a certain extent, and the content of the positive antibody in the blood sample can be quantitatively detected through a standard curve drawn by the method; and when the quantity of the positive antibodies in the blood sample is large, the positive antibodies in the blood sample are combined with the recombinant RBD antigen coated on the T line of the NC membrane before the RBD antibodies fluorescently marked by the quantum dots, the combination effect is strong, the recombinant RBD antigen is sealed, the fluorescence of the T line disappears at the moment, and the blood sample can only be qualitatively determined to be positive.
Drawings
FIG. 1 is a schematic structural diagram of a conventional test strip;
FIG. 2 is a schematic view of the testing process of the improved reagent strip of the present invention;
FIG. 3 is a schematic diagram of the test results of the improved reagent strip of the present invention when used for testing antibody-negative serum;
FIG. 4 is a schematic diagram of the detection result of the improved reagent strip of the present invention for detecting antibody-positive serum.
Detailed Description
The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention.
Example 1 preparation of QM-RBD antibody and QM-Rabbit IgG
Adjusting an S1 microsphere buffer system: 0.2ml of MES buffer solution with the concentration of 20mmol/L, pH =6 is mixed and diluted with 0.2ml of carboxyl quantum dot microsphere solution with the concentration of 1 mu mol/L to obtain a microsphere buffer system. The volume of the microsphere buffer system was 0.4mL, the final concentration of the carboxyl quantum dot microspheres was 0.5 μmol/L, and the final buffer system was 10mmol/L, pH =6 MES.
S2 microsphere activation: 4ul of 20mg/mL EDC-HCl (CAS: 25952-53-8) and 4ul of 20mg/mL NHSS (CAS: 6066-82-6) are quickly mixed, reacted at 37 ℃ for 15min, 8000g of the mixture is centrifuged for 20min, the supernatant is discarded, the precipitate is resuspended to 0.2mL volume by using a 0.4mL microsphere buffer system prepared in the step S1 (the microsphere concentration is recovered to about 1 mu mol/L), and the mixture is mixed;
s3 antibody-coupled microspheres: step S2, immediately adding 0.08mg of antibody to be labeled after microsphere activation, quickly mixing uniformly, and reacting for 1h at 37 ℃;
s4 wash and block: adding 20 mu L of sealing agent, mixing uniformly, reacting for 0.5h at 37 ℃, centrifuging for 15min at 8000g, discarding the supernatant, and suspending and dispersing the precipitate by using buffer solution to finish the coupling.
In step S2, the molar ratio of EDC, NHS and microspheres is about 2000:3000: 1.
In the step S4, the sealant formulation: 10mg BSA was dissolved in 1mL 100mM glycine solution. The buffer in step S4 may be a simple buffer such as PB, or may contain a stabilizer such as BSA, but the ionic strength is preferably low to facilitate colloidal stabilization.
According to the above preparation process, when the antibody to be labeled added in step S3 is rabbit IgG or RBD antibody, quantum dot fluorescence-labeled rabbit IgG or quantum dot fluorescence-labeled RBD antibody, respectively labeled as QM-rabbit IgG or QM-RBD antibody, is prepared.
Example 2: preparation of detection kit
1) Mixing the quantum dot fluorescence-labeled RBD antibody prepared in the embodiment 1 and QM-rabbit IgG, spraying the mixture on a release pad of a kit, and drying at 37 ℃ for 12h for later use;
2) respectively diluting the recombined RBD antigen and the goat anti-rabbit secondary antibody GAR into 0.2 mg/mL by using coating diluent (150mM PB, pH 7.4), respectively and uniformly spraying and scribing on an NC membrane detection line area (marked as a T line area) and a control line area (marked as a C line area) by using the amount of membrane liquid of 30 mu L/30cm, drying at 37 ℃ for 12h, and sealing for later use.
