CN114578046A - Novel coronavirus (SARS-CoV-2) antibody detection reagent - Google Patents
Novel coronavirus (SARS-CoV-2) antibody detection reagent Download PDFInfo
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Classifications
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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
-
- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- 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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- G—PHYSICS
- G01—MEASURING; TESTING
- 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 discloses a new application of poly-ApoA I in a new crown antibody immunity detection reagent, and the addition of poly-ApoA I can obviously reduce the background signal value of the detection reagent so as to effectively improve the sensitivity of the new crown antibody detection reagent.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a novel coronavirus (SARS-CoV-2) IgM/IgG antibody detection reagent.
Background
Apolipoprotein ApoA I (apolipoprotein ai) consists of 243 amino acid residues, has a molecular mass of 28.3KD, is mainly synthesized in the liver, is an important component of high-density lipoprotein (HDL), and is also a main executor of HDL function. ApoA I plays an important role in the transport and metabolism of cholesterol, anti-atherosclerosis, anti-endotoxin, anti-inflammatory, anti-viral and inhibition of tissue damage caused by acute phase inflammatory reactions.
The novel coronavirus (SARS-CoV-2) is a large virus family known to cause the common cold and more severe diseases such as Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS). The novel coronavirus is a new strain of coronavirus which has not been discovered in human body before, and the transmission path of the novel coronavirus is direct transmission, aerosol transmission and contact transmission, and the infectivity is extremely strong, so that the novel coronavirus becomes very important for the diagnosis of the novel coronavirus.
The virus is mainly detected by adopting a fluorescence quantitative PCR method to detect nucleic acid, and the positive rate of the nucleic acid detection is easily reduced due to the difference between the type and the collection mode of a specimen in the actual work; meanwhile, the nucleic acid detection time is long, the requirement on the biological safety level of a laboratory is high, and the method is inconvenient for being widely developed by primary hospitals. After the virus infects human body, the IgM antibody is generated in about 5-7 days, while the IgG antibody can be generated in about 10-15 days. Therefore, the detection of the content of IgG and IgM of SARS-CoV-2 in human serum is of great significance for the general investigation and accurate diagnosis of novel pneumonia.
At present, a plurality of new crown IgG/IgM rapid detection kits are available on the market, and indirect detection is mostly adopted, but due to the defects of the methodology and the nonspecific adsorption of antigens, the defects of high background value and poor sensitivity exist generally. Therefore, the technology for reducing the IgG/IgM detection background signal and improving the reagent sensitivity has important application value.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to solve the problems of low detection sensitivity and inaccurate detection result caused by high background in the existing novel crown detection antibody test reagent. The invention discovers that the addition of poly-ApoA I in the detection reagent can effectively reduce the background signal of the new corona detection reagent and improve the sensitivity of the detection reagent.
ApoAI is the protein fraction of plasma lipoproteins, a protein that binds and transports blood lipids to various tissues of the body for metabolism and utilization. ApoAI consists of 243 amino acid residues, is a single polypeptide chain, and has a molecular weight of 28.3 KD.
Poly-ApoAI refers to a polymeric form of ApoAI having a molecular weight of greater than 76.6 KD.
Polymeric apoai can be polymerized by physical or chemical means to form polymeric forms from apoai monomers. For example, the polymer can be obtained by heat-treating ApoAI, the polymer can be obtained by irradiating ApoAI with radiation, and the polymer can be obtained by adding a protein denaturing agent.
Unexpectedly, it has been found that the addition of poly-ApoA i to the antibody reagent for detecting neocorona can significantly reduce the background signal of detection and increase the detection sensitivity.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
the invention provides an application of poly-ApoAI in preparing a novel coronavirus (SARS-CoV-2) antibody detection reagent.
The invention provides an application of adding poly-ApoAI in preparing a novel coronavirus (SARS-CoV-2) antibody detection reagent, which can reduce the interference of background in detection, reduce noise and improve detection sensitivity.
In some embodiments of the present invention, poly-ApoAI can be added to the sample dilution in the reagent for detecting the antibody of the novel coronavirus (SARS-CoV-2) or can be added to the labeling solution.
In some embodiments of the invention, the working concentration of poly-ApoAI is between 0.01mg/ml and 2mg/ml, in some embodiments 0.01mg/ml, 0.02mg/ml, 0.04mg/ml, 0.08mg/ml, 1.6mg/ml, 1.7mg/ml, 1.8mg/ml, 1.9mg/ml, 2.0 mg/ml; in other embodiments, the working concentration of apoAI is from 0.1mg/ml to 1.5mg/ml, and in some embodiments the working concentration of apoAI is 0.1mg/ml, 0.2mg/ml, 0.3mg/ml, 1.5mg/ml, 1.4mg/ml, 1.3 mg/ml; in other embodiments, the working concentration of ApoAI is 0.4mg/ml to 1.2mg/ml, and in some experiments the working concentration of ApoAI is 0.4mg/ml, 0.5mg/ml, 0.6mg/ml, 0.7mg/ml, 0.8mg/ml, 0.9mg/ml, 1.0mg/ml, 1.1mg/ml, 1.2 mg/ml.
