CN111505283A - Novel coronavirus antibody detection kit, detection method and application - Google Patents
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Abstract
The invention discloses a novel coronavirus antibody detection kit, a detection method and application, aiming at solving or improving the technical problems of short RNA storage time, easy degradation, easy occurrence of false negative, poor specificity, poor reliability and the like in the prior art, wherein the kit comprises an R1 reagent, an R2 reagent, a magnetic particle reagent and a chemiluminescent substrate; the detection method comprises the following steps: the fusion protein of N protein and S protein is used as a compound for detection, and the magnetic particle chemiluminescence method is used for detecting the novel coronavirus antibody through SA cascade amplification. The invention has the obvious advantages of high sensitivity, strong specificity, large flux, high speed, high automation degree and the like; the detection sample is serum or plasma, the sample is easy to collect, the safety is high, the antibody in the sample is not easy to degrade, and the storage life is longer.
Description
Technical Field
The invention belongs to the technical field of antibody detection, and particularly relates to a novel coronavirus antibody detection kit, a detection method and application.
Background
The 2019-nCoV virus is also an orthosense RNA virus wrapped by outer membrane glycoprotein, but belongs to a new branch in a virus evolutionary tree and is a brand-new coronavirus. The research shows that the virus has obvious human-borne characteristics, has higher infectivity compared with SARS virus outbreak in 2003, has an average infection latency of 4.8 days, has an average time from the symptom outbreak to virus isolation of 2.9 days, is lower than 4.2 days of SARS, and needs to adopt more strict epidemic prevention measures than SARS. According to research, the genome of the 2019-nCoV virus has 79 percent of similarity with SARS, but at the protein level, the genome has high similarity. Through three-dimensional structure simulation, the spinous process protein (S protein) of the 2019-nCoV virus is found to have a receptor binding site similar to that of SARS virus, and probably also to infect humans by binding with ACE2 protein on human cells.
Patients infected with the new coronavirus have early mild symptoms including dry cough, fever, hypodynamia, dizziness, chest distress and the like, are serious in disease, and rapidly develop acute respiratory failure in later period, so that other complications of the patients are caused, and the patients die. The current definite diagnosis of patients with new coronavirus mainly comprises the examination of clinical symptoms such as fever, the CT examination of lung inflammation, the monitoring of serum biochemical indexes, the detection of nucleic acid RNA and the diagnosis of clinicians. Therefore, more specific and reliable detection means are required. In the existing nucleic acid detection means, a sample needs to be collected by a pharyngeal swab or alveolar lavage fluid, so that the safety problems of aerosol pollution and the like exist, the challenge is great for medical personnel, and meanwhile, the nucleic acid detection also has the defects that RNA is easy to degrade and false negative results easily occur.
Disclosure of Invention
In order to solve or improve at least one of the technical problems of short storage time, easy degradation, easy occurrence of false negative, poor specificity, poor reliability and the like of RNA in the prior art, the invention aims to provide a novel coronavirus antibody detection kit, a detection method and application.
The technical scheme adopted by one purpose of the invention is as follows:
a novel coronavirus antibody detection kit comprises an R1 reagent, an R2 reagent, a magnetic particle reagent and a chemiluminescent substrate.
Further, the test kit also comprises a quality control agent and a calibration agent for calibrating the curve; the calibrator and the quality control agent are both buffer solutions.
Furthermore, the R2 reagent contains alkaline phosphatase-labeled streptavidin with the concentration of 0.05-1 mu g/m L.
Furthermore, the R1 reagent contains the Biotin-labeled 2019-nCoV antigen complex with the concentration of 0.05-2 mug/m L.
Furthermore, the cleaning concentrated solution is used for preparing the cleaning solution.
Further, the magnetic particle reagent contains the magnetic particles coated with the Anti-human IgM antibody, wherein the concentration of the magnetic particles is 0.05-5 mu g/m L.
