CN113030469B

CN113030469B - Novel coronavirus detection method

Info

Publication number: CN113030469B
Application number: CN202110290165.7A
Authority: CN
Inventors: 路瑶; 刘昆; 黄炜
Original assignee: Hangzhou Jingbai Biotechnology Co ltd; GUIZHOU ACADEMY OF TESTING AND ANALYSIS
Current assignee: Hangzhou Jingbai Biotechnology Co ltd; GUIZHOU ACADEMY OF TESTING AND ANALYSIS
Priority date: 2021-03-18
Filing date: 2021-03-18
Publication date: 2024-04-09
Anticipated expiration: 2041-03-18
Also published as: CN113030469A

Abstract

The invention relates to a novel coronavirus detection method, which comprises the replacement of an antibody reserve buffer solution; preparing magnetic beads; labeling biotin with an antibody; preparing a standard substance; performing on-machine analysis; the detection method has the advantages that the virus characteristic protein with extremely low abundance value can be detected in a short time, 96 samples can be analyzed in one hour (288 samples can be obtained if the full-automatic HD-1 model flux is adopted), and meanwhile, sample loading and processing are automatically carried out in a closed instrument during sample loading analysis, so that the infection risk caused by contact of a tester and a detection sample is greatly reduced. The method not only can carry out qualitative detection, but also can carry out accurate quantitative detection, can improve the detection sensitivity of novel coronavirus infection to the greatest extent, and can also ensure the credibility 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 for non-diagnostic purposes.

Background

At present, the false negative rate of the nucleic acid detection serving as the most important detection index is too high, a certain number of asymptomatic patients (carriers of viruses) exist, and missed detection conditions frequently occur, so that the spread of the viruses and the development of epidemic situations are caused. Therefore, the establishment of a new detection technique is significant in reducing the false negative rate of detection. Meanwhile, a virus detection technology with higher sensitivity is developed while a targeted new coronavirus vaccine is developed, and the virus infection carrying risk can be determined earlier and faster, so that the flowing transmission path of the crowd is cut off, and the exact effect of preventing and screening new coronavirus infection carrying prevention and control is achieved.

At present, the false negative rate of the new coronavirus detected by a fluorescent quantitative PCR detection method is usually up to 40%, on one hand, because the sampling standardization of the detection material is insufficient, for example, when the virus is not diffused to the lower respiratory tract yet, the inaccuracy of the detection result can be reduced by adopting a sampling method of a sampling cavity swab, and the sampling cavity swab is accompanied with sampling pain or uncomfortable feeling and can lead to rejection of a subject; on the other hand, the detection limit is reduced due to the loss of the target fragment with nucleic acid extraction and PCR. However, the current detection methods (such as colloidal gold method) based on antibodies can only be qualitative and not quantitative, and an infected person may not generate enough antibodies due to the inactive or tolerability of the immune system, so that false negatives are caused; and colloidal gold itself has a certain false positive rate.

In the conventional ELISA reaction, the antigen-antibody reaction system is usually 100. Mu.l, and a large amount of fluorescent signal is generated and diluted in 100. Mu.l reaction environment, so that the greatest problem at present is that the detection sensitivity is not high, and the highest is usually in the picogram (pg/ml) scale.

In view of the above problems, the inventors put the target on the antigen for detecting a novel coronavirus, i.e., the novel coronavirus itself, to enhance the specificity and sensitivity of the detection method. The method is simple to operate, has extremely strong openness and flexibility, can detect the antigen to be detected in the sample only by specifically recognizing an antibody pair of a certain antigen and an antigen standard to be detected and configuring a standard curve, and 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 immune complex with double antibody sandwich, which ensures the specificity of the reaction. When in operation, one of the antibody pairs is coated on the antibody binding site of the magnetic beads with the diameter of 2.7 mu m according to certain coating conditions, the other is added after being marked by biotin for incubation, and avidin-coupled enzyme and enzyme reaction substrates are added after incubation is finished to form immune complexes added into a reaction chip. Each chip is provided with 238000 small holes, the diameter of each small hole is 4.2 mu m, only one magnetic bead just can fall into the small holes, after the magnetic beads fall into the small holes under the action of gravity, the system pushes a layer of oil on the surface of the chip, on one hand, the redundant magnetic beads are removed, and on the other hand, fluorescent signals are locked in the small holes (signal diffusion and cross reaction are not easy to occur). Under the condition, fluorescent molecules generated by the enzyme molecule catalytic substrate are locked in a small-hole reaction system, the magnetic beads containing the target antigen emit fluorescence because of enzyme, the proportion of the number of the magnetic beads containing fluorescence to the number of all the magnetic beads falling on the whole chip surface is positively correlated with the concentration of the target antigen in the sample, and the computer can accurately calculate the concentration of the target antigen in the sample to be detected.

Disclosure of Invention

The invention provides a novel coronavirus detection method for non-diagnostic purposes, which comprises the following steps: (1) The antibody reserve buffer is replaced by a magnetic bead connecting buffer; (2) preparation of magnetic beads; (3) antibody-labeled biotin; (4) configuration of a standard substance; and (5) performing on-machine analysis.

The step of replacing the antibody stock buffer solution with the magnetic bead connection buffer solution comprises the following steps:

1) Measuring the concentration of MM05 antibody and R001 antibody with an ultra-micro spectrophotometer, and calculating the required volume of each antibody according to the required amount of coated antibody of 100 mug and the measured concentration of the antibody;

2) Adding the MM05 antibody and the R001 antibody with required volumes into a filter respectively, adding magnetic bead connection buffer solution to 450-550 mu l, putting into a centrifuge, centrifuging for 3-8min, discarding bottom liquid, repeating the above washing steps twice, adding 400-500 mu l of magnetic bead connection buffer solution each time, putting the filter into a clean centrifuge tube upside down, centrifuging for 1-3min, and collecting liquid;

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

4) The volume of the prepared R001 antibody was 150. Mu.l, and the volume of the MM05 antibody was 300. Mu.l, and stored on ice for use.

