CN112505326A - Kit for detecting neutralizing antibody of new coronavirus - Google Patents
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Abstract
The invention provides a novel detection kit for a coronavirus neutralizing antibody. The kit is based on BAS-HTRF technology and mainly comprises: biotin-labeled hACE2, neocoronavirus spike protein RBD-Tag1, energy donor Streptavidin-Eu cryptate, energy receptor MAb Anti-Tag1-d2 and neocoronavirus neutralizing antibody. The method combines the BAS technology and the HTRF technology, is used for screening the novel coronavirus neutralizing antibody, can realize screening within 3 hours, is simple to operate, and does not need to carry out plate washing processes for many times. The combination of BAS and HTRF greatly improves the reaction sensitivity, and both systems can reduce nonspecific interference to the maximum extent, and are suitable for the detection of serum samples. The method can realize high-throughput detection, and has important significance for solving the detection of the neutralizing antibody of the new coronavirus in a large batch of samples.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a detection kit for a neutralizing antibody of a novel coronavirus.
Background
Since 2019, a novel coronavirus (SARS-CoV-2) has caused infection of millions of people worldwide, and development of a therapeutic agent and a prophylactic vaccine for COVID-19 (novel coronavirus pneumonia) has been actively carried out in various countries in the world. Due to the high pathogenicity and infectivity of SARS-CoV-2, all tests using live virus to assess the efficacy of related products must be performed in a biosafety third-level laboratory, which has largely hampered the development of vaccines and related drugs. Therefore, there is an urgent need for a safe, highly operable and easily standardized method for evaluating the antiviral effects of vaccines or drugs in preclinical and clinical stages. Among other things, detection of neutralizing antibodies against SARS-CoV-2 helps to understand the status of the protective immune response in COVID-19 patients and asymptomatic carriers.
SARS-CoV-2 is a single-stranded RNA virus with a genome length of approximately 29.8 kb. Two thirds of the genome are non-structural genes, which encode primarily enzymes associated with viral replication, while the remaining third, in turn, encodes four structural proteins: spinous process protein (S), small envelope protein (E), matrix protein (M) and nucleocapsid protein (N). Wherein the S protein contains the binding domain of the viral Receptor (RBD), which mediates the processes of viral attachment and entry into cells, and the coronavirus S protein is the major protein that stimulates the immune system to produce neutralizing antibodies after infection of the human body by the virus.
At present, the commonly used screening method of molecular level is enzyme-linked immunosorbent assay (ELISA), the main process is to coat spinous process protein RBD on a 96-hole ELISA plate, wash off redundant protein after incubating overnight, add the antibody to be detected and Human ACE2 protein, wash after incubating, add horseradish peroxidase (HRP) labeled secondary antibody, wash after incubating, add HRP color reagent finally, stop after reacting for half an hour, read on an ELISA reader, determine OD450And judging whether the antibody is a neutralizing antibody or not according to the signal and the shade of the color. The ELISA detection process has multiple steps and long time, needs to be subjected to plate washing, sample adding and color development for multiple times, cannot achieve high flux, and the coating protein can cover some binding sites to cause false negative results.
Disclosure of Invention
The invention aims to provide a novel detection kit for coronavirus neutralizing antibodies.
It is another object of the present invention to provide a novel method for detecting neutralizing antibodies to coronaviruses (including non-diagnostic and therapeutic purposes).
In order to achieve the object of the present invention, in a first aspect, the present invention provides a novel coronavirus neutralizing antibody detection kit based on BAS-HTRF technology, comprising: the kit comprises biotin-labeled hACE2 protein, protein-labeled neocoronavirus spike protein RBD, energy donor Streptavidin-Eu cryptate, energy receptor d2 or XL665 containing anti-protein-labeled antibody, a positive standard of a neutralizing antibody of the neocoronavirus, sample diluent and detection solution.