Further, the chemiluminescent substrate is 3- (2-spiroadamantane) -4-methoxy-4- (3-phosphoryloxy) -phenyl-1, 2-dioxetane disodium salt.
The other purpose of the invention adopts the technical scheme that:
a method for detecting a novel coronavirus antibody, comprising the steps of: the fusion protein of N protein and S protein is used as a compound for detection, and the magnetic particle chemiluminescence method is used for detecting the novel coronavirus antibody through SA cascade amplification.
Further, the method for detecting the novel coronavirus antibody comprises the following steps:
and (3) generating a complex: reacting the magnetic particles coated with the Anti-human IgM antibody, the new coronavirus antibody in the sample and the biotin-labeled antigen to obtain a mixture containing a compound;
generation of solid phase antibody-biotin antigen-SA-AP complex: cleaning the mixture, and removing unbound biotinylated antigen and other substances to obtain a complex; reacting the complex with alkaline phosphatase-labeled streptavidin to obtain an unbound solid-phase antibody-biotin antigen-SA-AP complex containing SA-AP;
and (3) detection: washing the solid phase antibody-biotin antigen-SA-AP complex containing the unbound SA-AP to remove the unbound SA-AP to obtain a solid phase antibody-biotin antigen-SA-AP complex; and (3) reacting the solid-phase antibody-biotin antigen-SA-AP compound with AMPPD to detect.
Still another object of the present invention adopts the technical scheme that:
the kit or the detection method is used for the application of the novel coronavirus antibody.
The invention has the beneficial effects that: the invention relates to a novel coronavirus antibody detection kit, a detection method and application, which are obtained by improving a traditional capture method and a magnetic particle chemiluminescence detection method; the detection method has the obvious advantages of high sensitivity, strong specificity, large flux, high speed, high automation degree and the like; simultaneously, the immune antibody IgM and IgG detection is combined, so that the initial screening of infected patients and the clinical confirmation of positive patients can be realized, and the monitoring after tracking can be realized; in addition, the detection sample is serum or plasma, the sample is easy to collect, the safety is high, the antibody in the sample is not easy to degrade, and the storage life is longer; the kit and the detection method of the invention can be used for detecting the novel coronavirus antibody.
Drawings
FIG. 12019-nCoV IgM antibody magnetic particle chemiluminescence principle diagram.
FIG. 2 is a schematic diagram of a conventional capture method.
Detailed Description
The invention is further explained below with reference to the drawings and the specific embodiments.
Preferably, the reagent R1 in the following examples is prepared from Biotin-labeled 2019-nCoV antigen complex and buffer solution thereof, wherein the concentration of the Biotin-labeled 2019-nCoV antigen complex is 0.05-2 μ g/M L, and the buffer solution comprises 0.01-0.1M of Tris (hydroxymethyl) aminomethane (Tris) which contains 0.5-5% by mass of Bovine Serum Albumin (BSA), 0.1-2% by mass of casein, 0.05-0.5% by mass of Tween 20(Tween-20) and 0.5-5% by mass of trehalose.
Preferably, the reagent R2 in the following examples is prepared from alkaline phosphatase-labeled streptavidin with a concentration of 0.05-1 μ g/M L and a buffer solution thereof, wherein the buffer solution comprises 0.01-0.1M Tris (hydroxymethyl) aminomethane (Tris) containing 0.1-5mM Mg2+0.01-1mM Zn2+Bovine Serum Albumin (BSA) with the mass fraction of 0.5-5%, Tween 20(Tween-20) with the mass fraction of 0.05-0.5%, trehalose with the mass fraction of 0.5-5% and glycerol with the mass fraction of 0.5-5%.
Preferably, the magnetic particle reagent is prepared by magnetic particles coated with Anti-human IgM antibody and a buffer solution thereof in the following embodiments, wherein the concentration of the magnetic particle reagent is 0.05-5 μ g/m L.
