CN113917138A - Novel coronavirus IgG antibody level detection kit - Google Patents
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Abstract
The invention provides a novel coronavirus IgG antibody level detection kit, which comprises an enzyme label plate coated with recombinant RBD protein. The kit adopts the recombinant new coronavirus RBD protein as a coating material, the RBD protein is a specific region for generating a neutralizing antibody, and an IgG antibody detected by the kit has better correlation with the neutralizing antibody, can be used as a substitute means of the neutralizing antibody, greatly improves the detection time efficiency, and has high detection efficiency, strong specificity and high sensitivity.
Description
Technical Field
The invention belongs to the technical field of biological detection, relates to a novel coronavirus IgG antibody level detection kit, and particularly relates to a novel coronavirus IgG antibody level detection kit and a detection method.
Background
The common signs of the novel coronavirus (SARS-CoV-2) are fever, hypodynamia, dry cough and dyspnea gradually, while part of patients have slight symptoms, even patients without clinical symptoms exist, the novel coronavirus has the characteristic of human transmission, the incubation period is generally 1 to 14 days, the incubation period is infectious, asymptomatic infectors can also become infection sources, the common infection and the spread are mainly achieved through respiratory droplets and close contact infection, and the population is generally susceptible. The fatality rate of the new coronavirus epidemic situation is about 2 percent at present, asymptomatic infectors are about 40 to 45 percent, the virus epidemic rate is high, the transmission is fast, the prevention and control difficulty is high, and the epidemic situation caused by the new coronavirus epidemic situation seriously affects various industries. In the face of new coronavirus with large epidemic range and high transmission rate, effective vaccination is a key method for epidemic prevention.
At present, a common detection kit for a new coronavirus antibody adopts whole virus coating, so that the specificity is weak, and the false positive probability is high, so that the novel detection kit for the coronavirus IgG antibody level, which has strong specificity and high sensitivity, is provided.
Disclosure of Invention
The present invention is directed to solving at least one of the above problems in the prior art. Therefore, the invention provides a novel coronavirus IgG antibody level detection kit and a detection method, the kit provided by the invention has low requirements on experimental environment, high detection efficiency, strong specificity and high sensitivity, and can replace a neutralizing antibody for detection.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides a novel coronavirus IgG antibody level detection kit, which comprises an ELISA plate coated with a recombinant RBD protein.
The kit provided by the invention adopts the recombinant RBD protein as a coating and the RBD protein as a specific region for generating the neutralizing antibody, so that the IgG antibody and the neutralizing antibody detected by the detection kit provided by the invention have better correlation, can be used as a substitute means of the neutralizing antibody, and can greatly improve the detection time efficiency.
Meanwhile, compared with full virus coating, the specificity of the recombinant RBD protein is stronger, the experimental background is clean, the false positive probability of the experiment can be reduced, and a more accurate antibody titer result can be obtained.
As a preferred embodiment of the present invention, the method for preparing the recombinant RBD protein comprises:
(A) constructing a plasmid containing an RBD protein gene sequence;
(B) and (3) carrying out protein expression on the plasmid obtained in the step (A) by using 293F cells, and then purifying to obtain the recombinant RBD protein.
The RBD protein gene of the invention is numbered QHD43416.1 in GEN BANK.
As a preferred technical scheme of the invention, the preparation method of the ELISA plate coated with the recombinant RBD protein comprises the following steps:
(a) uniformly mixing the recombinant RBD protein and the coating solution, placing the mixture in holes of an ELISA plate for incubation, and then washing the plate;
(b) and (b) sealing each hole of the ELISA plate obtained in the step (a) by using a sealing solution, washing the plate, and drying to obtain the ELISA plate coated with the recombinant RBD protein.
As a preferable technical scheme of the invention, the coating solution is 0.01M phosphate buffer salt solution.
As a preferred technical scheme of the invention, the blocking solution is 0.01M phosphate buffer salt solution containing 10% of calf serum or 10% of bovine serum albumin.
As a preferred technical scheme of the invention, the washing solution used for washing the plate is phosphate buffered saline solution containing Tween 20.
In a preferred embodiment of the present invention, the concentration of the solution of the recombinant RBD protein mixed with the coating solution is 0.5-2. mu.g/mL, such as 0.6. mu.g/mL, 0.8. mu.g/mL, 1.0. mu.g/mL, 1.2. mu.g/mL, 1.5. mu.g/mL, 1.8. mu.g/mL, etc.
