CN113009154A - One-step method novel magnetic microsphere detection kit for coronavirus neutralizing antibody and application thereof - Google Patents
One-step method novel magnetic microsphere detection kit for coronavirus neutralizing antibody and application thereof Download PDFInfo
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Abstract
The invention discloses a one-step method novel magnetic microsphere detection kit for coronavirus neutralizing antibodies, which consists of magnetic microspheres coupled with recombinant human ACE2 receptor protein (ACE2-hFc), RBD neutralizing antibody standard solution used as positive control, negative control, sample diluent, RBD protein (HRP-RBD) enzyme conjugate, 20 multiplied concentrated washing solution and luminescent solution, wherein the magnetic microspheres are arranged in a box body. The invention also discloses application of the kit in detecting biological samples containing the novel coronavirus neutralizing antibody. Experiments prove that the kit has high stability, strong selectivity, high detection speed, low cost and easy operation, overcomes the defects of high laboratory environment requirement, long operation time and the like when detecting the neutralizing antibody in the prior art, and shortens the operation time from 120 minutes to 20 minutes. Has great clinical application prospect.
Description
Technical Field
The invention relates to detection of a novel coronavirus (SARS-CoV-2) neutralizing antibody, in particular to a one-step method novel coronavirus neutralizing antibody magnetic microsphere detection kit and application thereof, belonging to the technical field of clinical examination.
Background
The pandemic of new coronary pneumonia (COVID-19) caused by the new coronary virus (SARS-CoV-2) is the most serious challenge human faces for over a century. The common signs of the novel coronavirus, namely SARS-CoV-2, after infection are respiratory symptoms, fever, cough, shortness of breath, dyspnea and the like. In more severe cases, the infection can lead to pneumonia, severe acute respiratory syndrome, renal failure, and even death.
Research has shown that coronavirus S protein is a key factor in virus virulence, is a key part determining virus virulence, tissue tropism and host range, and is also a main target for neutralizing antibodies and vaccine design. And the most direct detection method is the detection of the neocorona neutralizing antibody. Because, neutralizing antibodies that recognize the RBD region (receptor binding domain) on the S (spike) protein of the new coronavirus block the binding of the new coronavirus to the ACE2 receptor on human cells, rendering the new coronavirus incapable of infecting human cells. In addition, neutralizing antibodies can interact with other immune components, such as complement, phagocytic cells, natural killer cells, and the like. These effector responses may help clear the virus and infected cells. The higher the concentration of neutralizing antibody, the better the protection in general. Therefore, one of the indicators for early prediction of vaccine quality is to see how much neutralizing antibody is produced in human body.
The traditional method of detecting neutralizing antibodies is a live virus or pseudovirus neutralization assay. The test method needs a professional cell culture process, has high requirements on the experimental grade, long period and complex operation, and is not suitable for high-throughput screening of common vaccine inoculation people.
The neutralizing antibody titer can reflect the index of the antibody level in the blood sample, namely the quality or the strength of the immune response in the human body after vaccination. With the marketing of novel coronavirus vaccines, rapid, reliable, stable, and safe evaluation methods are needed for neutralizing antibodies in people vaccinated with new corona vaccines and in people recovering from new corona infections. At present, competitive inhibition detection methods related to enzyme-linked immunity of novel coronavirus neutralizing antibodies (CN202010919164.X, CN202010952237.5) exist, but the experimental detection methods disclosed in the above patent patents all belong to enzyme-linked immunosorbent assay, and have the defects of long operation time (2-3 hours), manual operation and the like in experimental operation. Because the infectivity of the new coronavirus is very strong, the manual operation and the reaction time are increased, and the possibility that the operator is infected is correspondingly increased. In addition, in the competitive inhibition method, due to the influence of the sample adding time and the sample adding interval, a false positive result is easily caused on the final detection result, and the judgment of the final neutralizing antibody content is influenced.