Preferably, the replacement of the antibody stock buffer of (1) with a magnetic bead ligation buffer comprises the following steps:

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

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

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

1) Washing the magnetic beads: taking 0.13ml of the magnetic bead stock solution after the replacement which is oscillated for more than 30s in a centrifuge tube, rapidly centrifuging, and absorbing and discarding supernatant; adding 250-350 mu L of magnetic bead cleaning buffer solution, carrying out vortex oscillation for 3-8s, carrying out rapid centrifugation, absorbing and discarding supernatant, repeating the magnetic bead washing step twice, and absorbing and discarding supernatant to obtain the magnetic beads washed by the buffer solution;

2) Conversion of magnetic beads: adding 250-350 mu L of magnetic bead connection buffer solution into the magnetic beads washed by the buffer solution, carrying out vortex oscillation for 3-8s, 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 3-8s, 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 small bottle containing 10mg of EDC, vortexing for 5-10s, taking 9 mu l of magnetic bead after buffer solution replacement, vortexing and mixing for 8-12s, mixing and incubating a centrifuge tube at 2-8 ℃ for 25-35min, rapidly centrifuging, taking away supernatant, respectively adding 300 mu l of replaced antibody MM05 solution and 150 mu l of 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, taking away supernatant, adding 300 mu l of magnetic bead cleaning buffer solution, vortexing and shaking for 3-8s, rapidly centrifuging, taking away supernatant, repeating the magnetic bead cleaning step again, adding 300 mu l of magnetic bead sealing buffer solution, vortexing and incubating for 3-8s at room temperature for 40-50min, rapidly centrifuging, taking away supernatant, adding 300 mu l of magnetic bead cleaning buffer solution, taking away MM and rapidly centrifuging, taking away supernatant, adding 300 mu l of magnetic bead cleaning buffer solution, mixing and incubating for 3-8s, rapidly centrifuging, taking away supernatant, adding 300 mu l of magnetic bead cleaning buffer solution, and rapidly shaking for 3-8s, and storing the magnetic bead coupling after vortexing at 3-5 s.

Preferably, the step of preparing the magnetic beads of (2) comprises:

1) Washing the magnetic beads: taking 0.13ml of the magnetic bead stock solution after the replacement which is oscillated for more than 30s in a centrifuge tube, rapidly centrifuging, and absorbing and discarding supernatant; adding 300 μl of magnetic bead cleaning buffer solution, vortex oscillating for 5s, rapidly centrifuging, removing supernatant, repeating the magnetic bead washing step twice, and removing supernatant to obtain magnetic beads washed by the buffer solution;

2) Conversion of magnetic beads: adding 300 μl 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 the supernatant, repeating the magnetic bead replacement step twice, carrying out rapid centrifugation, absorbing and discarding the supernatant, adding 291 μl 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 small bottle containing 10mg of EDC, vortexing for 8s, taking 9 mu l of magnetic bead after buffer solution replacement, vortexing and mixing for 10s, incubating the centrifuge tube at 5 ℃ for 30min, rapidly centrifuging, absorbing and discarding supernatant, respectively adding 300 mu l of replaced antibody MM05 solution and 150 mu l of replaced antibody R001 solution, vortexing and shaking for 10s, mixing and incubating the centrifuge tube at 5 ℃ for 2h, rapidly centrifuging, absorbing and discarding supernatant, adding 300 mu l of magnetic bead cleaning buffer, vortexing for 5s, rapidly centrifuging, absorbing and discarding supernatant, repeating the magnetic bead washing step again, adding 300 mu l of magnetic bead sealing buffer, vortexing for 5s, incubating at room temperature for 45min, rapidly centrifuging, absorbing and discarding supernatant, adding 300 mu l of magnetic bead cleaning buffer, oscillating for 5s, rapidly centrifuging, absorbing and discarding supernatant, and obtaining coupled magnetic bead Beading 05, and storing at 4 ℃.

The step of labeling biotin by the antibody in the step (3) is as follows:

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

2) Washing the filter membrane with 50 μl of biotinylation buffer 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 concentration and volume of the displaced antibodies were measured.

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

1) Adding a required volume of antibody R001 into a centrifugal filter, adding a biotinylation reaction buffer solution to 500 mu L, centrifuging for 5min, discarding bottom liquid, repeating the washing step twice, adding 450 mu L of biotinylation reaction buffer solution each time for washing, putting the filter into a clean centrifuge tube reversely, centrifuging for 2min, and collecting the antibody;

2) Washing the filter membrane with 50 μl of biotinylation buffer for 6 times, inverting the filter in a centrifuge tube, centrifuging for 2min, and collecting antibody-Biotin conjugate R001-Biotin solution;

3) The concentration and volume of the displaced antibodies were measured.

The configuration steps of the standard substance (4) are as follows:

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

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

The on-machine analysis step of (5) is as follows:

1) Taking a special 96-well plate for machine feeding, respectively adding 100ul of standard curve solution or sample to be tested, repeating for several times, repeating the steps to obtain a plurality of holes, adding Beads-MM05 25ul and R001-Biotin 20ul into each hole, incubating for 35min and 15s at 30 ℃ on a micro-plate shaking machine, then placing the 96-well plate on a plate washer, starting a 2-step washing procedure, washing unbound antigen or antibody, adding 100ul SBG into each hole, placing on the micro-plate shaking machine at 30 ℃ for 5min and 15s, then placing on the plate washer to continue starting the next step of established washing operation, washing excessive unbound enzyme, placing a plate for 10min after the plate washer procedure is finished, and finishing the plate washer procedure;

2) Opening the SR-X Analyzer, opening a Run menu key of the main interface, entering a panel setting interface, and setting operation parameters: firstly naming the program, then respectively carrying out operations of selecting a hole site-selecting a kit matching program-naming and setting dilution factors on corresponding hole sites according to the hole sites of the sample adding holes, clicking a "Use Layout" key to confirm the panel setting, adding all reagent consumables such as optical discs and 96 hole plates, emptying the waste boxes, and clicking a "Start" key to run 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 diagnostic.

