CN115184344A - Novel coronavirus IgG antibody detection method suitable for novel biological sample oral fluid and application thereof - Google Patents
Novel coronavirus IgG antibody detection method suitable for novel biological sample oral fluid and application thereof Download PDFInfo
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Abstract
The invention discloses an oral fluid sample collecting and processing method and a sampling quality control method, provides a magnetic particle chemiluminescence method for a novel coronavirus IgG antibody capable of detecting oral fluid, and establishes a critical value for detecting the novel coronavirus IgG antibody by the oral fluid. The invention discloses a detection method for detecting novel coronavirus IgG antibody in an oral fluid sample by using a magnetic particle chemiluminescence method for the first time, and a large amount of experimental data prove that the detection method has high sensitivity and specificity; the oral liquid collection is noninvasive, safe, convenient and efficient, and the infection risk of medical staff is reduced.
Description
Technical Field
The application belongs to the field of virus detection, particularly relates to a novel coronavirus detection method and application thereof, and more particularly relates to a novel coronavirus IgG antibody detection method suitable for novel biological sample oral fluid and application thereof.
Background
The novel coronavirus and MERS-CoV, SARS-CoV, which belong to the family of coronaviridae, are enveloped single-stranded positive-strand RNA viruses, the genome length of which is about 26000-32000bp, and the most commonly known RNA viruses.
The serological diagnosis of IgM and IgG detection of suspected cases of coronavirus pneumonia and the SARS-CoV-2 specific antibody detection after large-scale new coronavirus vaccination need to collect blood samples for detection, and blood collection has certain traumatism and potential infection risk, and the compliance of a person to be collected is poor, and the operation of professional medical personnel and professional blood collection places are needed.
Oral fluids (Oral fluids) are a mixture of saliva and Oral mucosal exudates, and are the first line of defense of the body against infection by pathogenic microorganisms. Saliva is mainly secreted by three pairs of large salivary glands, namely submandibular gland, parotid gland and sublingual gland in the oral cavity. The oral mucosa exudate refers to capillary exudate rich in gingival connective tissues, so that IgG, igA, igM and other components are similar to blood components, and therefore, the mixed oral fluid sample is more suitable for detecting novel coronavirus antibodies than a saliva sample.
In the early stage of the new crown epidemic situation, saliva is used for detecting antibodies of the new crown viruses abroad, and the detection method is mainly an enzyme-linked immunosorbent assay (ELISA). The disadvantages are that saliva contains few antibodies, and the detection sensitivity is low; the ELISA detection method has low sensitivity and specificity, and has the advantages of complex operation, long time consumption and low automation degree. And saliva specimen collection cannot be quality controlled.
The detection method is a colloidal gold method, and the technical defects of the colloidal gold are that the sensitivity is low, the quantitative detection cannot be carried out, and the types of IgG and IgM antibodies cannot be distinguished.
At present, the common methods for detecting the novel coronavirus antibody approved by the Chinese drug administration include ELISA, colloidal gold, fluorescence immunochromatography and chemiluminescence, but the methods are based on a detection method of a serum sample. There is no novel coronavirus antibody detection reagent for oral fluid [1].
Disclosure of Invention
The invention aims to provide a novel coronavirus IgG antibody sensitive and high-throughput detection method suitable for novel biological sample oral fluid.
The invention provides a noninvasive, safe and convenient oral liquid for detecting a novel coronavirus antibody, overcomes the invasiveness of blood collection, reduces the infection risk of medical personnel, and also makes up the defect of poor saliva detection sensitivity.
Furthermore, the invention provides a chemiluminescence method for detecting novel coronavirus IgG antibodies in oral fluid, which has the characteristics of high sensitivity, high specificity, rapidness, accuracy, automation, suitability for large-batch sample detection and the like, and overcomes the defects of low sensitivity and incapability of automation of colloidal gold; overcomes the defects of low sensitivity and specificity, complex operation and long time consumption of the ELISA method. And a quality control method for oral fluid collection and detection is established, so that the defect that quality control cannot be realized in saliva collection is overcome.