The magnetic particle chemiluminescence technology is a new analysis method combining a magnetic separation technology, a chemiluminescence technology and an immunoassay technology. The technology makes full use of the rapid and easy automation of the magnetic separation technology, the high sensitivity of the chemiluminescence technology and the specificity of immunoassay, and is the most popular labeling immunoassay technology at present.
In the prior art, the preparation of a magnetic microsphere detection kit for a novel one-step method coronavirus neutralizing antibody based on a magnetic particle chemiluminescence method and the application thereof are not reported on the basis of the principle that the interaction between horseradish peroxidase-labeled RBD protein (HRP-RBD) and recombinant human ACE2 receptor protein (ACE2-hFc) can be blocked by a SARS-CoV-2RBD neutralizing antibody.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a one-step method novel coronavirus neutralizing antibody magnetic microsphere detection kit and application thereof.
The invention relates to a one-step method novel magnetic microsphere detection kit for coronavirus neutralizing antibodies, which consists of magnetic microspheres coupled with recombinant human ACE2 receptor protein (ACE2-hFc), RBD neutralizing antibody standard solution serving as positive control, negative control, sample diluent, an RBD protein (HRP-RBD) enzyme conjugate, 20 multiplied concentrated washing solution and luminous solution, wherein the magnetic microspheres are arranged in a box body;
the method is characterized in that:
the magnetic microsphere is coupled with recombinant human ACE2 receptor protein (ACE2-hFc) with the concentration of 5-100 mug/mg; the working concentration of the magnetic microspheres is 0.2-0.5mg/mL, and the diluent is a preservation solution;
the standard solution of the RBD neutralizing antibody is as follows: diluting the sample diluent with RBD neutralizing antibody solution with the concentration of 1000 ng/ml;
the negative control is a negative serum of a normal human new coronavirus SARS-CoV-2 antibody;
the formula of the sample diluent is as follows: sterilized 1000mL of pure water containing 8g of NaCl and NaH2PO4·2H2O 0.2g、 Na2HPO4·12H2O2.9 g, SDS 1g, sodium caseinate 5g, TritonX-1000.1 mL, Proclin 3001 mL, pH 7.4;
the RBD enzyme conjugate is horse radish peroxidase-labeled recombinant RBD protein (HRP-RBD); the using amount of the HRP-labeled RBD is just saturated when the RBD is combined with the coating antigen, the using titer concentration of the RBD enzyme conjugate is 1:10000, and the diluent is an enzyme conjugate diluent; the preparation method of the RBD enzyme conjugate comprises the following steps: the RBD protein is labeled by a sodium periodate method and is combined with horseradish peroxidase. The preparation method is not limited to this method, and other protein labeling methods are also possible.
The formula of the 20 multiplied concentrated washing solution is as follows: sterilized 50mL double distilled water containing 8.0g NaCl and NaH2PO4·2H2O 0.2g、 Na2HPO4·12H2O2.9 g and Tween 200.5 mL;
the luminescent solution comprises solution A and solution B, and the mixed solution of 3.0mmol/L luminol solution and 0.3mmol/L paraiodophenol solution in equal volume ratio is named as solution A; hydrogen peroxide with the concentration of 7.5mmol/L is named as solution B; the solution A and solution B were mixed at a volume ratio of 1: 1.