Alternatively, the kit comprises: the kit comprises biotin-labeled neocoronavirus spike protein RBD, protein-labeled hACE2, energy donor Streptavidin-Eu cryptate, energy receptor d2 or XL665 containing anti-protein-labeled antibody, a positive standard of a neutralizing antibody of the neocoronavirus, a sample diluent and a detection solution.
Wherein the detection solution is PPI Europium detection buffer from Cisbio Bioassays, Cat 61DB9 RDF.
The energy donor Streptavidin-Eu cryptate was from Cisbio Bioassays, Cat 610 SAKLA.
Energy receptor d2 was obtained from Cisbio Bioassays under the designation 61 HISDLA.
The protein tag is selected from 6HIS, GST, FLAG, and the corresponding energy receptor containing Anti-protein tag antibody is selected from MAb Anti 6HIS-d2 or MAb Anti 6HIS-XL665, MAb Anti GST-d2 or MAb Anti GST-XL665, MAb Anti FLAG-d2 or MAb Anti FLAG-XL 665. Preferably, the protein tag is 6HIS and the energy receptor containing the Anti-protein tag antibody is MAb Anti 6HIS-d2 or MAb Anti 6HIS-XL 665.
Wherein the sample diluent is PBS containing 0-0.02% v/v Tween-20 and 0.1-0.5% BSA, and pH7.4. Preferably, the sample diluent is PBS containing 0.02% v/v Tween-20 and 0.1% BSA, pH 7.4.
The preparation method of the positive standard substance of the neutralizing antibody of the novel coronavirus comprises the following steps: the new coronavirus S1 protein is used to immunize mice, and the neutralizing antibody is obtained by separating and purifying the serum of the mice.
The kit further comprises a white 96-well plate and/or a 384-well plate.
In a second aspect, the present invention provides a novel method for detecting neutralizing antibodies to coronaviruses (including non-diagnostic and therapeutic purposes) comprising the steps of:
1) preparing a biotin-labeled hACE2 protein with a certain concentration, a new coronavirus spike protein RBD with a certain concentration and a new coronavirus neutralizing antibody positive standard solution with different concentrations by using the sample diluent;
2) adding 2 mu L of positive standard substance solution of the neutralizing antibody of the new coronavirus, 4 mu L of 5 mu g/mL biotin-labeled hACE2 solution and 4 mu L of 5 mu g/mL protein-labeled new coronavirus spike protein RBD solution into a white pore plate in sequence, and reacting for 15 minutes at room temperature;
3) diluting energy donor Streptavidin-Eu cryptate and energy receptor d2 containing anti-protein tag antibody with detection solution according to a certain proportion, mixing in equal volume, adding 10 mu L of the mixed solution into each hole, reacting at room temperature for 2 hours, reading by using an enzyme-linked immunosorbent assay, and respectively obtaining fluorescent signal values corresponding to 665nm and 620nm, and marking as F665And F620And calculate F665/F620A ratio; then, according to the concentration of the positive standard solution of the neutralizing antibody of the new coronavirus and the corresponding F665/F620Establishing a regression equation according to the ratio, and drawing a standard curve;
4) replacing the positive standard solution of the new coronavirus neutralizing antibody in the step 2) with a sample solution to be detected, and obtaining F of the sample solution to be detected by adopting the same method665/F620Substituting the ratio into the standard curve so as to judge the neutralization effect of the new coronavirus neutralizing antibody in the sample solution to be detected;
in the present invention, the amino acid sequence of the hACE2 protein is shown in GenBank: NP _068576.1, amino acid sequence of new coronavirus spike protein RBD see GenBank: QHD 43416.1.
The biotin-labeled hACE2 protein is obtained by adopting an in-vivo enzyme labeling mode and is labeled by biotin single-point fixed points.
The new coronavirus spike protein RBD is a purified recombinant protein.
