CN114062677A - Kit for detecting neutralizing antibody of new coronavirus and preparation method thereof - Google Patents
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Abstract
The invention relates to the technical field of medical detection, in particular to a kit for detecting a neutralizing antibody of a new coronavirus and a preparation method thereof. The kit detects the neutralizing antibody in a serum or plasma sample by adopting the technical principle that the enzyme-labeled neutralizing antibody competes with the antibody in the sample to be detected for binding with the S protein antigen. The invention provides a full-automatic high-flux detection method based on the kit, the method is based on a full-automatic immunoassay instrument platform, can realize the rapid, quantitative, high-flux and automatic detection of the neutralizing antibody of the new coronavirus, can carry out detection in a clinical laboratory, reports results within 20 minutes, and can detect 200 samples per hour.
Description
Technical Field
The invention relates to the technical field of medical detection, in particular to a kit for detecting a neutralizing antibody of a new coronavirus and a preparation method thereof.
Background
The novel coronavirus (SARS-CoV-2) belongs to the genus of coronavirus beta, is a single-stranded positive-strand RNA virus, the virus particles have typical coronavirus characteristics, the diameter is 100-150 nm, the virus particles are spherical, elliptical or polymorphic, and the surface of an envelope is covered with a unique edge formed by rodlike protrusions with wide intervals. The infection source is mainly a patient infected by the novel coronavirus, and the transmission through respiratory droplets and close contact is a main transmission path. The incubation period for viral infection is 1-14 days, mostly 3-7 days. It is mainly manifested by fever, dry cough, and asthenia, and a few patients are accompanied by nasal obstruction, watery nasal discharge, pharyngalgia, myalgia, diarrhea, etc. Mild patients only manifest low fever, mild fatigue, etc., without the manifestation of pneumonia, severe patients mostly develop dyspnea and/or hypoxemia after one week of onset, and severe patients can rapidly progress to acute respiratory distress syndrome. A major outbreak of new coronaviruses has led to more than 5000 million infections worldwide, with over 100 and over ten thousand deaths. The new coronavirus has the characteristic of high human transmissibility and is still infectious in the latent period, so that once community infection occurs, the infection speed is very high, and the prevention and control difficulty is very high. Until now, no effective specific medicine for the new coronavirus exists, and the patient mainly depends on the immune system of the patient to eliminate the virus, so that the aim of curing is fulfilled; vaccines are the most effective and specific drugs recognized globally to be able to control this global outbreak.
The main structural proteins of the novel coronavirus comprise spike protein (S protein) on an envelope, envelope protein M, matrix protein E and nucleocapsid protein N protein, wherein the S protein is a key protein when the virus invades host cells, the membrane outer structure of the S protein comprises 1273 amino acids, the amino acid position at 645 can be cut into S1 and S2 proteins by enzyme, and the S1 protein has a Receptor Binding Domain (RBD) which can be combined with ACE2 receptors on the host cells to mediate the invasion of the virus into the host cells.
Following infection with the new coronavirus, the human immune system can produce a variety of antibodies against the virus. As a neutralizing antibody with antiviral activity after infection and vaccine immunization, the neutralizing antibody only accounts for a small part of antibodies secreted by B cells, the blood titer of the neutralizing antibody is important, the content of the neutralizing antibody in a subject is the most important index for evaluating the effectiveness of the novel corona vaccine, and the neutralizing antibody can be used as a concomitant detection product of the vaccine to assist in confirming that the subject is continuously and effectively protected. Principle of body protection by neutralizing antibodies: binds to the new coronavirus surface protein, blocks the combination of the virus protein and a cell surface specific receptor ACE-2, and further prevents the virus from invading cells (non-neutralizing antibodies do not have the function). When neutralizing Antibody titers in antibodies are insufficient, there is a possibility that Antibody-dependent enhancement (ADE) type diseases are worsened: the virus binds to Fc receptors on the cell surface via antibodies, which in turn infect a wider variety of cells, including macrophages, leading to a disturbed immune response.