As a specific embodiment of the invention, the preparation method of the ELISA plate coated with the recombinant RBD protein comprises the following steps:
(a) diluting the recombinant RBD protein and 0.01M PBS according to a final concentration of 0.5-2 mug/mL, uniformly mixing, adding a 96-hole enzyme label plate, incubating at 100 muL/hole for 16-20h at 2-8 ℃ or incubating at 37 ℃ for 3h, washing the plate three times by using a phosphate buffer solution containing Tween 20, and patting to dry;
(b) and (b) sealing each hole of the ELISA plate obtained in the step (a) by using 0.01M phosphate buffer saline solution containing 10% calf serum at 37 ℃ for 1-3h, then washing the plate by using phosphate buffer saline solution containing Tween 20 for three times, and patting the plate dry to obtain the ELISA plate coated with the recombinant RBD protein.
As a preferable technical scheme, the kit further comprises an HRP labeled antibody of goat anti-human IgG, an enzyme diluent, a negative control, a sample diluent, a developing solution and a stopping solution.
In a preferred embodiment of the present invention, the enzyme diluent is a lotion containing calf serum and casein.
In a preferred embodiment of the present invention, the negative control is a sample diluent.
In a preferred embodiment of the present invention, the sample diluent is 0.01M phosphate buffered saline containing bovine serum or bovine serum albumin.
In a preferred embodiment of the present invention, the stop solution is a 2M sulfuric acid solution.
As a specific embodiment of the invention, the kit comprises the following components:
i. an ELISA plate coated with recombinant RBD protein, a 96-well plate in specification;
HRP-labeled antibody to goat anti-human IgG;
an enzyme diluent: a lotion containing 10% calf serum and 1% casein;
negative control: a sample diluent;
v. sample diluent: 0.01M phosphate buffered saline containing 10% calf serum or containing 10% bovine serum albumin;
color developing solution: a commercially available A/B solution;
vii, stop solution: 2M sulfuric acid solution.
In a second aspect, the present invention provides a novel method for detecting the level of coronavirus IgG antibodies, the method comprising:
(1) preparing an ELISA plate coated with recombinant RBD protein;
(2) diluting a sample to be detected, and then placing the sample in each hole of the ELISA plate obtained in the step (1) for incubation;
(3) adding HRP (horse radish peroxidase) labeled antibody of goat anti-human IgG into each hole of the ELISA plate obtained in the step (2) for incubation;
(4) adding a developing solution into each hole of the ELISA plate obtained in the step (3) for developing, and then adding a stop solution to terminate the reaction;
(5) the OD value of each well of the microplate at the wavelength of 450nm (630nm is the reference wavelength) is measured by a microplate reader.
As a specific embodiment of the present invention, the detection method of the present invention includes the following steps:
(1) diluting the recombinant RBD protein and 0.01M PBS according to a final concentration of 0.5-2 mug/mL, uniformly mixing, adding a 96-hole enzyme label plate, incubating at 100 muL/hole for 16-20h at 2-8 ℃ or incubating at 37 ℃ for 3h, washing the plate three times by using a phosphate buffer solution containing Tween 20, and patting to dry;
(2) and (2) sealing each hole of the ELISA plate obtained in the step (1) by using 0.01M phosphate buffer saline solution containing 10% calf serum at 37 ℃ for 1-3h, then washing the plate by using phosphate buffer saline solution containing Tween 20 for three times, and patting the plate dry to obtain the ELISA plate coated with the recombinant RBD protein.
(3) Diluting the serum to be detected by using a sample diluent, diluting the serum before the novel coronavirus vaccine immunization according to the ratio of 1:40, 1:80 and 1:160, and diluting the serum after the immunization according to the ratio of 1:160 and 1: 320. diluting 6-8 times in a gradient manner at a ratio of 1:640, 1:1280, 1:2560, 1:5120 and the like, then placing the diluted solution in each hole of an enzyme label plate coated with recombinant RBD protein for incubation at 37 ℃ for 0.5-2h, then washing the plate for 3 times by using phosphate buffer saline solution containing Tween 20, and drying by beating;
(4) adding HRP labeled antibody diluent of goat anti-human IgG into each hole of the ELISA plate obtained in the step (3), incubating for 0.5-2h, washing the plate for 5 times by using phosphate buffer solution containing Tween 20, and drying by beating;
(5) 50 mu L of color development liquid A, B is added into each hole of the ELISA plate obtained in the step (4), color development is carried out for 15min at room temperature, and then 50 mu L of stop solution is added to stop reaction;
(6) the OD value of each hole of the microplate at the wavelength of 450nm is measured by using a microplate reader, and 630nm is used as a reference.