The present invention provides another novel method for detecting neutralizing antibodies to coronavirus (including non-diagnostic and therapeutic purposes) comprising the steps of:
1) preparing a biotin-labeled new coronavirus spike protein RBD with a certain concentration, a new coronavirus spike protein hACE2 with a certain concentration and a new coronavirus neutralizing antibody positive standard solution with different concentrations by using the sample diluent;
2) adding 2 mu L of new coronavirus neutralizing antibody positive standard solution, 4 mu L of 5 mu g/mL biotin-labeled new coronavirus spike protein RBD solution and 4 mu L of 5 mu g/mL protein-labeled hACE2 solution into a white pore plate in sequence, and reacting for 15 minutes at room temperature;
3) diluting energy donor Streptavidin-Eu cryptate and energy receptor d2 containing anti-protein tag antibody with detection solution according to a certain proportion, mixing in equal volume, adding 10 mu L of the mixed solution into each hole, reacting at room temperature for 2 hours, reading by using an enzyme-linked immunosorbent assay, and respectively obtaining fluorescent signal values corresponding to 665nm and 620nm, and marking as F665And F620And calculate F665/F620A ratio; then, according to the concentration of the positive standard solution of the neutralizing antibody of the new coronavirus and the corresponding F665/F620Establishing a regression equation according to the ratio, and drawing a standard curve;
4) replacing the positive standard solution of the new coronavirus neutralizing antibody in the step 2) with a sample solution to be detected, and obtaining F of the sample solution to be detected by adopting the same method665/F620Substituting the ratio into the standard curve so as to judge the neutralization effect of the new coronavirus neutralizing antibody in the sample solution to be detected.
The method combines the BAS technology and the HTRF technology (namely the BAS-HTRF technology) to screen the new coronavirus neutralizing antibody, the screening can be realized within 3 hours by the technology, the operation is simple, and the plate washing process is not required for many times.
The Biotin-Avidin amplification System (bast-Avidin System, BAS) is a kind of biological amplification System. The combination between avidin and biotin has very high affinity, and the streptavidin molecule consists of four identical peptide chains, wherein each peptide chain can be combined with one biotin, so that one streptavidin molecule has four binding sites of the biotin molecule and can be simultaneously combined with biotinylated macromolecular derivatives and labels in a multivalent form. Therefore, the BAS has a multi-stage amplification effect, so that the sensitivity of the detection method can be greatly improved when the BAS is applied.
The homogeneous Time-Resolved Fluorescence (HTRF) technique combines two techniques, Fluorescence Resonance Energy Transfer (FRET) and Time Resolved Fluorescence (TRF). The intermolecular interaction process is detected with high specificity and high efficiency by utilizing the principle that the half-life period of the rare earth elements is long and the fluorescence signals are transmitted by the Donor and the Acceptor in a solution system through mutual combination and approach.
The detection and analysis principle of the invention is as follows: HTRF is a homogeneous reaction, with all components participating in the reaction in a white, shallow well plate, containing: biotin-labeled hACE2, neocoronavirus spike protein RBD-Tag1, neocoronavirus neutralizing antibody, energy donor Streptavidin-Eu cryptate and energy receptor MAb Anti-Tag1-d 2. Wherein the biotin-labeled hACE2 is combined with energy donor Streptavidin-Eu cryptate, the new coronavirus spike 1 is combined with an energy receptor MAb Anti-Tag1-d2, and the energy donor and the energy receptor are close to each other due to the combination of hACE2 and RBD, so that signals are detected. The new coronavirus neutralizing antibody blocks the reaction of hACE2 and RBD, so that the distance between an energy donor and an acceptor is enlarged, and the signal is weakened. Since the contents of the biotin-labeled hACE2 and the new coronavirus spike protein RBD-Tag1 in each hole are constant, when the concentration of the new coronavirus neutralizing antibody to be detected is higher, the content of the new coronavirus spike protein RBD bound on the biotin-labeled hACE2 is lower, the signal is weaker, and the inhibition rate is higher; on the contrary, when the concentration of the neutralizing antibody of the new coronavirus to be detected is lower, the signal is stronger, and the inhibition rate is lower. According to the standard curve made by using known new coronavirus neutralizing antibody concentration detection, the neutralizing capacity of the new coronavirus neutralizing antibody to be detected and whether protective antibody and titer are generated after the vaccine is given can be calculated.