In the one-step method novel magnetic microsphere detection kit for coronavirus neutralizing antibodies, the preparation method of the magnetic microspheres comprises the following steps:
1.1) preparing a coupling buffer: preparing 0.01mol/L MES buffer solution, and adjusting the pH value to 6.0;
MES 1.95g
the purified water is fixed to the volume of 1000mL
Filtering for sterilization, and storing at 4 deg.C;
1.2) preparing a washing liquid, wherein the formula and the preparation method of the washing liquid are as follows:
filtering for sterilization, and storing at 4 deg.C;
1.3) preparing a preservation solution, wherein the formula and the preparation method of the preservation solution are as follows:
filtering for sterilization, and storing at 4 deg.C;
1.4) coupling operation
(1) Activation of carboxyl magnetic microspheres: taking 10mg carboxyl magnetic microspheres, adding 500 mu L EDC/NHS solution to activate for 30min at room temperature, carrying out magnetic separation, and washing the magnetic microspheres by coupling buffer solution to obtain activated magnetic microspheres;
wherein the EDC is: 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, said NHS being: the solution of the N-hydroxysuccinimide is prepared by using a coupling buffer solution as a solvent, the concentration of the N-hydroxysuccinimide is 2mg/mL, and the EDC solution and the NHS solution are uniformly mixed in an equal volume ratio before use to obtain an EDC/NHS solution;
(2) coupling of carboxyl magnetic microspheres with ACE 2-hFc: adding 1-10mg/mL ACE2-hFc into the activated magnetic microspheres to make the concentration of the magnetic microsphere coupling ACE2-hFc be 5-100 mug/mg, reacting for 3h at room temperature, magnetically separating, washing with a washing solution to obtain immune magnetic microspheres, and adding a preservation solution to preserve at 4 ℃ for later use; when in use, the magnetic microspheres are diluted by using the preservation solution to ensure that the working concentration of the magnetic microspheres is 0.2-0.5 mg/mL.
In the one-step method novel magnetic microsphere detection kit for coronavirus neutralizing antibodies, the following steps are carried out: the recombinant human ACE2 receptor protein (ACE2-hFc) is a recombinant protein obtained by fusing human ACE2 protein expressed by CHO cells and humanized Fc; the RBD protein is a recombinant protein of a novel coronavirus SARS-CoV-2 full-length RBD expressed by CHO cells; the RBD neutralizing antibody is formed by neutralizing antibody variable region sequences from determined Xinguan convalescent patients, fusing with humanized Fc, and performing recombinant expression by CHO cells.
In the one-step method novel magnetic microsphere detection kit for coronavirus neutralizing antibodies, the following steps are carried out: the magnetic microspheres are coupled with recombinant human ACE2 receptor protein (ACE2-hFc) with the concentration of 60-100 mug/mg preferably; the working concentration of the magnetic microspheres is preferably 0.3-0.5mg/mL, and the diluent is a preservation solution.
In the one-step method novel magnetic microsphere detection kit for coronavirus neutralizing antibodies, the formula of the enzyme conjugate diluent is as follows: sterilized 1000mL of pure water containing 8g of NaCl and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O2.9 g, surfactant S93 g, casein sodium salt 5g, BSA 10g, Proclin 3001 mL.
The invention discloses application of a one-step method novel magnetic microsphere detection kit for coronavirus neutralizing antibodies in detection of biological samples containing the novel coronavirus neutralizing antibodies.
Among them, the preferred method for detecting the sample containing the novel coronavirus neutralizing antibody is as follows:
a) adding a sample of 5 mu L, HRP-RBD 50uL into the reaction cup by using a full-automatic magnetic particle chemiluminescence analyzer;
b) then adding 50 mu L of magnetic microsphere magnetic particle suspension coupled with recombinant human ACE2 receptor protein (ACE2-hFc) and 50 mu L of sample diluent into the reaction cup, wherein the concentration of the magnetic microsphere magnetic particle suspension is 0.2-0.5 mg/mL; the positive control and the negative control are carried out in the same operation step;
c) mixing, and incubating at 37 deg.C for 15 min;
d) washing with cleaning solution for 4-5 times by using a magnetic separation frame or a magnetic separator;
e) adding 50 mu L of luminescent substrate A liquid and luminescent substrate B liquid into each reaction cup;
f) detecting the luminous intensity 1-5 minutes after mixing;
g) reading RLU values of the sample, the positive control and the negative control;
h) the positive and negative cutoff detected by the SARS-CoV-2 neutralizing antibody can be used to account for the inhibition rate;
wherein, the negative reference substance and RLU of the sample in the single detection are used for calculating the antibody inhibition rate in the serum of the sample to be detected, and the positive reference substance RLU is only used for judging the detection effectiveness; the method for calculating the antibody inhibition rate in the sample to be detected comprises the following steps:
inhibition ═ 100% (1-sample RLU/negative control RLU) ×
And (4) result judgment standard:
the inhibition rate is more than or equal to 20%: positive, inhibition < 20%: and (4) negativity.
