CN113030469A - Novel coronavirus detection method - Google Patents

Abstract

The invention relates to a novel coronavirus detection method, which comprises the steps of replacing an antibody stock buffer solution; preparing magnetic beads; labeling biotin with an antibody; configuring a standard product; performing on-machine analysis; the detection method has the advantages that the virus characteristic protein with the extremely low abundance value can be detected in a short time, 96 samples can be analyzed within one hour (the flux can reach 288 if a full-automatic HD-1 machine type is adopted), and the samples are automatically loaded and processed in a closed instrument during the on-machine analysis, so that the infection risk caused by the contact of testers and the detected samples is greatly reduced. The method can not only carry out qualitative detection, but also carry out accurate quantitative detection, can improve the detection sensitivity of the novel coronavirus infection to the greatest extent, and can also ensure the reliability of the detection result.

Description

Novel coronavirus detection method

Technical Field

The invention belongs to the technical field of molecular biology detection, and particularly relates to a novel coronavirus detection method.

Background

Since the discovery and confirmation of a new coronavirus (hereinafter referred to as "new crown") in 11 months in 2019, the impact of the virus on global epidemic has been unattractive. By 2021, hundreds of millions of cases of the disease are diagnosed, and the detection, prevention and control situation is still a huge test for the global epidemic prevention and control business. At present, the false negative rate of nucleic acid detection which is the most important detection index is too high, a certain number of asymptomatic patients (virus carriers) exist, and the condition of missed detection is frequent, so that the virus spreads and epidemic situation develops. Therefore, establishing a new detection technology is significant for reducing the false negative rate of detection. Meanwhile, when a targeted new coronavirus vaccine is developed, a virus detection technology with higher sensitivity is developed, and virus infection carrying risks can be determined earlier and faster, so that the flow propagation path of people is cut off, and the carrying prevention and control effects of the new coronary pneumonia are exactly prevented and screened.

At present, the false negative rate of the new coronavirus detected by the fluorescence quantitative PCR detection method reaches 40%, on one hand, because the sampling standardization of the detection material is not enough, for example, when the virus is not spread to the lower respiratory tract, the inaccuracy of the detection result is reduced by adopting the sampling method of taking the oral swab, and the rejection of the subject is caused by the pain or discomfort of taking the nasal swab; on the other hand, the detection limit is also lowered due to the loss of target fragments with nucleic acid extraction and PCR. While current antibody-based detection methods (e.g., colloidal gold) are only qualitative and not quantitative, infected individuals may not produce enough antibody due to immune system inactivity or tolerance, resulting in false negatives; and the colloidal gold has a certain false positive rate.

In the traditional ELISA reaction, the antigen-antibody reaction system is usually 100 μ l, and a large amount of fluorescence signals are generated and then diluted in a reaction environment of 100 μ l, so that the greatest problem at present is that the detection sensitivity is not high, and the highest detection sensitivity is usually in picogram level (pg/ml).

In response to the above problems, the inventors placed targets on the antigen for detecting the new corona virus, i.e. the new corona virus itself, enhancing the specificity and sensitivity of the detection method. The method is developed by referring to the existing Single Molecule immune array (SimoA) technical platform, is simple to operate, has extremely strong openness and flexibility, and can detect the antigen to be detected in a sample by only needing an antibody pair capable of specifically recognizing a certain antigen and an antigen standard to be detected and configuring a standard curve, wherein the sample can be serum, plasma, cerebrospinal fluid, urine, saliva, cell lysate and the like. The technology uses the principle of traditional ELISA to form a double-antibody sandwich immune complex, and ensures the specificity of the reaction. During operation, one of the antibody pairs is coated on an antibody binding site of a magnetic bead with the diameter of 2.7 mu m according to a certain coating condition, the other antibody pair is labeled by biotin and then added for incubation, and after the incubation is finished, avidin coupled enzyme and enzyme reaction substrate are added to form an immune complex added into the reaction chip. Each chip is provided with 238000 pores, the diameter of each pore is 4.2 μm, and only one magnetic bead can fall into the pore, and after the magnetic bead falls into the pore based on the action of gravity, the system can push a layer of oil on the surface of the chip, on one hand, redundant magnetic beads are removed, and most importantly, fluorescence signals are locked in the pores (signal diffusion and cross reaction are not easy to occur). Under the condition, fluorescent molecules generated by the enzyme molecules catalyzing the substrate are locked in the small-hole reaction system, magnetic beads containing target antigens emit fluorescence due to the enzyme, the ratio of the number of the magnetic beads containing the fluorescence to the number of all the magnetic beads falling on the surface of the whole chip is in positive correlation with the concentration of the target antigens in the sample, and the computer can accurately calculate the concentration of the target antigens in the sample to be detected.

Disclosure of Invention

The invention provides a novel coronavirus detection method, which comprises the following steps: (1) replacing the antibody storage buffer solution with a magnetic bead connection buffer solution; (2) preparing magnetic beads; (3) labeling biotin with an antibody; (4) configuring a standard product; (5) and (4) performing computer analysis.

The step of replacing the antibody storage buffer solution with the magnetic bead connection buffer solution is as follows:

1) the concentrations of MM05 antibody and R001 antibody were measured with a spectrophotometer, and the volume required for each antibody was calculated from the amount of 100. mu.g of the coated antibody and the measured antibody concentration;

2) respectively adding MM05 antibody and R001 antibody with required volumes into a filter, respectively adding magnetic bead connection buffer solution to 550 mu l of the buffer solution, placing the filter into a centrifuge, centrifuging for 3-8min, discarding the bottom liquid, repeating the washing steps twice, adding 500 mu l of magnetic bead connection buffer solution of 400 mu l of the buffer solution each time, inversely placing the filter into a clean centrifuge tube, centrifuging for 1-3min, and collecting the liquid;

3) then adding 45-55 mul of magnetic bead connection buffer solution to wash the filter membrane for 4-8 times, inverting the filter in the step 2) in a centrifuge tube, centrifuging for 1-3min, and collecting liquid;

4) a150. mu.l volume of the prepared R001 antibody and a 300. mu.l volume of the MM05 antibody were stored on ice until use.

Preferably, the step of replacing the antibody stock buffer solution (1) with a magnetic bead ligation buffer solution comprises:

1) the concentrations of MM05 antibody and R001 antibody were measured with a spectrophotometer, and the volume required for each antibody was calculated from the amount of 100. mu.g of the coated antibody and the measured antibody concentration;

2) respectively putting MM05 antibody and R001 antibody with required volumes into a filter, respectively adding magnetic bead connection buffer solution to 500 mu l, putting the filter into a centrifuge, centrifuging for 5min, discarding bottom liquid, repeating the washing steps twice, adding 450 mu l of magnetic bead connection buffer solution each time, inversely putting the filter into a clean centrifuge tube, centrifuging for 2min, and collecting liquid;

3) then adding 50 mu L of magnetic bead connecting buffer solution to wash the filter membrane for 6 times, inverting the filter in the step 2) in a centrifuge tube, centrifuging for 2min, and collecting liquid;

4) a150. mu.l volume of the prepared R001 antibody and a 300. mu.l volume of the MM05 antibody were stored on ice until use.

The preparation steps of the magnetic beads (2) are as follows:

1) washing of magnetic beads: taking 0.13ml of the displaced magnetic bead stock solution which is vibrated for more than 30s, quickly centrifuging, and sucking and removing supernatant; adding 250 and 350 mu L of magnetic bead cleaning buffer solution, carrying out vortex oscillation for 3-8s, carrying out rapid centrifugation, sucking and discarding supernatant, repeating the magnetic bead washing step twice, and sucking and discarding supernatant to obtain the magnetic beads washed by the buffer solution;

2) conversion of magnetic beads: adding 250-;

3) adding pre-cooled 1ml of magnetic bead connection buffer solution into a vial containing 10mg of EDC, vortexing for 5-10s, taking 9 mul of the magnetic beads after being added with the buffer solution for replacement, vortexing and mixing for 8-12s, mixing and incubating the centrifuge tube at 2-8 ℃ for 25-35min, rapidly centrifuging, sucking off the supernatant, respectively adding 300 mul of the replaced antibody MM05 solution and 150 mul of the replaced antibody R001 solution, vortexing and shaking for 8-12s, mixing and incubating the centrifuge tube at 2-8 ℃ for 1.5-2.5h, rapidly centrifuging, sucking off the supernatant, adding 300 mul of magnetic bead cleaning buffer solution, vortexing and shaking for 3-8s, rapidly centrifuging, sucking off the supernatant, repeating the magnetic bead cleaning step again, adding 300 mul of magnetic bead sealing buffer solution, vortexing and shaking for 3-8s, and incubating at room temperature for 40-50min, and (3) quickly centrifuging, removing supernatant, adding 300 mu l of magnetic bead cleaning buffer solution, carrying out vortex oscillation for 3-8s, quickly centrifuging, removing supernatant, adding 300 mu l of magnetic bead diluent, carrying out vortex oscillation for 3-8s, quickly centrifuging to obtain magnetic bead Beads-MM05 solution coupled with the antibody, and storing at 3-5 ℃.

