阅读说明：本技术 一种针对SARS-CoV-2 RBD结构域的特异性中和抗体竞争法ELISA试剂盒 (Specific neutralizing antibody competition method ELISA kit aiming at SARS-CoV-2 RBD structural domain ) 是由 徐建华 张嘉琪 刘万里 石亚玲 王大伟 何金勇 邓敏 于 2020-11-18 设计创作，主要内容包括：本发明公开了一种针对SARS-CoV-2 RBD结构域的特异性中和抗体竞争法ELISA试剂盒,该试剂盒含有包被有人ACE2蛋白的固相载体、生物素标记的RBD蛋白。该试剂盒可用于提高新冠检测率,一定程度上弥补核酸检测假阴性带来的漏检问题；可用于检测新冠康复病人体内中和抗体含量,以判断是否可以复工复产及是否存在再次感染风险；还可有效地监测临床研究中的疫苗接种反应及作为后期疫苗效果的评价指标；中和抗体测定可支持强化监测措施,如普遍检测、活动性病例发现、接触者追踪和联系群集。(The invention discloses a specific neutralizing antibody competition ELISA kit aiming at SARS-CoV-2 RBD structure domain, which comprises a solid phase carrier coated with human ACE2 protein and biotin-labeled RBD protein. The kit can be used for improving the detection rate of new coronas and making up the problem of missed detection caused by false negative of nucleic acid detection to a certain extent; can be used for detecting the content of the neutralizing antibody in the body of a patient with new coronary rehabilitation to judge whether the patient can carry out repeated work and production and whether the patient has the risk of secondary infection; the vaccine inoculation reaction in clinical research can be effectively monitored and used as an evaluation index of the later vaccine effect; neutralizing antibody assays may support intensive monitoring measures such as general detection, active case finding, contacter tracking, and contact clustering.)

1. A novel competitive enzyme-linked immunoassay kit for a coronavirus neutralizing antibody is characterized by comprising a solid phase carrier coated with human ACE2 protein and biotin-labeled RBD protein.

2. The competitive enzyme-linked immunoassay kit according to claim 1, wherein the kit further comprises a color developing solution, a washing solution, a stop solution, a sample diluent, an enzyme-labeled secondary antibody, and a SARS-CoV-2 neutralizing antibody.

3. The competitive enzyme-linked immunoassay kit according to claim 1, wherein the antibody inhibition rate of the kit is represented by the formula: inhibition rate (1-OD value to be measured/negative control OD value) × 100%.

4. The competitive enzyme-linked immunoassay kit according to claim 2, wherein if the inhibition rate is less than 35.93%, the sample is free of neutralizing antibodies; the inhibition rate is higher than 35.93%, and the neutralizing antibody exists in the sample.

5. The competitive enzyme-linked immunoassay kit according to claim 1, wherein the test sample is serum.

6. The competitive enzyme-linked immunoassay kit according to claim 1, wherein the enzyme-labeled secondary antibody is HRP-streptavidin.

7. The competitive enzyme-linked immunoassay kit of claim 1, wherein the washing solution is a PBST solution containing 0.05% (v/v) tween and 0.5% (w/v) BSA.

8. The competitive enzyme-linked immunoassay kit according to claim 1, wherein the sample diluent is a PBS solution containing 0.5% (w/v) BSA and has a pH of 7.4.

9. The competitive enzyme-linked immunoassay kit according to claim 1, wherein the color-developing solution: contains 2mg/ml TMB, 50mmol/L disodium hydrogen phosphate, 24.3mmol/L citric acid, 0.75% (v/v) hydrogen peroxide.

10. The competitive ELISA kit of claim 1 wherein the stop solution is 2mol/L H₂SO₄。

Technical Field

The invention relates to the technical field of immunodetection, in particular to an ELISA kit for a specific neutralizing antibody competition method aiming at a SARS-CoV-2 RBD structural domain.

Background

The novel coronavirus (SARS-CoV-2) is a novel beta coronavirus (beta-coronavirus) which is from the subgenus Sarbecovirus, is circular or elliptical, has a diameter of 60-140 nm, and is in a crown-like shape under an electron microscope. Has a similar receptor binding domain to SARS-CoV, and relies on the human cell surface ACE2 protein as a cell entry receptor. The envelope spike protein (S) mediates receptor binding and membrane fusion and is also the primary target of antibodies.

The novel coronavirus neutralizing antibody is an antibody produced in blood of a patient suffering from the novel coronavirus pneumonia several days or about one week after the infection. Neutralizing antibodies (nabs) are typically raised against the S protein, and the Receptor Binding Domain (RBD) of the S protein (spike protein) on the surface of the virus exhibits direct neutralizing activity against virus entry into cells, and is the primary target of neutralizing antibodies (Abs), preventing efficient binding of the spike protein to the surface a CE2 receptor. In the method of vaccination using inactivated virus, it was demonstrated that the S protein of SARS-CoV-2 is an immunodominant viral antigen via the Receptor Binding Domain (RBD).