By the technical scheme, the invention at least has the following advantages and beneficial effects:
the invention combines the BAS technology and the HTRF technology, greatly improves the reaction sensitivity, can reduce nonspecific interference to the maximum extent by both systems, and is suitable for the detection of serum samples.
In the invention, the biotin-labeled hACE2 protein is obtained by adopting an in-vivo enzyme labeling mode, is labeled by biotin single points and has the characteristics of small batch difference and good repeatability.
Thirdly, the method is simple to operate, fast and effective; the experiment process does not need coating, repeated sample adding and plate washing, and only needs direct reading after sample adding and incubation.
And (IV) high-throughput detection can be realized. The method can be carried out in both white 96-well plates and 384-well plates, and has important significance for solving the detection of the neutralizing antibody of the new coronavirus in a large batch of samples.
Drawings
FIG. 1 is a graph showing the binding between the novel coronavirus spike proteins RBD and hACE2 in accordance with a preferred embodiment of the present invention.
FIG. 2 is a graph showing the inhibition curve of a reference sample of neutralizing antibodies against the novel coronavirus in the preferred embodiment of the present invention.
FIG. 3 is a comparison of the inhibition curves of reference samples for the biotin labeling of hACE2 or RBD assay for novel coronavirus neutralizing antibodies in the preferred embodiment of the present invention.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
The percent in the present invention means mass percent unless otherwise specified; but the percent of the solution, unless otherwise specified, refers to the grams of solute contained in 100mL of the solution.
Example 1 novel coronavirus neutralizing antibody detection kit based on BAS-HTRF technology
This example provides a novel coronavirus neutralizing antibody detection kit based on BAS-HTRF technology, the kit comprising:
(1) the biotin-labeled hACE2 is in a freeze-dried powder form, and the specification is 10 ug;
(2) the new coronavirus spike protein RBD with HIS label is lyophilized powder with specification of 10ug, and is from Acrobiosystems, Cat.No. SPD-C52H 1;
(3) energy donor Streptavidin-Eu cryptate from Cisbio Bioassays, Cat 61 HISDLA;
(4) energy receptor MAb Anti-6HIS-d2 from Cisbio Bioassays, cat # 61 HISDLA;
(5) new coronavirus neutralizing antibody (new coronavirus neutralizing antibody reference substance) in lyophilized powder form with specification of 10ug, from Acrobiosystems, Cat. No. S1N-M122;
(6) a 96-well white, shallow-well plate;
(7) sample diluent: PBS containing 0.02% v/v Tween-20 and 0.1% BSA, pH7.4, with a specification of 12 mL;
(8) detection liquid: PPI Europium detection buffer from Cisbio Bioassays, cat # 61DB9RDF, 5 mL.
Example 2 binding experiment of the novel coronavirus spike protein RBD and biotin-labeled hACE2
The binding assay included the following steps:
(1) preparing a protein solution: the biotin-labeled hACE2 protein was diluted to 2. mu.g/mL (final concentration 0.5. mu.g/mL), and the HIS-tagged novel coronavirus spike protein RBD was diluted two-fold from 3. mu.g/mL (final concentration 0.75. mu.g/mL). The sample diluent used was from the kit of example 1.
(2) Sample adding: prepared protein was added to a white assay plate at 5. mu.L/well each and incubated at room temperature for 15 minutes.
