CN111781354B - Novel coronavirus neutralizing antibody titer detection ELISA kit - Google Patents
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
Abstract
The invention provides a novel ELISA kit for detecting the titer of a coronavirus neutralizing antibody, which comprises: an enzyme label plate coated with biotin-streptavidin labeled human ACE2 protein, horseradish peroxidase labeled novel coronavirus RBD protein, novel coronavirus neutralizing antibody positive control, coating liquid, washing liquid, diluent, confining liquid, developing liquid, stopping liquid and the like. The kit has the characteristics of low cost, simple operation, high sensitivity, high specificity and high accuracy, and can be used for batch and rapid detection of novel coronavirus neutralizing antibodies.
Description
Technical Field
The invention relates to the technical field of immunoassay, in particular to a novel ELISA kit for detecting the titer of a coronavirus neutralizing antibody.
Background
The novel coronavirus SARS-CoV-2 (namely 2019-nCoV) is a virus newly discovered in 2019 and can cause human viral pneumonia or lung infection. The coronavirus genome is encoded as spinous process protein, envelope protein, membrane protein and nucleocapsid protein in sequence. The spinous process protein is the most important surface membrane protein of coronavirus, and contains two subunits, S1 subunit and S2 subunit. Wherein S1 mainly contains Receptor Binding Domain (RBD) responsible for recognizing cell receptors. The spike protein of SARS-CoV-2 interacts with the human Angiotensin-converting enzyme 2 (ACE 2) protein to infect human respiratory epithelial cells. The spinous process protein is responsible for the combination and membrane fusion of the new coronavirus and host cell membrane receptors, and is an important action site of the new coronavirus neutralizing antibody and a key target spot of vaccine design.
The novel corona neutralizing antibody can block the combination of virus spike protein and human ACE2 protein, so as to prevent virus from infecting human respiratory epithelial cell. In the actual development process, antibodies with neutralizing capacity need to be screened so as to achieve the purpose of treatment. In addition, the development of new corona vaccines must also be able to successfully induce neutralizing antibodies (protective antibodies) as a prerequisite for effective prevention of new corona infections. Therefore, there is a need to develop efficient and convenient methods for detecting novel coronavirus neutralizing antibodies.
The current methods for detecting neutralizing antibodies against novel coronaviruses are mainly live virus or pseudovirus neutralization assays. Due to the complex cell experiment process and the limitation of factors such as safety, reagent consumables and the like, the existing method is not suitable for high-throughput large-scale screening work of the new coronavirus neutralizing antibody.
Disclosure of Invention
The invention aims to provide an enzyme-linked immunosorbent assay (ELISA) detection kit which has the advantages of low cost, simple operation, high sensitivity, high specificity and high accuracy and is used for batch and rapid detection of novel coronavirus neutralizing antibodies.
To achieve the object of the present invention, in a first aspect, the present invention provides a novel coronavirus neutralizing antibody titer detection ELISA kit comprising: the kit comprises an enzyme label plate coated with streptavidin and biotin labeled human ACE2 protein, a horseradish peroxidase labeled novel coronavirus RBD protein, a novel coronavirus neutralizing antibody positive control (namely a novel coronavirus neutralizing antibody reference product), a coating solution, a washing solution, a diluent, a sealing solution, a developing solution, a stopping solution and the like.
Preferably, the concentration of streptavidin coated on the ELISA plate is 5ug/mL, the concentration of biotin-labeled human ACE2 protein is 2ug/mL, and the molar ratio of streptavidin to biotin-labeled human ACE2 protein is 1: 4.
The coating method comprises the following steps: the ELISA plate is coated overnight by streptavidin (abbreviated as SA) solution, and after the plate is washed by washing liquid, biotin-labeled human ACE2 protein is added, and after the plate is sealed by sealing liquid and washed, the plate is sealed and stored.
Preferably, in the horseradish peroxidase-labeled novel coronavirus RBD protein, the molar ratio of the horseradish peroxidase to the RBD protein is 4: 1.
The concentration of the horseradish peroxidase-labeled novel coronavirus RBD protein used was 50 ng/mL.
In the present invention, the labeling of the RBD protein is not limited to the labeling of the RBD protein of the coronavirus spike protein with horseradish peroxidase (HRP), and the RBD protein may be labeled with FITC, PE, or the like.
