WO2021217506A1

WO2021217506A1 - Application of specific iga and igm in early evaluation of risk of suffering from covid-19

Info

Publication number: WO2021217506A1
Application number: PCT/CN2020/087822
Authority: WO
Inventors: 刘志刚; 刘杰; 肖小军; 方章福; 刘晓宇
Original assignee: 深圳海博生物技术有限公司
Priority date: 2020-04-27
Filing date: 2020-04-29
Publication date: 2021-11-04
Also published as: CN111505310A

Abstract

Provided is an application of specific IgA and IgM as detection targets in early evaluation of a risk of an individual suffering from COVID-19, thereby being capable of evaluating, in an early stage, the risk that the individual suffers from COVID-19, enhancing the early warning and control of the individual, and being beneficial for controlling the propagation of COVID-19. Further provided are a reagent kit for evaluating, in an early stage, a risk of an individual suffering from COVID-19, and an application of a reagent for detecting specific IgA and IgM in the preparation of the reagent kit for early evaluation of the risk of the individual suffering from COVID-19.

Description

Application of specific IgA and IgM in the early evaluation of the risk of COVID-19

The present invention claims the priority of the prior application of the application number 202010345820.X with the title of "Application of Specific IgA and IgM in Early Evaluation of the Risk of COVID-19", which was submitted on April 27, 2020. The foregoing prior application The content of is incorporated into this text by way of introduction.

Technical field

This application relates to the field of biomedicine, in particular to the application of specific IgA and IgM in the early assessment of the risk of COVID-19.

Background technique

[Corrected in accordance with Rule 9.2 03.06.2020]
The World Health Organization has declared that the new coronavirus pneumonia (Corona Virus Disease 2019, COVID-19) is a global pandemic, but there is no gold standard for clinical testing of this infection.

At present, the problem of false negatives with more nucleic acid testing methods is more serious. The false negative rate of viral nucleic acid testing in suspected cases is relatively high. For patients diagnosed with COVID-19, the positive rate is only 30%-50%. The sampling process, nucleic acid extraction process and qPCR detection process of the nucleic acid detection process are prone to false negative problems.

In addition, the current serological testing methods for COVID-19 are only mentioned in the seventh edition of the trial version of the new coronavirus pneumonia diagnosis and treatment plan issued by the National Health Commission, that is, "serum new coronavirus-specific IgM antibodies and IgG antibodies are positive. The new coronavirus-specific IgG antibody in serum turns from negative to positive, or the recovery period is 4 times or more higher than that in the acute phase" is the serological basis for confirmed cases. Specific IgM antibodies and IgG antibodies can only reach the detection threshold in the middle and late stages of the disease, which is not conducive to the timely detection of COVID-19.

It can be seen that the early detection of COVID-19 is very urgent and significant.

Summary of the invention

In view of this, this application uses specific IgA and IgM as detection targets, which can evaluate the risk of individuals suffering from COVID-19 at an early stage, enhance individual early warning and prevention and control, and help control the spread of COVID-19 .

In the first aspect, this application provides the application of specific IgA and IgM as detection targets in the early evaluation of the risk of individuals suffering from COVID-19, which is 2-7 days after the individual has fever, respiratory symptoms or imaging features of pneumonia .

Optionally, the detection target is from at least one of serum, plasma and whole blood of the individual.

Optionally, the individual is a suspected COVID-19 case.

Optionally, when the specific IgA test is positive and the specific IgM test is negative, it is evaluated that the individual is at risk of contracting COVID-19.

Further, the evaluation includes: detecting the specific IgA and IgM of the individual by at least one of a chemiluminescence method, a protein chip method and an enzyme-linked immunoassay method, when the detection result of the specific IgA is higher than When the IgA cut-off value, the specific IgA is positive; when the detection result of the specific IgM is lower than the IgM cut-off value, the specific IgM is negative.