The invention discloses application of a one-step method novel magnetic microsphere detection kit for coronavirus neutralizing antibodies and application of indexes in neutralizing antibody evaluation of a new corona vaccine inoculated population and a new corona infected rehabilitation population.
The one-step method novel coronavirus neutralizing antibody magnetic microsphere detection kit provided by the invention is prepared based on a magnetic particle chemiluminescence method, realizes machine automation in the detection process, has the characteristics of high flux, high sensitivity, good specificity, good repeatability and the like, can be used for carrying out a rapid neutralizing antibody detection method in a biosafety secondary laboratory or a common laboratory, and is beneficial to epidemiological investigation of epidemic diseases, immune antibody monitoring and vaccine immunity efficacy evaluation.
The detection principle of the one-step method novel magnetic microsphere detection kit for the coronavirus neutralizing antibody is as follows: mimicking the virus neutralization process, the interaction between HRP-RBD and ACE2-hFc can be blocked by a SARS-CoV-2RBD neutralizing antibody. The preparation method comprises the steps of coating magnetic microspheres with recombinant human ACE2 receptor protein (ACE2-hFc), preparing enzyme conjugates with horseradish peroxidase-labeled RBD protein (HRP-RBD), sequentially adding a sample, the HRP-RBD and the recombinant human ACE2 receptor protein (ACE2-hFc) coated magnetic microspheres into a reaction cup by using a full-automatic magnetic particle immunoassay analyzer in one step, carrying out warm bath at 37 ℃ for 15 minutes, adding luminescent liquid after washing, carrying out warm bath for 3 minutes, and reading a luminescent value. The luminescence value is inversely proportional to the effective concentration of RBD neutralizing antibodies in the sample.
The invention has the beneficial effects that: the method has the advantages of high stability, high sensitivity, strong selectivity, high detection speed, low cost, easy operation and the like. The method overcomes the defects of complex operation, long operation time, easy influence of sample adding time on results and the like when detecting the novel coronavirus neutralizing antibody in the prior art, and shortens the operation time from 120 minutes to 20 minutes. Has great clinical application prospect.
Drawings
FIG. 1: the one-step method novel coronavirus neutralizing antibody magnetic microsphere detection kit disclosed by the invention is used for fitting a curve.
Detailed Description
The present invention will be described in detail with reference to the following detailed drawings and examples. The following examples are only preferred embodiments of the present invention, and it should be noted that the following descriptions are only for explaining the present invention and not for limiting the present invention in any form, and any simple modifications, equivalent changes and modifications made to the embodiments according to the technical spirit of the present invention are within the scope of the technical solution of the present invention.
In the following examples, materials, reagents and the like used were obtained commercially unless otherwise specified.
Example 1: preparation of one-step method novel magnetic microsphere detection kit for coronavirus neutralizing antibody
Preparing magnetic microspheres:
1.1) preparing a coupling buffer: preparing 0.01mol/L MES buffer solution, and adjusting the pH value to 6.0;
MES 1.95g
the purified water is fixed to the volume of 1000mL
Filtering for sterilization, and storing at 4 deg.C;
1.2) preparing a washing liquid, wherein the formula and the preparation method of the washing liquid are as follows:
filtering for sterilization, and storing at 4 deg.C;
1.3) preparing a preservation solution, wherein the formula and the preparation method of the preservation solution are as follows:
filtering for sterilization, and storing at 4 deg.C;
1.4) coupling operation
(1) Activation of carboxyl magnetic microspheres: taking 10mg carboxyl magnetic microspheres, adding 500 mu L EDC/NHS solution to activate for 30min at room temperature, carrying out magnetic separation, and washing the magnetic microspheres by coupling buffer solution to obtain activated magnetic microspheres;
wherein the EDC is: 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, said NHS being: the solution of the N-hydroxysuccinimide is prepared by using a coupling buffer solution as a solvent, the concentration of the N-hydroxysuccinimide is 2mg/mL, and the EDC solution and the NHS solution are uniformly mixed in an equal volume ratio before use to obtain an EDC/NHS solution;
(2) coupling of carboxyl magnetic microspheres with ACE 2-hFc: adding 1-10mg/mL ACE2-hFc into the activated magnetic microspheres to make the concentration of the magnetic microsphere coupling ACE2-hFc be 5-100 mug/mg, reacting for 3h at room temperature, magnetically separating, washing with washing solution to obtain immune magnetic microspheres, adding a preservation solution, preserving at 4 ℃ for later use; when in use, the magnetic microspheres are diluted by a preservation solution to ensure that the working concentration of the magnetic microspheres is 0.2-0.5 mg/mL.