In the invention, the positive control of the novel coronavirus neutralizing antibody is obtained from a Xinguan recovered patient, the variable region sequence of the Xinguan recovered patient is determined, and the positive control is formed by recombination, and the positive control is purchased from Shanghai Xiangyao biological technology limited.
Preferably, the coatingThe solution is sodium carbonate-sodium bicarbonate buffer solution, and comprises the following specific components: 15 mM Na2CO3，35 mM NaHCO3，pH 9.6。
Preferably, the washing solution is: 20mM Tris, 150mM NaCl, 0.5% v/v Tween-20, pH 7.4.
Preferably, the diluent is a washing solution containing 0.5% bovine serum albumin.
Preferably, the color developing solution is: 50mM Na2HPO4·12H2O +25 mM citric acid (C)6H8O7）+40 mg/mL TMB+3% v/v H2O2，pH5.5。
Preferably, the stop solution is a 1M sulfuric acid solution.
In the present invention, the microplate may be a 96-well plate.
Horseradish peroxidase-labeled novel coronavirus RBD protein and novel coronavirus neutralizing antibody positive control, preferably in a freeze-dried powder form in the kit. The lyophilized powder contains 10% trehalose (lyophilized protectant).
In a second aspect, the present invention provides a novel method for ELISA detection of coronavirus neutralizing antibodies.
The detection principle of the kit provided by the invention is as follows: each experimental hole and each positive control hole on the enzyme label plate are coated with the same amount of biotin-streptavidin labeled human ACE2 protein, a negative hole (containing no biotin labeled human ACE2 protein) is arranged, a sample to be detected and a new coronavirus neutralizing antibody positive control and a constant amount of HRP labeled new crown spike protein RBD are added, the HRP labeled new crown spike protein RBD and a new crown neutralizing antibody to be detected compete with each other to react with a solid phase antigen (human ACE2 protein), and because the solid phase antigen in each hole and the added HRP labeled new crown spike protein RBD have constant content, when the concentration of the new crown neutralizing antibody to be detected is high, the content of the HRP labeled new crown spike protein RBD combined on the solid phase antigen is low, a developing solution is added after washing with the HRP, the developing reaction is light, the OD value detected by an enzyme label instrument is low, and the inhibition rate is high; on the contrary, when the concentration of the new crown neutralizing antibody to be detected is low, the detected OD value is high, and the inhibition rate is low. By detecting the drawn standard curve with the known concentration of the new coronavirus neutralizing antibody (positive control), the neutralizing capacity of the new coronavirus neutralizing antibody to be detected and whether the protective antibody and the titer are generated after the vaccine is administered can be calculated.
The criteria for determination of neutralizing antibodies are as follows:
neutralizing Activity: when the OD value of the sample to be tested is lower than that of the positive well, the sample is judged to be a neutralizing antibody and is indicated by "+".
Non-neutralizing Activity: when the OD value of the sample to be tested is equal to that of the positive hole, the non-neutralizing antibody is judged, and is indicated by a negative sign.
Invalidation: when the positive control well did not develop color or developed color but the OD value was less than 2.0, the test was judged to be invalid.
By the technical scheme, the invention at least has the following advantages and beneficial effects:
the kit uses biotin-streptavidin labeled human ACE2 protein as a coating antigen, uses an antibody in a rehabilitation patient body as a positive neutralizing antibody as a kit reference substance, and screens neutralizing antibodies with neutralizing capacity and titer of protective antibodies generated after vaccine administration by competing with new crown-spike protein RBD and combining with biotin labeled human ACE2 protein.
The detection kit provided by the invention can accurately and sensitively detect different matrixes, such as the neutralizing capacity of the new corona neutralizing antibody in serum and the antibody titer, the pretreatment process of the sample is simple, the time consumption is low, a large number of samples can be detected simultaneously, and the sample detection cost is far lower than that of the traditional live virus or pseudovirus detection method. The invention has important significance for detecting the neutralizing antibody of the new coronavirus in a large batch of samples.
And (III) the protein reagent is a freeze-dried component, has long storage time and no radioactive pollution in liquid.
In the invention, the biotin-labeled human ACE2 protein is obtained by adopting an in-vivo enzyme labeling mode, is a biotin single-point fixed-point label, has the characteristics of small batch difference and good repeatability, and has a good space structure and higher sensitivity after being combined with SA fixed points in an enzyme label plate.
In the invention, the new coronavirus spike protein RBD is preferably marked by horseradish peroxidase, and the operation is simple.