Preferably, the buffers used in the following examples to formulate calibrators and quality control agents include: tris at a concentration of 0.01-0.1M; the Tris contains 0.5-5% of Bovine Serum Albumin (BSA), 0.05-0.5% of Tween 20(Tween-20) and 0.5-5% of sucrose.
In the invention, the (AMPPD) is 3- (2-spiral adamantane) -4-methoxy-4- (3-phosphoryl-oxy) -phenyl-1, 2-dioxane disodium salt; (AP) is alkaline phosphatase; (SA) is streptavidin.
The buffers used for R1, R2, magnetic particle reagents, calibrators, and quality control agents in the following examples may be replaced by other conventional buffers.
Example 1
A novel coronavirus antibody detection kit comprises an R1 reagent, an R2 reagent, a magnetic particle reagent and a chemiluminescent substrate.
Preferably, the R1 reagent contains Biotin-labeled 2019-nCoV antigen complex with the concentration of 0.05 μ g/m L, and the R2 reagent contains alkaline phosphatase-labeled streptavidin with the concentration of 0.05 μ g/m L, and the kit adopts the principle shown in FIG. 1 for detection.
Fig. 2 shows the detection principle of the conventional capture method. Comparing fig. 1 and fig. 2, it can be seen that the conventional capture method employs magnetic particles of Anti-human IgM antibody, and new coronavirus antibody in the sample to react with antigen which is not labeled by biotin, so as to generate AP-antigen complex for detection; the invention adopts the magnetic particles coated with Anti-human IgM antibody, the new coronavirus antibody in the sample and the biotin-labeled antigen to react to generate a compound, and then the compound of SA-AP is added to react to generate a solid phase antibody-biotin antigen-SA-AP compound for detection.
Example 2
A novel coronavirus antibody detection kit comprises an R1 reagent, an R2 reagent, a magnetic particle reagent and a chemiluminescent substrate.
Preferably, the R1 reagent contains Biotin-labeled 2019-nCoV antigen complex with the concentration of 2 mug/m L, and the R2 reagent contains alkaline phosphatase-labeled streptavidin with the concentration of 1 mug/m L, the kit adopts the principle shown in figure 1 for detection, and figure 2 shows the detection principle of the traditional capture method.
Preferably, the kit further comprises a quality control agent and a calibrator for calibrating the curve; the calibrator and the quality control agent are both buffer solutions.
Comparing fig. 1 and fig. 2, it can be seen that the conventional capture method employs magnetic particles of Anti-human IgM antibody, and new coronavirus antibody in the sample to react with antigen which is not labeled by biotin, so as to generate AP-antigen complex for detection; the invention adopts the magnetic particles coated with Anti-human IgM antibody, the new coronavirus antibody in the sample and the biotin-labeled antigen to react to generate a compound, and then the compound of SA-AP is added to react to generate a solid phase antibody-biotin antigen-SA-AP compound for detection.
Example 3
A novel coronavirus antibody detection kit comprises an R1 reagent, an R2 reagent, a magnetic particle reagent and a chemiluminescent substrate.
Preferably, the R1 reagent contains Biotin labeled 2019-nCoV antigen complex with the concentration of 1 mug/m L, and the R2 reagent contains alkaline phosphatase labeled streptavidin with the concentration of 0.08 mug/m L, and the kit adopts the principle shown in figure 1 for detection.
Comparing fig. 1 and fig. 2, it can be seen that the conventional capture method employs magnetic particles of Anti-human IgM antibody, and new coronavirus antibody in the sample to react with antigen which is not labeled by biotin, so as to generate AP-antigen complex for detection; the invention adopts the magnetic particles coated with Anti-human IgM antibody, the new coronavirus antibody in the sample and the biotin-labeled antigen to react to generate a compound, and then the compound of SA-AP is added to react to generate a solid phase antibody-biotin antigen-SA-AP compound for detection.
Preferably, the kit further comprises a quality control agent and a calibrator for calibrating the curve; the calibrator and the quality control agent are both buffer solutions.
Preferably, the kit further comprises a washing concentrate for preparing the washing solution.