Preferably, the method of the present invention comprises the steps of:
collecting oral cavity liquid samples: repeatedly brushing the gum part for 90 seconds up, down, left and right by using an oral liquid sampling brush until the brush head is completely soaked, putting the sampling brush back into the sampling tube, covering the tube cap,
storing, and sending to a laboratory for sample processing within 24 h. Preparation of a specimen treatment liquid: comprises the following components of fetal bovine serum, PBS buffer solution with PH =7.4, gentamicin and streptomycin. Preferably, a red dye may be additionally added thereto. Taking 100mL of sample treatment solution as an example, the preparation method comprises the following steps: 10% fetal calf serum, 1% gentamicin, 1% streptomycin and 0.5% red dye, the balance is PBS; specifically, 10mL of fetal bovine serum, 1mL of gentamicin (50 mg/mL), 1mL of penicillin streptomycin (10000U/mL of penicillin, 10000ug/mL of streptomycin), and 88% of PBS (pH = 7.2).
Treatment of oral liquid specimens: adding 0.5-1mL of specimen treatment liquid into a sampling tube, holding a sampling handle part by hand, repeatedly extruding a sponge brush head on the inner wall of the tube until the sponge brush head is squeezed to be dry, inversely inserting the sponge brush head into a tube cap of the sampling tube, covering the tube cap, centrifuging at 1500rpm for 1min, collecting the liquid in the sampling tube into a 2mL sterile tube, and immediately detecting; if the detection is not immediate, the sample is stored at-20 ℃.
Oral liquid sample collection quality control: selecting an oral fluid component human Total IgG antibody (Total IgG) as a quality control index, detecting the Total IgG in a sample while detecting the novel coronavirus IgG antibody, calculating a reference value range of the human population oral fluid Total IgG by detecting a large number of human population oral fluid samples, and determining a judgment standard of an oral fluid qualified specimen: and when the Total IgG concentration of the oral fluid is greater than or equal to the critical value of 0.356-0.497 mu g/mL, the oral fluid is a qualified sample, and when the Total IgG concentration is less than the critical value, the oral fluid is unqualified for sampling, and the oral fluid is rejected, so that the false negative result of detection is reduced.
Magnetic particle chemiluminescence establishment: selecting a novel coronavirus spike protein (S) receptor binding region (RBD region) as an antigen, labeling the antigen with Fluorescein Isothiocyanate (FITC), labeling a magnetic bead with an anti-FITC antibody, binding an antigen-antibody complex on the magnetic bead through the combination of the anti-FITC antibody and the FITC, precipitating in an external magnetic field, removing supernatant, cleaning the precipitated complex, adding a mouse anti-human IgG monoclonal antibody labeled with alkaline phosphatase to form a magnetic particle-S protein RBD antigen-novel coronavirus RBD IgG antibody-alkaline phosphatase labeled mouse anti-human IgG monoclonal antibody complex, adding a substrate solution, and catalyzing the substrate solution to emit light to form a light-emitting reaction. The luminescence value of each specimen was measured by a chemiluminescence immunoassay analyzer. The luminous value of the specimen is positively correlated with the concentration of the novel coronavirus antibody, so that the novel coronavirus IgG antibody in the oral fluid specimen is detected.
Establishment of the Cut-off value (Cut-off value): 197 blank healthy people who are not inoculated with the new coronary pneumonia vaccine (serum new coronary virus IgG antibodies are all negative), 161 subjects inoculated with the new coronary pneumonia vaccine (serum IgG antibodies are all positive), new coronary virus IgG antibodies of oral fluid of the subjects are detected, and the SPSS21 software is used for drawing ROC curves. The cut-off value is determined to be 86397.5-94530 through calculation.