Drawings
FIG. 1 is a graph of the standard inhibition curve for the detection of neutralizing antibodies against the novel coronavirus as plotted in the preferred embodiment of the present invention.
Detailed Description
Protein components used in the invention, such as SA, HRP marked new crown spike protein RBD and biotin marked human ACE2 protein, are expressed by human HEK293 cells, and can maintain natural protein structure. SA, coating amount of biotin-labeled human ACE2 protein, and selection by using chessboard method according to sensitivity and detection range as indexes.
Optimization of usage amount of HRP-labeled novel crown spike protein RBD: adding HRP-labeled new crown spike protein RBD with different concentrations into the ELISA plate, and selecting the using amount of the HRP-labeled new crown spike protein RBD as the amount just saturated when the HRP-labeled new crown spike protein RBD is combined with the coating antigen.
The invention adopts a competitive ELISA method, the new coronavirus neutralizing antibody can block the combination of the new crown spike protein RBD and the human ACE2 protein, and the sensitivity, the specificity, the accuracy and the precision are optimized.
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or the conditions as recommended by the manufacturer's instructions.
Example 1 construction of novel coronavirus neutralizing antibody detection enzyme-linked immunosorbent assay kit
The novel coronavirus neutralizing antibody titer detection ELISA kit provided by the embodiment comprises the following components:
(1) an ELISA plate pre-coated with biotin-streptavidin labeled human ACE2 protein, the specification of which is 96-well plate;
(2) horseradish peroxidase (HRP) -labeled novel crown spike protein RBD is in a freeze-dried powder form, and the specification is 10 ug;
(3) the new coronavirus neutralizing antibody reference substance is purchased from Shanghai Xiang Yao biological technology limited company, and is in the form of lyophilized powder with the specification of 20 ug;
(4) concentrate (10 ×) wash: 20mM Tris, 150mM NaCl, 0.5% v/v Tween-20, pH7.4, and the specification is 60 mL;
(6) TMB color development liquid: 50mM Na2HPO4·12H2O +25 mM citric acid +40 mg/mL TMB +3% v/v H2O2pH5.5, and the specification is 12 mL;
(7) stopping liquid: 1 mol/L sulfuric acid solution with the specification of 7 mL.
EXAMPLE 2 method of operating the kit
The vacuum packaging bag is opened and the microplate is taken out and equilibrated at room temperature for 5 minutes for use. Preparing 0 mu g/mL, 0.16 mu g/mL, 0.31 mu g/mL, 0.63 mu g/mL, 1.25 mu g/mL, 2.5 mu g/mL, 5 mu g/mL, 10 mu g/mL of a standard solution of a new coronavirus neutralizing antibody, adding 50 mu L of the treated sample to each well, repeating the standard sample and the sample for 3 times, adding 50 mu L of horseradish peroxidase (HRP) -labeled new coronavirus RBD diluted to 0.12ug/mL, and incubating for 60 minutes at 37 ℃; pouring out the liquid in the hole, washing for 3 times by using diluted lotion, and inversely placing the ELISA plate on water-absorbent paper for patting dry; taking single-component TMB appearing liquid, adding 100 mu L of the TMB appearing liquid into each hole, developing the color for 10-15 minutes in a dark place, adding 50 mu L of stop solution into each hole to stop the reaction, and measuring the OD value of each hole at the position with the wavelength of 450nm on an enzyme-labeling instrument. The experimental period was 2 h.
Marking the OD value-blank hole containing the positive hole of 0 mug/mL as B0, and marking the OD value obtained by subtracting the blank hole from the rest holes as B; with B/B0The percentage of the values is taken as the ordinate and the corresponding reference concentration value is taken as the abscissa, and a four-parameter fitting equation is used: y = (A-D)/[1 + (x/C) ^ B]+ D, 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 standard inhibition curve of the detection of the novel coronavirus neutralizing antibody is shown in figure 1. The four parameters of the measured curve are respectively: a = 14.38, B = -2.114, C = 1.742, D = 104.1, R2=0.9993。
Example 3 accelerated test of kit
The kit of example 2 was placed at 370C, preserving, respectively taking the kits which are placed for 0, 7, 14, 21 and 28 days, taking the reference substance of the neutralizing antibody of the new coronavirus as a fitting curve, and calculating the IC of the curve at different times50The value is obtained. The results of the measurements at different times are shown in Table 1:
from the above results, it can be seen that IC50The change of the value is not large, and the kit can be stored for at least more than 12 months at the temperature of-20 ℃.