Example 4
A novel coronavirus antibody detection kit comprises an R1 reagent, an R2 reagent, a magnetic particle reagent and a chemiluminescent substrate.
Preferably, the reagent R1 contains Biotin-labeled 2019-nCoV antigen complex with the concentration of 0.05 mu g/m L, the reagent R2 contains alkaline phosphatase-labeled streptavidin with the concentration of 0.05g/m L, and the magnetic particle reagent contains magnetic particles coated with Anti-human IgM antibody with the concentration of 0.05 mu g/m L.
The kit adopts the principle shown in figure 1 for detection.
Comparing fig. 1 and fig. 2, it can be seen that the conventional capture method employs magnetic particles of Anti-human IgM antibody, and new coronavirus antibody in the sample to react with antigen which is not labeled by biotin, so as to generate AP-antigen complex for detection; the invention adopts the magnetic particles coated with Anti-human IgM antibody, the new coronavirus antibody in the sample and the biotin-labeled antigen to react to generate a compound, and then the compound of SA-AP is added to react to generate a solid phase antibody-biotin antigen-SA-AP compound for detection.
Preferably, the kit further comprises a quality control agent and a calibrator for calibrating the curve; the calibrator and the quality control agent are both buffer solutions; the kit also comprises a cleaning concentrated solution for preparing a cleaning solution.
Example 5
A novel coronavirus antibody detection kit comprises an R1 reagent, an R2 reagent, a magnetic particle reagent and a chemiluminescent substrate.
Preferably, the reagent R1 contains Biotin-labeled 2019-nCoV antigen complex with the concentration of 2 mug/m L, the reagent R2 contains alkaline phosphatase-labeled streptavidin with the concentration of 1 mug/m L, the magnetic particle reagent contains magnetic particles coated with Anti-human IgM antibody with the concentration of 5 mug/m L, the chemiluminescent substrate is 3- (2-spiral adamantane) -4-methoxy-4- (3-phosphoryl-oxygen acyl) -phenyl-1, 2-dioxide cyclohexane disodium salt, and the kit adopts the principle shown in figure 1 for detection.
Comparing fig. 1 and fig. 2, it can be seen that the conventional capture method employs magnetic particles of Anti-human IgM antibody, and new coronavirus antibody in the sample to react with antigen which is not labeled by biotin, so as to generate AP-antigen complex for detection; the invention adopts the magnetic particles coated with Anti-human IgM antibody, the new coronavirus antibody in the sample and the biotin-labeled antigen to react to generate a compound, and then the compound of SA-AP is added to react to generate a solid phase antibody-biotin antigen-SA-AP compound for detection.
Preferably, the kit further comprises a quality control agent and a calibrator for calibrating the curve; the calibrator and the quality control agent are both buffer solutions; the kit also comprises a cleaning concentrated solution for preparing a cleaning solution.
Example 6
A method for detecting a novel coronavirus antibody, comprising the steps of: the fusion protein of N protein and S protein is used as a compound for detection, and the magnetic particle chemiluminescence method is used for detecting the novel coronavirus antibody through SA cascade amplification.
Example 7
A method for detecting a novel coronavirus antibody, comprising the steps of:
and (3) generating a complex: reacting the magnetic particles coated with the Anti-human IgM antibody, the new coronavirus antibody in the sample and the biotin-labeled antigen to obtain a mixture containing a compound;
generation of solid phase antibody-biotin antigen-SA-AP complex: cleaning the mixture, and removing unbound biotinylated antigen and other substances to obtain a complex; reacting the complex with alkaline phosphatase-labeled streptavidin to obtain an unbound solid-phase antibody-biotin antigen-SA-AP complex containing SA-AP;
and (3) detection: washing the solid phase antibody-biotin antigen-SA-AP complex containing the unbound SA-AP to remove the unbound SA-AP to obtain a solid phase antibody-biotin antigen-SA-AP complex; and (3) reacting the solid-phase antibody-biotin antigen-SA-AP compound with AMPPD to detect.
