CN113009154B - Novel one-step method coronavirus neutralizing antibody magnetic microsphere detection kit and application thereof - Google Patents
Novel one-step method coronavirus neutralizing antibody magnetic microsphere detection kit and application thereof Download PDFInfo
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Classifications
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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Abstract
The invention discloses a novel one-step method coronavirus neutralizing antibody magnetic microsphere detection kit, which consists of magnetic microspheres coupled with recombinant human ACE2 receptor protein (ACE 2-hFc), RBD neutralizing antibody standard solution serving as positive control, negative control, sample diluent, RBD protein (HRP-RBD) enzyme conjugate, 20 multiplied by concentrated washing solution and luminous solution, wherein the magnetic microspheres are arranged in the kit body. The invention also discloses application of the kit in detecting biological samples containing novel coronavirus neutralizing antibodies. 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
New coronapneumonia (COVID-19) pandemic caused by the new coronavirus (SARS-CoV-2) has been the most serious challenge faced by humans for centuries. The common signs of 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.
Studies have shown that coronavirus S protein is a key factor in viral virulence, a key component in determining viral virulence, tissue tropism and host range, and is also a primary target for neutralizing antibody and vaccine design. And evaluating the effect after vaccination, the most direct detection method is detection of the new crown neutralizing antibody. Because, neutralizing antibodies that recognize the RBD region (receptor binding domain) on the S (spike) protein of the new coronavirus block ACE2 receptor binding on the new coronavirus and human cells, the new coronavirus cannot infect human cells. In addition, neutralizing antibodies can interact with other immune components, such as complement, phagocytes, natural killer cells, and the like. Both of these effector responses can help to clear viruses and infected cells. The higher the concentration of neutralizing antibodies, the better the protection generally. Thus, one of the early indicators of vaccine quality is to see how much neutralizing antibodies it can induce in humans.
Traditional methods for detecting neutralizing antibodies are live or pseudovirus neutralization assays. The test method needs a professional cell culture process, has high requirements on experimental grades, has long period and complex operation, and is not suitable for high-throughput screening of common vaccinated people.
The titer of the neutralizing antibody can reflect the index of the antibody level in the blood sample, i.e. the quality or intensity of the immune response in the human body after vaccination. With the market of new coronavirus vaccines, a rapid, reliable, stable and safe evaluation method is needed for evaluating neutralizing antibodies in the population vaccinated with the new coronavirus vaccine and the recovered population after the new coronavirus infection. At present, a novel method for detecting the competitive inhibition of coronavirus neutralizing antibody enzyme-linked immunity (CN202010919464. X, CN 202010952237.5) exists, but the experimental detection methods disclosed in the special application belong to the enzyme-linked immunity method, and have the defects of long operation time (2-3 hours), manual operation and the like in the experimental operation. Because the new coronavirus has strong infectivity, the manual operation and the reaction time are increased, and the possibility of the operators being infected is correspondingly increased. In addition, in the competitive inhibition method, false positive results are easily caused to the final detection result due to the influence of the sample adding time and the sample adding interval, and the final judgment of the content of the neutralizing antibody is influenced.
The magnetic particle chemiluminescence technology is an emerging analysis method combining a magnetic separation technology, a chemiluminescence technology and an immunoassay technology. The technology fully utilizes the rapid and easy automation of the magnetic separation technology, the high sensitivity of the chemiluminescence technology and the specificity of the immunoassay, and is the most popular marking immunoassay technology at present.
In the prior art, the principle that the interaction between horseradish peroxidase labeled RBD protein (HRP-RBD) and recombinant human ACE2 receptor protein (ACE 2-hFc) can be blocked by SARS-CoV-2RBD neutralizing antibody is searched, and a one-step method novel coronavirus neutralizing antibody magnetic microsphere detection kit based on a magnetic particle chemiluminescence method and application thereof are prepared.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a novel one-step method coronavirus neutralizing antibody magnetic microsphere detection kit and application thereof.