Preferably, the preparation step of the magnetic beads (2) is:

1) washing of magnetic beads: taking 0.13ml of the displaced magnetic bead stock solution which is vibrated for more than 30s, quickly centrifuging, and sucking and removing supernatant; adding 300 mul of magnetic bead cleaning buffer solution, carrying out vortex oscillation for 5s, carrying out rapid centrifugation, sucking and discarding supernatant, repeating the magnetic bead cleaning step twice, and sucking and discarding supernatant to obtain magnetic beads washed by the buffer solution;

2) conversion of magnetic beads: adding 300 mu l of magnetic bead connection buffer solution into the magnetic beads washed by the buffer solution, carrying out vortex oscillation for 5s, carrying out rapid centrifugation, absorbing and discarding supernatant, repeating the magnetic bead replacement step twice, carrying out rapid centrifugation, absorbing and discarding supernatant, adding 291 mu l of magnetic bead connection buffer solution, carrying out vortex oscillation for 5s, carrying out rapid centrifugation, and placing on ice to obtain the magnetic beads replaced by the buffer solution;

3) adding precooled 1ml of magnetic bead connection buffer solution into a vial containing 10mg of EDC, vortexing for 8s, taking 9 mul of the magnetic bead to be added into the magnetic bead replaced by the buffer solution, vortexing and mixing for 10s, incubating the centrifuge tube at 5 ℃ for 30min, quickly centrifuging, discarding supernatant, respectively adding 300 mul of the replaced antibody MM05 solution and 150 mul of the replaced antibody R001 solution, vortexing and shaking for 10s, incubating the centrifuge tube at 5 ℃ for 2h, quickly centrifuging, discarding supernatant, adding 300 mul of magnetic bead cleaning buffer solution, vortexing and shaking for 5s, rapidly centrifuging, discarding supernatant, repeating the magnetic bead washing step again, adding 300 mul of magnetic bead sealing buffer solution, vortexing and shaking for 5s, incubating at room temperature for 45min, quickly centrifuging, discarding supernatant, adding 300 mul of magnetic bead cleaning buffer solution, vortexing and shaking for 5s, quickly centrifuging, discarding supernatant, adding 300 mul of magnetic bead diluent, carrying out vortex oscillation for 5s, and carrying out rapid centrifugation to obtain a magnetic bead Beads-MM05 solution coupled with the antibody, and storing at 4 ℃.

The (3) labeling biotin by the antibody comprises the following steps:

1) adding the antibody R001 with the required volume into a centrifugal filter, then adding biotinylation reaction buffer solution to 500 mu l, centrifuging for 3-8min, discarding the bottom liquid, repeating the washing step twice, adding 400-500 mu l biotinylation reaction buffer solution for washing each time, inverting the filter, putting into a clean centrifugal tube, centrifuging for 1-3min, and collecting the antibody;

2) washing the filter membrane with 50 μ l of biotinylation reaction buffer solution for 5-8 times, inverting the filter in a centrifuge tube, centrifuging for 1-3min, and collecting antibody-Biotin conjugate R001-Biotin solution;

3) the antibody concentration and volume after displacement were measured.

Preferably, the step (3) of labeling biotin with the antibody comprises the following steps:

1) adding the antibody R001 with the required volume into a centrifugal filter, then adding biotinylation reaction buffer solution to 500 mu L, centrifuging for 5min, discarding the bottom liquid, repeating the washing step twice, adding 450 mu L of biotinylation reaction buffer solution for washing each time, then placing the filter in a clean centrifugal tube in an inverted manner, centrifuging for 2min, and collecting the antibody;

2) then washing the filter membrane for 6 times by using 50 mu l of biotinylation reaction buffer solution, inverting the filter in a centrifuge tube, centrifuging for 2min, and collecting an antibody-Biotin conjugate R001-Biotin solution;

3) the antibody concentration and volume after displacement were measured.

The step of (4) configuring the standard substance is as follows:

1) taking the replaced antibody freeze-dried powder, adding 400ul of nucleic-free water for dissolving, and preserving at-80 ℃;

2) the standard curve dilution gradient is: 0, 0.1pg/ml, 1pg/ml, 10pg/ml, 100pg/ml, 1000 pg/ml.

The step of (5) computer analysis comprises the following steps:

1) taking a special 96-pore plate for the on-machine, respectively adding 100ul of standard curve solution or a sample to be tested, repeating for several times, adding Beads-MM 0525 ul and R001-Biotin 20ul in each pore site, incubating for 35min at 30 ℃ on a microplate shaker for 15s, then putting the 96-pore plate on a plate washer to start a washing program of a 2-step method, washing away unbound antigens or antibodies, adding 100ul of SBG in each pore site, putting the mixture on the microplate shaker for 30 ℃ and incubating for 5min for 15s, then putting the mixture on the plate washer to continuously start the next established washing operation, washing away excessive unbound enzymes, and after the program operation of the plate washer is finished, putting the plate for 10min to be dried, namely the on-machine;

2) opening SR-X Analyzer, opening 'Run' menu key of main interface, entering flat setting interface, setting operation parameters: firstly naming the program, then respectively carrying out operations of selecting hole sites, selecting a kit matching program, naming and setting dilution times on corresponding hole sites according to the hole sites of the sample adding holes, clicking a 'Use Layout' key to confirm the setting of a flat plate, adding all reagent consumables such as compact discs and 96-hole plates, emptying a waste box, and clicking a 'Start' key to operate the program;

3) after the operation is finished, the instrument generates a data report.

The MM05 antibody is a capture antibody, and the R001 antibody is a detection antibody.

The detection method of the invention is a novel coronavirus detection method which is not the purpose of diagnosis.

The invention has the following advantages:

1. the invention can detect the characteristic protein of the virus with extremely low abundance value in extremely short time, can analyze 96 samples (the flux can reach 288 if adopting a full-automatic HD-1 machine type) in one hour, and the sample on-machine analysis can automatically sample and process in a closed instrument, thereby greatly reducing the infection risk caused by the contact of testers and the detected samples.

2. The detection method has high sensitivity, ensures extremely low sensitivity, can ensure the reliability of a detection result, has detection capability greatly higher than that of a fluorescence PCR nucleic acid detection kit, and can not only carry out qualitative detection but also carry out accurate quantitative detection compared with an antibody detection kit (colloidal gold method).

3. The invention takes MM05 as a capture antibody and R001 as a detection antibody, and the technical effect of the development of the SiMoA computer program by adopting a 2-step method is good. The lower limit of detection (LOD) determined on the machine reaches 0.0552pg/ml, the lower limit of quantification (LLOQ) reaches 0.1940pg/ml, the range of quantification is 0.194-853pg/ml, and at the lower limit of quantification, the signal-to-noise value is 2.03.

4. The invention adopts the test of the incorporation/recovery rate, the result of the incorporation/recovery rate of manual 4-fold dilution after plasma incorporation is ideal, the average plasma incorporation/recovery rate reaches 113%, and the result is between 80% and 120% of an acceptable range.

5. After optimization by adding Helper beads, the invention finds that the detection sensitivity can be improved to the maximum extent by adding 70% of Helper beads, the lower limit of detection is reduced from 0.055pg/ml to 0.0157pg/ml, the lower limit of quantification is reduced from 0.194pg/ml to 0.068pg/ml, the range of quantification is 0.068-384pg/ml, the signal-to-noise value is 2.24 at the lower limit of quantification, the sensitivity is extremely high, the false negative rate can be obviously lowered, and the detection kit is applicable to various detection samples, and is suitable for qualitatively detecting N protein of 2019-nCOV virus in pneumonia cases, suspected aggregated suspected patients and the like infected by novel coronavirus or samples of blood, nasopharyngeal swabs, sputum and the like of other cases in vitro.