Based on the traditional capture method and the magnetic particle chemiluminescence detection method, the fusion protein of the N protein and the S protein is used as a compound for detection, so that the detection sensitivity is improved; furthermore, the detection sensitivity of the traditional capture method is enhanced by using the cascade amplification effect of SA; the kit is developed by using a magnetic particle chemiluminescence method, the detection time is short, the result is obtained within 20 minutes, the repeatability is good, and the full-automatic operation can be realized.
EXAMPLE 8 evaluation of the Performance of the New coronavirus antibody detection kit
The novel coronavirus antibody detection kit comprises 2019-nCoV antigen complex labeled by Biotin, a reagent containing Streptavidin (SA) labeled by Alkaline Phosphatase (AP), a reagent containing magnetic particles coated with Anti-humanIgM antibody, and 3- (2-spiral adamantane) -4-methoxy-4- (3-phosphoryl-oxy) -phenyl-1, 2-dioxane disodium salt; as shown in tables 1 and 2 below.
Main component of table 12019-nCoV IgM antibody detection kit
Preferably, the reagent R1 contains Biotin-labeled 2019-nCoV antigen complex with the concentration of 1 mu g/m L, the reagent R2 contains alkaline phosphatase-labeled streptavidin with the concentration of 1 mu g/m L, the magnetic particle reagent contains magnetic particles coated with Anti-human IgM antibody with the concentration of 1 mu g/m L, and the chemiluminescent substrate is 3- (2-helical adamantane) -4-methoxy-4- (3-phosphoryl-benzoyl) -phenyl-1, 2-dioxane disodium salt.
The detection method of the embodiment adopts the reagent in the kit, and the detection is carried out by the following steps:
and (3) generating a complex: reacting the magnetic particles coated with the Anti-human IgM antibody, the new coronavirus antibody in the sample and the biotin-labeled antigen to obtain a mixture containing a compound;
generation of solid phase antibody-biotin antigen-SA-AP complex: cleaning the mixture, and removing unbound biotinylated antigen and other substances to obtain a complex; reacting the complex with alkaline phosphatase-labeled streptavidin to obtain an unbound solid-phase antibody-biotin antigen-SA-AP complex containing SA-AP;
and (3) detection: washing the solid phase antibody-biotin antigen-SA-AP complex containing the unbound SA-AP to remove the unbound SA-AP to obtain a solid phase antibody-biotin antigen-SA-AP complex; and (3) reacting the solid-phase antibody-biotin antigen-SA-AP compound with AMPPD to detect.
The kit adopts the principle shown in figure 1, and 58 samples clinically diagnosed as 2019-nCoV and 113 random healthy human body samples are measured.
The operation flow of the sample detection is that 20u L sample, 50u L R1 reagent and 50u L magnetic particle reagent (M) are mixed evenly and then incubated for 5 minutes and washed for 3 times, then 100u L R2 reagent is added and incubated for 5 minutes and washed for 3 times, and then 200u L substrate is added to react for 5 minutes for detection.
The analysis of the detection results is specifically as follows:
(1) positive rate of agreement
Positive compliance rate: the kit of the invention measures 58 samples clinically diagnosed as 2019-nCoV. The result shows that the IgG kit positive coincidence rate is 98.3 percent (table 2), and the IgM kit positive coincidence rate is 84.5 percent (table 3), which indicates that the kit has high clinical positive detection rate.
TABLE 22019 clinical sample alignment of nCoV IgG assay kit
TABLE 32019-nCoV IgM kit clinical sample alignment
(2) Negative rate of agreement
Negative coincidence rate: the kit of the invention measures 113 random physical examination samples of healthy people. The result shows that the negative coincidence rate of the IgG kit is 100 percent (113/113) and the negative coincidence rate of the IgM kit is 100 percent (113/113), which indicates that the clinical negative coincidence rate of the kit is high.