Preparation of positive control: mixing 5 parts of samples with positive oral liquid detection results, inactivating at 56 ℃ for 30 minutes, adding the treatment solution to dilute to a working concentration, subpackaging and storing at-20 ℃.
Preparation of negative control: sample treatment liquid 1:100 diluted fetal calf serum, subpackaged and stored at-20 ℃.
The specific detection steps are as follows: (1) sample adding: adding 50-80 μ L oral liquid sample and 40-60 μ L FITC labeled novel coronavirus recombinant antigen into a reaction tube; if the sample contains the novel coronavirus RBD-IgG antibody, forming a compound RBD-IgG antibody-S protein RBD antigen with the FITC-labeled novel coronavirus recombinant antigen; (2) adding magnetic beads and incubating: adding 30-50 μ L of magnetic microparticle-anti-Fluorescein Isothiocyanate (FITC) antibody (same as above), and incubating at 37 deg.C for 15-20 min to form RBD-IgG antibody-S protein RBD antigen-magnetic particle complex; washing with the cleaning solution for 3 times to remove free components; (3) Adding alkaline phosphataseAntibody marking: labeling 40-60 μ L of alkaline phosphatase with mouse anti-human IgG monoclonal antibody
Adding the mixture into a reaction tube to form an alkaline phosphatase labeled antibody-RBD-IgG antibody-S protein RBD antigen-magnetic particle compound; (4) incubation: incubating at 37 deg.C for 15-20 min, washing with cleaning solution for 3 times, and washing off free components; and (5) detecting: adding 100 mu L of substrate, catalyzing substrate liquid by alkaline phosphatase to emit light, detecting in a dark place, and reacting for 5 seconds to read a chemiluminescence value.
Advantageous effects
Compared with the traditional antibody detection method, the method has the advantages that the blood sample collection required by the traditional antibody detection method is traumatic and has potential infection risks, the method is noninvasive in sampling, safe and convenient, the comfort is higher for the old and children, the collection can be carried out automatically, and the method is favorable for sample collection of special crowds and antibody screening of large crowds. Meanwhile, one oral liquid can detect not only the antibody but also the nucleic acid and the antigen, so that the method is efficient, saves and saves medical resources.
The method takes oral fluid as a detection sample, has the advantages of no wound, safety, convenience and high efficiency, overcomes the wound and potential infection risk of blood collection, and reduces the infection risk of medical personnel. The magnetic particle chemiluminescence method has high detection sensitivity and specificity, and is suitable for large-scale detection.
After sampling, the oral liquid is absorbed in the sponge brush, no liquid component exists, and no scattering risk exists in the transportation process. Meanwhile, the sample treatment method is simple and convenient, is easy to operate, and is favorable for popularization and use in basic public health institutions.
The existing commercial new coronavirus serum antibody detection kit approved by the national drug administration has poor detection capability on oral fluid samples, and the sensitivity (positive compliance rate) is 6.28% (13/207): the detection method provided by the invention has higher consistency with the detection result of the commercial detection reagent, and can supplement the defect that the commercial kit can not detect the oral fluid of the novel biological specimen.
Drawings
Fig. 1 shows that the magnetic particle chemiluminescence method of the present invention simultaneously detects paired serum and oral fluid of 161 subjects, and the correlation analysis of the semi-quantitative antibody detection results shows that the antibody detection results of the serum and oral fluid samples are highly correlated, and the correlation coefficient is r =0.856.
FIG. 2 shows the results of the detection of IgG antibodies against coronavirus from serum and oral fluid samples using commercial reagents and methods of the present product.
Examples
In order to make the content of the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the present invention is not limited thereto.
Example 1 test experiment of oral fluid at different collection times:
different subjects were selected, tested at different sampling times: the oral fluid sampling brush is used for repeatedly brushing the gum part up, down, left and right for 60s,90s,120s and 150s respectively, and the detection results of the novel coronavirus IgG antibody of the oral fluid at different sampling time are as follows
Therefore, the detection result, the sampling efficiency and the aggregation time of large-scale sampling are comprehensively considered, and the brushing time 90s is selected as the optimum sampling time of the oral liquid.