Further, the evaluation includes: detecting the individual specific IgA and IgM by a colloidal gold method, and when the specific IgA test is positive and the specific IgM test is negative, then evaluating that the individual has COVID -19 risk.

Optionally, the early stage is 3 to 5 days after the individual has fever, respiratory symptoms or imaging features of pneumonia.

This application uses specific IgA and IgM as detection targets for early assessment of the risk of individuals suffering from COVID-19, thereby improving the early warning, prevention and control of the individual’s illness, which is conducive to the control of the spread of COVID-19.

In the second aspect, this application provides a kit for early assessment of the risk of individuals suffering from COVID-19, using specific IgA and IgM as the detection targets. The early stage is after the individual has fever, respiratory symptoms, or imaging features of pneumonia. ~7 days.

Optionally, it also includes reagents for detecting the specific IgA and IgM. Further, it includes a reagent for detecting the specific IgA and IgM by at least one of a chemiluminescence method, a protein chip method, an enzyme-linked immunoassay method, and a colloidal gold method.

Optionally, the individual is a suspected COVID-19 case.

This application provides a kit for early assessment of the risk of individuals suffering from COVID-19, and the early assessment of the risk of COVID-19, which can then have an early warning effect on the individual, which is conducive to the prevention and control of COVID-19.

In the third aspect, this application provides the application of reagents for detecting specific IgA and IgM in the preparation of kits for early assessment of the risk of individuals suffering from COVID-19, the early stage being that the individual has fever, respiratory symptoms or pneumonia imaging 2 to 7 days after characteristics.

Optionally, the reagent for detecting specific IgA and IgM includes a reagent for detecting the specific IgA and IgM by at least one of a chemiluminescence method, a protein chip method, an enzyme-linked immunoassay method, and a colloidal gold method.

This application uses specific IgA and IgM as detection targets to evaluate the risk of individuals suffering from COVID-19 at an early stage, which is of great significance for the early warning and prevention and control of individuals, and has important reference value for the auxiliary diagnosis of COVID-19, and improves The speed of the diagnosis of COVID-19 has been improved, while avoiding large-scale spread.

Description of the drawings

In order to explain the embodiments of the present invention or the technical solutions in the prior art more clearly, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art. The specific embodiments described here are only used to explain the present invention, but not used to limit the present invention.

Fig. 1 is the cumulative seroconversion curve of COVID-19 patients after the onset in the examples of the application.

Detailed ways

The technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative work shall fall within the protection scope of this application.

This application provides the application of specific IgA and IgM as detection targets in the early evaluation of the risk of individuals suffering from COVID-19, which is 2-7 days after the individual has fever, respiratory symptoms or imaging features of pneumonia.

The inventor of the present application found that the detection results of IgA and IgM are biological indicators closely related to COVID-19, and the appearance of IgA is earlier than IgM. Therefore, by using specific IgA and IgM as detection targets, individuals can be evaluated at an early stage. The risk of COVID-19 disease has important guiding significance for the treatment, prevention and control of patients, and the control of the spread of COVID-19.

In the implementation of the present application, the application in the early evaluation of the risk of an individual suffering from COVID-19 can be, but not limited to, the application in the early evaluation of the individual’s risk of COVID-19 and/or the application of the early evaluation of the individual’s COVID-19 risk. -19 Application in risk system.

In the embodiment of the present application, the risk of individuals suffering from COVID-19 is evaluated at an early stage through the correlation between specific IgA and IgM.

In the embodiment of the present application, the detection target comes from at least one of the individual's serum, plasma, and whole blood. Furthermore, anticoagulants are added to plasma and whole blood to avoid coagulation. Furthermore, the anticoagulant includes at least one of ethylenediaminetetraacetic acid, heparin, and sodium citrate.