The invention has the following advantages:

1. the invention can detect the characteristic proteins of viruses with extremely low abundance values in extremely short time, can analyze 96 samples in one hour (288 samples can be obtained if the full-automatic HD-1 model flux is adopted), and simultaneously, the sample loading analysis is automatically carried out in a closed instrument, thereby greatly reducing the infection risk caused by the contact of test staff and the detection samples.

2. The detection method has high sensitivity, ensures extremely low sensitivity, can ensure the reliability of detection results, has detection capability greatly stronger than that of a fluorescent PCR nucleic acid detection kit, and can perform qualitative detection and accurate quantitative detection compared with the antibody detection kit (colloidal gold method).

3. The invention uses MM05 as capture antibody and R001 as detection antibody, and the technological effect developed by the SiMoA on-machine program by adopting a '2-step method' is good. The upper machine measured a lower limit of detection (LOD) of 0.0552pg/ml, a lower limit of quantification (LLOQ) of 0.1940pg/ml, and a range of quantification of 0.194-853 pg/ml, at which the signal to noise value was 2.03.

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

5. After the addition of the Helper beads is optimized, the detection sensitivity of the invention can be improved to the greatest extent by adding 70% of the Helper beads, the detection lower limit is reduced from 0.055pg/ml to 0.0157pg/ml, the quantitative lower limit is reduced from 0.194pg/ml to 0.068pg/ml, the quantitative range is 0.068-384pg/ml, the signal to noise value is 2.24 at the quantitative lower limit, the sensitivity is extremely high, the false negative rate can be remarkably low, and the detection kit is applicable to detecting various samples, and is suitable for qualitatively detecting N proteins of 2019-nCOV viruses in samples of novel pneumonia suspected cases, suspected aggregated patients and the like of coronavirus infection or blood, nasopharyngeal swabs, sputum and the like of other cases in vitro.

Drawings

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

fig. 2 is an enlarged view of a SiMoA reaction disc and schematic illustration of enzymatic reaction [ note: A. a schematic diagram of the bottom of the optical disc; B. a local microscopic enlarged view of the optical disk main reaction area unit; C. a small hole scanning electron microscope enlarged view in the reaction area unit; D. the reaction area unit is small Kong Poumian, and the antigen-antibody reaction and the sealing oil push the magnetic beads to fall into the holes; E. sealing oil seals the small holes where the magnetic beads fall and eliminates redundant magnetic bead schematic diagrams on the surface of the micropore area (the magnetic bead holes combined with the antigen to be detected show fluorescence, and the holes without the antigen to be detected and the magnetic beads do not emit fluorescence); F. schematic diagram of magnetic bead and antigen-antibody binding; G. diagram of parts in figure F ];

FIG. 3HB1 standard curve;

FIG. 4HB2 standard curve;

FIG. 5HB3 standard curve;

FIG. 6HB4 standard curve.

Detailed Description

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

Example 1

1. The stock buffer was replaced with magnetic bead ligation buffer:

1.1 measuring the concentration of MM05 antibody and R001 antibody respectively by using an ultra-micro spectrophotometer, and calculating the required volume of each antibody according to the required amount of coated antibody of 100 mug and the measured concentration of the antibody;

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

1.3 adding 50 μl 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 the 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 for use.

2. Preparation of magnetic beads:

2.1, taking 0.13ml of the magnetic bead stock solution after the replacement which is oscillated for more than 30s, putting the magnetic bead stock solution into a centrifuge tube, rapidly centrifuging, and absorbing and discarding supernatant; adding 300 μl of magnetic bead cleaning buffer solution, vortex oscillating for 5s, rapidly centrifuging, removing supernatant, repeating the magnetic bead washing step twice, and removing supernatant to obtain magnetic beads washed by the buffer solution;

2.2 adding 300 μl 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 the supernatant, repeating the magnetic bead replacement step twice, carrying out rapid centrifugation, absorbing and discarding the supernatant, adding 291 μl 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 adding precooled 1ml of magnetic bead connection buffer solution into a small bottle containing 10mg of EDC, vortexing for 8s, taking 9 mu l of magnetic bead after buffer solution replacement, vortexing for 10s, mixing and incubating a centrifuge tube at 5 ℃ for 30min, rapidly centrifuging, absorbing supernatant, respectively adding 300 mu l of a replaced antibody MM05 solution and 150 mu l of a replaced antibody R001 solution, vortexing for 10s, mixing and incubating the centrifuge tube at 5 ℃ for 2h, rapidly centrifuging, absorbing supernatant, adding 300 mu l of magnetic bead cleaning buffer, vortexing for 5s, rapidly centrifuging, absorbing supernatant, repeating the magnetic bead washing step once again, adding 300 mu l of magnetic bead sealing buffer, vortexing for 5s, incubating at room temperature for 45min, rapidly centrifuging, absorbing supernatant, adding 300 mu l of magnetic bead cleaning buffer, vortexing for 5s, rapidly centrifuging, absorbing supernatant, adding 300 mu l of magnetic bead diluent, rapidly centrifuging to obtain magnetic bead cleaning buffer of coupled antibody, and storing the magnetic bead solution at 4 ℃ at Beading temperature.