(II) preparation of a kit solution:
the sample diluent formula is:
adjusting pH to 7.4, filtering for sterilization, and storing at 4 deg.C;
the formula of 20 × concentrated washing solution is: sterilized 50mL double distilled water containing 8.0g NaCl and NaH2PO4.2H2O 0.2g、Na2HPO4·12H2O2.9 g and Tween 200.5 mL;
the negative control is the negative serum of the antibody of the normal human new coronavirus SARS-CoV-2;
RBD neutralizing antibody standard solutions (positive controls) were: diluting the sample diluent with RBD neutralizing antibody solution with the concentration of 1000 ng/ml;
the preparation method of the recombinant human ACE2 receptor protein comprises the steps of utilizing human ACE2 protein expressed by CHO cells and recombinant ACE2-hFc fused with humanized Fc; the preparation process is a method well known to those skilled in the biological field.
The coating amount of the recombinant human ACE2-hFc protein is selected by a chessboard method according to the sensitivity and the detection range.
The RBD protein preparation method is a recombinant protein of a novel coronavirus SARS-CoV-2 full-length RBD expressed by CHO cells; the preparation process is a method well known to those skilled in the biological field.
The RBD neutralizing antibody is prepared by neutralizing antibody variable region sequence from new crown rehabilitation patient and fusing with humanized Fc, and performing recombinant expression by CHO cell.
The preparation method of the RBD enzyme conjugate comprises the following steps: the RBD protein is labeled by a sodium periodate method and is combined with Horse Radish Peroxidase (HRP). The preparation method is not limited to this method, and other protein labeling methods are also possible.
Optimization of usage amount of HRP-labeled novel crown spike protein RBD: adding HRP-labeled RBD with different concentrations into a magnetic microsphere solution of ACE2-hFc, and selecting the using amount of the HRP-labeled RBD as the just saturated amount of the HRP-labeled RBD combined with the coating antigen. The preferred use titer concentration is 1: 10000.
The formulation of the enzyme conjugate diluent was: sterilized 1000mL of pure water containing 8g of NaCl and NaH2PO4·2H2O 0.2g、 Na2HPO4·12H2O2.9 g, surfactant S93 g, casein sodium salt 5g, BSA 10g, Proclin 3001 mL;
the luminescent solution comprises solution A and solution B, and the mixed solution of 3.0mmol/L luminol solution and 0.3mmol/L p-iodophenol solution in equal volume ratio is named as solution A; hydrogen peroxide with the concentration of 7.5mmol/L is named as solution B; the solution A and solution B were mixed at a volume ratio of 1: 1.
(III) a one-step method novel magnetic microsphere detection kit for coronavirus neutralizing antibodies, which consists of magnetic microspheres coupled with recombinant human ACE2 receptor protein (ACE2-hFc), RBD neutralizing antibody standard solution serving as positive control, negative control, sample diluent, RBD protein (HRP-RBD) enzyme conjugate, 20 Xconcentrated washing solution and luminescent solution, wherein the magnetic microspheres are arranged in a box body;
wherein: the magnetic microsphere is coupled with recombinant human ACE2 receptor protein (ACE2-hFc) with the concentration of 5-100 mug/mg; the working concentration of the magnetic microspheres is 0.2-0.5mg/mL, and the diluent is a preservation solution; preferably: the magnetic microsphere is coupled with recombinant human ACE2 receptor protein (ACE2-hFc) with the concentration of 60-100 mug/mg; the working concentration of the magnetic microspheres is 0.3-0.5mg/mL, and the diluent is a preservation solution. The RBD enzyme conjugate is horse radish peroxidase-labeled recombinant RBD protein (HRP-RBD); the dosage of the HRP-labeled RBD is just saturated when the RBD-labeled RBD is combined with the coating antigen, the titer concentration of the RBD enzyme conjugate is 1:10000, and the diluent is the diluent of the enzyme conjugate.