Preparing a detection reagent strip by adopting the prepared release pad and an NC membrane coating a recombinant RBD antigen on the T line area and a goat anti-rabbit anti-GAR antibody on the C line area:
1) preparation of a conventional reagent strip (structure shown in FIG. 1):
the reagent strip comprises a bottom lining, an NC film, a sample pad, a release pad and absorbent paper. The sample pad, the release pad, the NC film and the absorbent paper are assembled on the bottom liner in an overlapping mode, the release pad and the absorbent paper are respectively overlapped at the left end and the right end of the NC film, the sample pad is overlapped at the left end of the release pad, the test paper board is formed after assembly, and the test paper board is cut into strips with the width of 3.78 mm. In the comparison of fig. 1, the T line and the C line are arranged on the NC film at intervals from left to right, the T line is close to the release pad, and the C line is close to the absorbent paper.
Referring to fig. 1, when the conventional reagent strip prepared as described above is assembled in a kit, there is a sample application hole through which a blood sample is dropped onto a sample pad for detection, and the blood sample is diluted with PBS buffer.
2) The preparation of the improved reagent strip of the invention comprises the following steps:
the reagent strip comprises a bottom lining, an NC film, a first sample pad, a second sample pad, a release pad and absorbent paper. The sample pad II, the release pad, the sample pad I, the NC film and the absorbent paper are sequentially overlapped and assembled on the bottom liner from left to right, the sample pad I and the absorbent paper are respectively overlapped at the left end and the right end of the NC film, a detection area is formed on the surface of the NC film, and a detection line T line close to the sample pad I and a control line C line close to the absorbent paper are arranged on the NC film; the right end of the release pad is overlapped on the left end of the first sample pad, and the second sample pad is overlapped on the left end of the release pad. After assembly, a test paper board is formed and cut into strips with the width of 3.78 mm. The T line and the C line are arranged on the NC film at left and right intervals, the T line is close to the sample pad I, and the C line is close to the absorbent paper.
When the improved reagent strip is assembled in a kit, the improved reagent strip is provided with two sample adding holes, a blood sample is added to one drop of a sample pad through the sample adding holes for detection, and PBS buffer solution is added to the other drop of the sample pad through the sample adding holes to serve as a chromatographic pushing solution. The structure of the improved reagent strip is schematically shown in figure 2.
When the improved reagent strip is used for detecting antibody negative serum, a schematic diagram of the detection result is shown in FIG. 3. As can be seen from FIG. 3, the quantum dot fluorescently labeled RBD antibody was bound to the T-line of the reagent strip, and a fluorescent band was visualized on the T-line.
When the improved reagent strip is used for detecting antibody positive serum, a schematic diagram of a detection result is shown in FIG. 4. The positive antibody in the serum is combined on the T line of the reagent strip, and the quantum dot fluorescence labeled RBD antibody is finally chromatographed on absorbent paper.
Example 3:
method for quantitatively detecting novel coronavirus antibody in human blood when improved reagent strip prepared based on example 2 is assembled in kit
1) Standard curve: the new coronavirus antibody RBD is taken as a standard substance, and PBS with pH =7.4 is used for preparing standard working solutions with the concentrations of 50, 100, 200, 400 and 800 ng/ml respectively, and a blank is PBS buffer with pH = 7.4. Respectively spotting the reagent strips, adding 30ul of standard working solution to one drop of the sample pad through the sample adding hole, then immediately adding 90ul of PBS buffer solution to the second drop of the sample pad through the sample adding hole to serve as chromatography pushing solution, displaying a fluorescence strip on a T line after about 15min of chromatography, and measuring the fluorescence value of the T line by using a dry fluorescence chromatography analyzer; setting 5 repeats for each concentration, drawing a standard curve by taking the average fluorescence value as a vertical coordinate and the concentration as a horizontal coordinate, and performing linear regression to obtain a linear equation;
2) detecting a blood sample: diluting human blood to 2.5 times of the original volume by PBS buffer solution with pH =7.4, then dripping 30ul of PBS buffer solution on the first sample pad through the first sample adding hole, then dripping 90ul of PBS buffer solution on the second sample pad through the second sample adding hole as chromatography propelling liquid, measuring the T-line fluorescence value by a dry fluorescence chromatography analyzer after chromatography for 15min, and substituting the measured T-line fluorescence value into the linear equation in the step 1) to calculate the content of the new coronavirus antibody in the blood sample. The detection is carried out in Hangzhou areas, 20 healthy people who are not injected with the novel coronavirus vaccine and 20 healthy people who are injected with the novel coronavirus vaccine generated by the three-needle Anhui Zhifei technology company Limited are detected by the kit developed by the technical scheme of the invention, and when the blood of 20 people who are not injected with the vaccine is detected, the fluorescence value on the T line is very strong, and the results are negative. The blood of 20 persons injected with the vaccine is detected, positive antibodies are basically detected, when the positive antibodies are detected, the fluorescence value on the T line is obviously weakened, the content of the antibodies in the blood can be presumed according to a standard curve drawn by the method, and the result accuracy is high.