(3) Adding a detection reagent: diluting energy donor Streptavidin-Eu cryptate and energy receptor MAb Anti 6HIS-d2 with detection solution at a ratio of 1:100, mixing in equal volume, adding 10 μ L of the above mixed solution into each well, incubating at room temperature (20-30 deg.C) for 2 hr, reading to obtain fluorescent light with wavelength of 665nm and 620nm, respectivelyOptical signal value, noted F665And F620。
(4) Calculating Ratio = (F) by adopting formula665/F620) X 10000, the higher the ratio, the stronger the binding of hACE2 protein to RBD protein.
Example 3 blocking assay for neutralizing antibodies to New coronavirus
The blocking experiment included the following steps:
(1) preparing a protein and antibody solution: the neutralizing antibody against the new coronavirus was diluted two-fold from 100. mu.g/mL (final concentration of 10. mu.g/mL), the biotin-labeled human ACE2 protein was diluted to 5. mu.g/mL (final concentration of 1. mu.g/mL), and the HIS-tagged new coronavirus spike protein RBD was diluted to 5. mu.g/mL (final concentration of 1. mu.g/mL).
(2) Sample adding: adding 2 mu L of new coronavirus neutralizing antibody, 4 mu L of biotin label ACE2 and 4 mu L of new coronavirus spinous process protein RBD with HIS label into a white light pore plate in sequence, setting a positive signal pore (without new coronavirus neutralizing antibody) and a blank signal pore (without new coronavirus neutralizing antibody and new coronavirus spinous process protein RBD with HIS label), and reacting for 15 minutes at room temperature.
(3) Adding a detection reagent: diluting energy donor Streptavidin-Eu cryptate and energy receptor MAb Anti 6HIS-d2 with detection solution according to a ratio of 1:100, mixing in equal volume, adding 10 μ L of the mixed solution into each hole, incubating for 2 hours at room temperature (20-30 ℃), reading, obtaining fluorescence signal values corresponding to 665nm and 620nm respectively, and recording as F665And F620。
(4) Calculating Ratio = (F) by adopting formula665/F620) X 10000, the lower the ratio, the stronger the neutralization effect.
Example 4 different Buffer composition verification
This example verifies the blocking experiment under three different buffer combinations, as follows:
combination 1: the sample diluent and the energy donor/energy acceptor diluent are PBS containing 0.02% v/v Tween-20 and 0.1% BSA, and the pH value is 7.4;
and (3) combination 2: both the sample dilutions and the energy donor/energy acceptor dilutions were PPI Europium detection buffer from Cisbio Bioassays, cat # 61DB9 RDF;
and (3) combination: the sample diluent was PBS containing 0.02% v/v Tween-20 and 0.1% BSA, pH7.4, and the energy donor/energy acceptor diluent was PPI Europium detection buffer from Cisbio Bioassays.
As can be seen from Table 1, by IC50Comparing, if the sample diluent and the detection solution both use the PPI Europium detection buffer sold in the market, the detection sensitivity is about 2 times lower than that of the other two groups; if the sample diluent and the detection solution are both PBS solution with 0.02% v/v Tween-20 and 0.1% BSA, the fluorescence intensity corresponding to pH7.4 and 620nm is too high to exceed the detection limit of the instrument (<260000), 665nm, so that the Ratio value is low; if self-prepared PBS (0.02% v/v Tween-20 and 0.1% BSA) is used, pH7.4 is used as a sample diluent, and PPI Europium detection buffer is used as an energy donor and energy acceptor diluent, the sensitivity and Ratio value of the reaction can be obviously improved.
EXAMPLE 5 establishment of Standard Curve
Setting the value obtained by subtracting the Ratio of the blank hole from the Ratio of the positive hole containing 0 mug/mL as B0, and setting the value obtained by subtracting the Ratio of the blank hole from the Ratio of the rest experimental holes as B; with B/B0The percentage of values is the ordinate, the corresponding standard concentration value is the abscissa, and a four-parameter fitting equation is used: y = (A-D)/[1 + (x/C) ^ B]+ D represents the standard inhibition curve for detection of neutralizing antibodies against the novel coronavirus. The four parameters of the measured curve are respectively: a = 97.99, B = -1.652, C = 1.126, D = -5.517, R2=0.9966。
Wherein, A: estimating an asymptote on the curve;
d: estimating the value of an asymptote under a curve;
b: the slope of the curve;
c: maximum binding half the corresponding dose.