The invention also provides a novel coronavirus (SARS-CoV-2) antibody detection reagent, which comprises a sample diluent and a marking solution, wherein the sample diluent and/or the marking solution contain poly-ApoAI.
In the novel coronavirus (SARS-CoV-2) antibody detection reagent provided by the invention, the working concentration of poly-ApoAI is 0.01mg/ml-2mg/ml, and in some embodiments, the working concentration of poly-ApoAI is 0.01mg/ml, 0.02mg/ml, 0.04mg/ml, 0.08mg/ml, 1.6mg/ml, 1.7mg/ml, 1.8mg/ml, 1.9mg/ml, 2.0 mg/ml; in other embodiments, the working concentration of apoAI is from 0.1mg/ml to 1.5mg/ml, and in some embodiments the working concentration of apoAI is 0.1mg/ml, 0.2mg/ml, 0.3mg/ml, 1.5mg/ml, 1.4mg/ml, 1.3 mg/ml; in other embodiments, the working concentration of ApoAI is 0.4mg/ml to 1.2mg/ml, and in some experiments the working concentration of ApoAI is 0.4mg/ml, 0.5mg/ml, 0.6mg/ml, 0.7mg/ml, 0.8mg/ml, 0.9mg/ml, 1.0mg/ml, 1.1mg/ml, 1.2 mg/ml.
The term "working concentration" as used herein refers to the concentration used in the actual assay.
The invention also provides a detection kit for detecting SARS-CoV-2 antibody by lateral immunochromatography, the kit comprises a lateral immunochromatography card and a sample diluent, and the sample diluent contains poly-ApoAI.
The invention also provides an immunoassay kit for detecting the novel coronavirus (SARS-CoV-2) antibody, and a sample diluent in the kit contains poly-ApoAI.
The invention also provides an immunoassay kit for detecting the novel coronavirus (SARS-CoV-2) antibody, wherein the marking liquid in the kit contains poly-ApoAI.
Detailed Description
The invention is further illustrated by the following examples:
unless otherwise indicated, all chemical reagents used in the examples of the present invention were analytical grade and were obtained from Sigma-aldrich. The chemiluminescence detector is a Bincho BHP9507 chemiluminescence detector of Beijing Bincho photon technology corporation in China.
Example 1 preparation of poly-apoai:
1. 50L of serum is collected, 250ml of 10% polyanion and 2mol/L of 2500ml calcium chloride are added, the mixture is evenly mixed and placed for 30 minutes at 4000 rpm and centrifuged for 20 minutes, and supernatant fluid is collected.
2. Adding guanidine hydrochloride into the supernatant to 6mol/L, keeping the temperature at 37 ℃ for 3 hours, centrifuging to separate the apolipoprotein, dialyzing with physiological saline to remove the guanidine hydrochloride, adjusting the density to 1.21g/cm3 with KBr, centrifuging again, collecting the solution at the lower third section of the centrifuge tube, dialyzing with physiological saline to remove KBr, and dialyzing with 6mol/L urea.
3. Performing gradient chromatography with DEAE-Sepharose CL6B column, collecting the main peak which is pure ApoAI, dialyzing to remove urea, measuring protein content with Shimadzu-UV 2550 spectrophotometer at 280nm wavelength to obtain total ApoAI content of about 25g, and adjusting ApoAI concentration to 10 g/ml.
4. Incubating 10G/ml ApoAI in a water bath at 37 ℃ for 10h, performing gradient chromatography by using Sepharose G-25 column, and collecting the main peak, namely the polymer ApoAI.
Example 2 testing of multimeric ApoA i in the detection of chemiluminescent novel coronavirus (SARS-CoV-2) IgG antibodies:
1. a set of chemiluminescent kits containing poly-apoai in the sample diluent and a set of conventional chemiluminescent kits not containing poly-apoai were prepared using conventional preparation methods known to those skilled in the art.
| Components | Ingredient/use |
| R1 | Sample dilutions (Experimental group containing 0.5mg/ml poly-ApoAI) |
| R2 | Magnetic bead coated neocorona N or S antigen |
| R3 | Alkaline phosphatase-labeled anti-human IgG antibody |
| Chemiluminescent substrate | AMPPD |
| Cleaning concentrate | For preparing cleaning solution (PBST) |
| Quality control product | Sheep polyclonal antibody cross-linked with human antibody |
R1 can be prepared by conventional formulation, and contains phosphate buffer pair, inorganic salt, surfactant, and antiseptic.