In the test OD value result, the OD value of a negative control hole (sample diluent) is not higher than 0.05, 2.1 times of the OD value of the negative control hole is taken as a cutoff value, the positive judgment standard of a single dilution multiple is that the OD value is not less than 0.105, and the titer of the sample to be detected is the highest dilution multiple reaching positive conversion.
The novel coronavirus consists of four proteins: spike protein S, membrane protein M, envelope protein E, and nucleocapsid protein N, wherein a Receptor Binding Domain (RBD) contained in spike protein S is a key site for binding to a target cell.
The vaccine inoculation is one of key means for epidemic prevention, the evaluation index of the effectiveness of the vaccine inoculation is antibody level, and common SARS-CoV-2 antibody detection methods include colloidal gold method, enzyme linked immunosorbent assay (ELISA), chemiluminescence immunoassay (CLIA), neutralization titer detection and the like. The titer of the neutralizing antibody is an evaluation index of common viral vaccines, the new coronavirus has the characteristics of high pathogenicity and high infectivity, the detection of the related neutralizing antibody needs to be carried out by adopting live virus in laboratories above the P3 grade, the requirements on detection environment and operation of experimenters are high, and the high requirement on detection conditions has certain limitation on the evaluation of clinical serum. The ELISA method allows quantitative detection compared to the colloidal gold method. Compared with CLIA method, ELISA cost is lower, and no special chemiluminescence detection instrument is needed. Compared with the neutralization titer, the detection of the ELISA antibody does not need to contact live virus, does not need a P3 laboratory, and the experimental condition is easy to realize. In addition, the period of the detection of the neutralization titer is 4 to 7 days, and the period of the detection of the ELISA antibody is 3 to 5 hours.
The kit provided by the invention adopts the recombinant new coronavirus RBD protein as a coating to detect the IgG antibody, and the detected IgG antibody has better correlation with a neutralizing antibody generated by whole virus vaccine immunization, can be used as a substitute means of the neutralizing antibody, and greatly improves the detection time efficiency. The recombinant RBD protein of the coating substance adopted in the detection method provided by the invention has stronger specificity and clean experimental background, can reduce the false positive probability of the experiment and obtain more accurate antibody titer results. Moreover, the invention adopts a one-step indirect method, and the method is simple and rapid.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the specific embodiments are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The raw materials used in the following examples of the invention were sourced as follows:
the recombinant RBD protein is provided by Beijing Kexing vitamin technology Co., Ltd (hereinafter referred to as "Kexing Zhongwei"), and the preparation method is shown in example 1;
the reagent for experiment is prepared by Kexing Zhongwei laboratory, the coating solution is 0.01M phosphate buffer salt solution, the sample diluent and the sealing solution are 0.01M sulfate buffer salt solution containing 10% calf serum, and the washing solution is PBST20The stop solution is 2M sulfuric acid solution, and the enzyme diluent is lotion containing 10% calf serum and 1% casein;
the color developing solution is a commercially available A/B solution which is purchased from Dunpang Biotechnology Limited company;
the 96-hole enzyme label plate is purchased from Shenzhen Jinlau actual Co., Ltd;
calf serum was purchased from national sea bioengineering, ltd, langzhou;
goat anti-human IgG HRP secondary antibody was purchased from Sino Biological Inc (LOT: SSA002, HO13AP 1001);
the serum is from Kexing medium vitamin production
Volunteers immunized with the novel inactivated coronavirus vaccine (Vero cells).
Example 1.
This example provides a method for preparing recombinant RBD protein, as follows:
(A) constructing a plasmid containing an RBD protein gene sequence;
(B) and (3) carrying out protein expression on the plasmid obtained in the step (A) by using 293F cells, culturing for 3-5 days, and purifying by adopting affinity chromatography to obtain the recombinant RBD protein.
Example 2.
This example used the recombinant RBD protein prepared in example 1 to detect levels of novel coronavirus IgG antibodies in a sample as follows:
(1) diluting the recombinant RBD protein and 0.01M PBS according to a final concentration of 1 mu g/mL, uniformly mixing, adding a 96-hole enzyme label plate, 100 mu L/hole, incubating at 5 ℃ for 18h, washing the plate for three times by using a phosphate buffer solution containing Tween 20, and patting to dry;
(2) and (2) sealing each hole of the ELISA plate obtained in the step (1) by using 0.01M phosphate buffer saline solution containing 10% calf serum at 37 ℃ for 2h, then washing the plate by using phosphate buffer saline solution containing Tween 20 for three times, and patting the plate dry to obtain the ELISA plate coated with the recombinant RBD protein.