Drawings

FIG. 1 is a schematic diagram of the "sandwich method" principle of ELISA antigen-antibody reaction;

fig. 2 is an enlarged view of the SiMoA reaction disc and a schematic diagram of enzymatic reaction [ note: A. a bottom schematic view of the optical disc; B. local microscopic magnification of the main reaction area unit of the optical disc; C. the enlarged view of a scanning electron microscope of a small hole in the reaction area unit; D. the antigen-antibody reaction in the section diagram of the small hole in the reaction area unit and the condition that the sealing oil pushes the magnetic bead to fall into the hole are schematically shown; E. the sealing oil seals the small hole in which the magnetic bead falls and removes the redundant magnetic bead schematic diagram on the surface of the micro-hole area (the hole site of the magnetic bead combined with the antigen to be detected shows fluorescence, and the hole sites without the antigen to be detected and the magnetic bead do not emit fluorescence); F. the binding schematic diagram of the magnetic beads and the antigen antibody; G. parts of diagram F ];

FIG. 3HB1 standard curve;

FIG. 4HB2 standard curve;

FIG. 5HB3 standard curve;

FIG. 6HB4 standard curve.

Detailed Description

MM05 was used as the capture antibody and R001 as the detection antibody.

Example 1

1. Replacing the storage buffer solution with a magnetic bead connection buffer solution:

1.1 measuring the concentrations of MM05 antibody and R001 antibody, respectively, with an ultramicro spectrophotometer, calculating the volume required for each antibody based on the amount of 100. mu.g required for coating the antibody and the measured antibody concentration;

1.2 respectively putting MM05 antibody and R001 antibody with required volumes into a filter, respectively adding magnetic bead connection buffer solution to 500 mu l, putting the filter into a centrifuge, centrifuging for 5min, discarding bottom liquid, repeating the washing steps twice, adding 450 mu l of magnetic bead connection buffer solution each time, inversely putting the filter into a clean centrifugal tube, centrifuging for 2min, and collecting liquid;

1.3, adding 50 mul of magnetic bead connecting buffer solution to wash the filter membrane for 6 times, inverting the filter in the step 2) in a centrifuge tube, centrifuging for 2min, and collecting liquid;

1.4A volume of 150. mu.l of the prepared R001 antibody and a volume of 300. mu.l of the MM05 antibody were stored on ice until use.

2. Preparation of magnetic beads:

2.1 taking 0.13ml of the magnetic bead stock solution after the replacement and the shaking for more than 30s, quickly centrifuging, and sucking and removing supernatant; adding 300 mul of magnetic bead cleaning buffer solution, carrying out vortex oscillation for 5s, carrying out rapid centrifugation, sucking and discarding supernatant, repeating the magnetic bead cleaning step twice, and sucking and discarding supernatant to obtain magnetic beads washed by the buffer solution;

2.2 adding 300 mul of magnetic bead connection buffer solution into the magnetic beads washed by the buffer solution, carrying out vortex oscillation for 5s, carrying out rapid centrifugation, absorbing and discarding supernatant, repeating the magnetic bead replacement step twice, carrying out rapid centrifugation, absorbing and discarding supernatant, adding 291 mul of magnetic bead connection buffer solution, carrying out vortex oscillation for 5s, carrying out rapid centrifugation, and placing on ice to obtain the magnetic beads replaced by the buffer solution;

2.3 Add 1ml of precooled magnetic bead connection buffer into a vial containing 10mg EDC, vortex for 8s, add 9. mu.l of the buffer-exchanged magnetic beads, vortex and shake for 10s, mix and incubate the centrifuge tube at 5 ℃ for 30min, centrifuge rapidly, discard the supernatant, add 300. mu.l of the exchanged antibody MM05 solution and 150. mu.l of the exchanged antibody R001 solution, vortex and shake for 10s, mix and incubate the centrifuge tube at 5 ℃ for 2h, centrifuge rapidly, discard the supernatant, add 300. mu.l of magnetic bead washing buffer, vortex and shake for 5s, centrifuge rapidly, discard the supernatant, repeat the magnetic bead washing step again, add 300. mu.l of magnetic bead blocking buffer, vortex and shake for 5s, centrifuge at room temperature for 45min, centrifuge rapidly, discard the supernatant, add 300. mu.l of magnetic bead washing buffer, vortex and shake for 5s, centrifuge rapidly, discard the supernatant, adding 300 mul of magnetic bead diluent, carrying out vortex oscillation for 5s, and carrying out rapid centrifugation to obtain a magnetic bead Beads-MM05 solution coupled with the antibody, and storing at 4 ℃.

3. Antibody-labeled biotin:

adding the antibody R001 with the required volume into a centrifugal filter, then adding biotinylation reaction buffer solution to 500 mu l, centrifuging for 5min, discarding the bottom liquid, repeating the washing step twice, adding 450 mu l biotinylation reaction buffer solution for washing each time, then placing the filter in a clean centrifugal tube in an inverted manner, centrifuging for 2min, and collecting the antibody; the filter was washed 6 times with 50. mu.l of biotinylation reaction buffer. Inverting the filter in a centrifuge tube, centrifuging for 2min, and collecting an antibody-Biotin conjugate R001-Biotin solution; the antibody concentration and volume after displacement were measured.

4. Configuration of standards

Taking the replaced antibody freeze-dried powder, adding 400ul of nucleic-free water for dissolving, and preserving at-80 ℃; the standard curve dilution gradient is: 0, 0.1pg/ml, 1pg/ml, 10pg/ml, 100pg/ml, 1000 pg/ml.

5. Analysis on computer

Taking a special 96-pore plate for an on-computer, respectively adding 100ul of standard curve solution or a sample to be detected, repeating for several times, wherein several holes need to be repeated, adding Beads-MM 0525 ul and R001-Biotin 20ul into each hole, incubating for 35min15s at 30 ℃ on a microplate shaker, then putting the 96-pore plate on a plate washer, starting a washing program of a 2-step method, washing away unbound antigens or antibodies, adding 100ul of SBG into each hole, incubating for 5min15s at 30 ℃ on the microplate shaker, then putting the plate washer on the next step of established washing operation, washing away excessive unbound enzymes, after the plate washer program is operated, putting the plate for 10min to be dried, opening SR-X Analyzer, opening a 'Run' menu key of a main interface, entering the plate setting interface, and setting operation parameters: firstly naming the program, then respectively carrying out operations of selecting hole sites, selecting a kit matching program, naming and setting dilution times on corresponding hole sites according to the hole sites of the sample adding holes, clicking a 'Use Layout' key to confirm the setting of a flat plate, adding all reagent consumables such as compact discs and 96-hole plates according to the operations, emptying a waste box, clicking a 'Start' key to operate the program, and copying data and reports generated by an instrument after the operation is finished.

Example 2

1. Replacing antibody stock buffer with magnetic bead connection buffer:

1.1 measuring the concentrations of MM05 antibody and R001 antibody, respectively, with an ultramicro spectrophotometer, calculating the volume required for each antibody based on the amount of 100. mu.g required for coating the antibody and the measured antibody concentration;

1.2 respectively putting MM05 antibody and R001 antibody with required volumes into a filter, respectively adding magnetic bead connection buffer solution to 450 mu l, putting the filter into a centrifuge, centrifuging for 3min, discarding bottom liquid, repeating the washing steps twice, adding 400 mu l of magnetic bead connection buffer solution each time, inversely putting the filter into a clean centrifugal tube, centrifuging for 1min, and collecting liquid;

1.3, adding 45 mul of magnetic bead connecting buffer solution to wash the filter membrane for 4 times, inverting the filter in the step 2) in a centrifuge tube, centrifuging for 1min, and collecting liquid;

1.4A volume of 150. mu.l of the prepared R001 antibody and a volume of 300. mu.l of the MM05 antibody were stored on ice until use.

2. Preparation of magnetic beads:

2.1 taking 0.13ml of the magnetic bead stock solution after the replacement and the shaking for more than 30s, quickly centrifuging, and sucking and removing supernatant; adding 250 mu l of magnetic bead cleaning buffer solution, carrying out vortex oscillation for 3s, carrying out rapid centrifugation, sucking and discarding supernatant, repeating the magnetic bead washing step twice, and sucking and discarding supernatant to obtain magnetic beads washed by buffer solution;

2.2 adding 250 mul of magnetic bead connection buffer solution into the magnetic beads washed by the buffer solution, carrying out vortex oscillation for 3s, carrying out rapid centrifugation, absorbing and discarding supernatant, repeating the magnetic bead replacement step twice, carrying out rapid centrifugation, absorbing and discarding supernatant, adding 291 mul of magnetic bead connection buffer solution, carrying out vortex oscillation for 3s, carrying out rapid centrifugation, and placing on ice to obtain the magnetic beads replaced by the buffer solution;

2.3 Add 1ml of precooled magnetic bead ligation buffer into a vial containing 10mg EDC, vortex for 5s, add 9. mu.l of the buffer-exchanged magnetic beads, vortex and shake for 8s, incubate the centrifuge tube at 2 ℃ for 25min, centrifuge rapidly, discard the supernatant, add 300. mu.l of the exchanged antibody MM05 solution and 150. mu.l of the exchanged antibody R001 solution, vortex and shake for 8s, incubate the centrifuge tube at 2 ℃ for 1.5h, centrifuge rapidly, discard the supernatant, add 300. mu.l of magnetic bead washing buffer, vortex and shake for 3s, centrifuge rapidly, discard the supernatant, repeat the magnetic bead washing step again, add 300. mu.l of magnetic bead blocking buffer, vortex and shake for 3s, incubate at room temperature for 40min, centrifuge rapidly, discard the supernatant, add 300. mu.l of magnetic bead washing buffer, vortex and shake for 3s, centrifuge rapidly, discard the supernatant, add 300. mu.l of magnetic bead diluent, vortexing for 3s, and rapidly centrifuging to obtain antibody-coupled magnetic bead Beads-MM05 solution, and storing at 3 deg.C.