The new corona vaccine induced neutralizing antibody detection is evaluated by a virus infection verification method and an immune blocking verification method. And (3) virus infection verification adopts a principle that the neutralizing antibody is used for preventing the virus from infecting cells, the condition that the number of the virus-infected cells is reduced after the neutralizing antibody is added is observed, and the blocking effect is calculated. Because of the high infectivity and pathogenicity of the new coronavirus, infection verification by using the new coronavirus must be carried out in a laboratory with more than three levels of biosafety, and is limited by laboratory conditions and virus sources. In addition, there are often some differences in live virus detection results from different laboratories due to the different virus strains, culture conditions, and evaluation criteria for the results. Although the use of a pseudovirus system for neutralizing antibodies can reduce the biosafety risk, the whole process still requires culturing cells and viruses, and results cannot be reported rapidly in batches.
The immune blocking verification has two technical principles, one is to utilize a principle that a neutralizing antibody blocks the combination of virus RBD protein and a receptor ACE-2, and the titer of the neutralizing antibody is calculated by adding the neutralizing antibody and detecting the reduction amount of the RBD protein combined on the ACE-2, wherein the Beijing Baipusai biological science and technology limited company applies an invention patent adopting the method, and the patent name is as follows: a novel coronavirus neutralizing antibody titer detection ELISA kit patent, application No. 202010919164.X, can rapidly detect neutralizing antibodies in a conventional laboratory in batches, but because ACE2 is a substance which has certain physiological activity with human serum, the content of the ACE2 is different in different populations, the ACE2 can be over-expressed in certain hypertensive populations, and the detection result is interfered. In addition, the method only detects the neutralizing antibody capable of blocking the binding of RBD and ACE2, and in the existing research, the neutralizing antibody in human bodies after the infection of the new coronavirus has the epitope of the neutralizing antibody on the NTD structure of the S protein and the S2 protein except the epitope on the RBD protein, so that the neutralizing antibody cannot be detected by blocking the binding of the RBD protein and the receptor ACE-2. The other method is to calculate the titer of the neutralizing antibody by using the principle that the known monoclonal antibody with the neutralizing activity of the novel coronavirus competitively binds with the antibody in the sample to be tested and detecting the reduction amount of the antibody bound on the S protein after adding the known neutralizing antibody, and the method can also detect the neutralizing antibody in a routine laboratory in batch and fast without interference of ACE2 in the sample.
The novel coronavirus SARS-CoV-2 is highly infectious and pathogenic, and conventional detection of neutralizing antibodies requires collection of live virus and cells, manipulation by highly skilled personnel in the P3 laboratory through complex laboratory procedures, which are both inconvenient and biologically safe, and take several days to obtain results. In addition, there are often some differences in live virus detection results from different laboratories due to the different virus strains, culture conditions, and evaluation criteria for the results. Although the use of a pseudovirus system for neutralizing antibodies can reduce the biosafety risk, the whole process still requires culturing cells and viruses, and results cannot be reported rapidly in batches.
Application number 202010919164.X, filed by Beijing Baipusais Biotechnology GmbH, entitled: a novel ELISA kit for detecting the titer of a neutralizing antibody of a coronavirus, which utilizes the principle that the neutralizing antibody blocks the combination of a virus RBD protein and a receptor ACE-2, detects the reduction amount of the RBD protein combined on the ACE-2 after the neutralizing antibody is added to calculate the titer of the neutralizing antibody, can rapidly detect the neutralizing antibody in a conventional laboratory in batches, but because the ACE2 is a physiologically active substance in human serum, the content of the ACE2 is different in different populations, the ACE can be excessively expressed in certain hypertensive populations, and the detection result is interfered. In addition, the method only detects the neutralizing antibody capable of blocking the binding of RBD and ACE2, and in the existing research, the neutralizing antibody in human bodies after the infection of the new coronavirus has the epitope of the neutralizing antibody on the NTD structure of the S protein and the S2 protein except the epitope on the RBD protein, so that the neutralizing antibody cannot be detected by blocking the binding of the RBD protein and the receptor ACE-2.
Disclosure of Invention
In view of this, the invention provides a kit for detecting a novel coronavirus neutralizing antibody and a preparation method thereof. The kit can detect the new coronavirus S protein RBD, NTD and S2 related neutralizing antibodies without being interfered by ACE2 in a sample, and has the advantages of high quantification, high accuracy, good specificity and high detection speed.