2. The detection is carried out by the following steps:
(1) 10ul of serum sample or 10ul of quality control, 50ul of R1 and 50ul of R2 were added to the reaction cup;
(2) incubating at 37 ℃ for 5min, washing for four times, and adding 100ul of R3;
(3) incubate 5min at 37 ℃ and wash four times before adding chemiluminescent substrate readings.
3. And (3) detection results:
from the results, the background signal value of the negative serum of the group added with the polyapoai is reduced by 40-50%, the signal-to-noise ratio (P/N) is improved by more than 90%, and the sensitivity of the reagent is obviously improved.
Example 3 testing of multimeric ApoAI in the detection of chemiluminescent novel coronavirus (SARS-CoV-2) IgM antibody:
1. a set of chemiluminescent kits containing poly-apoai in the sample diluent and a set of conventional chemiluminescent kits not containing poly-apoai were prepared using conventional preparation methods known to those skilled in the art.
R1 can be prepared by conventional formulation, and contains Tris buffer pair, inorganic salt, surfactant, and antiseptic.
2. The detection is carried out by the following steps:
(1) 10ul of serum sample or 10ul of quality control, 50ul of R1 and 50ul of R2 were added to the reaction cup;
(2) incubating at 37 ℃ for 5min, washing for four times, and adding 100ul of R3;
(3) incubate 5min at 37 ℃ and wash four times before adding chemiluminescent substrate readings.
3. And (3) detection results:
from the results, the background signal and the quality control signal of the negative serum of the group added with the polyapaoi are obviously reduced, the negative serum sample is reduced by 77-78%, the signal-to-noise ratio is improved by 43-55%, and the sensitivity of the reagent is obviously improved.
Example 4 testing of different concentrations of polyapoai in the detection of chemiluminescent novel coronavirus (SARS-CoV-2) IgG or IgM antibodies:
dilutions containing varying concentrations of poly-APOAI were prepared and tested in the chemiluminescent novel coronavirus (SARS-CoV-2) IgG or IgM antibody assay as described in example 3 or example 4 above, with the following results:
IgM detection Signal to noise ratio (P/N):
in IgM detection, the addition of poly-APOAI of 0.01mg/ml-2.0mg/ml in a sample diluent can reduce the luminous values of negative serum and quality control substances, and compared with the addition of no poly-APOAI, the signal-to-noise ratio is improved, and the sensitivity of a detection reagent is enhanced.
IgG assay Signal to noise ratio (P/N):
in IgG detection, the addition of 0.01-2.0 mg/ml of polyaPAOAI in the sample diluent can reduce the luminous value of negative serum and improve the luminous value of quality control products, and compared with the addition of no polyaPAOAI, the signal-to-noise ratio is improved, and the sensitivity of the detection reagent is enhanced.
Example 5 application of PolyAPOAI in assay reagents for the lateral Immunochromatographic detection of antibodies to the novel coronavirus (SARS-CoV-2)
A novel coronavirus (2019-nCoV) IgM/IgG antibody detection kit (colloidal gold method) which is available on the market (manufacturer A) is purchased, a sample diluent in the kit is divided into 2 parts, poly APOAI is added into one part to be 1mg/ml, and the other part is not processed at all. The serum which is negative in the detection of 300 human nucleic acids is detected, and the result is as follows:
| negative result | Positive results | Rate of accuracy | |
| Control | 285 | 15 | 95％ |
| Polymeric APOAI | 297 | 3 | 99％ |
From the results, it can be seen that the addition of polyapaoi in the sample diluent can improve the accuracy of detecting novel coronavirus (2019-nCoV) IgM/IgG by colloidal gold, and effectively reduce the false positive results.
EXAMPLE six assay for detecting novel coronavirus (SARS-CoV-2) IgG antibody by adding poly-APOAI to the labeling solution.
1. A set of chemiluminescent kits containing poly-ApoAI in the labeling solution (R3) and a set of conventional chemiluminescent kits not containing poly-ApoAI were prepared using conventional preparation methods known to those skilled in the art.
R1 can be prepared by conventional formulation, and contains carbonate buffer pair, inorganic salt, surfactant, and antiseptic.
2. The detection is carried out by the following steps:
(1) 10ul of serum sample or 10ul of quality control, 50ul of R1 and 50ul of R2 were added to the reaction cup;
(2) incubating at 37 ℃ for 5min, washing for four times, and adding 100ul of R3;
(3) incubate 5min at 37 ℃ and wash four times before adding chemiluminescent substrate readings.