Method for detecting antibodies to the novel coronavirus in human blood when the conventional reagent strip prepared in example 2 was assembled in a kit:
1) detection and qualification: taking a new coronavirus antibody RBD as a standard substance, preparing a working solution with the concentration of 100 ng/ml by PBS (phosphate buffer solution) with the pH =7.4, spotting a conventional reagent strip, dripping 90ul of the working solution onto a sample pad through a sample adding hole, and displaying a fluorescence strip on a T line after chromatography for about 3 min;
2) detecting a blood sample: diluting human blood with PBS (pH = 7.4) buffer solution to 5 times of original volume, adding 90ul dropwise to the sample pad through the sample adding hole, and performing chromatography for 3min, and then determining the fluorescence value of T line with a dry fluorescence chromatography analyzer. We detect in Hangzhou region, 20 healthy people who have not injected the novel coronavirus vaccine and 20 healthy people's blood who have injected the novel coronavirus vaccine that three-needle Anhui Zhifei technology limited company produced in the whole course are got, detect with the kit that conventional reagent strip equipment as shown in figure 1, when 20 people's blood detection of not injecting the vaccine, the fluorescence value on the T line is very strong, and the result is negative. The blood test of 20 persons injected with vaccine can basically detect only weak positive results (because the T-line fluorescence cannot be well disappeared even if the blood sample contains relatively high concentration of antibody in the conventional scheme).
According to the detection result, compared with the detection method of the conventional reagent strip, the chromatography speed of the detection method of the improved reagent strip is obviously reduced, because the detection method of the invention firstly drops the blood sample on the first sample pad to ensure that the blood sample firstly chromatographs a certain region, and then immediately drops the PBS buffer solution on the second sample pad as the chromatography pushing solution, when the chromatography pushing solution pushes the quantum dot fluorescence labeling RBD antibody to advance to the NC membrane through the chromatography region of the blood sample, the blood sample on the first sample pad has certain chromatography resistance to the passing chromatography pushing solution.
Claims (8)
1. A detection kit for quantitatively detecting a new coronavirus antibody in human blood is characterized by comprising an immunochromatography reagent strip, wherein the immunochromatography reagent strip comprises a bottom lining, an NC membrane, a first sample pad, a second sample pad, a release pad and absorbent paper; the sample pad II, the release pad, the sample pad I, the NC film and the absorbent paper are sequentially overlapped and assembled on the bottom liner from left to right, the sample pad I and the absorbent paper are respectively overlapped at the left end and the right end of the NC film, a detection area is formed on the surface of the NC film, and a detection line T line close to the sample pad I and a control line C line close to the absorbent paper are arranged on the NC film; the right end of the release pad is laminated on the left end of the first sample pad, and the second sample pad is laminated on the left end of the release pad;
coupling rabbit IgG to a carboxyl quantum dot microsphere to obtain QM-rabbit IgG; coupling the RBD antibody to a carboxyl quantum dot microsphere to obtain a QM-RBD antibody;
and mixing the QM-RBD antibody and the QM-rabbit IgG, spraying the mixture on a release pad of an immunochromatography reagent strip, coating a recombinant RBD antigen on an NC membrane of a T line area, and coating a goat anti-rabbit secondary antibody GAR on an NC membrane of a C line area.