The invention provides a kit for detecting a neutralizing antibody of a novel coronavirus, which comprises:
a kit for detecting neutralizing antibodies against a novel coronavirus, comprising:
magnetic particle suspension coated by novel coronavirus S protein, sample diluent and enzyme conjugate working solution;
the magnetic particle suspension coated by the novel coronavirus S protein consists of magnetic particles coated by the novel coronavirus S protein and a preservation solution 1;
the concentration of the magnetic particles coated by the novel coronavirus S protein is 1-10 ug/ml;
the preservation solution 1 is a Tris buffer solution containing 1% BSA and 0.1% P300 preservative;
the enzyme conjugate is a chemiluminescent enzyme-labeled novel coronavirus neutralizing antibody.
In some embodiments, the S protein is an S protein recombinantly expressed from a HEK293 cell gene.
The preparation method of the magnetic particle suspension coating the novel coronavirus S protein comprises the following steps:
dissolving EDC and NHS in 0.1M MES (pH 5.0) buffer, adding magnetic particles; lightly shaking the mixture at room temperature for reaction for 30 minutes; adsorbing the magnetic particles at the bottom by a magnet, and removing the supernatant; then 0.1MMES (pH 5.0) buffer solution is added, and the magnetic particles are resuspended; repeating the above operations for 2 times to obtain activated magnetic particles;
adding the S protein into the activated magnetic particles; then 0.1M MES (pH 5.0) buffer was added to the reaction mixture to a final volume of 1 ml; lightly shaking the mixture at room temperature for reaction for 60 minutes; adsorbing the magnetic particles to the bottom with a magnet, removing the supernatant, washing 3 times with 0.02M PBS pH 8.0; and adding a preservation solution 2 to obtain the magnetic particle suspension coated with the novel coronavirus S protein.
In some embodiments, the enzyme conjugate working fluid comprises an enzyme conjugate and a preservation fluid 2;
the preservation solution 2 is Tris buffer solution containing 20% of calf serum and 0.1% of P300 preservative.
In some embodiments, the neutralizing antibody against the novel coronavirus in the enzyme conjugate working solution is a murine anti-monoclonal antibody, a neutralizing antibody produced by B cells extracted from convalescent patients, or a humanized monoclonal antibody, and the concentration of the neutralizing antibody against the novel coronavirus in the enzyme conjugate working solution is 1 to 5 mg/L.
In some embodiments, each 1L of the sample diluent consists of water and:
5.80g of disodium hydrogen phosphate dodecahydrate, 0.59g of sodium dihydrogen phosphate dihydrate, 9.00g of sodium chloride, 10g of BSA, 201ml of Tween-and 0.1-0.3% (w/w) of preservative.
In the present invention, the chemiluminescent enzyme is horseradish peroxidase, acridinium ester, alkaline phosphatase, isoluminol or ruthenium terpyridyl, including but not limited to horseradish peroxidase, and in some embodiments, the chemiluminescent enzyme is horseradish peroxidase.
The kit also comprises a calibrator comprising a calibrator matrix solution and a novel coronavirus neutralizing antibody;
the calibrator matrix solution is normal human serum added with 0.1% of protein protective agent and 0.1% of P300 preservative;
the novel coronavirus neutralizing antibody is a mouse anti-monoclonal antibody, a neutralizing antibody produced by B cells extracted from convalescent patients or a humanized monoclonal antibody.
When the kit is used for detecting the new coronavirus neutralizing antibody in a sample to be detected, the sample diluent, the magnetic particle suspension and the enzyme conjugate can be added into a reaction cup for reaction at the same time, or the sample, the sample diluent and the magnetic particle can be reacted for a period of time firstly, and then the enzyme conjugate is added for reaction.
The invention also provides a preparation method of the kit, which comprises the following steps:
dissolving EDC and NHS in 0.1M MES (pH 5.0) buffer, adding magnetic particles; lightly shaking the mixture at room temperature for reaction for 30 minutes; adsorbing the magnetic particles at the bottom by a magnet, and removing the supernatant; then 0.1MMES (pH 5.0) buffer solution is added, and the magnetic particles are resuspended; repeating the above operations for 2 times to obtain activated magnetic particles;
adding the S protein into the activated magnetic particles; then 0.1M MES (pH 5.0) buffer was added to the reaction mixture to a final volume of 1 ml; lightly shaking the mixture at room temperature for reaction for 60 minutes; adsorbing the magnetic particles to the bottom with a magnet, removing the supernatant, washing 3 times with 0.02M PBS pH 8.0; adding a preservation solution 1 to obtain the magnetic particle suspension coated with the novel coronavirus S protein;
mixing activated chemiluminescent enzyme with a new coronavirus neutralizing antibody, reacting at 2-8 ℃ overnight, adding a sodium borohydride reductase conjugate, dialyzing to remove unreacted reagents, adding 50% by volume of glycerol, storing at-20 ℃, and diluting with a Tris buffer solution containing 20% calf serum and 0.1% P300 preservative when in use to obtain an enzyme conjugate working solution;
the sample diluent and the calibrator matrix solution are prepared according to the formula.