(3) Diluting the serum to be detected by using a sample diluent, diluting the serum before the novel coronavirus vaccine immunization according to the ratio of 1:40, 1:80 and 1:160, and diluting the serum after the immunization according to the ratio of 1:160 and 1: 320. diluting in a gradient of 1:640, 1:1280, 1:2560 and 1:5120, placing in each hole of an enzyme label plate coated with recombinant RBD protein, incubating for 1h at 37 ℃, washing the plate for 3 times by using phosphate buffered saline containing Tween 20, and drying by beating;
(4) adding HRP (horse radish peroxidase) labeled antibody diluent of goat anti-human IgG into each hole of the ELISA plate obtained in the step (3), incubating for 1h, washing the plate for 5 times by using phosphate buffer solution containing Tween 20, and drying by beating;
(5) 50 mu L of color development liquid A, B is added into each hole of the ELISA plate obtained in the step (4), color development is carried out for 15min at room temperature, and then 50 mu L of stop solution is added to stop reaction;
(6) the OD value of each hole of the microplate at the wavelength of 450nm is measured by using a microplate reader, and 630nm is used as a reference.
In the test OD value result, the OD value of a negative control hole (sample diluent) is not higher than 0.05, 2.1 times of the OD value of the negative control hole is taken as a cutoff value, the positive judgment standard of a single dilution multiple is that the OD value is not less than 0.105, and the titer of the sample to be detected is the highest dilution multiple reaching positive conversion.
Serum RBD IgG antibody titer before and after immunization of 10 volunteers new crown vaccine was detected by the detection method provided in example 2, and the results are shown in tables 1 and 2:
TABLE 1
TABLE 2
As can be seen from tables 1 and 2, the IgG antibody titer of the serum before and after immunization of the new corona vaccine can be detected by the method established by the invention, and the serum titer after immunization is increased by 4 times compared with that before immunization, namely positive conversion. The IgG antibody titer of the serum before immunization is less than 40, so that the IgG titer of the serum after immunization is more than or equal to 160, and the IgG titer is positive conversion.
Comparative example 1
This comparative example provides a method for detecting neutralizing antibodies using a viral suspension, as follows:
serum samples collected from vaccine immunized volunteers were inactivated at 56 ℃ for 0.5h and serially diluted in cell culture medium in two steps. The diluted serum was mixed with 100CCID50The virus suspensions were mixed in a 96 well plate at a ratio of 1:1, 5% CO at 36.5 deg.C2Incubate in incubator for 2 hours. The product is obtained by mixing (1-2). times.104Vero cells were added to the serum virus mixture at 5% CO2Incubate at 36.5 ℃ for 5 days in an incubator. The cytopathic effect of the samples was recorded under the microscope, with the end titer being the reciprocal of the highest dilution of serum that did not produce cytopathic effect.
The serum before and after immunization of the new corona vaccine is detected by the methods provided in example 2 and comparative example 1 respectively, and the specificity and sensitivity of the two methods are compared.
Among these, 30 volunteer sera were tested, and among the 30 serum of the new corona vaccine volunteer, there were 10 placebo groups and 20 vaccine groups.
The titers of the neutralizing antibody before and after immunization and the RBD IgG antibody detected by the methods of example 2 and comparative example 1 are shown in tables 3 and 4, respectively;
TABLE 3
TABLE 4
The positive conversion rates of the neutralizing antibody and the IgG antibody of one group of different time points are counted from the tables 3 and 4, and the results are shown in the table 5;
TABLE 5
As can be seen from tables 3-5, 30 new coronal vaccine volunteer sera had 10 placebo groups and 20 vaccine groups, and both the neutralizing antibody and IgG antibody in the placebo group were negative; the neutralizing antibody and the IgG antibody of the serum before the immunization of the vaccine group are negative, and the specificity of the detection method provided by the invention is 100%. From positive conversion samples of two antibodies after vaccine immunization, the sensitivity of the detection method provided by the invention in 0 day of immunization 28 days is 56%, the sensitivity of blood collection in 0 and 28 day of immunization 35 days is 85%, and the sensitivities in 0 and 28 day of immunization 42 days and 56 days are both 100%.
And counting the positive conversion rates of the neutralizing antibody and the IgG antibody at each time point before and after the immunization of the vaccine, wherein the positive conversion rates of the two antibodies at each time point are consistent, and the difference is within 10 percent, which shows that the detection method provided by the invention is well matched with the detection method of the neutralizing antibody, can replace the detection of the neutralizing antibody, and is used as a high-efficiency new crown vaccine antibody evaluation method.