2. A method for quantitatively detecting the antibody of the neocoronavirus in the human blood based on the detection kit of claim 1, which is characterized by comprising the following operation steps:
1) making a standard curve: preparing a series of standard working solutions with different concentrations by using a pH buffer solution by using a monoclonal antibody of the recombinant new coronavirus RBD as a standard substance, wherein a blank reference solution is the pH buffer solution; respectively spotting the immunochromatography reagent strips of the detection kit, dripping standard working solution or blank control solution onto a sample pad I of the immunochromatography reagent strip, then dripping pH buffer solution onto a sample pad II to serve as chromatography pushing solution, measuring a fluorescence value of a T line by using a dry fluorescence chromatography analyzer after chromatography for a period of time, drawing a standard curve by taking an average fluorescence value as a vertical coordinate and taking the concentration of a standard substance as a horizontal coordinate, and performing linear regression to obtain a linear equation;
2) detecting a blood sample: dripping a blood sample on a sample pad I of the immunochromatography reagent strip, then dripping a pH buffer solution on a sample pad II to serve as a chromatography pushing solution, measuring a T-line fluorescence value by using a dry fluorescence chromatography analyzer after chromatography is carried out for a period of time, and substituting the measured T-line fluorescence value into the linear equation in the step 1), so that the content of the new coronavirus antibody in the blood sample can be calculated.
3. The method for quantitatively detecting the antibody against the novel coronavirus in the human blood as set forth in claim 2, wherein the pH buffer is PBS buffer of pH =7.4 or MES buffer of pH =6 in step 1) or step 2).
4. The method according to claim 2, wherein in step 1), 20-40 ul, preferably 30ul of the standard working solution or the blank control solution is dropped onto the first sample pad of the immunochromatographic reagent strip; the volume of the pH buffer solution dropped on the second sample pad is 80-100 ul, preferably 90 ul; and (3) measuring the T-line fluorescence value by using a dry fluorescence chromatographic analyzer after the chromatography time is 12-20 min, preferably 15 min.
5. The method for quantitatively detecting the antibodies to the neocoronavirus in the human blood according to claim 2, wherein in the step 2), the sample volume of the blood sample is 20-40 ul, preferably 30 ul; the volume of the pH buffer solution dropped on the second sample pad is 80-100 ul, preferably 90 ul; and (3) measuring the T-line fluorescence value by using a dry fluorescence chromatographic analyzer after the chromatography time is 12-20 min, preferably 15 min.
6. The method for quantitatively detecting antibodies to the novel coronavirus in human blood according to claim 2, wherein in the step 2), the blood sample is whole blood, plasma or serum; when the blood sample is whole blood or plasma, the blood sample is diluted to 3 to 5 times the original volume with a pH buffer solution and then used for measurement.
7. The detection kit for quantitative determination of antibodies to neocoronaviruses in human blood as claimed in claim 1, wherein said carboxyl-based quantum dot microspheres are quantum dot fluorescent microspheres with carboxyl functional groups on the surface, and said QM-rabbit IgG or QM-RBD antibodies are prepared by the steps of: and activating by using an EDC/NHS cross-linking agent, covalently cross-linking the rabbit IgG or RBD antibody to the quantum dot fluorescent microsphere, and blocking redundant spark groups by using BSA and glycine to obtain the quantum dot fluorescence-labeled rabbit IgG or RBD antibody.
8. The kit of claim 7, wherein the QM-rabbit IgG or QM-RBD antibody is prepared by the following steps:
adjusting an S1 microsphere buffer system: mixing and diluting MES buffer solution with the concentration of 20mmol/L, pH =6 and carboxyl quantum dot microsphere solution with the concentration of 1 mu mol/L according to the volume ratio of 1:1 to obtain a microsphere buffer system;
s2 microsphere activation: quickly and uniformly mixing EDC-HCl solution with the concentration of 20mg/ml and NHS solution with the concentration of 20mg/ml according to the volume ratio of 1:1, reacting for 15min at 37 ℃, centrifuging for 20min at 8000g, discarding the supernatant, and resuspending the precipitate by using a microsphere buffer system prepared in the step S1;
s3 antibody-coupled microspheres: step S2, immediately adding rabbit IgG or RBD antibody after microsphere activation, rapidly mixing uniformly, and reacting for 1h at 37 ℃;
s4 wash and block: adding 20 mu L of sealing agent, mixing uniformly, reacting for 0.5h at 37 ℃, centrifuging for 15min at 8000g, discarding the supernatant, and suspending and dispersing the precipitate by using buffer solution to finish the coupling.
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