The method utilizes the principle that the known monoclonal antibody with the novel coronavirus neutralizing activity is in competitive combination with the antibody in a sample to be detected, and detects the reduction amount of the antibody bound on the S protein to calculate the titer of the neutralizing antibody after the known neutralizing antibody is added, can also detect the neutralizing antibody in a conventional laboratory in batch and quickly, is not interfered by ACE2 in the sample, can carry out detection in a clinical laboratory, can report results within 20 minutes at the fastest speed, and can detect 200 samples per hour.
The present invention can detect the titer of neutralizing antibodies in a sample by using a known novel coronavirus S protein comprising neutralizing antibodies that specifically bind to RBD, NTD, and S2 proteins, in competition with the neutralizing antibodies in the sample.
The kit realizes the automatic detection of samples on the chemiluminescence detectors of AutoLumo A2000, AutoLumo A2000 Plus B and AutoLumo A1000 of Anzhi organisms. The detection principle is as follows: the known novel coronavirus neutralizing antibody in the enzyme conjugate competes with an antibody in a sample to be detected for binding with the S protein antigen coated on the magnetic particles, if the sample contains the neutralizing antibody, the enzyme-labeled neutralizing antibody is blocked from binding with the S protein antigen coated on the magnetic particles, so that the detection signal value is reduced, and the content of the neutralizing antibody in the sample to be detected is in inverse proportion to the detection signal value.
The kit provided by the invention detects the neutralizing antibody in the serum or plasma sample by adopting the technical principle that the enzyme-labeled neutralizing antibody competes with the antibody in the sample to be detected for binding with the S protein antigen, and compared with the prior art, the kit provided by the invention has the following advantages:
1. the novel coronavirus neutralizing antibody detection kit (magnetic particle chemiluminescence method) is applied based on a full-automatic chemiluminescence platform, manual operation is avoided, and the result is stable and reliable.
2. The kit of the invention can preferably select a one-step reaction mode, and has short test time: the test result can be obtained within 17min, and the test throughput is high and is 200 tests/hour.
3. The kit is a quantitative kit, the linear range can reach 100 times of S/CO, the quantitative detection of samples with different antibody titers such as serum of a rehabilitee, different vaccination samples and the like can be realized, the dynamic detection of the neutralizing antibody level can be realized, and the detection effectiveness is greatly improved.
4. The kit can detect the neutralizing antibody containing the neo-corona RBD, NTD and S2 by detecting the neutralizing antibody, can quickly adjust the sequence of the enzyme-labeled neutralizing antibody by means of a mature recombinant antibody expression platform, and can quickly adapt to the change of the neutralizing antibody caused by virus variation.
5. The kit is a quantitative kit, can realize the quantitative detection of samples with different antibody titers such as serum of a rehabilitee, different vaccination samples and the like, can realize the dynamic detection of the level of the neutralizing antibody, and greatly improves the detection effectiveness.
Detailed Description
The invention provides a kit for detecting a neutralizing antibody of a new coronavirus and a preparation method thereof. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
The invention is further illustrated by the following examples:
EXAMPLE 1 preparation of the kit of the invention
Preparation of magnetic microparticle suspension: putting 200ul of magnetic particles with carboxyl on the surface in a glass bottle, adsorbing the magnetic particles at the bottom of the glass bottle by using a magnet, and removing the supernatant; 2ml of 0.02M PBS (pH 8.0) was added, and the above operation was repeated 3 times. Dissolving EDC and NHS in 0.1MMES (pH 5.0) buffer solution at a concentration of 20mg/ml, respectively, and adding 1ml of each solution into magnetic particles; lightly shaking the mixture at room temperature for reaction for 30 minutes; adsorbing the magnetic particles at the bottom by a magnet, and removing the supernatant; then 2ml of 0.1M MES (pH 5.0) buffer solution is added, and the magnetic particles are resuspended; the above operation was repeated 2 times. Adding an S protein antigen expressed by HEK293 cell gene recombination into the activated magnetic particles; then 0.1M MES (pH 5.0) buffer was added to the reaction mixture to a final volume of 1 ml; lightly shaking the mixture at room temperature for reaction for 60 minutes; the magnetic particles were adsorbed to the bottom with a magnet, the supernatant was removed, and washed 3 times with 2ml of 0.02M PBS (pH 8.0); 20ml of Tris buffer (pH 8.0) containing 1% BSA and 0.1% preservative P300 was added and stored at 2-8 ℃.