3. Antibody-labeled biotin:

adding the antibody R001 with the required volume into a centrifugal filter, then adding biotinylation reaction buffer solution to 500 mu L, centrifuging for 3min, discarding the bottom liquid, repeating the washing step twice, adding 400 mu L of biotinylation reaction buffer solution for washing each time, then placing the filter in a clean centrifugal tube in an inverted manner, centrifuging for 1min, and collecting the antibody; then washing the filter membrane for 5 times by using 50 mul of biotinylation reaction buffer solution, inverting the filter in a centrifuge tube, centrifuging for 1min, and collecting an antibody-Biotin conjugate R001-Biotin solution; the antibody concentration and volume after displacement were measured.

4. Configuration of standards

Taking the replaced antibody freeze-dried powder, adding 400ul of nucleic-free water for dissolving, and preserving at-80 ℃; the standard curve dilution gradient is: 0, 0.1pg/ml, 1pg/ml, 10pg/ml, 100pg/ml, 1000 pg/ml.

5. Analysis on computer

Taking a special 96-pore plate for an on-computer, respectively adding 100ul of standard curve solution or a sample to be detected, repeating for several times, wherein several holes need to be repeated, adding Beads-MM 0525 ul and R001-Biotin 20ul into each hole, incubating for 35min15s at 30 ℃ on a microplate shaker, then putting the 96-pore plate on a plate washer, starting a washing program of a 2-step method, washing away unbound antigens or antibodies, adding 100ul of SBG into each hole, incubating for 5min15s at 30 ℃ on the microplate shaker, then putting the plate washer on the next step of established washing operation, washing away excessive unbound enzymes, after the plate washer program is operated, putting the plate for 10min to be dried, opening SR-X Analyzer, opening a 'Run' menu key of a main interface, entering the plate setting interface, and setting operation parameters: firstly naming the program, then respectively carrying out operations of selecting hole sites, selecting a kit matching program, naming and setting dilution times on corresponding hole sites according to the hole sites of the sample adding holes, clicking a 'Use Layout' key to confirm the setting of a flat plate, adding all reagent consumables such as compact discs and 96-hole plates according to the operations, emptying a waste box, clicking a 'Start' key to operate the program, and copying data and reports generated by an instrument after the operation is finished.

Example 3

1. Replacing antibody stock buffer with magnetic bead connection buffer:

1.1 measuring the concentrations of MM05 antibody and R001 antibody, respectively, with an ultramicro spectrophotometer, calculating the volume required for each antibody based on the amount of 100. mu.g required for coating the antibody and the measured antibody concentration;

1.2 respectively putting MM05 antibody and R001 antibody with required volumes into a filter, respectively adding magnetic bead connection buffer solution to 550 mu l, putting the filter into a centrifuge, centrifuging for 8min, discarding bottom liquid, repeating the washing steps twice, adding 500 mu l of magnetic bead connection buffer solution each time, inversely putting the filter into a clean centrifugal tube, centrifuging for 3min, and collecting liquid;

1.3, adding 55 mul of magnetic bead connecting buffer solution to wash the filter membrane for 4-8 times, inverting the filter in the step 2) in a centrifuge tube, centrifuging for 3min, and collecting liquid;

1.4A volume of 150. mu.l of the prepared R001 antibody and a volume of 300. mu.l of the MM05 antibody were stored on ice until use.

2. Preparation of magnetic beads:

2.1 taking 0.13ml of the magnetic bead stock solution after the replacement and the shaking for more than 30s, quickly centrifuging, and sucking and removing supernatant; adding 350 mul of magnetic bead cleaning buffer solution, carrying out vortex oscillation for 8s, carrying out rapid centrifugation, sucking and discarding supernatant, repeating the magnetic bead cleaning step twice, and sucking and discarding supernatant to obtain magnetic beads washed by buffer solution;

2.2 adding 350. mu.l of magnetic bead connection buffer solution into the magnetic beads washed by the buffer solution, carrying out vortex oscillation for 8s, carrying out rapid centrifugation, sucking and discarding the supernatant, repeating the magnetic bead replacement step twice, carrying out rapid centrifugation, sucking and discarding the supernatant, adding 291. mu.l of magnetic bead connection buffer solution, carrying out vortex oscillation for 8s, carrying out rapid centrifugation, and placing on ice to obtain the magnetic beads replaced by the buffer solution;

2.3 Add 1ml of precooled magnetic bead ligation buffer into a vial containing 10mg EDC, vortex for 10s, add 9. mu.l of the buffer-exchanged magnetic beads, vortex and shake for 12s, mix and incubate the centrifuge tube at 2-8 ℃ for 35min, centrifuge rapidly, discard the supernatant, add 300. mu.l of the exchanged antibody MM05 solution and 150. mu.l of the exchanged antibody R001 solution, vortex and shake for 12s, mix and incubate the centrifuge tube at 8 ℃ for 2.5h, centrifuge rapidly, discard the supernatant, add 300. mu.l of magnetic bead washing buffer, vortex and shake for 8s, centrifuge rapidly, discard the supernatant, repeat the magnetic bead washing step again, add 300. mu.l of magnetic bead blocking buffer, vortex and shake for 8s, incubate at room temperature for 50min, centrifuge rapidly, discard the supernatant, add 300. mu.l of magnetic bead washing buffer, vortex and shake for 8s, centrifuge rapidly, discard the supernatant, adding 300 mul of magnetic bead diluent, carrying out vortex oscillation for 8s, and carrying out rapid centrifugation to obtain a magnetic bead Beads-MM05 solution coupled with the antibody, and storing at 5 ℃.

3. Antibody-labeled biotin:

adding the antibody R001 with the required volume into a centrifugal filter, then adding biotinylation reaction buffer solution to 500 mu l, centrifuging for 8min, discarding the bottom liquid, repeating the washing step twice, adding 500 mu l biotinylation reaction buffer solution for washing each time, then placing the filter in a clean centrifugal tube in an inverted manner, centrifuging for 3min, and collecting the antibody; the filter was washed 8 times with 50. mu.l of biotinylation reaction buffer. Inverting the filter in a centrifuge tube, centrifuging for 3min, and collecting an antibody-Biotin conjugate R001-Biotin solution; the antibody concentration and volume after displacement were measured.

4. Configuration of standards

Taking the replaced antibody freeze-dried powder, adding 400ul of nucleic-free water for dissolving, and preserving at-80 ℃; the standard curve dilution gradient is: 0, 0.1pg/ml, 1pg/ml, 10pg/ml, 100pg/ml, 1000 pg/ml.

5. Analysis on computer

Taking a special 96-pore plate for an on-computer, respectively adding a standard curve solution or 100ul of a sample to be detected, repeating for several times, wherein several holes need to be repeated, adding Beads-MM 0525 ul and R001-Biotin 20ul into each hole, incubating for 35min at 30 ℃ on a microplate shaker for 15s, then putting the 96-pore plate on a plate washer, starting a washing program of a 2-step method, washing away unbound antigens or antibodies, adding 100ul SBG into each hole, putting the hole washer on the microplate shaker for 30 ℃ and incubating for 5min15s, then putting the hole washer on a next set washing operation, washing away excessive unbound enzymes, after the plate washer program is finished, putting the plate for 10min to be dried, opening SR-X Analyzer, opening a 'Run' menu key of a main interface, entering the plate setting interface, and setting operation parameters: firstly naming the program, then respectively carrying out operations of selecting hole sites, selecting a kit matching program, naming and setting dilution times on corresponding hole sites according to the hole sites of the sample adding holes, clicking a 'Use Layout' key to confirm the setting of a flat plate, adding all reagent consumables such as compact discs and 96-hole plates according to the operations, emptying a waste box, clicking a 'Start' key to operate the program, and copying data and reports generated by an instrument after the operation is finished.