The binding curve of the new coronavirus spike protein RBD and hACE2 is shown in figure 1, and the inhibition curve of the new coronavirus neutralizing antibody reference product is shown in figure 2.
Example 6 Effect of the test substrate
The method can detect the neutralizing antibody of the new coronavirus in the serum and verify the IC of different serum matrixes (10%, 25%, 50%, 100%) to the IC50Interference of values. The IC of the curves in different matrices was calculated using the reference sample of neutralizing antibodies against the new coronavirus (i.e., neutralizing antibodies against the new coronavirus in the kit of example 1) as the fitted curve50The value is obtained. The results of the different matrix assays are shown in table 2:
from the above results, it can be seen that as the serum concentration increases, the IC is corrected50The influence of the value is small.
Example 7 Effect of Biotin labeling of different Components on the neutralizing Effect
In example 3, the hACE2 protein is labeled with biotin, and the RBD protein is a recombinant protein containing an HIS tag. The two component ratios were tested as in example 3 and the final neutralization effect was compared.
Wherein, the HillSlope represents the gradient of the curve, and the closer to-1, the more gentle the curve and the wider the linear range.
As shown in Table 3, the sensitivity of the biotin-labeled hACE2 and the sensitivity of the biotin-labeled RBD protein are consistent, but the linear range of the biotin-labeled RBD protein is narrow, which is not favorable for the quantitative detection of antibodies.
The results of comparison of the inhibition curves of reference products for the biotin labeling hACE2 or RBD assay for the novel coronavirus neutralizing antibodies are shown in FIG. 3.
Example 8 precision assay of the kit
The procedure of example 2 was repeated for 6 different days to obtainCalculating IC50The value is obtained. The measurement results are shown in table 4:
as can be seen from Table 4, the IC of the different day experiments50The change is very small, which indicates that the precision of the kit is good.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may 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 (7)
1. Kit for the detection of neutralizing antibodies against a novel coronavirus, characterized in that it is based on BAS-HTRF technology and comprises: biotin-labeled hACE2 protein, protein-labeled neocoronavirus spike protein RBD, energy donor Streptavidin-Eu cryptate, energy receptor d2 or XL665 containing anti-protein-labeled antibody, a positive standard of a neutralizing antibody of the neocoronavirus, sample diluent and detection solution; or,
the kit comprises: the kit comprises biotin-labeled neocoronavirus spike protein RBD, protein-labeled hACE2, energy donor Streptavidin-Eu cryptate, energy receptor d2 or XL665 containing anti-protein-labeled antibody, a positive standard of a neutralizing antibody of the neocoronavirus, a sample diluent and a detection solution.
2. The kit of claim 1, wherein the protein tag is selected from 6HIS, GST, FLAG and the corresponding Anti-protein tag antibody-containing energy receptor is selected from MAb Anti 6HIS-d2 or MAb Anti 6HIS-XL665, MAb Anti GST-d2 or MAb Anti GST-XL665, MAb Anti FLAG-d2 or MAb Anti FLAG-XL 665.
3. The kit of claim 1, wherein the sample diluent is PBS containing 0-0.02% v/v Tween-20 and 0.1-0.5% BSA, ph 7.4.
4. The kit of claim 1, wherein the positive standard for neutralizing antibodies against the novel coronavirus is prepared by a method comprising: the new coronavirus S1 protein is used to immunize mice, and the neutralizing antibody is obtained by separating and purifying the serum of the mice.