3. Antibody-labeled biotin:

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

4. Configuration of standards

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

5. On-line analysis

Taking a 96-well plate special for machine feeding, respectively adding 100ul of standard curve solution or sample to be tested, repeating for several times, repeating the steps to obtain a plurality of holes, adding Beads-MM05 ul and R001-Biotin 20ul into each hole, incubating for 35min and 15s at 30 ℃ on a micro-plate shaking table, then placing the 96-well plate on a plate washer, starting a 2-step washing program, washing unbound antigen or antibody, adding 100ul SBG into each hole, incubating for 5min and 15s at 30 ℃ on the micro-plate shaking table, then placing on the plate washer to continuously start the next step of established washing operation, washing excessive unbound enzyme, placing a plate for 10min after the operation of the plate washer program is finished, opening SR-X Analyzer, opening a Run menu key of a main interface, entering a plate setting interface, and setting operation parameters: firstly naming the program, then respectively carrying out operations of selecting a hole site-selecting a kit matching program-naming and setting dilution factors on corresponding hole sites according to the hole sites of the sample adding holes, clicking a "Use Layout" key to confirm the panel setting, adding all reagent consumables such as optical discs and 96 hole plates, emptying the waste boxes, clicking a "Start" key to run the program, and copying the instrument to generate data and reports after the operation is finished.

Example 2

1. The antibody stock buffer was replaced with magnetic bead ligation buffer:

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

1.3 adding 45 μl of magnetic bead connection 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 the liquid;

2. Preparation of magnetic beads:

2.1, taking 0.13ml of the magnetic bead stock solution after the replacement which is oscillated for more than 30s, putting the magnetic bead stock solution into a centrifuge tube, rapidly centrifuging, and absorbing and discarding supernatant; adding 250 μl of magnetic bead cleaning buffer solution, vortex oscillating for 3s, rapidly centrifuging, removing supernatant, repeating the magnetic bead washing step twice, and removing supernatant to obtain magnetic beads washed with the buffer solution;

2.2 adding 250 μl 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 the supernatant, repeating the magnetic bead replacement step twice, carrying out rapid centrifugation, absorbing and discarding the supernatant, adding 291 μl 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 adding precooled 1ml of magnetic bead connection buffer solution into a small bottle containing 10mg of EDC, vortexing for 5s, taking 9 mu l of magnetic Beads replaced by the buffer solution, vortexing and mixing for 8s, incubating a centrifuge tube at 2 ℃ for 25min, rapidly centrifuging, absorbing and discarding supernatant, respectively adding 300 mu l of replaced antibody MM05 solution and 150 mu l of replaced antibody R001 solution, vortexing and shaking for 8s, incubating the centrifuge tube at 2 ℃ for 1.5h, rapidly centrifuging, absorbing and discarding supernatant, adding 300 mu l of magnetic bead cleaning buffer, vortexing and shaking for 3s, rapidly centrifuging, absorbing and discarding supernatant, repeating the magnetic bead washing step again, adding 300 mu l of magnetic bead sealing buffer, vortexing and shaking for 3s, incubating at room temperature for 40min, rapidly centrifuging, absorbing and discarding supernatant, adding 300 mu l of magnetic bead cleaning buffer, vortexing for 3s, rapidly centrifuging, absorbing and discarding supernatant, adding 300 mu l of magnetic bead dilution, oscillating for 3s, rapidly centrifuging, and obtaining the magnetic bead cleaning buffer coupled with the antibody, wherein the magnetic bead is stored at the temperature of 05 ℃ for 3 ℃.

3. Antibody-labeled biotin:

adding a required volume of antibody R001 into a centrifugal filter, adding a biotinylation reaction buffer solution to 500 mu L, centrifuging for 3min, discarding bottom liquid, repeating the washing step twice, adding 400 mu L of biotinylation reaction buffer solution each time for washing, putting the filter into a clean centrifuge tube reversely, centrifuging for 1min, and collecting the antibody; washing the filter membrane 5 times with 50 μl of biotinylation reaction buffer, inverting the filter in a centrifuge tube, centrifuging for 1min, and collecting antibody-Biotin conjugate R001-Biotin solution; the concentration and volume of the displaced antibodies were measured.

4. Configuration of standards

5. On-line analysis

Example 3

1. The antibody stock buffer was replaced with magnetic bead ligation buffer:

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

1.3 adding 55 μl 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 3min, and collecting the liquid;

2. Preparation of magnetic beads:

2.1, taking 0.13ml of the magnetic bead stock solution after the replacement which is oscillated for more than 30s, putting the magnetic bead stock solution into a centrifuge tube, rapidly centrifuging, and absorbing and discarding supernatant; adding 350 μl of magnetic bead cleaning buffer solution, vortex oscillating for 8s, rapidly centrifuging, removing supernatant, repeating the magnetic bead washing step twice, and removing supernatant to obtain magnetic beads washed with the buffer solution;

2.2 adding 350 μl 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, absorbing and discarding the supernatant, repeating the magnetic bead replacement step twice, carrying out rapid centrifugation, absorbing and discarding the supernatant, adding 291 μl 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 adding precooled 1ml of magnetic bead connection buffer solution into a small bottle containing 10mg of EDC, vortexing for 10s, taking 9 mu l of magnetic Beads after buffer solution replacement, vortexing for 12s, mixing and incubating a centrifuge tube at 2-8 ℃ for 35min, rapidly centrifuging, taking away supernatant, respectively adding 300 mu l of a replaced antibody MM05 solution and 150 mu l of a replaced antibody R001 solution, vortexing for 12s, mixing and incubating the centrifuge tube at 8 ℃ for 2.5h, rapidly centrifuging, taking away supernatant, adding 300 mu l of magnetic bead cleaning buffer solution, vortexing for 8s, rapidly centrifuging, taking away supernatant, repeating the magnetic bead washing step again, adding 300 mu l of magnetic bead sealing buffer solution, vortexing for 8s, incubating at room temperature for 50min, rapidly centrifuging, taking away supernatant, adding 300 mu l of magnetic bead cleaning buffer solution, vortexing for 8s, rapidly centrifuging, taking away supernatant, adding 300 mu l of magnetic bead diluent, oscillating for 8s, rapidly centrifuging, and obtaining the magnetic bead-coupled antibody Beading for 5 s.