Example 4

1. Replacing antibody stock buffer with magnetic bead connection buffer:

1.1 measuring the concentrations of MM05 antibody and R001 antibody, respectively, with an ultramicro spectrophotometer, calculating the volume required for each antibody based on the amount of 100. mu.g required for coating the antibody and the measured antibody concentration;

1.2 respectively putting MM05 antibody and R001 antibody with required volumes into a filter, respectively adding magnetic bead connection buffer solution to 480 mu l, putting the filter into a centrifuge, centrifuging for 6min, discarding bottom liquid, repeating the washing steps twice, adding 420 mu l of magnetic bead connection buffer solution each time, inversely putting the filter into a clean centrifuge tube, centrifuging for 2min, and collecting liquid;

1.3, adding 48 mul of magnetic bead connecting buffer solution to wash the filter membrane for 5 times, inverting the filter in the step 2) in a centrifuge tube, centrifuging for 2min, and collecting liquid;

1.4A volume of 150. mu.l of the prepared R001 antibody and a volume of 300. mu.l of the MM05 antibody were stored on ice until use.

2. Preparation of magnetic beads:

2.1 taking 0.13ml of the magnetic bead stock solution after the replacement and the shaking for more than 30s, quickly centrifuging, and sucking and removing supernatant; adding 280 mul of magnetic bead cleaning buffer solution, carrying out vortex oscillation for 6s, carrying out rapid centrifugation, sucking and discarding supernatant, repeating the magnetic bead cleaning step twice, and sucking and discarding supernatant to obtain magnetic beads washed by the buffer solution;

2.2 adding 280 mul of magnetic bead connection buffer solution into the magnetic beads washed by the buffer solution, carrying out vortex oscillation for 4s, carrying out rapid centrifugation, absorbing and discarding supernatant, repeating the magnetic bead replacement step twice, carrying out rapid centrifugation, absorbing and discarding supernatant, adding 291 mul of magnetic bead connection buffer solution, carrying out vortex oscillation for 4s, carrying out rapid centrifugation, and placing on ice to obtain the magnetic beads after buffer solution replacement;

2.3 Add 1ml of precooled magnetic bead ligation buffer into a vial containing 10mg EDC, vortex for 6s, add 9. mu.l of the buffer-exchanged magnetic beads, vortex and shake for 9s, mix and incubate the centrifuge tube at 4 ℃ for 28min, centrifuge rapidly, discard the supernatant, add 300. mu.l of the exchanged antibody MM05 solution and 150. mu.l of the exchanged antibody R001 solution, vortex and shake for 9s, mix and incubate the centrifuge tube at 4 ℃ for 1.8h, centrifuge rapidly, discard the supernatant, add 300. mu.l of magnetic bead washing buffer, vortex and shake for 4s, centrifuge rapidly, discard the supernatant, repeat the magnetic bead washing step again, add 300. mu.l of magnetic bead blocking buffer, vortex and shake for 4s, incubate at room temperature for 48min, centrifuge rapidly, discard the supernatant, add 300. mu.l of magnetic bead washing buffer, vortex and shake for 4s, centrifuge rapidly, discard the supernatant, adding 300 mul of magnetic bead diluent, whirling and oscillating for 4s, and quickly centrifuging to obtain magnetic bead Beads-MM05 solution coupled with the antibody, and storing at 4 ℃.

3. Antibody-labeled biotin:

adding the antibody R001 with the required volume into a centrifugal filter, then adding biotinylation reaction buffer solution to 500 mu L, centrifuging for 4min, discarding the bottom liquid, repeating the washing step twice, adding 420 mu L of biotinylation reaction buffer solution for washing each time, then placing the filter in a clean centrifugal tube in an inverted manner, centrifuging for 2min, and collecting the antibody; the filter was washed 7 times with 50. mu.l of biotinylation reaction buffer. Inverting the filter in a centrifuge tube, centrifuging for 2min, and collecting an antibody-Biotin conjugate R001-Biotin solution; the antibody concentration and volume after displacement were measured.

4. Configuration of standards

Taking the replaced antibody freeze-dried powder, adding 400ul of nucleic-free water for dissolving, and preserving at-80 ℃; the standard curve dilution gradient is: 0, 0.1pg/ml, 1pg/ml, 10pg/ml, 100pg/ml, 1000 pg/ml.

5. Analysis on computer

Taking a special 96-pore plate for an on-computer, respectively adding a standard curve solution or 100ul of a sample to be detected, repeating for several times, wherein several holes need to be repeated, adding Beads-MM 0525 ul and R001-Biotin 20ul into each hole, incubating for 35min at 30 ℃ on a microplate shaker for 15s, then putting the 96-pore plate on a plate washer, starting a washing program of a 2-step method, washing away unbound antigens or antibodies, adding 100ul SBG into each hole, putting the hole washer on the microplate shaker for 30 ℃ and incubating for 5min15s, then putting the hole washer on a next set washing operation, washing away excessive unbound enzymes, after the plate washer program is finished, putting the plate for 10min to be dried, opening SR-X Analyzer, opening a 'Run' menu key of a main interface, entering the plate setting interface, and setting operation parameters: firstly naming the program, then respectively carrying out operations of selecting hole sites, selecting a kit matching program, naming and setting dilution times on corresponding hole sites according to the hole sites of the sample adding holes, clicking a 'Use Layout' key to confirm the setting of a flat plate, adding all reagent consumables such as compact discs and 96-hole plates according to the operations, emptying a waste box, clicking a 'Start' key to operate the program, and copying data and reports generated by an instrument after the operation is finished.

Example 5

1. Replacing antibody stock buffer with magnetic bead connection buffer:

1.1 measuring the concentrations of MM05 antibody and R001 antibody, respectively, with an ultramicro spectrophotometer, calculating the volume required for each antibody based on the amount of 100. mu.g required for coating the antibody and the measured antibody concentration;

1.2 respectively putting MM05 antibody and R001 antibody with required volumes into a filter, respectively adding magnetic bead connection buffer solution to 520 mu l, putting the filter into a centrifuge, centrifuging for 7min, discarding bottom liquid, repeating the washing steps twice, adding 480 mu l of magnetic bead connection buffer solution each time, inversely putting the filter into a clean centrifugal tube, centrifuging for 1min, and collecting liquid;

1.3, adding 52 mul of magnetic bead connecting buffer solution to wash the filter membrane for 4-8 times, inverting the filter in the step 2) in a centrifuge tube, centrifuging for 3min, and collecting liquid;

1.4A volume of 150. mu.l of the prepared R001 antibody and a volume of 300. mu.l of the MM05 antibody were stored on ice until use.

2. Preparation of magnetic beads:

2.1 taking 0.13ml of the magnetic bead stock solution after the replacement and the shaking for more than 30s, quickly centrifuging, and sucking and removing supernatant; adding 290 mu l of magnetic bead cleaning buffer solution, carrying out vortex oscillation for 7s, carrying out rapid centrifugation, sucking and discarding supernatant, repeating the magnetic bead washing step twice, and sucking and discarding supernatant to obtain magnetic beads washed by buffer solution;

2.2 adding 260. mu.l of magnetic bead connection buffer solution into the magnetic beads washed by the buffer solution, carrying out vortex oscillation for 4s, carrying out rapid centrifugation, sucking and discarding supernatant, repeating the magnetic bead replacement step twice, carrying out rapid centrifugation, sucking and discarding supernatant, adding 291. mu.l of magnetic bead connection buffer solution, carrying out vortex oscillation for 7s, carrying out rapid centrifugation, and placing on ice to obtain the magnetic beads after buffer solution replacement;

2.3 Add 1ml of precooled magnetic bead ligation buffer into a vial containing 10mg EDC, vortex for 9s, add 9. mu.l of the buffer-exchanged magnetic beads, vortex and shake for 11s, mix and incubate the centrifuge tube at 7 ℃ for 34min, centrifuge rapidly, discard the supernatant, add 300. mu.l of the exchanged antibody MM05 solution and 150. mu.l of the exchanged antibody R001 solution, vortex and shake for 9s, mix and incubate the centrifuge tube at 7 ℃ for 2.3h, centrifuge rapidly, discard the supernatant, add 300. mu.l of magnetic bead washing buffer, vortex and shake for 7s, centrifuge rapidly, discard the supernatant, repeat the magnetic bead washing step again, add 300. mu.l of magnetic bead blocking buffer, vortex and shake for 4s, incubate for 43min at room temperature, centrifuge rapidly, discard the supernatant, add 300. mu.l of magnetic bead washing buffer, vortex and shake for 5s, centrifuge rapidly, discard the supernatant, add 300. mu.l of magnetic bead diluent, vortexing for 5s, and rapidly centrifuging to obtain antibody-coupled magnetic bead Beads-MM05 solution, and storing at 4 deg.C.