According to the method provided in example 2, the elisa plate coated with the recombinant RBD protein was dried and sealed to form a dry plate, the reagents were divided into separate reagent detection cassettes, and the differences between the detection methods and the kits were investigated by comparison, see example 3.
Example 3 comparison of Wet and Dry plate assay kits made with recombinant RBD proteins of the invention
In this example, the recombinant RBD protein prepared in example 2 was used to prepare a novel coronavirus IgG antibody level detection kit, which was composed of the following components:
i. an ELISA plate (the ELISA plate obtained in the step (2) of the embodiment 2) coated with the recombinant RBD protein, and a 96-well plate with the specification;
a sheep anti-human HRP-labeled antibody;
enzyme dilutions: a lotion containing 10% calf serum and 1% casein;
negative control: a sample diluent;
v. sample diluent: 0.01M phosphate buffered saline containing 10% calf serum or containing 10% bovine serum albumin;
color developing solution: a commercially available A/B solution;
vii, stop solution: 2M sulfuric acid solution.
The test results are shown in Table 6;
TABLE 6
As is clear from Table 6, the detection kit (dry plate) prepared by the method of the present invention still did not affect the detection sensitivity, and the detection results of example 2 and example 3 were completely consistent.
According to the embodiment and the performance test result, the detection method and the detection kit provided by the invention are short in time consumption, the result is obtained in 3-4 hours, the requirement on the experimental environment is low, the operation can be carried out in an open laboratory, and the operation training of personnel is simple.
When the detection method and the kit are used for detecting the new coronavirus RBD IgG antibody, the antibody positive conversion rate is close to the neutralizing antibody positive conversion rate, the specificity of the method reaches 100 percent, and the sensitivity of serum detection is 100 percent after the vaccine is immunized for 2 weeks.
The detection method and the detection kit can be efficiently applied to the evaluation of serum antibodies after the immunization of the new coronavirus, and can be applied to the clinical screening of serology after the infection of the new coronavirus.
Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. A novel coronavirus IgG antibody level detection kit is characterized in that the kit comprises an enzyme label plate coated with recombinant RBD protein.
2. The kit of claim 1, wherein said recombinant RBD protein is produced by a method comprising:
(A) constructing a plasmid containing an RBD protein gene sequence;
(B) and (3) carrying out protein expression on the plasmid obtained in the step (A) by using 293F cells, and then purifying to obtain the recombinant RBD protein.
3. The kit of claim 1 or 2, wherein the preparation method of the ELISA plate coated with the recombinant RBD protein comprises the following steps:
(a) uniformly mixing the recombinant RBD protein and the coating solution, placing the mixture in holes of an ELISA plate for incubation, and then washing the plate;
(b) and (b) sealing each hole of the ELISA plate obtained in the step (a) by using a sealing solution, washing the plate, and drying to obtain the ELISA plate coated with the recombinant RBD protein.
4. The kit of claim 3, wherein the coating solution is 0.01M phosphate buffered saline;
and/or the confining liquid is 0.01M phosphate buffer saline solution containing 10% calf serum or containing 10% bovine serum albumin;
and/or the washing solution used for washing the plate is phosphate buffered saline solution containing Tween 20.
5. The kit according to claim 3 or 4, wherein the concentration of the solution obtained by uniformly mixing the recombinant RBD protein and the coating solution is 0.5-2 μ g/mL.
6. The kit according to any one of claims 1 to 5, characterized in that the kit further comprises an HRP-labeled antibody of goat anti-human IgG, an enzyme diluent, a negative control, a sample diluent, a developing solution and a stopping solution.
7. The kit of claim 6, wherein the enzyme diluent is a wash solution comprising calf serum and casein;
and/or, the negative control is a sample diluent.
8. The kit of claim 6 or 7, wherein the sample diluent is a 0.01M phosphate buffered saline solution containing calf serum or containing bovine serum albumin.
9. The kit according to any one of claims 6 to 8, wherein the stop solution is a 2M sulfuric acid solution.
10. A novel coronavirus IgG antibody level detection method, which is characterized by comprising the following steps:
(1) preparing an ELISA plate coated with recombinant RBD protein;
(2) diluting a sample to be detected, and then placing the sample in each hole of the ELISA plate obtained in the step (1) for incubation;
(3) adding HRP (horse radish peroxidase) labeled antibody of goat anti-human IgG into each hole of the ELISA plate obtained in the step (2) for incubation;
(4) adding a developing solution into each hole of the ELISA plate obtained in the step (3) for developing, and then adding a stop solution to terminate the reaction;
(5) and measuring the OD value of each hole of the ELISA plate at the wavelength of 450nm by using an ELISA reader, wherein 630nm is the reference wavelength.
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