In the implementation of this application, the individual is a suspected case of COVID-19. Determine whether an individual is a suspected COVID-19 case according to the diagnostic criteria for suspected cases in the "New Coronavirus Pneumonia Diagnosis and Treatment Method (Trial Seventh Edition)" issued by the National Health Commission, for example, by combining epidemiological history and clinical Comprehensive analysis of performance. By detecting the specific IgA and IgM in suspected cases, it has important reference value for the diagnosis or elimination of suspected cases at an early stage.

In the embodiment of the present application, when the specific IgA test is positive and the specific IgM test is negative, the individual is evaluated as being at risk of COVID-19. It is understandable that when the specific IgA test is positive and the specific IgM test is negative, the individual's risk of COVID-19 can be evaluated at an early stage, that is to say, the individual's risk of disease can be evaluated at an early stage, and then Individuals can be isolated at an early stage to avoid spreading. At the same time, lung CT testing and nucleic acid testing can be performed to further determine whether the individual has COVID-19 and improve the speed of COVID-19 diagnosis.

In the embodiment of the present application, the evaluation includes: detecting the specific IgA and IgM of the individual by at least one of the chemiluminescence method, the protein chip method and the enzyme-linked immunoassay method, when the detection result of the specific IgA is higher than the IgA cut-off value , Specific IgA is positive; when the detection result of specific IgM is lower than the IgM cut-off value, specific IgM is negative. Further, when the OD value of the specific IgA is higher than the IgA cut-off value, the specific IgA is positive; when the OD value of the specific IgM is lower than the IgM cut-off value, the specific IgM is negative. In the embodiment of the application, the cut-off value (cut-off value) is the average, percentage or minimum value of the amount or concentration of specific IgA and IgM in the normal population, and the cut-off value can also be determined by statistical analysis . In an embodiment of the present application, the average value of the amount or concentration of specific IgA and IgM in the normal population is used as the cut-off value. In another embodiment of the present application, the cut-off value is determined by statistical analysis.

In an embodiment of the present application, the detection can be performed by a two-step indirect immune chemiluminescence method. In one embodiment, at least one of the individual’s serum, plasma and whole blood is pretreated and incubated with magnetic beads; after washing, it is incubated with a chemiluminescent marker; and then a substrate solution is added for color development. Read the OD value at the wavelength. If there is SARS-CoV-2 IgA antibody or IgM antibody in the sample, it can form magnetic bead coating-SARS-CoV-2 IgA antibody-chemiluminescence marker complex or magnetic bead coating-SARS-CoV- 2 IgM antibody-chemiluminescence label complex, the luminescence intensity of the chemiluminescence label is positively correlated with the content of specific IgA antibody or IgM antibody. Optionally, the chemiluminescent label can be, but is not limited to, luminol, isoluminol, ruthenium terpyridine, or acridinium ester. Optionally, the chemiluminescence substrate liquid includes a pre-excitation liquid and an excitation liquid. For example, the pre-excitation liquid can be an H ₂ O ₂ solution, and the excitation liquid can be a NaOH solution. Further, the magnetic beads and the chemiluminescent label are respectively labeled with specific IgA and IgA antibodies. Optionally, the OD value can be read at 450nm to judge negative and positive. Optionally, pretreatment includes, but is not limited to, inactivation, dilution, and the like.

In an embodiment of the present application, after pretreatment of at least one of the individual’s serum, plasma and whole blood, it is placed in a protein chip for incubation, followed by washing and adding a tracer and a chromogenic agent to the specific Read the OD value at the wavelength to judge negative and positive. Optionally, spot spots of IgA antibody or IgM antibody of SARS-CoV-2 are provided in the protein chip. Further, the protein chip is also provided with quality control protein spots, and the quality control protein spots can be colored during the detection process. Furthermore, the protein chip is also provided with a blank control detection point. Further, the detection wavelength is selected according to the color developer.