Detection of neutralizing antibodies can improve diagnostic rates because of the proportion of false negative problems with real-time RT-PCR; antibody assays can support intensive monitoring measures such as universal detection, active case discovery, contacter tracking, and contact clustering; and the method can also effectively monitor the vaccination reaction in clinical research and be used as an evaluation index of the later vaccine effect.

The existing detection method for SARS-CoV-2 mainly comprises nucleic acid detection and serology detection, and the main method for nucleic acid detection comprises a fluorescence PCR method; the serological detection method mainly comprises the following steps: enzyme-linked immunosorbent assay, colloidal gold chromatography, and chemiluminescence assay. Although the nucleic acid detection has high sensitivity, strong specificity and high accuracy, the operation requirement is relatively high and the detection time is long. The antibody detection sensitivity is high, and the specificity is strong.

Currently, commercial reagents that utilize different viral antigens and detect different antibody classes are in use. However, the serological tests for SARS-CoV-2 currently available on the market are not always detectable in a reliable manner, and these methods currently only detect the presence of a stage of infection and do not allow confirmation of the presence of a risk of reinfection in the patient after healing, and of exposure and infection risk in the general public, which may prevent an appropriate population test for clinical or epidemiological purposes.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a specific neutralizing antibody competition method ELISA kit aiming at a SARS-CoV-2 RBD structural domain, aiming at a high immunogenic receptor binding structural domain (RBD) on a virus Spike soluble outer membrane structural domain (Spike), the in-vivo neutralizing antibody of a coronavirus infected patient is detected by adopting a competition ELISA method technology, and the method has the advantages of reliable theory, practical feasibility, reliable result in the aspects of accuracy and repeatability and can be completed only in a secondary biological safety laboratory. The immunoassay technology has the advantages of high sensitivity, strong specificity, simple operation, good result repeatability and less interference factors.

In order to achieve the purpose, the invention is realized by the following technical scheme:

coating human ACE2 protein on a microporous plate, adding biotin-labeled RBD into to-be-detected serum for reaction, adding reactants into the coated microporous plate for competitive combination, performing color reaction after HRP-streptavidin reaction, measuring absorbance value at 450/630nm, calculating the inhibition rate of a neutralizing antibody, and judging the neutralizing antibody of the new coronavirus in the serum or plasma.

The invention claims a novel competitive enzyme-linked immunoassay kit for coronavirus neutralizing antibodies, which is characterized by comprising a solid phase carrier coated with human ACE2 protein and biotin-labeled RBD protein.

The biotin-labeled RBD protein is expressed by c-terminal poly-histidine tag of DNA sequence coding SARS-COV-2spike protein (RBD); the purified protein is biotinylated in vitro; can specifically bind endogenous neutralizing antibody and ACE2 protein.

The solid phase carrier coated with the human ACE2 protein is a 96-well plate uniformly coated with the human ACE2 protein.

Neutralizing antibodies are produced by hybridomas produced by the fusion of B cells obtained by immunizing mice with purified recombinant SARS-CoV-2(2019nCoV) Spike RBD-MFC protein with mouse myeloma; can be specifically combined with RBD protein.

Preferably, the kit also comprises a developing solution, a washing solution, a stop solution, a sample diluent, an enzyme-labeled secondary antibody and a SARS-CoV-2 neutralizing antibody.

Wherein the neutralizing antibody of SARS-CoV-2 is a positive control. The neutralizing antibody of SARS-CoV-2 is secreted by hybridoma produced by fusion of B cell obtained by immunizing mouse with purified recombinant SARS-CoV-2(2019nCoV) Spike RBD-MFC protein and mouse myeloma.

Preferably, the antibody inhibition rate formula of the kit is as follows: inhibition rate (1-OD value to be measured/negative control OD value) × 100%.

More preferably, the inhibition is less than 35.93%, and the sample is free of neutralizing antibodies; if the inhibition rate is higher than 35.93%, the neutralizing antibody exists in the sample.

Preferably, the test sample is serum.

Preferably, the enzyme-labeled secondary antibody is HRP-streptavidin.

HRP-streptavidin is HRP-labeled streptavidin.

Preferably, the wash solution is a PBST solution containing 0.05% (v/v) Tween and 0.5% (w/v) BSA.

Preferably, the sample dilution is a PBS solution containing 0.5% (w/v) BSA at pH 7.4.