The invention relates to a novel one-step method coronavirus neutralizing antibody magnetic microsphere detection kit, which consists of magnetic microspheres coupled with recombinant human ACE2 receptor protein (ACE 2-hFc), RBD neutralizing antibody standard solution serving as positive control, negative control, sample diluent, RBD protein (HRP-RBD) enzyme conjugate, 20 multiplied by concentrated washing solution and luminous solution, wherein the magnetic microspheres are arranged in the kit;
the method is characterized in that:
the magnetic microsphere is coupled with recombinant human ACE2 receptor protein (ACE 2-hFc) with the concentration of 5-100 mug/mg; the working concentration of the magnetic microsphere is 0.2-0.5mg/mL, and the diluent is preservation solution;
the RBD neutralizing antibody standard solution is as follows: an RBD neutralizing antibody solution with a concentration of 1000ng/ml diluted by the sample diluent;
the negative control is the negative serum of the SARS-CoV-2 antibody of the new coronavirus of the normal human;
the formula of the sample diluent is as follows: the sterilized 1000mL pure water contains 8g NaCl and NaH 2 PO 4 ·2H 2 O 0.2g、 Na 2 HPO 4 ·12H 2 O2.9 g, SDS 1g, casein sodium salt 5g, triton X-100.1 mL, proclin300 1mL, pH 7.4;
the RBD enzyme conjugate is horseradish peroxidase-labeled recombinant RBD protein (HRP-RBD); the HRP-labeled RBD is used in an amount which is just saturated when the RBD is combined with the coating antigen, the titer concentration of the RBD enzyme conjugate is 1:10000, and the diluent is enzyme conjugate diluent; the preparation method of the RBD enzyme conjugate comprises the following steps: a conjugate of RBD protein marked by sodium periodate method and horseradish peroxidase. The preparation method is not limited to this method, and other protein labeling methods are also possible.
The formula of the 20 x concentrated washing liquid is as follows: the sterilized 50mL double distilled water contains 8.0g NaCl and NaH 2 PO 4 ·2H 2 O 0.2g、 Na 2 HPO 4 ·12H 2 2.9g of O and 0.5mL of Tween 20;
the luminous solution comprises solution A and solution B, wherein the mixed solution of luminol solution with the concentration of 3.0mmol/L and p-iodophenol solution with the equal volume ratio of 0.3mmol/L is named as solution A; the hydrogen peroxide with the concentration of 7.5mmol/L is named as solution B; when in use, the solution A and the solution B are mixed according to the volume ratio of 1:1.
In the magnetic microsphere detection kit for the novel coronavirus neutralizing antibody by the one-step method, the preparation method of the magnetic microsphere comprises the following steps:
1.1 Preparing a coupling buffer solution: namely, preparing 0.01mol/L MES buffer solution, and adjusting the pH value to 6.0;
MES 1.95g
the volume of the purified water is fixed to 1000mL
Filtering and sterilizing, and preserving at 4 ℃;
1.2 Preparing a washing liquid, wherein the formula and the preparation method of the washing liquid are as follows:
filtering and sterilizing, and preserving at 4 ℃;
1.3 A preservation solution is prepared, and the formula and the preparation method of the preservation solution are as follows:
filtering and sterilizing, and preserving at 4 ℃;
1.4 Coupling operation)
(1) Activation of carboxyl magnetic microspheres: taking 10mg of carboxyl magnetic microspheres, adding 500 mu L of EDC/NHS solution, activating for 30min at room temperature, performing magnetic separation, and washing the magnetic microspheres with coupling buffer solution to obtain activated magnetic microspheres;
wherein the EDC is: 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, said NHS being: n-hydroxysuccinimide, wherein the solution is prepared by using a coupling buffer solution as a solvent, the concentration of the coupling buffer solution is 2mg/mL, and the EDC and the NHS solution are uniformly mixed in equal volume ratio before use to obtain the EDC/NHS solution;
(2) Carboxyl magnetic microsphere is coupled with ACE 2-hFc: adding ACE2-hFc with the concentration of 1-10mg/mL into the activated magnetic microsphere, enabling the concentration of the magnetic microsphere coupled with the ACE2-hFc to be 5-100 mug/mg, reacting for 3 hours at room temperature, magnetically separating, washing with a washing solution to obtain the immune magnetic microsphere, and adding a preservation solution to preserve at 4 ℃ for later use; when in use, the preservation solution is diluted to ensure that the working concentration of the magnetic microsphere is 0.2-0.5mg/mL.