In an embodiment of the present application, after pretreatment of at least one of the individual's serum, plasma and whole blood, it is placed in an enzyme-labeled plate coated with an antigen, and after incubation and washing, an enzyme-labeled object is added, After incubation and washing, add a color reagent to incubate, and finally add a terminator to stop the reaction, and read the OD value at a specific wavelength to judge negative and positive. Furthermore, a regression relationship is usually established based on the concentration of the standard substance and the OD value, and then the concentration is calculated to judge negative and positive. In another embodiment, after pretreatment of at least one of the individual’s serum, plasma and whole blood, it is placed in an enzyme-labeled plate coated with an antigen, and after incubation and washing, a biotin secondary antibody is added, After incubation and washing, add avidin-HRP solution to incubate and wash, then add color reagent, keep the reaction away from light for up to 20 minutes; add a terminator to terminate the reaction; use a microplate reader to measure the OD value at a wavelength of 450nm. Furthermore, a regression relationship between the concentration of the standard and the OD value is usually established to calculate the concentration.

In the embodiment of this application, the evaluation includes: detecting individual-specific IgA and IgM by the colloidal gold method. When the specific IgA test is positive and the specific IgM test is negative, then the individual is assessed as being at risk of COVID-19. In one embodiment, it is detected by the colloidal gold method, and it is judged as positive when there is a color development result, and it is judged as negative when there is no color development result.

In the embodiment of the present application, the early stage is 2 to 5 days after the individual has fever, respiratory symptoms, or imaging features of pneumonia. Further, the early stage is 3 to 5 days after the individual has fever, respiratory symptoms or imaging features of pneumonia.

This application also provides a kit for early assessment of the risk of individuals suffering from COVID-19, using specific IgA and IgM as detection targets, and the early stage is 2-7 days after the individual has fever, respiratory symptoms or imaging features of pneumonia.

In the embodiment of the present application, reagents for detecting specific IgA and IgM are also included. Further, it includes a reagent for detecting specific IgA and IgM by at least one of a chemiluminescence method, a protein chip method, an enzyme-linked immunoassay method, and a colloidal gold method. In one embodiment, the reagents for detecting specific IgA and IgM by chemiluminescence methods include magnetic beads, chemiluminescence label substrate solutions, and the like. In one embodiment, the reagents for detecting specific IgA and IgM by the protein chip method include protein chips, tracers, and chromogenic agents. In one embodiment, the reagents for detecting specific IgA and IgM by the enzyme-linked immunoassay method include an enzyme-labeled plate coated with an antigen, an enzyme-labeled object, a color developing agent, and the like. In another embodiment, the reagents for detecting specific IgA and IgM by the enzyme-linked immunoassay method include an enzyme-labeled plate coated with an antigen, a biotin secondary antibody, avidin-HRP solution, a color reagent, and the like.

In the embodiment of the present application, it also includes detecting the specific IgA and IgM of the individual by at least one of the chemiluminescence method, the protein chip method and the enzyme-linked immunoassay method. When the detection result of the specific IgA is higher than the IgA cut-off value, Specific IgA is positive; when the detection result of specific IgM is lower than the IgM cut-off value, specific IgM is negative. Further, when the OD value of the specific IgA is higher than the IgA cut-off value, the specific IgA is positive; when the OD value of the specific IgM is lower than the IgM cut-off value, the specific IgM is negative. In the embodiment of the application, the cut-off value (cut-off value) is the average, percentage or minimum value of the amount or concentration of specific IgA and IgM in the normal population, and the cut-off value can also be determined by statistical analysis . In an embodiment of the present application, the average value of the amount or concentration of specific IgA and IgM in the normal population is used as the cut-off value. In another embodiment of the present application, the cut-off value is determined by statistical analysis.

This application also provides the application of reagents for the detection of specific IgA and IgM in the preparation of kits for early assessment of the risk of individuals suffering from COVID-19. In the early stage, the individuals have fever, respiratory symptoms, or imaging features of pneumonia after 2-7 sky.

In the embodiment of the present application, the reagent for detecting specific IgA and IgM includes a reagent for detecting specific IgA and IgM by at least one of a chemiluminescence method, a protein chip method, an enzyme-linked immunoassay method, and a colloidal gold method.