3. Antibody-labeled biotin:

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

4. Configuration of standards

5. On-line analysis

Taking a 96-well plate special for machine feeding, respectively adding a standard curve solution or a sample to be tested into 100ul, repeating for several times, repeating the steps to obtain a plurality of holes, adding Beads-MM05 25ul and R001-Biotin 20ul into each hole, incubating for 15s at 30 ℃ on a micro-plate shaking table, then placing the 96-well plate on a plate washer, starting a 2-step washing program to wash unbound antigen or antibody, adding 100ul SBG into each hole, placing the plate washer on the micro-plate shaking table at 30 ℃ for 5min for 15s, then placing the plate washer on the plate washer to continue starting the next step of established washing operation, washing excessive unbound enzyme, placing the plate for 10min after the operation of the plate washer program is finished, opening SR-X Analyzer, opening a Run menu key of a main interface, entering a plate setting interface, and setting operation parameters: firstly naming the program, then respectively carrying out operations of selecting a hole site-selecting a kit matching program-naming and setting dilution factors on corresponding hole sites according to the hole sites of the sample adding holes, clicking a "Use Layout" key to confirm the panel setting, adding all reagent consumables such as optical discs and 96 hole plates, emptying the waste boxes, clicking a "Start" key to run the program, and copying the instrument to generate data and reports after the operation is finished.

Example 4

1. The antibody stock buffer was replaced with magnetic bead ligation buffer:

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

1.3 adding 48 μl of magnetic bead connection 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 the liquid;

2. Preparation of magnetic beads:

2.1, taking 0.13ml of the magnetic bead stock solution after the replacement which is oscillated for more than 30s, putting the magnetic bead stock solution into a centrifuge tube, rapidly centrifuging, and absorbing and discarding supernatant; adding 280 μl of magnetic bead cleaning buffer solution, vortex oscillating for 6s, rapidly centrifuging, removing supernatant, repeating the magnetic bead washing step twice, and removing supernatant to obtain magnetic beads washed with the buffer solution;

2.2 adding 280 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, 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 4s, carrying out rapid centrifugation, and placing on ice to obtain the magnetic beads after buffer solution replacement;

2.3 adding precooled 1ml of magnetic bead connection buffer solution into a small bottle containing 10mg of EDC, swirling for 6s, taking 9 μl of magnetic Beads replaced by the buffer solution, swirling and shaking for 9s, mixing and incubating a centrifuge tube at 4 ℃ for 28min, rapidly centrifuging, absorbing and discarding supernatant, respectively adding 300 μl of a replaced antibody MM05 solution and 150 μl of a replaced antibody R001 solution, swirling and shaking for 9s, mixing and incubating the centrifuge tube at 4 ℃ for 1.8h, rapidly centrifuging, absorbing and discarding supernatant, adding 300 μl of magnetic bead cleaning buffer, swirling and shaking for 4s, rapidly centrifuging, absorbing and discarding supernatant, repeating the magnetic bead washing step again, adding 300 μl of magnetic bead sealing buffer, swirling and shaking for 4s, incubating at room temperature for 48min, rapidly centrifuging, absorbing and discarding supernatant, adding 300 μl of magnetic bead cleaning buffer, swirling and shaking for 4s, rapidly centrifuging, absorbing and discarding supernatant, adding 300 μl of magnetic bead diluent, shaking for 4s, rapidly centrifuging, and obtaining the magnetic bead-coupled antibody Beadis at 4 ℃ under the conditions of vortex-05 ℃.

3. Antibody-labeled biotin:

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

4. Configuration of standards

5. On-line analysis

Example 5

1. The antibody stock buffer was replaced with magnetic bead ligation buffer:

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

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

2. Preparation of magnetic beads:

2.1, taking 0.13ml of the magnetic bead stock solution after the replacement which is oscillated for more than 30s, putting the magnetic bead stock solution into a centrifuge tube, rapidly centrifuging, and absorbing and discarding supernatant; adding 290 μl of magnetic bead cleaning buffer solution, vortex oscillating for 7s, rapidly centrifuging, removing supernatant, repeating the magnetic bead washing step twice, and removing supernatant to obtain magnetic beads washed by the buffer solution;

2.2 adding 260 μl 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 the supernatant, repeating the magnetic bead replacement step twice, carrying out rapid centrifugation, absorbing and discarding the supernatant, adding 291 μl of magnetic bead connection buffer solution, carrying out vortex oscillation for 7s, carrying out rapid centrifugation, and placing on ice to obtain magnetic beads after buffer solution replacement;

2.3 adding precooled 1ml of magnetic bead connection buffer solution into a small bottle containing 10mg of EDC, vortexing for 9s, taking 9 mu l of magnetic Beads after buffer solution replacement, vortexing for 11s, mixing and incubating a centrifuge tube at 7 ℃ for 34min, rapidly centrifuging, taking away supernatant, respectively adding 300 mu l of a replaced antibody MM05 solution and 150 mu l of a replaced antibody R001 solution, vortexing for 9s, mixing and incubating the centrifuge tube at 7 ℃ for 2.3h, rapidly centrifuging, taking away supernatant, adding 300 mu l of magnetic bead cleaning buffer, vortexing for 7s, rapidly centrifuging, taking away supernatant, repeating the magnetic bead washing step again, adding 300 mu l of magnetic bead sealing buffer, vortexing for 4s, incubating at room temperature for 43min, rapidly centrifuging, taking away supernatant, adding 300 mu l of magnetic bead cleaning buffer, vortexing for 5s, rapidly centrifuging, taking away supernatant, adding 300 mu l of magnetic bead diluent, oscillating for 5s, rapidly centrifuging, obtaining the magnetic bead cleaning buffer solution coupled with the antibody, and storing the magnetic bead at the temperature of 05 ℃ to 4 ℃.