The magnetic microsphere detection kit for the novel coronavirus neutralizing antibody by the one-step method comprises the following steps: the recombinant human ACE2 receptor protein (ACE 2-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 recombinant expression of the variable region sequence of the neutralizing antibody from a new coronary rehabilitation patient obtained by measurement and fusion with humanized Fc through CHO cells.
The magnetic microsphere detection kit for the novel coronavirus neutralizing antibody by the one-step method comprises the following steps: the magnetic microsphere is coupled with recombinant human ACE2 receptor protein (ACE 2-hFc) with the concentration of preferably 60-100 mug/mg; the working concentration of the magnetic microsphere is preferably 0.3-0.5mg/mL, and the diluent is preservation solution.
In the magnetic microsphere detection kit for the novel coronavirus neutralizing antibody by the one-step method, the formula of the enzyme conjugate diluent is as follows: the sterilized 1000mL pure water contains 8g NaCl and NaH 2 PO 4 ·2H 2 O 0.2g、Na 2 HPO 4 ·12H 2 O2.9 g, surfactant S9 3g, casein sodium salt 5g, BSA 10g and Proclin300 mL.
The invention relates to an application of a one-step method novel coronavirus neutralizing antibody magnetic microsphere detection kit in detecting biological samples containing novel coronavirus neutralizing antibodies.
Among them, the preferred method for detecting a sample containing a novel coronavirus neutralizing antibody is:
a) Adding a sample 5 mu L, HRP-RBD 50uL into the reaction cup by using a full-automatic magnetic particle chemiluminescence analyzer;
b) Adding 50 mu L of magnetic microsphere magnetic particle suspension coupled with recombinant human ACE2 receptor protein (ACE 2-hFc) and 50 mu L of sample diluent into a reaction cup at the concentration of 0.2-0.5 mg/mL; positive control and negative control are performed in the same way;
c) After mixing, incubating for 15 minutes at 37 ℃;
d) Washing with a magnetic separation frame or a magnetic separator for 4-5 times;
e) Adding 50 mu L of luminous substrate A liquid and 50 mu L of luminous substrate B liquid into each reaction cup;
f) Detecting the luminous intensity 1-5 minutes after uniformly mixing;
g) Reading RLU values of the sample, the positive control and the negative control;
h) Positive and negative cutoff for SARS-CoV-2 neutralizing antibody detection can be used to interpret the inhibition rate;
wherein, the RLU of the negative control substance and the sample in the single detection is used for calculating the antibody inhibition rate in the serum of the sample to be detected, and the RLU of the positive control substance is only used for judging the detection effectiveness; the method for calculating the inhibition rate of the antibody in the sample to be detected comprises the following steps:
inhibition = (1-sample RLU/negative control RLU) ×100%
Result determination criteria:
the inhibition rate is more than or equal to 20 percent: positive, inhibition rate < 20%: negative.
The invention relates to an application of a one-step method novel coronavirus neutralizing antibody magnetic microsphere detection kit and an application of the index in neutralizing antibody evaluation of a new coronavirus vaccine inoculated population and a recovered population after new coronavirus infection.
The magnetic microsphere detection kit for the novel coronavirus neutralizing antibody by the one-step method is prepared based on a magnetic particle chemiluminescence method, fully realizes machine automation in the detection process, has the characteristics of high flux, high sensitivity, good specificity, good repeatability and the like, and can be used for carrying out a rapid neutralizing antibody detection method in a biosafety secondary laboratory or a common laboratory, thereby being beneficial to epidemiological investigation, immune antibody monitoring and vaccine immune efficacy evaluation.