3. And (3) detection results:
from the results, the background signal value of the negative serum of the group added with the polyapoai is reduced by 40-50%, the signal-to-noise ratio (P/N) is improved by more than 95%, and the sensitivity of the reagent is obviously improved.
EXAMPLE seventhly, the assay for detecting the novel coronavirus (SARS-CoV-2) IgM antibody by adding polyaPAOAI to the labeling solution
1. A panel of chemiluminescent kits containing poly-apoai in a labeling solution (R3) and a panel of conventional chemiluminescent kits not containing poly-apoai were prepared using conventional preparation methods known to those skilled in the art.
R1 can be prepared by conventional formulation, and contains phosphate buffer pair, inorganic salt, surfactant, and antiseptic.
2. The detection is carried out by the following steps:
(1) 10ul of serum sample or 10ul of quality control, 50ul of R1 and 50ul of R2 were added to the reaction cup;
(2) incubating at 37 ℃ for 5min, washing for four times, and adding 100ul of R3;
(3) incubate 5min at 37 ℃ and wash four times before adding chemiluminescent substrate readings.
3. And (3) detection results:
the results show that the background signal and the quality control signal of the negative serum of the group added with the polyapaoi are obviously reduced, the background value of the negative serum sample is reduced by more than 70 percent, the signal-to-noise ratio is improved by 43 to 55 percent, and the sensitivity of the reagent is obviously improved.
It can be seen from the above-mentioned results of the different methodologies that the addition of polyapoai in the sample diluent or in the labeling solution can improve the detection sensitivity or accuracy of the antibody without being affected by the detection methodology.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. The application of poly-ApoAI in preparing novel coronavirus (SARS-CoV-2) antibody detection reagent is disclosed.
2. The use according to claim 1, wherein the working concentration of poly-apoai is between 0.01mg/ml and 2 mg/ml;
preferably the working concentration of the ApoA i is 0.1mg/ml to 1.5 mg/ml;
preferably the working concentration of the ApoA i is between 0.4mg/ml and 1.2 mg/ml.
3. The use of claim 1, wherein the poly-apoai is added to a sample diluent or to a labeling solution in a reagent for detecting antibodies to the type coronavirus (SARS-CoV-2).
4. The use according to claim 1, wherein the addition of said multimeric ApoAI reduces the background interference of said novel coronavirus (SARS-CoV-2) antibody detection reagent.
5. Use according to claim 1, characterized in that the novel coronavirus (SARS-CoV-2) antibody assay is the detection of IgG or IgM or IgA antibodies.
6. A novel coronavirus (SARS-CoV-2) antibody detection sample diluent, wherein the sample diluent contains poly-ApoAI;
preferably the working concentration of the ApoA i is 0.01mg/ml to 2 mg/ml;
preferably the working concentration of the ApoA i is 0.1mg/ml to 1.5 mg/ml;
preferably, the working concentration of the ApoA i is between 0.4mg/ml and 1.2 mg/ml.
7. A novel coronavirus (SARS-CoV-2) antibody detection marker fluid is characterized in that the marker fluid contains poly-ApoAI;
preferably the working concentration of the ApoA i is 0.01mg/ml to 2 mg/ml;
preferably the working concentration of the ApoA i is 0.1mg/ml to 1.5 mg/ml;
preferably the working concentration of the ApoA i is between 0.4mg/ml and 1.2 mg/ml.
8. The detection kit for detecting the novel coronavirus (SARS-CoV-2) antibody by lateral immunochromatography is characterized by comprising a lateral immunochromatography card and a sample diluent, wherein the sample diluent contains poly-ApoAI.
9. An immunoassay kit for detecting a novel coronavirus (SARS-CoV-2) antibody, the kit comprises a sample diluent and a marking solution, and is characterized in that the sample diluent and/or the marking solution contain poly-ApoA I.
10. The kit of claim 9, wherein the working concentration of poly-apoai is 0.01mg/ml to 2 mg/ml;
preferably the working concentration of the ApoA i is 0.1mg/ml to 1.5 mg/ml;
preferably the working concentration of the ApoA i is between 0.4mg/ml and 1.2 mg/ml.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011368620.2A CN114578046A (en) | 2020-11-30 | 2020-11-30 | Novel coronavirus (SARS-CoV-2) antibody detection reagent |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011368620.2A CN114578046A (en) | 2020-11-30 | 2020-11-30 | Novel coronavirus (SARS-CoV-2) antibody detection reagent |
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| Publication Number | Publication Date |
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| CN114578046A true CN114578046A (en) | 2022-06-03 |
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|---|---|---|---|
| CN202011368620.2A Pending CN114578046A (en) | 2020-11-30 | 2020-11-30 | Novel coronavirus (SARS-CoV-2) antibody detection reagent |
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| Country | Link |
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| CN (1) | CN114578046A (en) |
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