Example 2 novel coronavirus antibody detection in oral fluid samples
(1) Collecting oral cavity liquid samples: repeatedly brushing the gum part for 90 seconds up, down, left and right by using an oral liquid sampling brush until the brush head is completely soaked, putting the sampling brush back into the sampling tube, covering a tube cap, preserving at-4 ℃, and conveying to a laboratory for sample treatment within 24 hours.
(2) Treatment of oral liquid specimens: adding 0.5-1mL of specimen treatment liquid into a sampling tube, holding a sampling handle part by hand, repeatedly extruding a sponge brush head on the inner wall of the tube until the sponge brush head is squeezed to be dry, inversely inserting the sponge brush head into a tube cap of the sampling tube, covering the tube cap, centrifuging at 1500rpm for 1min, collecting the liquid in the sampling tube into a 2mL sterile tube, and immediately detecting; if the detection is not immediate, the sample is stored at-20 ℃.
(3) Selecting a novel recombinant protein having neutralizing activity in the spike protein (S) RBD region of coronavirus [2](Sino
Cat: 40592-V08B) as a detection antigen and labeled with Fluorescein Isothiocyanate (FITC).
(4) Novel coronavirus recombinant antigen labeling: FITC was dissolved in DMSO to give a 20mg/ml solution, and stored in the dark. Mixing the recombinant antigen of the new coronavirus with a FITC solution in a ratio of 1:1.5-1, adding the mixture into a brown glass bottle according to the molar ratio of 1.0, fully and uniformly mixing, and carrying out acid-base balance and purification on a labeled product to obtain a novel FITC-labeled coronavirus recombinant antigen; stored under dark conditions at-20 deg.C [3] [4].
(5) Magnetic particle labeling: magnetic beads of carboxyl groups
And (3) performing magnetic separation and washing for 3 times, removing a supernatant, and adding a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution and an N-hydroxysuccinimide solution to activate carboxyl on the surfaces of the magnetic beads. Activated carboxyl magnetic beads and anti-FITC antibodies are mixed according to the ratio of 100:1, and keeping a uniformly mixed state after coupling; washing with 0.01M PBS for 3 times, and diluting to desired volume to obtain magnetic microparticle-Fluorescein Isothiocyanate (FITC) antibody.
(6) Sample preparation, namely adding 50-80 mu L of oral liquid to be detected, uniformly mixing for 5 seconds by using a vortex mixer, and standing for 15 minutes to start an experiment; a negative control and a positive control are set simultaneously.
(7) Sample adding: adding 40-60 μ L FITC labeled novel coronavirus recombinant antigen and 50-80 μ L oral liquid sample processed by the above steps into a reaction tube; if the sample contains the novel coronavirus RBD-IgG antibody, forming a compound RBD-IgG antibody-S protein RBD antigen with the FITC-labeled novel coronavirus recombinant antigen;
(8) adding magnetic beads and incubating: adding 30-50 μ L of magnetic microparticle-anti-Fluorescein Isothiocyanate (FITC) antibody (same as above), and incubating at 37 deg.C for 15-20 min to form RBD-IgG antibody-S protein RBD antigen-magnetic particle complex; cleaning solution (Yu tool No. 20200040) for 3 times to remove free components;
(9) adding a secondary antibody: labeling 40-60 μ L of alkaline phosphatase with mouse anti-human IgG monoclonal antibody
Adding the mixture into a reaction tube to form an alkaline phosphatase labeled antibody-RBD-IgG antibody-S protein RBD antigen-magnetic particle compound;
incubation at r: incubating at 37 deg.C for 15-20 min, washing with washing solution for 3 times, and washing off free components;
and (3) detection: adding 100 mu L of substrate, catalyzing substrate liquid by alkaline phosphatase to emit light, detecting in a dark place, and reacting for 5 seconds to read a chemiluminescence value.