The detection principle of the magnetic microsphere detection kit for the novel coronavirus neutralizing antibody by the one-step method is as follows: mimicking the viral neutralization process, the interaction between HRP-RBD and ACE2-hFc may be blocked by SARS-CoV-2RBD neutralizing antibodies. Preparing enzyme conjugate by recombinant human ACE2 receptor protein (ACE 2-hFc) coated magnetic microsphere and horseradish peroxidase labeled RBD protein (HRP-RBD), in one-step reaction, sequentially adding a sample, HRP-RBD and recombinant human ACE2 receptor protein (ACE 2-hFc) coated magnetic microsphere into a reaction cup by using a full-automatic magnetic particle immunoassay instrument, carrying out warm bath for 15 minutes at 37 ℃, washing, adding luminous liquid, carrying out warm bath for 3 minutes, and reading luminous value. The luminescence value is inversely proportional to the effective concentration of RBD neutralizing antibodies in the sample.
The beneficial effects of the invention are as follows: 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 that the operation is complex, the operation time is long, the result is easily influenced by the sample adding time and the like when the novel coronavirus neutralizing antibody is detected 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 of the invention is used for fitting a curve to the magnetic microsphere detection kit for the neutralizing antibody of the coronavirus.
Detailed Description
The present invention will be described in detail with reference to the following drawings and examples. The following examples are only preferred embodiments of the present invention, and it should be noted that the following descriptions are merely for explaining the present invention, and are not limited in any way, and any simple modification, equivalent variation and modification of the embodiments according to the technical principles of the present invention fall within the scope of the technical solutions of the present invention.
In the following examples, materials, reagents and the like used, unless otherwise specified, were obtained commercially.
Example 1: the invention relates to a preparation method of a novel magnetic microsphere detection kit for neutralizing antibodies of coronaviruses by a one-step method
Preparation of magnetic microspheres:
1.1 Preparing a coupling buffer solution: namely, preparing 0.01mol/L MES buffer solution, and adjusting the pH value to 6.0;
MES 1.95g
the volume of the purified water is fixed to 1000mL
Filtering and sterilizing, and preserving at 4 ℃;
1.2 Preparing a washing liquid, wherein the formula and the preparation method of the washing liquid are as follows:
filtering and sterilizing, and preserving at 4 ℃;
1.3 A preservation solution is prepared, and the formula and the preparation method of the preservation solution are as follows:
filtering and sterilizing, and preserving at 4 ℃;
1.4 Coupling operation)
(1) Activation of carboxyl magnetic microspheres: taking 10mg of carboxyl magnetic microspheres, adding 500 mu L of EDC/NHS solution, activating for 30min at room temperature, performing magnetic separation, and washing the magnetic microspheres with coupling buffer solution to obtain activated magnetic microspheres;
wherein the EDC is: 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, said NHS being: n-hydroxysuccinimide, wherein the solution is prepared by using a coupling buffer solution as a solvent, the concentration of the coupling buffer solution is 2mg/mL, and the EDC and the NHS solution are uniformly mixed in equal volume ratio before use to obtain the EDC/NHS solution;
(2) Carboxyl magnetic microsphere is coupled with ACE 2-hFc: adding ACE2-hFc with the concentration of 1-10mg/mL into the activated magnetic microsphere, enabling the concentration of the magnetic microsphere coupled with the ACE2-hFc to be 5-100 mug/mg, reacting for 3 hours at room temperature, magnetically separating, washing with a washing solution to obtain the immune magnetic microsphere, and adding a preservation solution to preserve at 4 ℃ for later use; when in use, the magnetic microsphere is diluted by preservation solution to ensure that the working concentration of the magnetic microsphere is 0.2-0.5mg/mL.
(II) preparation of a kit solution:
the sample diluent formula is:
adjusting pH to 7.4, filtering, sterilizing, and preserving at 4deg.C;
the formula of the 20 x concentrated washing solution is: the sterilized 50mL double distilled water contains 8.0g NaCl and NaH 2 PO 4. 2H 2 O 0.2g、Na 2 HPO 4 ·12H 2 2.9g of O and 0.5mL of Tween 20;
the negative control is the negative serum of SARS-CoV-2 antibody of normal human new coronavirus;
the RBD neutralizing antibody standard solution (positive control) was: an RBD neutralizing antibody solution with a concentration of 1000ng/ml diluted by a sample diluent;
the preparation method of the recombinant human ACE2 receptor protein is to utilize the fusion of the human ACE2 protein expressed by CHO cells and humanized Fc to recombinant ACE2-hFc; the preparation process is a well known method to those skilled in the biological arts.