Preparation of sample diluent: preparing 1000ml of sample diluent, 5.80g of disodium hydrogen phosphate dodecahydrate, 0.59g of sodium dihydrogen phosphate dihydrate, 9.00g of sodium chloride, 10g of BSA and 201ml of Tween-201, diluting the sample to 1000ml with deionized water, and adding 0.1% preservative P300.
Preparation of enzyme conjugates: horse Radish Peroxidase (HRP) is activated by a conventional modified sodium periodate method, and a monoclonal antibody capable of neutralizing the novel coronavirus is added, and the neutralizing antibody can effectively block the infection of the virus to cells through the verification of a classical virus neutralizing experiment. The neutralizing antibody may be a murine monoclonal antibody, a neutralizing antibody produced by B cells extracted from convalescent patients, or a humanized monoclonal antibody. Reacting at 2-8 ℃ overnight, adding a sodium borohydride reductase conjugate, dialyzing to remove unreacted reagents, adding 50% glycerol by volume, and storing at-20 ℃. When in use, the extract is diluted in Tris buffer solution containing 20% calf serum and 0.1% P300 preservative according to the proportion of 1:2000 and stored at the temperature of 2-8 ℃.
Preparation of a calibrator: the neutralizing antibody of the novel coronavirus is added into the calibrator matrix solution. Neutralizing antibodies include murine monoclonal antibodies, polyclonal animal sera raised against neocorona S, neutralizing antibodies produced by B cells extracted from the serum of the convalescent person, humanized recombinant neutralizing antibodies. The calibrator matrix solution is normal human serum supplemented with 0.1% protein protectant and 0.1% P300 preservative.
Example 2 preparation of a kit of the invention
Preparation of magnetic microparticle suspension: putting 200ul of magnetic particles with carboxyl on the surface in a glass bottle, adsorbing the magnetic particles at the bottom of the glass bottle by using a magnet, and removing the supernatant; 2ml of 0.02M PBS (pH 8.0) was added, and the above operation was repeated 3 times. Dissolving EDC and NHS in 0.1M acetic acid (pH 4.5) buffer solution at a concentration of 20mg/ml, respectively, and adding 1ml of each solution into the magnetic particles; lightly shaking the mixture at room temperature for reaction for 30 minutes; adsorbing the magnetic particles at the bottom by a magnet, and removing the supernatant; then 2ml of 0.1M acetic acid (pH 5.0) buffer solution is added, and the magnetic particles are resuspended; the above operation was repeated 2 times. Adding an S protein antigen expressed by HEK293 cell gene recombination into the activated magnetic particles; then 0.02MPBS (pH 7.4) buffer solution is added to the mixture until the final volume is 1 ml; lightly shaking the mixture at room temperature for reaction for 60 minutes; the magnetic particles were adsorbed to the bottom with a magnet, the supernatant was removed, and washed 3 times with 2ml of 0.02M PBS (pH 7.4); 20ml of Tris buffer (pH 7.4) containing 1% BSA and 0.1% preservative P300 was added and stored at 2-8 ℃.
Preparation of sample diluent: preparing 1000ml of sample diluent, 5.80g of disodium hydrogen phosphate dodecahydrate, 0.59g of sodium dihydrogen phosphate dihydrate, 9.00g of sodium chloride, 10g of BSA and 201ml of Tween-201, diluting the sample to 1000ml with deionized water, and adding 0.1% preservative P300.
Preparation of enzyme conjugates: horse Radish Peroxidase (HRP) is activated by a conventional modified sodium periodate method, and a monoclonal antibody capable of neutralizing the novel coronavirus is added, and the neutralizing antibody can effectively block the infection of the virus to cells through the verification of a classical virus neutralizing experiment. The neutralizing antibody may be a murine monoclonal antibody, a neutralizing antibody produced by B cells extracted from convalescent patients, or a humanized monoclonal antibody. Reacting at 2-8 ℃ overnight, adding a sodium borohydride reductase conjugate, dialyzing to remove unreacted reagents, adding 50% glycerol by volume, and storing at-20 ℃. When in use, the extract is diluted in Tris buffer solution containing 20% calf serum and 0.1% P300 preservative according to the proportion of 1:2000 and stored at the temperature of 2-8 ℃.