Establishment of the Cut-off value (Cut-off value): 197 blank healthy people (serum new coronavirus IgG antibody is negative) without new coronarism vaccine are selected, 161 subjects (serum IgG antibody is positive) are inoculated with the new coronarism vaccine, new coronarism IgG antibody of oral fluid is detected, SPSS21 software is used for drawing ROC curve, the area under the curve is shown in the figure,
area under the curve
Testing result variables:
| area of | Standard error of a | And (6) progressive Sig. b | Asymptotic 95% confidence interval | |
| Lower limit of | Upper limit of | |||
| .989 | .004 | .000 | .981 | .997 |
a. Under the nonparametric assumption
b. Zero hypothesis: real area =0.5
According to the sensitivity and specificity of the method at different cutoff values, corresponding cut-off values are calculated. Through calculation, when the cut-off value 86397.5-94530 is selected, the detection performance of the experiment is the best, the cut-off value is a critical value of the method, and the sensitivity is
95.0 to 96.9 percent and specificity of 94.4 to 94.9 percent.
The ROC curve analysis result shows that the area under the curve is 0.989, which indicates that the detection method has higher accuracy.
And (3) judging the detection result: the sample luminous value/critical value is greater than or equal to 1, the detection result is judged to be positive, the ratio is less than 1,
the detection result is judged to be negative.
Quality control of oral fluid sample collection and detection: selecting human Total IgG antibody (Total IgG) as quality control index, and selecting commercial detection kit
The human Total IgG antibody detection kit (magnetic particle chemiluminescence method) detects Total IgG in a sample while detecting novel coronavirus IgG antibody, and 215 non-inoculated samples are collectedBlank healthy population (serum new coronavirus IgG antibody is negative) of the new coronary pneumonia vaccine and a third test subject (serum IgG antibody is positive) of 161 inoculated new coronary pneumonia vaccine oral fluid, and the Total IgG reference value range of the oral fluid of the population is calculated. Obtaining a critical value of 0.356-0.497 mug/mL of Total IgG, wherein when the Total IgG detection value is higher than the critical value, the oral fluid specimen is qualified, and the detection result of the novel coronavirus IgG antibody is correct; and when the Total IgG detection value is lower than the critical value, the sampling is determined to be unqualified, and the detection result of the novel coronavirus IgG antibody in the oral liquid cannot be adopted. Unqualified samples are removed through quality control, and the existence of false negative results is reduced.
Quality control of the detection method: the positive control and the negative control are simultaneously established under the calculated critical value, the experiment is established, and the result can be adopted.
Example 3 comparison of the Effect of the oral liquid sample detection method and the commercial serum detection kit for detecting novel coronavirus antibodies
The existing commercial new coronavirus serum IgG antibody detection kit approved by the national drug administration has poor detection capability on oral fluid samples and low positive rate.
In the embodiment, a commercial new coronavirus serum antibody IgG detection kit (national mechanical Standard: 20203400183) is used as a control reagent, and IgG antibody detection is respectively carried out on 207 parts of blood and oral liquid paired samples, and the results show that:
the sensitivity (positive coincidence rate) is 6.28% (13/207), so that the traditional serum antibody detection reagent has poor sensitivity when detecting an oral liquid sample, and is not suitable for detecting the oral liquid antibody.
The detection method of the invention can detect not only oral cavity liquid antibody, but also serum antibody, and the detection result of the detection serum antibody has better consistency with the detection result of a commercial serum antibody detection kit approved by the national drug administration.
A commercial serum antibody detection reagent (national mechanical Standard: 20203400183) and the oral liquid new coronavirus IgG antibody detection reagent are adopted to simultaneously detect 162 parts of new coronavirus IgG antibodies in a serum sample, and the results show that: the commercial serum antibody detection kit (national mechanical Standard: 20203400183) detects 161 positive cases and 1 negative case; the oral liquid new coronavirus IgG antibody detection reagent is 161 positive cases and 1 negative case.