Preferably, the color developing solution: contains 2mg/ml TMB, 50mmol/L disodium hydrogen phosphate, 24.3mmol/L citric acid, 0.75% (v/v) hydrogen peroxide.

Preferably, the stop solution is 2mol/L H₂SO₄。

Most preferably, a specific neutralizing antibody competition ELISA kit for SARS-CoV-2 RBD domain,

a, make up

A 96-well plate coated with human ACE2 protein, 0.2 ng/well (the preparation method comprises coating human ACE2 protein capable of combining with RBD protein in a microplate, configuring into 2ug/ml, 100 μ l/well, coating on an enzyme-linked plate, standing overnight at 4 deg.C, and sealing);

20ng of biotin-labeled RBD protein per well;

enzyme-labeled secondary antibody: HRP-streptavidin;

color development liquid: contains 3,3,5,5' -Tetramethylbenzidine (TMB) 2mg/ml, disodium hydrogen phosphate 50mmol/L, citric acid 24.3mmol/L, and hydrogen peroxide 0.75% (v/v);

stopping liquid: containing 2mol/L H₂SO₄；

Sample diluent: PBS solution containing 0.5% (w/v) BSA, pH 7.4;

washing liquid: PBST solution containing 0.05% (v/v) Tween and 0.5% (w/v) BSA.

Second, use method

1. Sample treatment: adding 1.0 μ l (20ng/μ l) biotin-labeled RBD into 100 μ l of serum sample to be tested, and incubating at 37 deg.C for 60 min;

2. sample incubation: the sample obtained in step 1 was added to a 96-well plate coated with human ACE2 protein for competitive binding, and 100. mu.l/well was added and incubated at 37 ℃ for 60 min.

3. Reacting with an enzyme-labeled secondary antibody: after washing the plate, 100. mu.l of HRP-streptavidin was added to each well for reaction at 37 ℃ for 30 min.

4. TMB color development: after the plate is thoroughly washed, 100 mul of substrate color developing solution is added into each hole, and the plate is developed for 15-20 min in a dark place at 37 ℃.

5. And (4) terminating: adding 50 mul of stop solution, measuring the light absorption value at 450/630nM by an enzyme-linked immunosorbent assay, wherein the color depth in the microporous plate is in negative correlation with the concentration of the substance to be measured.

6. Calculating an inhibition rate: inhibition rate (1-OD value to be measured/negative control OD value) × 100%.

Third, interpretation of results

If the inhibition rate is lower than 35.93%, the sample has no neutralizing antibody; the inhibition rate is higher than 35.93%, and the neutralizing antibody exists in the sample.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a specific neutralizing antibody competition method ELISA kit aiming at SARS-CoV-2 RBD structure domain, the kit can be used for improving the new crown detection rate and making up the problem of missed detection caused by false negative of nucleic acid detection to a certain extent; can be used for detecting the content of the neutralizing antibody in the body of a patient with new coronary rehabilitation to judge whether the patient can carry out repeated work and production and whether the patient has the risk of secondary infection; the vaccine inoculation reaction in clinical research can be effectively monitored and used as an evaluation index of the later vaccine effect; neutralizing antibody assays may support intensive monitoring measures such as general detection, active case finding, contacter tracking, and contact clustering.

Drawings

FIG. 1 is a schematic diagram of the experimental principle and operation of the kit.

Detailed Description

The invention is described in further detail below with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

Example 1 detection of Biotin-labeled RBD neutralization Effect

First, experiment method

To evaluate the neutralizing effect of biotin-labeled RBD protein, the neutralizing effect was measured between different concentrations of biotinylated RBD and different concentrations of neutralizing antibody. The specific operation is as follows:

1. preparation of ELISA plates coated with human ACE 2: coating human ACE2 protein capable of combining with RBD protein in a microplate, configuring into 2 μ g/ml and 100 μ l/well, coating on an enzyme-linked plate, standing at 4 deg.C overnight, and sealing;

2. sample treatment: adopting a healthy human serum sample, adding different amounts of biotin-labeled RBD and neutralizing antibody for binding reaction (the specific dosage of each hole is shown in table 1), and incubating for 60min at 37 ℃;

3. sample incubation: adding the reactant in the step 2 into the ELISA plate coated with human ACE2 prepared in the step 1 for competitive combination, adding samples at 100 mul/hole, and incubating for 60min at 37 ℃;

4. reaction with a secondary antibody: after washing the plate, adding 100 mul HRP-streptavidin into each hole for reaction, and reacting for 30min at 37 ℃;

5. TMB color development: after the plate is thoroughly washed, 100 mul of color development solution (containing 2mg/ml TMB, 50mmol/L disodium hydrogen phosphate, 24.3mmol/L citric acid and 0.75% hydrogen peroxide) is added into each hole, and the plate is developed for 15-20 min in a dark place at 37 ℃;

6. and (4) terminating: adding a stop solution (containing 2mol/L H)₂SO₄) Measuring the light absorption value at 450/630nm by an enzyme-labeling instrument with 50 μ l, wherein the color depth in the microporous plate is in negative correlation with the concentration of the substance to be measured;

7. calculating an inhibition rate: inhibition rate ═ 100% (1-sample OD value/negative control OD value).