The coating amount of the recombinant human ACE2-hFc protein is selected by adopting a chessboard method according to the sensitivity and the detection range as an index.
The RBD protein preparation method is a recombinant protein of novel coronavirus SARS-CoV-2 full-length RBD expressed by CHO cells; the preparation process is a well known method to those skilled in the biological arts.
The RBD neutralizing antibodies were prepared by recombinant expression in CHO cells using neutralizing antibody variable region sequences from the new coronary rehabilitation patients assayed and fused with humanized Fc.
The preparation method of the RBD enzyme conjugate comprises the following steps: a conjugate of RBD protein labeled by sodium periodate method and horseradish peroxidase (HRP). The preparation method is not limited to this method, and other protein labeling methods are also possible.
Optimization of HRP-labeled novel coronal spike protein RBD usage: HRP-labeled RBD was added to the ACE2-hFc magnetic microsphere solution at various concentrations, and the HRP-labeled RBD was selected to be used in an amount that was just saturated with the binding of the coating antigen. The preferred use titer concentration is 1:10000.
The formulation of the enzyme conjugate dilutions was: the sterilized 1000mL pure water contains 8g NaCl and NaH 2 PO 4 ·2H 2 O 0.2g、 Na 2 HPO 4 ·12H 2 O2.9 g, surfactant S9 3g, casein sodium salt 5g, BSA 10g and Proclin300 mL;
the luminous solution comprises solution A and solution B, wherein the mixed solution of luminol solution with the concentration of 3.0mmol/L and p-iodophenol solution with the equal volume ratio of 0.3mmol/L is named as solution A; the hydrogen peroxide with the concentration of 7.5mmol/L is named as solution B; when in use, the solution A and the solution B are mixed according to the volume ratio of 1:1.
The novel coronavirus neutralizing antibody magnetic microsphere detection kit comprises magnetic microspheres coupled with recombinant human ACE2 receptor protein (ACE 2-hFc) arranged in a kit body, RBD neutralizing antibody standard solution serving as positive control, negative control, sample diluent, RBD protein (HRP-RBD) enzyme conjugate, 20 multiplied by concentrated washing liquid and luminous liquid;
wherein: the magnetic microsphere is coupled with recombinant human ACE2 receptor protein (ACE 2-hFc) with the concentration of 5-100 mug/mg; the working concentration of the magnetic microsphere is 0.2-0.5mg/mL, and the diluent is preservation solution; preferably: the magnetic microsphere is coupled with recombinant human ACE2 receptor protein (ACE 2-hFc) with the concentration of 60-100 mug/mg; the working concentration of the magnetic microsphere is 0.3-0.5mg/mL, and the diluent is preservation solution. The RBD enzyme conjugate is horseradish peroxidase-labeled recombinant RBD protein (HRP-RBD); the HRP-labeled RBD is used in an amount which is just saturated in its binding to the coating antigen, the RBD enzyme conjugate is used in a titer concentration of 1:10000, and the diluent is an enzyme conjugate diluent.
Example 2: the invention relates to an application of a one-step method novel coronavirus neutralizing antibody magnetic microsphere detection kit in detecting biological samples containing novel coronavirus neutralizing antibodies.