Example 4 kit component Freeze-thaw experiments
Reconstituting the lyophilized components of the kit of example 2 (the HRP-labeled neocoronatine RBD was reconstituted to 100ug/mL with deionized water, the new coronavirus neutralizing antibody positive control was diluted to 200ug/mL with deionized water), split-charging each tube at a rate greater than 5ug, storing at-20 ℃, freezing and thawing for 1, 2, and 3 times (freezing and thawing at-70 ℃ and then returning to room temperature), using the reference sample of the neocoronaviruse neutralizing antibody as a fitting curve, and calculating IC of different time curves50The value is obtained. The results of the measurements at different times are shown in Table 2:
from the above results, it can be seen that IC50The change of the value is not large, and the freeze-dried powder components in the kit have no influence on active ingredients in three times of freeze thawing at-20 ℃.
Example 5 kit matrix Effect experiment
The kit of example 2 can detect neutralizing antibodies against the new coronavirus in serum and verify the IC in different serum matrices (Buffer, 10%, 20%, 50%, 100% human serum)50The interference of (2). Calculating IC of curves in different matrixes by taking a reference substance of the neutralizing antibody of the new coronavirus as a fitting curve50The value is obtained. The results of the different matrix measurements are shown inTable 3:
from the above results, it can be seen that IC50The change of the value is not large, and the detection of the kit in different matrixes has no influence.
Example 6 precision assay of kit
The new coronavirus neutralizing antibody in the kit of example 2 was subjected to 6 consecutive assay batches, each of which was at least 2 replicates, according to the kit protocol. Calculating IC of curves in different matrixes by taking a reference substance of the neutralizing antibody of the new coronavirus as a fitting curve50The value is obtained. The results of the different matrix assays are shown in table 4:
from the above results, it can be seen that the inhibition rate and IC were different for different days50The change is small, which indicates that the precision of the kit is good.
Example 7 kit accuracy experiment
The new coronavirus neutralizing antibody in the kit of example 2 is respectively set with three concentrations of 5ug/ml, 2.5ug/ml and 0.6ug/ml, high, medium and low according to the kit operation method, and the new coronavirus neutralizing antibody reference is used as a fitting standard curve to calculate the values of different quality control concentrations. The results of the different quality control values are shown in Table 5:
from the above results, the accuracy of the kit was 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 (3)
1. Novel coronavirus neutralizing antibody titer detection ELISA kit, characterized in that, the kit comprises: the kit comprises an enzyme label plate coated with streptavidin and biotin labeled human ACE2 protein, a horseradish peroxidase labeled novel coronavirus RBD protein, a novel coronavirus neutralizing antibody positive control, a coating solution, a washing solution, a diluent, a confining solution, a developing solution and a stopping solution;
the concentration of streptavidin coated on the ELISA plate is 5ug/mL, the concentration of biotin-labeled human ACE2 protein is 2ug/mL, and the molar ratio of the streptavidin to the biotin-labeled human ACE2 protein is 1: 4;
the enzyme label plate coating method comprises the following steps: the ELISA plate is coated with streptavidin solution overnight, and after the plate is washed by washing liquid, biotin-labeled human ACE2 protein is added, and the plate is sealed and washed by sealing liquid;
in the novel coronavirus RBD protein marked by horseradish peroxidase, the molar ratio of the horseradish peroxidase to the RBD protein is 4: 1;
the use concentration of the novel coronavirus RBD protein marked by horseradish peroxidase is 50 ng/mL; the novel coronavirus RBD protein marked by horseradish peroxidase is in a freeze-dried powder form in the kit, and the freeze-dried powder contains 10% of trehalose;
the coating solution is a sodium carbonate-sodium bicarbonate buffer solution, and comprises the following specific components: 15 mM Na2CO3，35 mM NaHCO3，pH 9.6；
The washing solution is as follows: 20mM Tris, 150mM NaCl, 0.5% v/v Tween-20, pH7.4;
the diluent is a washing liquid containing 0.5 percent of bovine serum albumin;
the color development liquid is as follows: 50mM Na2HPO4·12H2O +25 mM citric acid +40 mg/mL TMB +3% H2O2，pH5.5。
2. The kit according to claim 1, wherein the stop solution is a 1M sulfuric acid solution.
3. The kit of claim 1 or 2, wherein the microplate is a 96-well plate.
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