Preparation of a calibrator: the neutralizing antibody of the novel coronavirus is added into the calibrator matrix solution. Neutralizing antibodies include murine monoclonal antibodies, polyclonal animal sera raised against neocorona S, neutralizing antibodies produced by B cells extracted from the serum of the convalescent person, humanized recombinant neutralizing antibodies.
EXAMPLE 3 method of handling the kit
Placing the sample container in an instrument mating sample rack; loading a sample rack, and inputting sample information in an instrument software interface; selecting "run" to start the test, the instrument will perform the following operations:
1) the sample rack is transported to a sample sucking position, and the reaction container is loaded to a sample loading position.
2) Dispensing of 50. mu.L of sample or 50. mu.L of calibrator, 20. mu.L of magnetic microparticle suspension, and 50. mu.L of sample diluent was completed.
3) The reaction solution was mixed well and incubated at 37 ℃ for 15 minutes.
4) After the incubation is completed, the reaction solution is washed and separated by using a washing solution.
5) Dispensing of 50. mu.L of enzyme conjugate was completed.
6) The reaction solution was mixed well and incubated at 37 ℃ for 15 minutes.
7) After the incubation is completed, the reaction solution is washed and separated by using a washing solution.
8) Dispensing of 50. mu.L of the substrate A solution and 50. mu.L of the substrate B solution was completed.
9) And (4) uniformly mixing the reaction solution, and detecting the luminous intensity.
10) The instrument automatically reports the test results.
EXAMPLE 4 method of handling the kit
Placing the sample container in an instrument mating sample rack; loading a sample rack, and inputting sample information in an instrument software interface; selecting "run" to start the test, the instrument will perform the following operations:
1) the sample rack is transported to a sample sucking position, and the reaction container is loaded to a sample loading position.
2) Dispensing of 30 μ L of sample or 30 μ L of calibrator, 20 μ L of magnetic microparticle suspension, 50 μ L of sample diluent, and 50 μ L of enzyme conjugate was completed.
3) The reaction solution was mixed well and incubated at 37 ℃ for 15 minutes.
4) After the incubation is completed, the reaction solution is washed and separated by using a washing solution.
5) Dispensing of 50. mu.L of the substrate A solution and 50. mu.L of the substrate B solution was completed.
6) And (4) uniformly mixing the reaction solution, and detecting the luminous intensity.
7) The instrument automatically reports the test results.
Example 5
By using a new crown neutralizing antibody Detection Kit (SARS-CoV-2 neutralizationbiotody Detection Kit) purchased from Kinsley as a control Kit, and adopting the control Kit and the Kit of the invention in the embodiment 1 to detect 50 samples of hypertensive patients and 60 samples of myocardial infarction patients which are not infected with new crown viruses and are not inoculated with new crown vaccines in parallel according to the method of the embodiment 4, the abnormal high expression of ACE2 can exist in the two groups, and the result shows that the control Kit detects 2 positive samples, the specificity is 98.2%, the scheme does not detect positive samples, and the specificity is 100%.
In addition, the test results of the comparative document 1, in which the ACE2 proteins were added in series to the negative samples, and the test results of the comparative document 1 and the test results of the test kit of the present invention were tested in parallel, showed that the test results of the comparative document 1 were disturbed and the concentrations thereof tended to increase as the concentration of ACE2 added to the samples increased, whereas the test results of the present invention were not substantially changed and not disturbed, and the results are shown in table 1.
TABLE 1
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.
Claims (9)
1. A kit for detecting neutralizing antibodies against a novel coronavirus, comprising:
magnetic particle suspension coated by novel coronavirus S protein, sample diluent and enzyme conjugate working solution;
the magnetic particle suspension coated by the novel coronavirus S protein consists of magnetic particles coated by the novel coronavirus S protein and a preservation solution 1;
the concentration of the magnetic particles coated by the novel coronavirus S protein is 1-10 ug/ml;
the preservation solution 1 is a Tris buffer solution containing 1% BSA and 0.1% P300 preservative;
the enzyme conjugate is a chemiluminescent enzyme-labeled novel coronavirus neutralizing antibody.