3. Antibody-labeled biotin:

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

4. Configuration of standards

5. On-line analysis

Experimental example: to prove the scientificity and rationality of the invention, the following experimental study of methodology was performed

1. The invention relies on technology and detection principle

1.1 Ejection technique

Is developed on the prior single molecule immune array (Single Molecule Arrays, siMoA) technical platform. SimoA is based on enzyme-linked immune reaction (Enzyme Linked Immunoreactivity, ELISA), and utilizes array-arranged micro-pore (as shown in a B diagram in fig. 2) chips to capture and detect antigen with extremely low concentration by matching with micro magnetic beads, so that the aim of detecting the antigen is achieved, and the detection effect approaches that of directly detecting single protein molecules. The chip is integrated on a specially designed optical disc (shown as a diagram in fig. 2), 24 chips/channels are arranged on each optical disc, each channel is used for detecting one sample, and multiple marker molecules can be detected simultaneously in one reaction.

1.2 principle of detection

The method comprises the steps of taking magnetic beads with the diameter of 2.7 mu m as a carrier, distributing 250000 binding sites on the surface of each magnetic bead, loading a capture antibody on the sites for capturing an antigen to be detected, combining a specific detection antibody with the antigen to be detected (sandwich method, as shown in figure 1), combining biotin marks and avidin coupled enzyme on the detection antibody, and decomposing a substrate in a reaction solution to generate fluorescence. Thus, the fluorophores generated by the enzyme are limited in a very small reaction pore (the diameter is 4.2 μm and the volume is 50 fl) (as shown in a graph C in FIG. 2), and the unit fluorescence intensity of the fluorescent molecules can be further highlighted due to the much smaller reaction area compared with the conventional fluorescent molecules, so that the enzyme-labeled molecules with very low concentration can be detected.

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

2 summary 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 protocol

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

Table 1 on-machine experimental materials table

Connection scheme	Connecting magnetic beads	Attachment of biotin
			Connection scheme 1	R001	MM05
Connection scheme 2	MM05	R001

The results of preliminary pre-experiment measurement show that: the MM05 protein is used as a capture antibody, the capture antibody is coated on the surface of a magnetic bead, the R001 protein is used as a detection antibody by using biotin labeling, and the detection limit and the quantitative limit are lower when the kit quantifies the level of the N protein, so that the optimal coating scheme of the antibody pair is determined.

2.3.2 determination of the treatment steps for the test samples and the control

2.3.2.1MM05 antibody and R001 antibody stock buffer were replaced with magnetic bead ligation buffer;

2.3.2.2 preparation of magnetic beads: comprises preparing two magnetic Beads of Beads-MM05 and Beads-R001;

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

2.3.2.4 standard configuration;

2.3.3 on-machine analysis

2.3.3.1 determination of the on-machine test protocol

After standard configuration, two different coating schemes 2.3.2.2 and 2.3.2.3 were tested, respectively, and on-machine testing was performed using "2-Step" and "3-Step", and on-machine test schemes are shown in table 2.

Table 2"2-Step" and "3-Step" on-machine test tables

Name of upper computer program	Antibodies for use in connection with magnetic beads	Antibodies for attachment of biotin	Elution mode
				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-machine analysis

Taking a 96-well plate special for on-machine, respectively adding 100ul of standard curve solution or sample to be tested, repeating each standard twice, adding coated magnetic beads and antibodies into each hole site, incubating for 15s at 30 ℃ on a micro-pore plate shaking machine, then placing the 96-well plate on a plate washer, eluting according to a scheme shown in the table 2, washing off unbound antigens or antibodies, adding 100ul of SBG into each hole site, placing on the micro-pore plate shaking machine for incubation at 30 ℃ for 5min15s, then placing on the plate washer, continuously starting the next step of established washing operation, washing off excessive unbound enzymes, placing a plate for 10min after the operation of the plate washer is finished, opening an SR-X Analyzer, opening a 'Run' menu key of a main interface, entering a plate setting interface, and setting operation parameters: firstly naming the program, then respectively carrying out operations of selecting a hole site-selecting a kit matching program-naming and setting dilution factors on corresponding hole sites according to the hole sites of the sample adding holes, clicking a "Use Layout" key to confirm the panel setting, adding all reagent consumables such as optical discs and 96 hole plates, emptying the waste boxes, clicking a "Start" key to run the program, and copying the instrument to generate data and reports after the operation is finished. The standard curve, standard point basic information, curve matching coefficient and detection capability parameters of the four schemes are respectively shown in fig. 3, fig. 4, fig. 5, fig. 6, table 3, table 4, table 5, table 6, table 7, table 8, table 9, table 10, table 11, table 12, table 13 and table 14.

TABLE 3HB1 standard point basic information Table

Note that: the machine uses calipers A-F for 6 standard spots, 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 the wells containing the magnetic beads in the array dense wells.

Table 4HB1 Curve matching coefficient Table

The detection range data is obtained through calculation of a built-in analysis program of a computer and is shown in the following table:

TABLE 5HB1 detectability parameter Table

TABLE 6 HB2 standard dot basic information Table

Note that: the machine uses calipers A-F for 6 standard spots, 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 the concentration value of protein is measured, and AEB is determined by the average fluorescence intensity of the wells containing the magnetic beads in the array close wells.