Second, Experimental methods

The results are shown in Table 1.

Table 1:

the detection result shows that compared with the test without the addition of the neutralizing antibody, the biotin-labeled RBD has higher inhibition rate on the neutralizing antibody, namely the biotin-labeled RBD selected by the competitive ElISA kit has better neutralizing effect.

And the method finds that when each hole is coated with 0.2ng of human ACE2 protein, the dosage of biotin-labeled RBD protein and the dosage of SARS-CoV-2 neutralizing antibody are respectively 10-40 ng and 73.75-1180 ng, and the biotin-labeled RBD has higher inhibition rate on the SARS-CoV-2 neutralizing antibody.

Example 2 specific neutralizing antibody competition ELISA kit against the RBD domain of the New coronavirus

The components of the ELISA kit for the specific neutralizing antibody competition method aiming at the RBD structural domain of the novel coronavirus are as follows:

A. a 96-well plate coated with human ACE2 protein, 0.2 ng/well (the preparation method comprises coating human ACE2 protein capable of combining with RBD protein in a microplate, configuring into 2ug/ml, 100 μ l/well, coating on an enzyme-linked plate, standing overnight at 4 deg.C, and sealing);

B. 20ng of biotin-labeled RBD protein per well;

C. enzyme-labeled secondary antibody: HRP-streptavidin;

D. color development liquid: contains 3,3,5,5' -Tetramethyllbenzidine (TMB) of 2mg/ml, disodium hydrogen phosphate of 50mmol/L, citric acid of 24.3mmol/L and 0.75% (v/v);

E. stopping liquid: containing 2mol/L H₂SO₄；

F. Sample diluent: PBS solution containing 0.5% (w/v) BSA, pH 7.4;

G. washing liquid: PBST solution containing 0.05% (v/v) Tween and 0.5% (w/v) BSA.

Secondly, the use method of the ELISA kit for the specific neutralizing antibody competition method aiming at the RBD domain of the new coronavirus, namely the ELISA detection method for the specific neutralizing antibody competition method aiming at the RBD domain of the new coronavirus (the experimental operation diagram of the kit is shown in figure 1), comprises the following steps:

1. sample treatment: adding 1.0 μ l (20ng/μ l) biotin-labeled RBD into 100 μ l of serum sample to be tested, and incubating at 37 deg.C for 60 min;

2. sample incubation: the sample obtained in step 1 was added to a 96-well plate coated with human ACE2 protein for competitive binding, and 100. mu.l/well was added and incubated at 37 ℃ for 60 min.

3. Reacting with an enzyme-labeled secondary antibody: after washing the plate, 100. mu.l of HRP-streptavidin was added to each well for reaction at 37 ℃ for 30 min.

4. TMB color development: after the plate is thoroughly washed, 100 mul of substrate color developing solution is added into each hole, and the plate is developed for 15-20 min in a dark place at 37 ℃.

5. And (4) terminating: adding 50 mul of stop solution, measuring the light absorption value at 450/630nM by an enzyme-linked immunosorbent assay, wherein the color depth in the microporous plate is in negative correlation with the concentration of the substance to be measured.

6. Calculating an inhibition rate: inhibition rate (1-OD value to be measured/negative control OD value) × 100%.

EXAMPLE 3 detection of clinical samples

First, experiment method

1. To test the effect of the kit in the new coronary patients, clinical samples were tested using the kit of example 2. In this example, 14 patients with new crown infection after hospitalization for a period of time were included according to the diagnosis and treatment protocol for new coronavirus pneumonia, and 7 healthy patients were also included as controls.

2. The result of the detection

The detection result is shown in table 1, the inhibition rate of the new crown infected patient is compared with that of the healthy patient, the p value is less than 0.05, and the kit provided by the invention can obviously distinguish the new crown infected patient from the healthy patient according to the inhibition rate. When CUT OFF is calculated from NC +2SD (NC is a healthy control, [19.7 ± 7.98]), CUT OFF is calculated to be 19.97+2 × 7.98 as 35.93, i.e., when the inhibition rate is less than 35.93%, it can be considered that no neutralizing antibody is present in the patient, and when the inhibition rate is more than 35.93%, it can be considered that a neutralizing antibody is present in the patient.

Table 2:

the above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.