5. The kit of any one of claims 1 to 4, wherein the kit further comprises a white 96-well plate and/or a 384-well plate.
6. A method for detecting neutralizing antibodies against novel coronaviruses for non-diagnostic and therapeutic purposes, comprising the steps of:
1) preparing a biotin-labeled hACE2 protein with a certain concentration, a new coronavirus spike protein RBD with a certain concentration and a new coronavirus neutralizing antibody positive standard solution with different concentrations by using the sample diluent;
2) adding 2 mu L of positive standard substance solution of the neutralizing antibody of the new coronavirus, 4 mu L of 5 mu g/mL biotin-labeled hACE2 solution and 4 mu L of 5 mu g/mL protein-labeled new coronavirus spike protein RBD solution into a white pore plate in sequence, and reacting for 15 minutes at room temperature;
3) diluting energy donor Streptavidin-Eu cryptate and energy receptor d2 containing anti-protein tag antibody with detection solution according to a certain proportion, mixing in equal volume, adding 10 mu L of the mixed solution into each hole, reacting at room temperature for 2 hours, reading by using an enzyme-linked immunosorbent assay, and respectively obtaining fluorescent signal values corresponding to 665nm and 620nm, and marking as F665And F620And calculate F665/F620A ratio; then, according to the concentration of the positive standard solution of the neutralizing antibody of the new coronavirus and the corresponding F665/F620Establishing a regression equation according to the ratio, and drawing a standard curve;
4) replacing the positive standard solution of the new coronavirus neutralizing antibody in the step 2) with a sample solution to be detected, and obtaining a sample to be detected by adopting the same methodProduct solution F665/F620Substituting the ratio into the standard curve so as to judge the neutralization effect of the new coronavirus neutralizing antibody in the sample solution to be detected;
wherein the protein tag, positive standard of neutralizing antibody of new coronavirus, biotin-labeled hACE2 protein, new coronavirus spike protein RBD containing protein tag, energy donor Streptavidin-Eu cryptate, energy acceptor d2 containing anti-protein tag antibody and sample diluent are from the kit of any one of claims 1-5.
7. A method for detecting neutralizing antibodies against novel coronaviruses for non-diagnostic and therapeutic purposes, comprising the steps of:
1) preparing a biotin-labeled new coronavirus spike protein RBD with a certain concentration, a new coronavirus spike protein hACE2 with a certain concentration and a new coronavirus neutralizing antibody positive standard solution with different concentrations by using the sample diluent;
2) adding 2 mu L of new coronavirus neutralizing antibody positive standard solution, 4 mu L of 5 mu g/mL biotin-labeled new coronavirus spike protein RBD solution and 4 mu L of 5 mu g/mL protein-labeled hACE2 solution into a white pore plate in sequence, and reacting for 15 minutes at room temperature;
3) diluting energy donor Streptavidin-Eu cryptate and energy receptor d2 containing anti-protein tag antibody with detection solution according to a certain proportion, mixing in equal volume, adding 10 mu L of the mixed solution into each hole, reacting at room temperature for 2 hours, reading by using an enzyme-linked immunosorbent assay, and respectively obtaining fluorescent signal values corresponding to 665nm and 620nm, and marking as F665And F620And calculate F665/F620A ratio; then, according to the concentration of the positive standard solution of the neutralizing antibody of the new coronavirus and the corresponding F665/F620Establishing a regression equation according to the ratio, and drawing a standard curve;
4) replacing the positive standard solution of the new coronavirus neutralizing antibody in the step 2) with a sample solution to be detected, and obtaining F of the sample solution to be detected by adopting the same method665/F620The ratio is substituted into the standard curve to judgeNeutralizing effect of the new coronavirus neutralizing antibody in the sample solution to be detected;
wherein the protein tag, positive standard of neutralizing antibody of new coronavirus, biotin-labeled new coronavirus spike protein RBD, hACE2 containing protein tag, energy donor Streptavidin-Eu cryptate, energy acceptor d2 containing anti-protein tag antibody and sample diluent are from the kit of any one of claims 1-5.
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