Among them, the preferred method for detecting a sample containing a novel coronavirus neutralizing antibody is:
a) Adding a sample 5 mu L, HRP-RBD 50uL into the reaction cup by using a full-automatic magnetic particle chemiluminescence analyzer;
b) Adding 50 mu L of magnetic microsphere magnetic particle suspension coupled with recombinant human ACE2 receptor protein (ACE 2-hFc) and 50 mu L of sample diluent into a reaction cup at the concentration of 0.2-0.5 mg/mL; positive control and negative control are performed in the same way;
c) After mixing, incubating for 15 minutes at 37 ℃;
d) Washing with a magnetic separation frame or a magnetic separator for 4-5 times;
e) Adding 50 mu L of luminous substrate A liquid and 50 mu L of luminous substrate B liquid into each reaction cup;
f) Detecting the luminous intensity 1-5 minutes after uniformly mixing;
g) Reading RLU values of the sample, the positive control and the negative control;
h) Positive and negative cutoff for SARS-CoV-2 neutralizing antibody detection can be used to interpret the inhibition rate;
wherein, the RLU of the negative control substance and the sample in the single detection is used for calculating the antibody inhibition rate in the serum of the sample to be detected, and the RLU of the positive control substance is only used for judging the detection effectiveness; the method for calculating the inhibition rate of the antibody in the sample to be detected comprises the following steps:
inhibition = (1-sample RLU/negative control RLU) ×100%
Result determination criteria:
the inhibition rate is more than or equal to 20 percent: positive, inhibition rate < 20%: negative.
Example 3: the invention relates to a one-step method for detecting linearity, accuracy and precision of a novel coronavirus neutralizing antibody magnetic microsphere detection kit
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, respectively, with sample dilutions.
(2) And (3) testing a calibration product: the prepared calibration product is detected by the method described in the example 2 to obtain an RLU value;
(3) Establishment of a standard curve: and generating a calibration curve of the batch of reagents by adopting a proper fitting mode according to the preparation concentration of the calibrator and the RLU value. See table 1 and fig. 1.
TABLE 1 linearity of the kit
| 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 100ng/mL RBD neutralizing antibody standard was tested in parallel 10 times and the statistics are shown in Table 2.
Table 2: precision test results
Precision: CV% = 3.02%
Example 4: the invention relates to an application of a one-step method novel coronavirus neutralizing antibody magnetic microsphere detection kit in screening clinical samples of injection vaccines
200 cases of clinical injection new crown vaccine immune samples and 500 cases of non-injection new crown vaccine samples are used for comparison with clinical cell neutralization experiments, statistical indexes of the degree of coincidence or difference between the measurement results of the kit and the neutralization experimental results are calculated, and the evaluation method is shown in table 3.
TABLE 3 comparison of the inventive kit with cell neutralization assay
Sensitivity calculation: 190/(190+7) ×100% =96.4%.
Specificity calculation: 499/(499+4) ×100% =99.2%
Total compliance rate: (190+499)/(190+7+499+4) ×100% =98.4%
The test shows that the kit for detecting the novel coronavirus neutralizing antibody and the detection method thereof have good consistency with the neutralizing antibody detection gold standard, can be used for evaluating the production 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 (5)
1. A novel one-step method coronavirus neutralizing antibody magnetic microsphere detection kit comprises magnetic microspheres coupled with recombinant human ACE2 receptor protein ACE2-hFc, RBD neutralizing antibody standard solution as positive control, negative control, sample diluent, RBD protein HRP-RBD enzyme conjugate, 20X concentrated washing solution and luminous solution;
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 microsphere is 0.2-0.5mg/mL, and the diluent is preservation solution;
the RBD neutralizing antibody standard solution is as follows: an RBD neutralizing antibody solution with a concentration of 1000ng/ml diluted by a sample diluent;
the negative control is the negative serum of the SARS-CoV-2 antibody of the new coronavirus of the normal human;
the formula of the sample diluent is as follows: the sterilized 1000mL pure water contains 8g NaCl and NaH 2 PO 4 ·2H 2 O 0.2g、Na 2 HPO 4 ·12H 2 O2.9 g, SDS 1g, casein sodium salt 5g, triton X-100.1 mL, proclin300 1mL, pH 7.4;
the RBD enzyme conjugate is horseradish peroxidase-labeled recombinant RBD protein HRP-RBD; the HRP-labeled RBD is used in an amount which is just saturated when the RBD is combined with the coating antigen, the titer concentration of the RBD enzyme conjugate is 1:10000, and the diluent is enzyme conjugate diluent;
the formula of the 20 x concentrated washing liquid is as follows: the sterilized 50mL double distilled water contains 8.0g NaCl and NaH 2 PO 4 ·2H 2 O 0.2g、Na 2 HPO 4 ·12H 2 2.9g of O and 0.5mL of Tween 20;
the luminous solution comprises a solution A and a solution B, wherein a mixed solution of luminol solution with the concentration of 3.0mmol/L and a solution of p-iodophenol with the equal volume ratio is named as the solution A; the hydrogen peroxide with the concentration of 7.5mmol/L is named as solution B; when in use, the solution A and the solution B are mixed according to the volume ratio of 1:1.