3. Antibody-labeled biotin:

adding the antibody R001 with the required volume into a centrifugal filter, then adding biotinylation reaction buffer solution to 500 mu L, centrifuging for 7min, discarding the bottom liquid, repeating the washing step twice, adding 490 mu L of biotinylation reaction buffer solution for washing each time, then placing the filter in a clean centrifugal tube in an inverted manner, centrifuging for 1min, and collecting the antibody; the filter was washed 6 times with 50. mu.l of biotinylation reaction buffer. Inverting the filter in a centrifuge tube, centrifuging for 3min, and collecting an antibody-Biotin conjugate R001-Biotin solution; the antibody concentration and volume after displacement were measured.

4. Configuration of standards

Taking the replaced antibody freeze-dried powder, adding 400ul of nucleic-free water for dissolving, and preserving at-80 ℃; the standard curve dilution gradient is: 0, 0.1pg/ml, 1pg/ml, 10pg/ml, 100pg/ml, 1000 pg/ml.

5. Analysis on computer

Taking a special 96-pore plate for an on-computer, respectively adding 100ul of standard curve solution or a sample to be detected, repeating for several times, wherein several holes need to be repeated, adding Beads-MM 0525 ul and R001-Biotin 20ul into each hole, incubating for 35min15s at 30 ℃ on a microplate shaker, then putting the 96-pore plate on a plate washer, starting a washing program of a 2-step method, washing away unbound antigens or antibodies, adding 100ul of SBG into each hole, incubating for 5min15s at 30 ℃ on the microplate shaker, then putting the plate washer on the next step of established washing operation, washing away excessive unbound enzymes, after the plate washer program is operated, putting the plate for 10min to be dried, opening SR-X Analyzer, opening a 'Run' menu key of a main interface, entering the plate setting interface, and setting operation parameters: firstly naming the program, then respectively carrying out operations of selecting hole sites, selecting a kit matching program, naming and setting dilution times on corresponding hole sites according to the hole sites of the sample adding holes, clicking a 'Use Layout' key to confirm the setting of a flat plate, adding all reagent consumables such as compact discs and 96-hole plates according to the operations, emptying a waste box, clicking a 'Start' key to operate the program, and copying data and reports generated by an instrument after the operation is finished.

Experimental example: to demonstrate the scientificity and rationality of the present invention, the following experimental studies of methodology were carried out

1. The invention relies on the technology and the detection principle

1.1 relying on the technology

Developed based on the existing Single Molecule immune array (SimoA) technology platform. The SiMoA is based on Enzyme Linked immunosorbent assay (ELISA), and uses a microwell (as shown in fig. 2B) chip arranged in an array to capture and detect an antigen with an extremely low concentration in combination with a micro magnetic bead, thereby achieving a detection effect close to that of directly detecting a single protein molecule. The chips are integrated on specially designed optical discs (such as A diagram in FIG. 2), each optical disc is provided with 24 chips/channel, each channel is used for detecting one sample, and a plurality of marker molecules can be simultaneously detected by one reaction.

1.2 principle of detection

Magnetic beads with the diameter of 2.7 microns are used as carriers, about 250000 binding sites are distributed on the surface of each magnetic bead, capture antibodies can be loaded on the binding sites to capture antigens to be detected, specific detection antibodies are combined with the antibodies to be detected (a sandwich method, as shown in figure 1), biotin labels and avidin coupled enzymes are combined on the detection antibodies, and substrates in a reaction solution can be decomposed to generate fluorescence. Therefore, the fluorophore generated by the enzyme is confined in the reaction pore (diameter 4.2 μm, volume 50fl) with very small volume (as shown in figure 2, panel C), and the unit fluorescence intensity of the fluorescent molecule is more prominent due to the much smaller reaction area than that of the conventional one, so that the enzyme-labeled molecule with very low concentration can be detected.

After the reaction is finished, the photographing system irradiates the chip reaction area with fluorescence and white light respectively, and converts the optical image into a digital signal for interpretation: the number of the hole sites containing fluorescence is positively correlated with the proportion of the number of all the magnetic beads falling into the reaction hole area and the concentration of the antigen to be detected in the sample, and the distribution of the magnetic beads capturing one or more antigens in the array holes accords with Poisson distribution. Thus, the protein concentration is measured by the Average number of Enzyme-labeled magnetic beads (AEB), which is determined by the Average fluorescence intensity of the wells containing the beads in the array. The background intensity of the fluorescence photography is generally in the range of 0.005-0.01, near the bottom layer of poisson noise (also called "shot noise"), which can highlight the existence of fluorescence signal, so that the receiver can capture the fluorescence signal more easily, determine the existence of magnetic beads (as shown in E diagram in FIG. 2) and enzyme activity.

Disclosure of the invention

2.1 materials and reagents

2.2 instrumentation

2.3 confirmation of detection method

2.3.1 confirmation of Capture and detection antibodies and coating protocols

The present invention refers to an antibody coupled to a magnetic bead as a Capture antibody (Capture antibody) and a biotin-labeled antibody as a detection antibody (Detector antibody). In the preliminary experiment, two computer experimental materials were first prepared according to the steps of magnetic bead preparation and antibody-linked biotin, as shown in table 1:

TABLE 1 on-machine experiment Material Table

Connection scheme	Connecting magnetic beads	Linking biotin
			Connection scheme
1	R001	MM05
			Connection scheme 2	MM05	R001

The results of preliminary experiments show that: the MM05 protein is used as a capture antibody and coated on the surface of magnetic beads, the R001 protein is labeled by biotin and used as a detection antibody, and the detection limit and the quantification limit are lower when the kit quantifies the N protein level, so that the optimal coating scheme of the antibody pair is determined.

2.3.2 determination of the procedure for treating the test sample and the control

2.3.2.1MM05 antibody and R001 antibody stock buffer are replaced by magnetic bead connection buffer;

2.3.2.2 preparation of magnetic beads: comprises preparing two magnetic Beads, i.e. Beads-MM05 and Beads-R001;

2.3.2.3 antibody-labeled biotin: comprises the preparation of two Biotin connecting antibodies of R001-Biotin and MM 05-Biotin);

2.3.2.4 configuration of standards;

2.3.3 on-Board analysis

2.3.3.1 determination of on-machine test protocol

After the configuration of the standards, 2.3.2.2 and 2.3.2.3 were tested separately for two different coating protocols, using "2-Step" and "3-Step" for the on-machine test, see Table 2.

TABLE 2 "2-Step" and "3-Step" on-machine test charts

Name of computer program	Antibodies for linking magnetic beads	Antibodies for biotin attachment	Elution method
				HB1	R001	MM05	2-Step
HB2	R001	MM05	3-Step
				HB3	MM05	R001	2-Step
HB4	MM05	R001	3-Step

2.3.3.2 on-board analysis

Taking a special 96-well plate for an upper computer, respectively adding 100ul of standard curve solution or a sample to be detected, repeating each standard twice, adding coated magnetic beads and antibodies into each hole, incubating the well plate on a microplate shaker at 30 ℃ for 35min15s, then placing the 96-well plate on a plate washer, eluting according to a scheme shown in a table 2, washing away unbound antigens or antibodies, then adding 100ul of SBG into each hole, incubating the well plate on the microplate shaker at 30 ℃ for 5min15s, then placing the well plate on the plate washer, continuously starting the next set washing operation, washing away excessive unbound enzymes, placing the plate 10min for drying after the program operation of the plate washer is finished, opening SR-X Analyzer, opening a 'Run' key of a main interface, entering the plate setting interface, and setting operation parameters: firstly naming the program, then respectively carrying out operations of selecting hole sites, selecting a kit matching program, naming and setting dilution times on corresponding hole sites according to the hole sites of the sample adding holes, clicking a 'Use Layout' key to confirm the setting of a flat plate, adding all reagent consumables such as compact discs and 96-hole plates according to the operations, emptying a waste box, clicking a 'Start' key to operate the program, and copying data and reports generated by an instrument after the operation is finished. The standard curves, the standard point basic information, the curve matching coefficients and the detection capability parameters of the four schemes are respectively shown in the figures 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14.

TABLE 3HB1 Standard Point basic information Table

Note: the machine uses 6 standard spots of calibers A-F, and each spot is repeated twice, wherein, AEB (average number of enzyme labels per bead) refers to the average number of magnetic beads marked by luciferase (biotin) and is used for measuring and calculating the concentration value of protein, and AEB is determined by the average fluorescence intensity of wells containing magnetic beads in array wells.