Example 2: the invention discloses application of a one-step method novel magnetic microsphere detection kit for coronavirus neutralizing antibodies in detection of biological samples containing the novel coronavirus neutralizing antibodies.
Among them, the preferred method for detecting the sample containing the novel coronavirus neutralizing antibody is as follows:
a) adding a sample of 5 mu L, HRP-RBD 50uL into the reaction cup by using a full-automatic magnetic particle chemiluminescence analyzer;
b) then adding 50 mu L of magnetic microsphere magnetic particle suspension coupled with recombinant human ACE2 receptor protein (ACE2-hFc) and 50 mu L of sample diluent into the reaction cup, wherein the concentration of the magnetic microsphere magnetic particle suspension is 0.2-0.5 mg/mL; the positive control and the negative control are carried out in the same operation step;
c) mixing, and incubating at 37 deg.C for 15 min;
d) washing with cleaning solution for 4-5 times by using a magnetic separation frame or a magnetic separator;
e) adding 50 mu L of luminescent substrate A liquid and luminescent substrate B liquid into each reaction cup;
f) detecting the luminous intensity 1-5 minutes after mixing;
g) reading RLU values of the sample, the positive control and the negative control;
h) the positive and negative cutoff detected by the SARS-CoV-2 neutralizing antibody can be used to account for the inhibition rate;
wherein, the negative reference substance and RLU of the sample in the single detection are used for calculating the antibody inhibition rate in the serum of the sample to be detected, and the positive reference substance RLU is only used for judging the detection effectiveness; the method for calculating the antibody inhibition rate in the sample to be detected comprises the following steps:
inhibition ═ 100% (1-sample RLU/negative control RLU) ×
And (4) result judgment standard:
the inhibition rate is more than or equal to 20%: positive, inhibition < 20%: and (4) negativity.
Example 3: the one-step method novel magnetic microsphere detection kit for the coronavirus neutralizing antibody detects linearity, accuracy and precision
1) Linearity of the kit
(1) Preparing a calibration product: RBD neutralizing antibodies were diluted to 10ng/mL, 20ng/mL, 50ng/mL, 100ng/mL, 250ng/mL, 500ng/mL, 1000ng/mL with sample dilutions, respectively.
(2) And (3) testing a calibration sample: detecting the prepared calibration substance by the method described in the embodiment 2 to obtain an RLU value;
(3) establishment of a standard curve: a calibration curve for the batch of reagents was generated using a suitable fit based on the prepared concentration of the calibrator and the RLU values. See table 1 and figure 1.
TABLE 1 kit linearity
| Concentration of | 0ng/ml | 10ng/ml | 20ng/ml | 50ng/ml | 100ng/ml | 250ng/ml | 500ng/ml | 1000ng/ml |
| RLU | 125093874 | 123920380 | 105139201 | 83559280 | 46201648 | 17920380 | 3210381 | 10384 |
| Inhibition rate | - | 1％ | 16％ | 33％ | 63％ | 86％ | 97％ | 100％ |
2) Precision test:
the standard substance of the RBD neutralizing antibody with the concentration of 100ng/mL is tested 10 times in parallel, and the result is shown in the table 2.
Table 2: results of precision test
Precision: CV% ═ 3.02%
Example 4: application of one-step method novel magnetic microsphere detection kit for coronavirus neutralizing antibody in clinical sample screening of injection vaccine
Comparing 200 cases of immune samples with clinical injection of new corona vaccines and 500 cases of immune samples without injection of new corona vaccines with clinical cell neutralization experiments, calculating statistical indexes of coincidence or difference degrees of the detection results and the neutralization experiments of the kit, and evaluating methods are shown in table 3.