2. The kit according to claim 1, wherein the S protein is an S protein expressed by HEK293 cell gene recombination.
3. The kit of claim 1, wherein the suspension of magnetic particles coated with the novel coronavirus S protein is prepared by:
dissolving EDC and NHS in 0.1M MES (pH 5.0) buffer, adding magnetic particles; lightly shaking the mixture at room temperature for reaction for 30 minutes; adsorbing the magnetic particles at the bottom by a magnet, and removing the supernatant; then 0.1MMES (pH 5.0) buffer solution is added, and the magnetic particles are resuspended; repeating the above operations for 2 times to obtain activated magnetic particles;
adding the S protein into the activated magnetic particles; then 0.1M MES (pH 5.0) buffer was added to the reaction mixture to a final volume of 1 ml; lightly shaking the mixture at room temperature for reaction for 60 minutes; adsorbing the magnetic particles to the bottom with a magnet, removing the supernatant, washing 3 times with 0.02M PBS pH 8.0; and adding the preservation solution 1 to obtain the magnetic particle suspension coated with the novel coronavirus S protein.
4. The kit according to claim 1, wherein the enzyme conjugate working solution comprises an enzyme conjugate and a preservation solution 2;
the preservation solution 2 is Tris buffer solution containing 20% of calf serum and 0.1% of P300 preservative.
5. The kit according to claim 1, wherein the neutralizing antibody against the neocoronaviruse in the working solution of the enzyme conjugate is a murine anti-2019-nCoV S monoclonal antibody, and the neutralizing antibody or humanized monoclonal antibody produced by B cells extracted from convalescent patients is at a concentration of 1-5 mg/L.
6. The kit according to claim 1,
each 1L of the sample diluent consisted of water and the following components:
5.80g of disodium hydrogen phosphate dodecahydrate, 0.59g of sodium dihydrogen phosphate dihydrate, 9.00g of sodium chloride, 10g of BSA, 201ml of Tween-and 0.1-0.3% (w/w) of preservative.
7. The kit of claim 1, wherein the chemiluminescent enzyme is horseradish peroxidase, acridinium ester, alkaline phosphatase, isoluminol, or ruthenium terpyridyl.
8. The kit of claim 1, further comprising a calibrator; the calibrator comprises calibrator matrix liquid and novel coronavirus neutralizing antibodies;
the calibrator matrix solution is normal human serum added with 0.1% of protein protective agent and 0.1% of P300 preservative;
the novel coronavirus neutralizing antibody is a mouse anti-monoclonal antibody, a neutralizing antibody produced by B cells extracted from convalescent patients or a humanized monoclonal antibody.
9. The method for preparing the kit according to any one of claims 1 to 8, comprising:
dissolving EDC and NHS in 0.1M MES (pH 5.0) buffer, adding magnetic particles; lightly shaking the mixture at room temperature for reaction for 30 minutes; adsorbing the magnetic particles at the bottom by a magnet, and removing the supernatant; then 0.1MMES (pH 5.0) buffer solution is added, and the magnetic particles are resuspended; repeating the above operations for 2 times to obtain activated magnetic particles;
adding the S protein into the activated magnetic particles; then 0.1M MES (pH 5.0) buffer was added to the reaction mixture to a final volume of 1 ml; lightly shaking the mixture at room temperature for reaction for 60 minutes; adsorbing the magnetic particles to the bottom with a magnet, removing the supernatant, washing 3 times with 0.02M PBS pH 8.0; adding a preservation solution 1 to obtain the magnetic particle suspension coated with the novel coronavirus S protein;
mixing activated chemiluminescent enzyme with a new coronavirus neutralizing antibody, reacting at 2-8 ℃ overnight, adding a sodium borohydride reductase conjugate, dialyzing to remove unreacted reagents, adding 50% by volume of glycerol, storing at-20 ℃, and diluting with a Tris buffer solution containing 20% calf serum and 0.1% P300 preservative when in use to obtain an enzyme conjugate working solution;
the sample diluent and the calibrator are prepared according to the formula composition.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117110606A (en) * | 2023-08-21 | 2023-11-24 | 广东省一鼎生物技术有限公司 | Method for detecting neutralizing antibodies of different types of HPV (human papilloma Virus) based on competition method |
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