(3) Precision: and respectively measuring the high-value, medium-value and low-value quality control substances twice a day, detecting in the afternoon, repeating for 4 times each time, measuring for 10 days totally, measuring each concentration for 80 times totally, calculating the coefficient of variation, and checking the measurement precision of the reagent in high-value, medium-value and low-value samples. The results show that the coefficient of variation is within 8 percent, and the repeatability is good.
(4) Stability: the 2019-nCoV IgG detection kit and the 2019-nCoV IgM detection kit (M, R2) are respectively placed at 37 ℃ for 7 days, and the signal retention rates of high, medium and low 3 concentration quality control are measured. The results are all more than 95 percent, and the reagent shows that the kit has good stability and meets the clinical requirements.
(5) Specificity: the detection results of bilirubin, hemoglobin, rheumatoid factors, lipid, RF and HAMA with different concentrations added to the serum with different concentration values of high, medium and low show that the additive has no influence on the detection result of the kit.
In conclusion, the kit or the detection method of the present embodiment can be used for the application of the novel coronavirus antibody.
The present invention is not limited to the above-described alternative embodiments, and various other forms of products can be obtained by anyone in light of the present invention. The above detailed description should not be taken as limiting the scope of the invention, which is defined in the claims, and which the description is intended to be interpreted accordingly.
Claims (10)
1. A novel coronavirus antibody detection kit is characterized in that: including R1 reagents, R2 reagents, magnetic particle reagents, and chemiluminescent substrates.
2. The kit for detecting a novel coronavirus antibody according to claim 1, wherein: also comprises a quality control agent and a calibrator for calibrating the curve; the calibrator and the quality control agent are both buffer solutions.
3. The kit for detecting the novel coronavirus antibody according to claim 1, wherein the reagent R2 contains streptavidin labeled with alkaline phosphatase at a concentration of 0.05-1 μ g/m L.
4. The kit for detecting the novel coronavirus antibody according to claim 1, wherein the R1 reagent comprises a Biotin-labeled 2019-nCoV antigen complex at a concentration of 0.05-2 μ g/m L.
5. The kit for detecting a novel coronavirus antibody according to claim 1, wherein: also comprises a cleaning concentrated solution for preparing the cleaning solution.
6. The kit for detecting the novel coronavirus antibody according to claim 1, wherein the magnetic particle reagent comprises Anti-human IgM antibody-coated magnetic particles at a concentration of 0.05-5 μ g/m L.
7. The kit for detecting a novel coronavirus antibody according to claim 1, wherein: the chemiluminescent substrate is 3- (2-spiral adamantane) -4-methoxy-4- (3-phosphorus oxygen acyl) -phenyl-1, 2-dioxetane disodium salt.
8. A method for detecting a novel coronavirus antibody, comprising the steps of: the fusion protein of N protein and S protein is used as a compound for detection, and the magnetic particle chemiluminescence method is used for detecting the novel coronavirus antibody through SA cascade amplification.
9. The method of claim 8, comprising the steps of:
and (3) generating a complex: reacting the magnetic particles coated with the Anti-human IgM antibody, the new coronavirus antibody in the sample and the biotin-labeled antigen to obtain a mixture containing a compound;
generation of solid phase antibody-biotin antigen-SA-AP complex: cleaning the mixture, and removing unbound biotinylated antigen and other substances to obtain a complex; reacting the complex with alkaline phosphatase-labeled streptavidin to obtain an unbound solid-phase antibody-biotin antigen-SA-AP complex containing SA-AP;
and (3) detection: washing the solid phase antibody-biotin antigen-SA-AP complex containing the unbound SA-AP to remove the unbound SA-AP to obtain a solid phase antibody-biotin antigen-SA-AP complex; and (3) reacting the solid-phase antibody-biotin antigen-SA-AP compound with AMPPD to detect.
10. Use of the kit according to any one of claims 1 to 7 or the detection method according to any one of claims 8 to 9 for novel coronavirus antibodies.
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