TABLE 3 comparison of the kit of the invention with the cell neutralization assay
And (3) sensitivity calculation: 190/(190+7) × 100% ═ 96.4%.
And (3) calculating the specificity: 499/(499+4) × 100%
The total coincidence rate is as follows: (190+499)/(190+7+499+4) × 100%
The tests show that the kit for detecting the novel coronavirus neutralizing antibody and the detection method thereof have good consistency with the detection gold standard of the neutralizing antibody, can be used for evaluating the generation of the neutralizing antibody after the novel coronavirus vaccine is inoculated, have good sensitivity and specificity, and are beneficial to accurately evaluating the vaccine effect clinically.
Claims (7)
1. A one-step method novel coronavirus neutralizing antibody magnetic microsphere detection kit comprises magnetic microspheres coupled with recombinant human ACE2 receptor protein (ACE2-hFc), RBD neutralizing antibody standard solution used as positive control, negative control, sample diluent, RBD protein (HRP-RBD) enzyme conjugate, 20 x concentrated washing solution and luminous solution, wherein the magnetic microspheres are arranged in a box body;
the method is characterized in that:
the magnetic microsphere is coupled with recombinant human ACE2 receptor protein (ACE2-hFc) with the concentration of 5-100 mug/mg; the working concentration of the magnetic microspheres is 0.2-0.5mg/mL, and the diluent is a preservation solution;
the standard solution of the RBD neutralizing antibody is as follows: diluting the sample diluent with RBD neutralizing antibody solution with the concentration of 1000 ng/ml;
the negative control is a negative serum of a normal human new coronavirus SARS-CoV-2 antibody;
the formula of the sample diluent is as follows: sterilized 1000mL of pure water containing 8g of NaCl and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O2.9 g, SDS 1g, sodium caseinate 5g, TritonX-1000.1 mL, Proclin 3001 mL, pH 7.4;
the RBD enzyme conjugate is horse radish peroxidase-labeled recombinant RBD protein (HRP-RBD); the using amount of the HRP-labeled RBD is just saturated when the RBD is combined with the coating antigen, the using titer concentration of the RBD enzyme conjugate is 1:10000, and the diluent is an enzyme conjugate diluent;
the formula of the 20 multiplied concentrated washing solution is as follows: sterilized 50mL double distilled water containing 8.0g NaCl and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O2.9 g and Tween 200.5 mL;
the luminescent solution comprises solution A and solution B, and the mixed solution of 3.0mmol/L luminol solution and 0.3mmol/L paraiodophenol solution in equal volume ratio is named as solution A; hydrogen peroxide with the concentration of 7.5mmol/L is named as solution B; the solution A and solution B were mixed at a volume ratio of 1: 1.
2. The one-step method magnetic microsphere detection kit for the novel coronavirus neutralizing antibody according to claim 1, wherein the preparation method of the magnetic microsphere comprises the following steps:
1.1) preparing a coupling buffer: preparing 0.01mol/L MES buffer solution, and adjusting the pH value to 6.0;
MES 1.95g
the purified water is fixed to the volume of 1000mL
Filtering for sterilization, and storing at 4 deg.C;
1.2) preparing a washing liquid, wherein the formula and the preparation method of the washing liquid are as follows:
filtering for sterilization, and storing at 4 deg.C;
1.3) preparing a preservation solution, wherein the formula and the preparation method of the preservation solution are as follows:
filtering for sterilization, and storing at 4 deg.C;
1.4) coupling operation
(1) Activation of carboxyl magnetic microspheres: taking 10mg carboxyl magnetic microspheres, adding 500 mu L EDC/NHS solution to activate for 30min at room temperature, carrying out magnetic separation, and washing the magnetic microspheres by coupling buffer solution to obtain activated magnetic microspheres;
wherein the EDC is: 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, said NHS being: the solution of the N-hydroxysuccinimide is prepared by using a coupling buffer solution as a solvent, the concentration of the N-hydroxysuccinimide is 2mg/mL, and the EDC solution and the NHS solution are uniformly mixed in an equal volume ratio before use to obtain an EDC/NHS solution;
(2) coupling of carboxyl magnetic microspheres with ACE 2-hFc: adding 1-10mg/mL ACE2-hFc into the activated magnetic microspheres to make the concentration of the magnetic microsphere coupling ACE2-hFc be 5-100 mug/mg, reacting for 3h at room temperature, magnetically separating, washing with a washing solution to obtain immune magnetic microspheres, and adding a preservation solution to preserve at 4 ℃ for later use; when in use, the magnetic microspheres are diluted by a preservation solution to ensure that the working concentration of the magnetic microspheres is 0.2-0.5 mg/mL.