TABLE 4HB 1 Curve matching coefficients Table

The data of the detection range obtained by the calculation of the built-in analysis program of the computer are shown in the following table:

TABLE 5HB 1 detection capability parameter Table

TABLE 6HB 2 Standard Point basic information Table

Note: the machine used 6 standard spots of calibers A-F, each spot was replicated twice, where the average number of luciferase (biotin) -labeled beads was used as the AEB (average number of enzyme label per bead) to measure the protein concentration, and the AEB was determined by the average fluorescence intensity of the wells containing the beads in the wells of the array.

TABLE 7 HB2 Curve matching coefficients Table

The data of the detection range obtained by the calculation of the built-in analysis program of the computer are shown in the following table:

TABLE 8 HB2 detectability parameter Table

TABLE 9 HB3 Standard Point basic information Table

Note: the machine used 6 standard spots of calibers A-F, each spot was replicated twice, where the average number of luciferase (biotin) -labeled beads was used as the AEB (average number of enzyme label per bead) to measure the protein concentration, and the AEB was determined by the average fluorescence intensity of the wells containing the beads in the wells of the array.

TABLE 10 HB3 Curve matching coefficients Table

The data of the detection range obtained by the calculation of the built-in analysis program of the computer are shown in the following table:

TABLE 11 HB3 detection capability parameter Table

TABLE 12 HB4 Standard Point basic information Table

Note: the machine used 6 standard spots of calibers A-F, each spot was replicated twice, where the average number of luciferase (biotin) -labeled beads was used as the AEB (average number of enzyme label per bead) to measure the protein concentration, and the AEB was determined by the average fluorescence intensity of the wells containing the beads in the wells of the array.

TABLE 13 HB4 Curve matching coefficients Table

The data of the detection range obtained by the calculation of the built-in analysis program of the computer are shown in the following table:

TABLE 14 HB4 detectability parameter Table

As a result: as can be seen from Table 11, the detection sensitivity was best in the on-machine assay, with a lower limit of detection (LOD) of 0.0552pg/ml, a lower limit of quantitation (LLOQ) of 0.1940pg/ml, a quantitation range of 0.194-853pg/ml, and a signal to noise value of 2.03 at the lower limit of quantitation.

In quantifying components in a sample, there is a key factor that affects the final assay result, even the occurrence of false positives, which are Matrix effects (Matrix effects). The matrix refers to the part of the sample except the target analyte, and because the components of the matrix can significantly interfere with the analysis process, the influence is called matrix effect, and the reduction of the matrix effect as much as possible is particularly important in an ELISA kit. Thus, this experiment used the "Spike/Recovery Assay" to examine whether the present technology is affected by matrix effects.

2.3.4 incorporation/recovery test

The test process is to incorporate the antigen to be tested with known concentration into the target sample with measured concentration, and the ratio of the concentration increase part to the incorporation concentration measured before and after incorporation is the recovery rate, and the recovery rate is presented in percentage. The closer the recovery rate is to 100%, the closer the experimental results are to the true values. The recovery (%) was calculated as:

test value (pg/ml)/expected value (pg/ml) × 100% ═ recovery%.

Optionally 6 samples of healthy volunteers, 3 sera (Serum) and 3 plasmas (Plasma) and 1 control of blank dilutions, were prepared for recovery measurements on pooled sera and Plasma with standard protein samples at both high and low concentrations, and the assay groupings and loading are shown in Table 15:

TABLE 15 incorporation/recovery design

The actual protein concentration after the addition of the sample was measured using the SimoA platform according to the dilution scheme of the above table, the test procedure was as in "2.3.3.2", and the incorporation/recovery rate of the present invention in each volunteer sample and at different dilution rates was obtained according to the theoretical value of the protein incorporated, and the results are shown in Table 16.

TABLE 16 incorporation/recovery experiments

Results of the incorporation/recovery experiments:

generally, a recovery of 80-120% of the sample is acceptable. The plasma doping/recovery rate obtained in the test of the invention is ideal, and the average value is 113%, which indicates that the influence of the blood matrix effect received by the detection kit is within an acceptable range.

2.3.4Homebrew Re-optimization

When the magnetic bead-Capture is added conventionally, the auxiliary magnetic beads (Helper beads) equipped by the SiMoA platform are added, so that the combination efficiency of the Capture antibody and the magnetic beads can be improved, and the sensitivity and the accuracy of the detection technology can be further improved.

The best protocol selected in 2.3.3.2, HB3, with the addition of unequal amounts of Helper beads, with the detection antibodies held constant, was designated as "HB-letter" for each run in the protocol, and the protocol is shown in Table 17:

TABLE 17 Re-optimization protocol for Homebrew

The optimization results of adding different amounts of Helper beads were tested according to the above scheme, the computer test method is the same as the "2.3.3.2" procedure, and the results are shown in Table 18.

TABLE 18 Reptimization results Table for Homebrew

Re-optimization result of Homebrew: as can be seen from table 18, HB4 having the highest detection sensitivity can improve the detection sensitivity of the present invention to the greatest extent when 70% Helper beads are added, and compared to table 11, the lower detection limit is reduced from 0.055pg/mL to 0.0157pg/mL, the lower quantification limit is reduced from 0.194pg/mL to 0.068pg/mL, the quantification range is 0.068-384pg/mL, and the signal-to-noise value at the lower quantification limit is 2.24, which has extremely high sensitivity, can significantly reduce the false negative rate, is applicable to various detection samples, and is suitable for in vitro auxiliary judgment of the N protein of 2019-nCOV virus in samples such as suspected cases of coronavirus infection, suspected aggregated disease cases, and the like, or in samples for detection of other cases such as blood, nasopharyngeal swab, sputum, and the like.

And (4) conclusion:

1. the invention takes MM05 as a capture antibody and R001 as a detection antibody, and the technical effect of the development of the SiMoA computer program by adopting a 2-step method is good. The lower limit of detection (LOD) determined on the machine reached 0.0552pg/ml, the lower limit of quantitation (LLOQ) reached 0.1940pg/ml, the range of quantitation was 0.194-853pg/ml, at the lower limit of quantitation, the value of signal to noise was 2.03.

2. The invention adopts the test of the doping/recovery rate, the doping/recovery rate result of manual 4-fold dilution after plasma doping is more ideal, the average plasma doping/recovery rate reaches 113%, and the result can be accepted within the range of 80% -120%.

3. After the technology is optimized by adding Helper beads, the detection sensitivity of the invention can be improved to the maximum extent by adding 70% of Helper beads, the lower limit of detection is reduced from 0.055pg/mL to 0.0157pg/mL, the lower limit of quantification is reduced from 0.194pg/mL to 0.068pg/mL, the range of quantification is 0.068-384pg/mL, the value of signal to noise is 2.24 at the lower limit of quantification, the sensitivity is extremely high, the false negative rate can be obviously lowered, and the detection kit is applicable to various detection samples, and is suitable for qualitatively detecting N protein of 2019-nCOV virus in pneumonia cases, suspected aggregated disease cases and the like infected by novel coronavirus or in blood, nasopharyngeal swab, sputum and other samples of other cases in vitro.

While the invention has been described in detail in the foregoing by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that certain changes and modifications may be made therein based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims (10)

1. A novel coronavirus detection method is characterized by comprising the following steps: (1) replacing the antibody storage buffer solution with a magnetic bead connection buffer solution; (2) preparing magnetic beads; (3) labeling biotin with an antibody; (4) configuring a standard product; (5) and (4) performing computer analysis.

2. The detection method according to claim 1, wherein the step of replacing the antibody stock buffer solution (1) with the magnetic bead ligation buffer solution comprises:

1) the concentrations of MM05 antibody and R001 antibody were measured with a spectrophotometer, and the volume required for each antibody was calculated from the amount of 100. mu.g of the coated antibody and the measured antibody concentration;

2) respectively adding MM05 antibody and R001 antibody with required volumes into a filter, respectively adding magnetic bead connection buffer solution to 550 mu l of the buffer solution, placing the filter into a centrifuge, centrifuging for 3-8min, discarding the bottom liquid, repeating the washing steps twice, adding 500 mu l of magnetic bead connection buffer solution of 400 mu l of the buffer solution each time, inversely placing the filter into a clean centrifuge tube, centrifuging for 1-3min, and collecting the liquid;

3) then adding 45-55 mul of magnetic bead connection buffer solution to wash the filter membrane for 4-8 times, inverting the filter in the step 2) in a centrifuge tube, centrifuging for 1-3min, and collecting liquid;

4) a150. mu.l volume of the prepared R001 antibody and a 300. mu.l volume of the MM05 antibody were stored on ice until use.