TABLE 7 HB2 Curve match coefficient Table

TABLE 8 HB2 detection Capacity parameter Table

Item	Detail
		Assumed LOD(multiplier)	0.3923(1.3)pg/ml
Native LOD(SD)	-(2.5)pg/ml
		LLOQ(concentration CV)	1.0900pg/ml
ULOQ(concentration CV)	903pg/ml
		Signal to noise at LLOQ	1.69
concentration(at 15AEB)	431.9pg/ml
		Assay Dynamic Range	3.3logs
Assay Range(pg/mL)	0.203-432pg/ml

TABLE 9 HB3 standard dot basic information Table

TABLE 10 HB3 Curve match coefficient Table

TABLE 11 HB3 detection Capacity parameter Table

Item	Detail
		Assumed LOD(multiplier)	0.0552(1.3)pg/ml
Native LOD(SD)	0.0833(2.5)pg/ml
		LLOQ(concentration CV)	0.194(20％)pg/ml
ULOQ(concentration CV)	4959(20％)pg/ml
		Signal to noise at LLOQ	2.03
concentration(at 15AEB)	853pg/ml
		Assay Dynamic Range	3.6logs
Assay Range(pg/mL)	0.194-853pg/ml

Table 12 HB4 standard point basic information table

TABLE 13 HB4 Curve match coefficient Table

TABLE 14 HB4 detection Capacity parameter Table

Item	Detail
		Assumed LOD(multiplier)	0.1397(1.3)pg/ml
Native LOD(SD)	-(2.5)pg/ml
		LLOQ(concentration CV)	0.552(20％)pg/ml
ULOQ(concentration CV)	-(20％)pg/ml
		Signal to noise at LLOQ	2.14
concentration(at 15AEB)	2228.4pg/ml
		Assay Dynamic Range	3.6logs
Assay Range(pg/mL)	0.552-2228.4pg/ml

Results: as is clear from Table 11, the detection sensitivity was optimal in the upper machine, the lower limit of detection (LOD) was 0.0552pg/ml, the lower limit of quantification (LLOQ) was 0.1940pg/ml, and the quantitative range was 0.194-853pg/ml, and the signal-to-noise value was 2.03 at the lower limit of quantification.

In quantifying the components in a sample, there is a critical factor that affects the final assay result, even if false positives occur, which is the Matrix effect (Matrix effects). The matrix refers to that portion of the sample other than the analyte of interest, and since matrix components can have significant interference with the analysis process, these effects are collectively referred to as matrix effects, and minimizing the matrix effects is particularly important in ELISA kits. Thus, this experiment uses the "Spike/Recovery Assay" to examine whether the technique of the present invention is affected by matrix effects.

2.3.4 incorporation/recovery test

The test procedure is to incorporate the antigen to be tested with known concentration into the target sample with measured concentration, and the ratio of the measured concentration increasing part to the concentration to be incorporated before and after the incorporation is the recovery rate, and the recovery rate is expressed in percentage. The closer the recovery rate is to 100%, the closer the experimental result is to the true value. The recovery (%) is calculated as:

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

Optionally 6 healthy volunteer samples, of which 3 Serum (Serum) and 3 Plasma (Plasma), and 1 blank diluent control, were prepared for both high and low concentration standard protein samples for mixed Serum and Plasma recovery determinations, test packets and loading conditions are shown in table 15:

TABLE 15 incorporation/recovery design scheme

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

TABLE 16 blending/recovery experiments

Experimental results of incorporation/recovery:

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

2.3.4 re-optimization of Homebrew

The magnetic beads-Capture are added conventionally, and meanwhile, auxiliary magnetic beads (Helper beads) equipped with a SiMoA platform are added, so that the binding efficiency of a Capture antibody and the magnetic beads can be improved, and the sensitivity and the accuracy of a detection technology are further improved.

The best protocol HB3 screened in 2.3.3.2 was supplemented with unequal amounts of Helper beads, with the detection antibody remaining unchanged, in an attempt to increase the detection sensitivity of the method, each time the on-machine program was designated as "HB-letter" format, test protocol see Table 17:

table 17 Homebrew re-optimization protocol

The optimization effect of adding different amounts of Helper beads was tested according to the protocol of the above table, the on-machine test method was the same as the procedure of "2.3.3.2", and the results are shown in table 18.

Table 18 Homebrew re-optimization results Table

Item	Detail
		Assumed LOD(multiplier)	0.0157pg/ml
Native LOD(standard deviation)	-
		LLOQ(concentration CV)	0.0680pg/ml
ULOQ(concentration CV)	2971pg/ml
		Signal to noise at LLOQ	2.24
concentration(at 15AEB)	383.8pg/ml
		Assay Dynamic Range	3.8logs
Assay Range(pg/mL)	0.068-384pg/ml

Re-optimization results for Homebrew: as is clear from Table 18, HB4 having the highest detection sensitivity can maximally improve the detection sensitivity of the present invention when 70% of Helper beads are added, and the lower limit of detection is decreased from 0.055pg/mL to 0.0157pg/mL, the lower limit of quantification is decreased from 0.194pg/mL to 0.068pg/mL, the quantitative range is 0.068-384pg/mL, and the value of signal to noise is 2.24 at the lower limit of quantification, so that the detection sensitivity is extremely high, the false negative rate can be remarkably low, and the detection sample is various, and the detection device is suitable for in vitro auxiliary determination of N protein of 2019-nCOV virus in samples such as suspected cases of novel coronavirus infection, suspected aggregated disease patients and the like or blood, nasopharyngeal swab, sputum and the like of other cases.

Conclusion:

1. the invention uses MM05 as capture antibody and R001 as detection antibody, and the technological effect developed by the SiMoA on-machine program by adopting a '2-step method' is good. The upper machine measured a lower limit of detection (LOD) of 0.0552pg/ml, a lower limit of quantification (LLOQ) of 0.1940pg/ml, a range of quantification of 0.194-853pg/ml, and a signal to noise value of 2.03 at the lower limit of quantification.

2. The invention adopts the test of the doping/recovery rate, the doping/recovery rate of manual 4-time dilution after the plasma is doped is ideal, the average plasma doping/recovery rate reaches 113%, and the acceptable range of the result is 80-120%.