3. The one-step method magnetic microsphere detection kit for novel coronavirus neutralizing antibodies according to claim 1, which is characterized in that: the recombinant human ACE2 receptor protein (ACE2-hFc) is a recombinant protein obtained by fusing human ACE2 protein expressed by CHO cells and humanized Fc; the RBD protein is a recombinant protein of a novel coronavirus SARS-CoV-2 full-length RBD expressed by CHO cells; the RBD neutralizing antibody is formed by neutralizing antibody variable region sequences from determined Xinguan convalescent patients, fusing with humanized Fc, and performing recombinant expression by CHO cells.
4. The one-step novel coronavirus neutralizing antibody magnetic microsphere detection kit as claimed in claim 1, wherein the magnetic microsphere is coupled with recombinant human ACE2 receptor protein (ACE2-hFc) at a concentration of 60-100 μ g/mg; the working concentration of the magnetic microspheres is 0.3-0.5mg/mL, and the diluent is a preservation solution.
5. The one-step method magnetic microsphere detection kit for novel coronavirus neutralizing antibodies according to claim 1, wherein the formula of the enzyme conjugate diluent is as follows: sterilized 1000mL of pure water containing 8g of NaCl and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O2.9 g, surfactant S93 g, casein sodium salt 5g, BSA 10g, Proclin 3001 mL.
6. Use of the one-step magnetic microsphere detection kit for novel coronavirus neutralizing antibodies according to claim 1 for detecting biological samples containing novel coronavirus neutralizing antibodies.
7. The use according to claim 6, wherein the method for detecting a sample containing neutralizing antibodies against a novel coronavirus is:
a) adding a sample of 5 mu L, HRP-RBD 50uL into the reaction cup by using a full-automatic magnetic particle chemiluminescence analyzer;
b) then adding 50 mu L of magnetic microsphere magnetic particle suspension coupled with recombinant human ACE2 receptor protein (ACE2-hFc) and 50 mu L of sample diluent into the reaction cup, wherein the concentration of the magnetic microsphere magnetic particle suspension is 0.2-0.5 mg/mL; the positive control and the negative control are carried out in the same operation step;
c) mixing, and incubating at 37 deg.C for 15 min;
d) washing with cleaning solution for 4-5 times by using a magnetic separation frame or a magnetic separator;
e) adding 50 mu L of luminescent substrate A liquid and luminescent substrate B liquid into each reaction cup;
f) detecting the luminous intensity 1-5 minutes after mixing;
g) reading RLU values of the sample, the positive control and the negative control;
h) the positive and negative cutoff detected by the SARS-CoV-2 neutralizing antibody can be used to account for the inhibition rate;
wherein, the negative reference substance and RLU of the sample in the single detection are used for calculating the antibody inhibition rate in the serum of the sample to be detected, and the positive reference substance RLU is only used for judging the detection effectiveness; the method for calculating the antibody inhibition rate in the sample to be detected comprises the following steps:
inhibition ═ 100% (1-sample RLU/negative control RLU) ×
And (4) result judgment standard:
the inhibition rate is more than or equal to 20%: positive, inhibition < 20%: and (4) negativity.
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