The calculation results in that,
sensitivity (positive coincidence) =161/161=100%
Specificity (negative compliance) =1/1=100%
Total coincidence rate =162/162=100%
Correlation analysis of the above data: the chemiluminescence method of the invention simultaneously detects the paired serum and oral fluid of 161 cases of subjects, and the correlation analysis of the semi-quantitative antibody detection result shows that the antibody detection results of the serum and the oral fluid samples are highly correlated, and the correlation coefficient is r =0.856 (figure 1).
In conclusion, the detection method of the new crown antibody and the commercialized kit approved by the drug administration for detecting the blood freshening crown antibody have high conformity, and the existing data show that the detection efficiency is completely equivalent.
The method can be simultaneously applied to the detection of oral fluid and blood samples, the detection is simultaneously carried out on two samples of the same case, the result shows that the oral fluid and the blood samples have good consistency, the consistency can be verified mutually, the accuracy of the detection result is ensured, and the oral fluid can replace the blood to carry out the detection of the new coronavirus IgG antibody.
The oral fluid and blood matched samples of 162 subjects are respectively detected by adopting the technology of the invention, and the results show that:
161 of 162 serum samples were positive for the new coronavirus IgG antibody, and 1 was negative; of 162 oral fluid samples, 156 of the new coronavirus IgG antibodies were positive and 6 of them were negative.
The calculation results in that,
sensitivity (positive coincidence) =156/161=96.89%
Specificity (negative compliance) =1/1=100%
Total coincidence rate =157/162=96.91%
In conclusion, the oral liquid new coronavirus IgG antibody detection reagent can be used for simultaneously detecting new coronavirus IgG antibodies of a serum sample and an oral liquid sample, and the detection efficiencies are basically the same (FIG. 2).
In conclusion, the oral liquid new coronavirus IgG antibody detection reagent can detect a blood sample and an oral liquid sample, the results of the two can be verified mutually, the sensitivity and the specificity are better, and the coincidence rate is higher, while the traditional serum antibody detection reagent (national mechanical standard: 20203400183) only detects a serum sample, and the sensitivity and the specificity are poorer when the oral liquid sample is detected.
It should be understood that equivalents and modifications to the disclosed embodiments and concepts may occur to one skilled in the art, and all such modifications and alterations are intended to fall within the scope of the appended claims. Reference:
[1]Antibody responses to SARS-CoV-2 in patients with COVID-19[J].Nature Medicine.
[2]Jm A,MD A,Ks A,et al.Improved production of SARS-CoV-2 spike receptor-binding domain(RBD)for serology assays[J].Protein Expression and Purification,2020.
[3] zhouyanchun, dingshi, tangyan, et al, magnetic particle chemiluminescence immunoassay is established to detect serum CTX [ J ]. International inspection journal of medicine, 2020,41 (18): 2236-2238,2243. DOI.
[4] A novel coronavirus IgM/IgG magnetic particle chemiluminescence immunoassay kit is CN202010416888.2[ P ].
Claims (9)
1. An oral liquid specimen treating liquid for detecting novel coronavirus IgG antibody of novel biological sample oral liquid is characterized by comprising the following components of fetal bovine serum, PBS buffer solution with pH =7.4, gentamicin and streptomycin.
2. The oral fluid specimen treatment solution according to claim 1, wherein the concentrations of the respective portions are: 10% of fetal calf serum, 1% of gentamicin, 1% of streptomycin and the balance of PBS, preferably, 0.5% of red dye can be added.