3. After the technology is added with the Helper beads to optimize, the detection sensitivity of the invention can be improved to the greatest extent by adding 70% of the Helper beads, the detection lower limit is reduced from 0.055pg/mL to 0.0157pg/mL, the quantitative lower limit is reduced from 0.194pg/mL to 0.068pg/mL, the quantitative range is 0.068-384pg/mL, the value of signal to noise is 2.24 at the quantitative lower limit, the sensitivity is extremely high, the false negative rate can be obviously lower, and the detection sample is various, and is suitable for qualitatively detecting N proteins of 2019-nCOV viruses in samples of novel pneumonia suspected cases, suspected aggregated disease patients and the like or blood, nasopharyngeal swab, sputum and the like of other cases in vitro.

While the invention has been described in detail in the foregoing general description, embodiments and experiments, it will be apparent to those skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims

1. A novel coronavirus detection method for non-diagnostic purposes, characterized in that the detection method comprises the steps of: (1) The antibody reserve buffer is replaced by a magnetic bead connecting buffer; (2) preparation of magnetic beads; (3) antibody-labeled biotin; (4) configuration of a standard substance; (5) on-machine analysis;

2) Adding the MM05 antibody and the R001 antibody with required volumes into a filter respectively, adding magnetic bead connection buffer solution to 450-550 mu l, putting into a centrifuge, centrifuging for 3-8min, discarding bottom liquid, repeating the washing step twice, adding 400-500 mu l of magnetic bead connection buffer solution each time, putting the filter into a clean centrifuge tube upside down, centrifuging for 1-3min, and collecting liquid;

4) The prepared R001 antibody is prepared into a volume of 150 mu l, and the MM05 antibody is prepared into a volume of 300 mu l and put on ice for standby;

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

3) Adding precooled 1ml of magnetic bead connection buffer solution into a small bottle containing 10mg of EDC, swirling for 5-10s, taking 9 mu l of magnetic bead after buffer solution replacement, swirling and vibrating and mixing for 8-12s, mixing and incubating a centrifuge tube at 2-8 ℃ for 25-35min, rapidly centrifuging, taking away supernatant, respectively adding 300 mu l of antibody MM05 solution after replacement and 150 mu l of antibody R001 solution after replacement, swirling and vibrating for 8-12s, mixing and incubating a centrifuge tube at 2-8 ℃ for 1.5-2.5h, rapidly centrifuging, taking away supernatant, adding 300 mu l of magnetic bead cleaning buffer solution, swirling and vibrating for 3-8s, rapidly centrifuging, taking away supernatant, repeating the magnetic bead cleaning step again, adding 300 mu l of magnetic bead sealing buffer solution, swirling and vibrating and incubating for 3-8s, rapidly centrifuging for 40-50min at room temperature, taking away supernatant, adding 300 mu l of magnetic bead cleaning buffer solution, taking away vortex and vibrating for 3-8s, rapidly centrifuging, taking away supernatant, adding 300 mu l of magnetic bead cleaning buffer solution, mixing and incubating for 3-8s, rapidly centrifuging, taking away supernatant, adding 300 mu l of magnetic bead cleaning buffer solution, and rapidly vibrating and storing the magnetic bead cleaning solution, and rapidly vibrating and storing the magnetic bead under the conditions at 3-8s, namely, performing vortex vibration and 3-5 s;

The step of labeling biotin by the antibody in the step (3) is as follows:

3) Measuring the concentration and volume of the displaced antibody;

the configuration steps of the standard substance (4) are as follows:

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

the on-machine analysis step of (5) is as follows:

1) Taking a special 96-well plate for machine feeding, respectively adding 100ul of standard curve solution or sample to be tested, repeating for several times, repeating the steps to obtain several holes, adding Beads-MM05 25ul and R001-Biotin 20ul into each hole, incubating for 35min and 15s at 30 ℃ on a micro-plate shaking machine, then placing the 96-well plate on a plate washing machine, starting a 2-step washing procedure, washing unbound antigen or antibody, adding 100ul SBG into each hole, incubating for 5min and 15s at 30 ℃ on the micro-plate shaking machine, then placing on the plate washing machine for continuous washing operation, washing excessive unbound enzyme, placing a plate for 10min after the plate washing machine is finished, and then machine feeding can be realized;

2) Opening the SR-X Analyzer, opening a Run menu key of the main interface, entering a panel setting interface, and setting operation parameters: firstly naming the program at this time, then respectively carrying out operations of selecting a hole site-selecting a kit matching program-naming and setting dilution factors on corresponding hole sites according to the hole sites of the sample adding holes, clicking a "Use Layout" key to confirm the panel setting, adding optical discs, 96 hole plates and all reagent consumables, emptying waste boxes, clicking a "Start" key to run the program;

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

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

3) Adding 50 μl 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 the liquid;

3. The method according to claim 1, wherein the step of preparing the magnetic beads of (2) is:

3) Adding precooled 1ml of magnetic bead connection buffer solution into a small bottle containing 10mg of EDC, swirling for 8s, taking 9 mu l of magnetic bead after buffer solution replacement, swirling and shaking for 10s, mixing and incubating a centrifuge tube at 5 ℃ for 30min, rapidly centrifuging, sucking and discarding supernatant, respectively adding 300 mu l of a replaced antibody MM05 solution and 150 mu l of a replaced antibody R001 solution, swirling and shaking for 10s, mixing and incubating the centrifuge tube at 5 ℃ for 2h, rapidly centrifuging, sucking and discarding supernatant, adding 300 mu l of magnetic bead cleaning buffer, swirling and shaking for 5s, rapidly centrifuging, sucking and discarding supernatant, repeating the magnetic bead washing step again, adding 300 mu l of magnetic bead sealing buffer, swirling and shaking for 5s, incubating at room temperature for 45min, rapidly centrifuging, sucking and discarding supernatant, adding 300 mu l of magnetic bead cleaning buffer, swirling and shaking for 5s, rapidly centrifuging, sucking and discarding supernatant, adding 300 mu l of magnetic bead diluent, swirling and shaking for 5s, obtaining magnetic bead washing buffer of coupled antibody, and storing at 4 ℃.

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

3) The concentration and volume of the displaced antibodies were measured.

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