2. The one-step method novel coronavirus neutralizing antibody magnetic microsphere detection kit according to claim 1, wherein the preparation method of the magnetic microsphere is as follows:
1.1 Preparing a coupling buffer solution: namely, preparing 0.01mol/L MES buffer solution, and adjusting the pH value to 6.0;
MES 1.95g
the volume of the purified water is fixed to 1000mL
Filtering and sterilizing, and preserving at 4 ℃;
1.2 Preparing a washing liquid, wherein the formula and the preparation method of the washing liquid are as follows:
NaCl 8.0g
NaH 2 PO 4 0.2g
Na 2 HPO 4 •12H 2 O 2.9g
tween 20.5 mL
The volume of the purified water is fixed to 1000mL
Filtering and sterilizing, and preserving at 4 ℃;
1.3 A preservation solution is prepared, and the formula and the preparation method of the preservation solution are as follows:
NaCl 8.0g
NaH 2 PO 4 ·2H 2 O 0.2g
Na 2 HPO 4 •12H 2 O 2.9g
sucrose 30g
BSA 10g
Casein sodium salt 10g
Tween 20.5 mL
The volume of the purified water is fixed to 1000mL
Filtering and sterilizing, and preserving at 4 ℃;
1.4 Coupling operation)
(1) Activation of carboxyl magnetic microspheres: taking 10mg of carboxyl magnetic microspheres, adding 500 mu L of EDC/NHS solution, activating for 30min at room temperature, performing magnetic separation, and washing the magnetic microspheres with coupling buffer solution to obtain activated magnetic microspheres;
wherein the EDC is: 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, said NHS being: n-hydroxysuccinimide, wherein the solution is prepared by using a coupling buffer solution as a solvent, the concentration of the coupling buffer solution is 2mg/mL, and the EDC and the NHS solution are uniformly mixed in equal volume ratio before use to obtain the EDC/NHS solution;
(2) Carboxyl magnetic microsphere is coupled with ACE 2-hFc: adding ACE2-hFc with the concentration of 1-10mg/mL into the activated magnetic microsphere, enabling the concentration of the magnetic microsphere coupled with the ACE2-hFc to be 5-100 mug/mg, reacting for 3 hours at room temperature, magnetically separating, washing with a washing solution to obtain the immune magnetic microsphere, and adding a preservation solution to preserve at 4 ℃ for later use; when in use, the magnetic microsphere is diluted by preservation solution to ensure that the working concentration of the magnetic microsphere is 0.2-0.5mg/mL.
3. The one-step method novel coronavirus neutralizing antibody magnetic microsphere detection kit according to claim 1, wherein: 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 recombinant expression of the variable region sequence of the neutralizing antibody from a new coronary rehabilitation patient obtained by measurement and fusion with humanized Fc through CHO cells.
4. The one-step method novel coronavirus neutralizing antibody magnetic microsphere detection kit according to claim 1, wherein the magnetic microsphere is coupled with recombinant human ACE2 receptor protein ACE2-hFc with the concentration of 60-100 μg/mg; the working concentration of the magnetic microsphere is 0.3-0.5mg/mL, and the diluent is preservation solution.
5. The one-step method of magnetic microsphere assay kit for neutralizing antibodies to coronaviruses according to claim 1, wherein the enzyme conjugate diluent is formulated as follows: the sterilized 1000mL pure water contains 8g NaCl and NaH 2 PO 4 ·2H 2 O 0.2g、Na 2 HPO 4 ·12H 2 O2.9 g, surfactant S9 3g, casein sodium salt 5g, BSA 10g and Proclin300 mL.
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