3. A method for treating an oral fluid specimen with the oral fluid specimen treatment liquid according to claim 1, comprising the steps of: the method comprises the following steps of: repeatedly brushing the oral cavity liquid sampling brush on the gum part for 90 seconds up, down, left and right, putting the gum part back into the sampling tube,
storing, and sending to a laboratory for sample treatment within 24 hours; secondly, treating the oral liquid: using 0.5-1mL of sample treatment fluid which is researched and developed to vibrate, uniformly mix, extrude, elute and centrifuge the sampling brush, finally collecting the oral cavity fluid into a 2mL sterile tube, and immediately detecting; if the detection is not immediate, the sample is stored at-20 ℃.
4. A quality control method for novel oral fluid collection of biological samples for novel coronavirus IgG antibody detection, characterized by comprising the steps of (1) performing sample processing by the method of claim 3; (2) Selecting an oral fluid component human Total IgG (Total IgG) antibody as a quality control index, calculating a reference value range of the human population oral fluid human Total IgG by detecting a large number of population oral fluid samples, and determining a judgment standard of the oral fluid qualified specimen: and when the concentration of the human total IgG in the oral fluid is greater than or equal to the critical value of 0.356-0.497 mu g/mL, rejecting the qualified sample and reducing the false negative detection result.
5. A novel coronavirus antibody detection kit comprising the oral fluid specimen treatment fluid according to claim 1.
6. Use of the oral fluid specimen treatment fluid according to claim 1 or the novel coronavirus antibody detection kit according to claim 5 for preparing a novel coronavirus antibody detection medicament.
7. Use according to claim 6, characterized in that the detection of the detection drug is based on magnetic particle chemiluminescence detection, comprising the following steps: (1) Selecting a recombinant protein with neutralizing activity in a novel coronavirus spike protein (S) RBD (receptor binding domain) region as an antigen, and labeling the antigen with Fluorescein Isothiocyanate (FITC); (2) Labeling magnetic beads with an anti-FITC antibody, binding an antigen-antibody complex on the magnetic beads through binding the anti-FITC antibody and FITC, and precipitating an immune complex in an external magnetic field; (3) Alkaline phosphatase is used for marking a mouse anti-human IgG monoclonal antibody, and an indirect method principle is adopted for detecting a novel coronavirus RBD-IgG antibody in a human oral fluid sample; (4) And judging the detection result according to the ratio of the chemiluminescence value of the sample to the critical value.
8. The use according to claim 7, wherein the threshold value (cut-off value) of the magnetic particle chemiluminescence detection method for novel coronavirus IgG antibodies in oral fluid is established in step (4) by detecting oral fluid in a blank population not inoculated with the new coronary pneumonia vaccine and a third immunized population inoculated with the new coronary pneumonia vaccine, and is 86397.5-94530.
9. Use according to claim 7, characterized in that the steps of steps (1) to (3) comprise in particular: (1) sample adding: adding 50-80 μ L oral liquid sample and 40-60 μ L FITC labeled novel coronavirus recombinant antigen into a reaction tube; if the sample contains the novel coronavirus RBD-IgG antibody, forming a compound RBD-IgG antibody-S protein RBD antigen with the FITC-labeled novel coronavirus recombinant antigen; (2) adding magnetic beads and incubating: addingAdding 30-50 μ L of magnetic microparticle-anti-Fluorescein Isothiocyanate (FITC) antibody (same as above), and incubating at 37 deg.C for 15-20 min to form RBD-IgG antibody-S protein RBD antigen-magnetic particle complex; washing with cleaning solution for 3 times to remove free components; (3) adding alkaline phosphatase labeled antibody: 40-60. Mu.L of alkaline phosphatase-labeled mouse anti-human IgG monoclonal antibody
Adding the mixture into a reaction tube to form an alkaline phosphatase labeled antibody-RBD-IgG antibody-S protein RBD antigen-magnetic particle compound; (4) incubation: incubating at 37 deg.C for 15-20 min, washing with cleaning solution for 3 times, and washing off free components; and (5) detecting: adding 100 mu L of substrate, catalyzing substrate liquid by alkaline phosphatase to emit light, detecting in a dark place, and reacting for 5 seconds to read a chemiluminescence value.
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