3. The detection method according to claim 2, wherein the step of replacing the antibody stock buffer with the magnetic bead ligation buffer in step (1) comprises:

1) the concentrations of MM05 antibody and R001 antibody were measured with a spectrophotometer, and the volume required for each antibody was calculated from the amount of 100. mu.g of the coated antibody and the measured antibody concentration;

2) respectively putting MM05 antibody and R001 antibody with required volumes into a filter, respectively adding magnetic bead connection buffer solution to 500 mu l, putting the filter into a centrifuge, centrifuging for 5min, discarding bottom liquid, repeating the washing steps twice, adding 450 mu l of magnetic bead connection buffer solution each time, inversely putting the filter into a clean centrifuge tube, centrifuging for 2min, and collecting liquid;

3) then adding 50 mul of magnetic bead connection buffer solution to wash the filter membrane for 6 times, inverting the filter in the step 2) in a centrifuge tube, centrifuging for 2min, and collecting liquid;

4) a150. mu.l volume of the prepared R001 antibody and a 300. mu.l volume of the MM05 antibody were stored on ice until use.

4. The detection method according to claim 1, wherein the preparation step of (2) the magnetic beads is:

1) washing of magnetic beads: taking 0.13ml of the displaced magnetic bead stock solution which is vibrated for more than 30s, quickly centrifuging, and sucking and removing supernatant; adding 250-;

2) conversion of magnetic beads: adding 250-;

3) adding pre-cooled 1ml of magnetic bead connection buffer solution into a vial containing 10mg of EDC, vortexing for 5-10s, taking 9 mul of the magnetic beads after being added with the buffer solution for replacement, vortexing and mixing for 8-12s, mixing and incubating the centrifuge tube at 2-8 ℃ for 25-35min, rapidly centrifuging, sucking off the supernatant, respectively adding 300 mul of the replaced antibody MM05 solution and 150 mul of the replaced antibody R001 solution, vortexing and shaking for 8-12s, mixing and incubating the centrifuge tube at 2-8 ℃ for 1.5-2.5h, rapidly centrifuging, sucking off the supernatant, adding 300 mul of magnetic bead cleaning buffer solution, vortexing and shaking for 3-8s, rapidly centrifuging, sucking off the supernatant, repeating the magnetic bead cleaning step again, adding 300 mul of magnetic bead sealing buffer solution, vortexing and shaking for 3-8s, incubating at room temperature for 40-50min, rapidly centrifuging, and (3) absorbing and removing the supernatant, adding 300 mu l of magnetic bead cleaning buffer solution, carrying out vortex oscillation for 3-8s, carrying out rapid centrifugation, absorbing and removing the supernatant, adding 300 mu l of magnetic bead diluent, carrying out vortex oscillation for 3-8s, carrying out rapid centrifugation to obtain magnetic bead Beads-MM05 solution coupled with the antibody, and storing at 3-5 ℃.

5. The detection method according to claim 4, wherein the preparation step of (2) the magnetic beads is:

1) washing of magnetic beads: taking 0.13ml of the displaced magnetic bead stock solution which is vibrated for more than 30s, quickly centrifuging, and sucking and removing supernatant; adding 300 mul of magnetic bead cleaning buffer solution, carrying out vortex oscillation for 5s, carrying out rapid centrifugation, sucking and discarding supernatant, repeating the magnetic bead cleaning step twice, and sucking and discarding supernatant to obtain magnetic beads washed by the buffer solution;

2) conversion of magnetic beads: adding 300 mu l of magnetic bead connection buffer solution into the magnetic beads washed by the buffer solution, carrying out vortex oscillation for 5s, carrying out rapid centrifugation, absorbing and discarding supernatant, repeating the magnetic bead replacement step twice, carrying out rapid centrifugation, absorbing and discarding supernatant, adding 291 mu l of magnetic bead connection buffer solution, carrying out vortex oscillation for 5s, carrying out rapid centrifugation, and placing on ice to obtain the magnetic beads replaced by the buffer solution;

3) adding precooled 1ml of magnetic bead connection buffer solution into a vial containing 10mg of EDC, vortexing for 8s, taking 9 mul of the magnetic bead to which the buffer solution is replaced, vortexing and mixing for 10s, mixing and incubating the centrifuge tube at 5 ℃ for 30min, quickly centrifuging, sucking off the supernatant, respectively adding 300 mul of the replaced antibody MM05 solution and 150 mul of the replaced antibody R001 solution, vortexing and oscillating for 10s, mixing and incubating the centrifuge tube at 5 ℃ for 2h, quickly centrifuging, sucking off the supernatant, adding 300 mul of magnetic bead cleaning buffer solution, vortexing and oscillating for 5s, quickly centrifuging, sucking off the supernatant, repeating the magnetic bead washing step again, adding 300 mul of magnetic bead sealing buffer solution, vortexing and oscillating for 5s, incubating at room temperature for 45min, quickly centrifuging, sucking off the supernatant, adding 300 mul of magnetic bead cleaning buffer solution, vortexing and oscillating for 5s, quickly centrifuging, sucking off the supernatant, adding 300 mul of magnetic bead diluent, carrying out vortex oscillation for 5s, and carrying out rapid centrifugation to obtain a magnetic bead Beads-MM05 solution coupled with the antibody, and storing at 4 ℃.

6. The detection method according to claim 1, wherein the step (3) of labeling biotin with an antibody comprises:

1) adding the antibody R001 with the required volume into a centrifugal filter, then adding biotinylation reaction buffer solution to 500 mu l, centrifuging for 3-8min, discarding the bottom liquid, repeating the washing step twice, adding 400-500 mu l biotinylation reaction buffer solution for washing each time, inverting the filter, putting into a clean centrifugal tube, centrifuging for 1-3min, and collecting the antibody;

2) washing the filter membrane with 50 μ l of biotinylation reaction buffer solution for 5-8 times, inverting the filter in a centrifuge tube, centrifuging for 1-3min, and collecting antibody-Biotin conjugate R001-Biotin solution;

3) the antibody concentration and volume after displacement were measured.

7. The detection method according to claim 6, wherein the step (3) of labeling biotin with an antibody comprises:

1) adding the antibody R001 with the required volume into a centrifugal filter, then adding biotinylation reaction buffer solution to 500 mu L, centrifuging for 5min, discarding the bottom liquid, repeating the washing step twice, adding 450 mu L of biotinylation reaction buffer solution for washing each time, then placing the filter in a clean centrifugal tube in an inverted manner, centrifuging for 2min, and collecting the antibody;

2) the filter was washed 6 times with 50. mu.l of biotinylation reaction buffer. Inverting the filter in a centrifuge tube, centrifuging for 2min, and collecting an antibody-Biotin conjugate R001-Biotin solution;

3) the antibody concentration and volume after displacement were measured.

8. The detection method according to claim 1, wherein the step of configuring the standard (4) is:

1) taking the replaced antibody freeze-dried powder, adding 400ul of nucleic-free water for dissolving, and preserving at-80 ℃;

2) the standard curve dilution concentration gradient is: 0, 0.1pg/mL, 1pg/mL, 10pg/mL, 100pg/mL, 1000 pg/mL.

9. The detection method according to claim 1, wherein the step of (5) on-machine analysis comprises:

1) taking a special 96-pore plate for an on-machine, respectively adding 100ul of standard curve solution or a sample to be tested, repeating for several times, adding Beads-MM 0525 ul and R001-Biotin 20ul into each pore site, incubating for 35min at 30 ℃ on a microplate shaker for 15s, then putting the 96-pore plate on a plate washer, starting a washing program of a 2-step method, washing away unbound antigens or antibodies, adding 100ul of SBG into each pore site, incubating for 5min at 30 ℃ on the microplate shaker for 15s, then putting the plate washer on a washing machine for continuous washing operation, washing away excessive unbound enzymes, and after the program operation of the plate washer is finished, putting the plate for 10min to be dried completely, thus obtaining the on-machine;

2) opening SR-X Analyzer, opening 'Run' menu key of main interface, entering flat setting interface, setting operation parameters: firstly naming the program, then respectively carrying out operations of selecting hole sites, selecting a kit matching program, naming and setting dilution times on corresponding hole sites according to the hole sites of the sample adding holes, clicking a 'Use Layout' key to confirm the setting of a flat plate, adding all reagent consumables such as compact discs and 96-hole plates, emptying a waste box, and clicking a 'Start' key to operate the program;

3) after the operation is finished, the instrument is copied to generate data and reports.

10. The detection method according to claim 1, wherein the antibodies are a MM05 antibody and a R001 antibody, the MM05 antibody is a capture antibody, and the R001 antibody is a detection antibody.

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