This application uses the detection of IgA and IgM to assist in the early diagnosis of whether an individual has COVID-19, and to assist in the early diagnosis of the risk of individuals suffering from COVID-19, thereby providing early diagnosis of COVID-19 This method has increased the speed of diagnosis of COVID-19, speeded up the treatment, prevention and control of patients, and avoided large-scale spread.

Example

In this application, 37 COVID-19 patients (experimental group) and 61 non-COVID-19 patients (control group) were tested and compared. The research was approved by the Ethics Committee of the First Affiliated Hospital of Guangzhou Medical University.

The serum samples of the above experimental group and control group were inactivated at 56°C for 30 minutes, and the chemiluminescence immunoassay kit and KAESER 6600 automatic chemiluminescence immunoassay analyzer (Chongqing Cosmai Biotechnology Co., Ltd.) were used to detect the experimental group and control group. The level of specific IgA, IgM and IgG antibodies against the spike protein (S protein) of SARS-CoV-2 in the serum samples. Among them, the chemiluminescence immunoassay kit adopts two-step indirect immunoassay. The serum sample is incubated with magnetic beads, the unbound material is washed by magnetic separation, the magnetic beads are collected and incubated with the acridinium ester label; the substrate solution is added to Color development, detect the optical density (OD) value at 450nm, and determine the cut-off value (also called cut-off value) by statistical analysis based on the average background reactivity of all SARS-CoV-2 negative serum samples in the study. For IgA, OD The cut-off value of is 3.48; for IgM, the cut-off value of OD is 0.78; for IgG, the cut-off value of OD is 1.71.

The seroconversion of anti-SARS-CoV-2 antibodies in COVID-19 patients was tested, that is, the spike protein (S Protein) protein-specific antibodies, the results are shown in Figure 1. It can be seen that the first seroconversion day of IgA is 2 days after the onset of symptoms, and the first seroconversion day of IgG and IgM is 5 days after the onset of symptoms. The onset or onset of symptoms means that the individual has fever, respiratory symptoms or pneumonia. Imaging features. At the same time, within 1-3 days of the onset of symptoms, two patients can detect specific IgA. At this time, no patient can detect specific IgG and IgM. On the 14th day of the onset of symptoms, the seroconversion rates of IgA, IgM and IgG were 90.32% (28/31), 77.41% (24/31) and 77.41% (24/31) respectively; according to the cumulative seroconversion curve, IgA, The median time for IgM and IgG were 13 days, 15 days, and 15 days, respectively.

It can be seen that serum IgA can be detected earlier than IgG and IgM antibodies. Specific IgA has important application value in the early evaluation of the risk of COVID-19. At the same time, combined with IgM, it can be used to evaluate COVID in the early stage. -19 Risk of illness. Antibodies of the IgG and IgM isotypes have been commonly used as detection antibodies in clinical practice, while the diagnostic value of IgA has been greatly ignored. This application improves the diagnostic value of IgA and has an early evaluation of the risk of COVID-19. Significance.

The above-disclosed are only the preferred embodiments of the application, and of course the scope of rights of the application cannot be limited by this. Those of ordinary skill in the art can understand all or part of the process for realizing the above-mentioned embodiments and make them in accordance with the claims of the application. The equivalent change of is still within the scope of the invention.

Claims

The application of specific IgA and IgM as detection targets in the early assessment of the risk of individuals suffering from COVID-19, characterized in that the early stage is 2-7 days after the individual has fever, respiratory symptoms or imaging features of pneumonia.
The application according to claim 1, wherein the detection target comes from at least one of serum, plasma and whole blood of the individual.
The application according to claim 1, wherein when the specific IgA test is positive and the specific IgM test is negative, the individual is evaluated as having a risk of COVID-19.
The application according to claim 3, wherein the evaluation comprises:

The specific IgA and IgM of the individual are detected by at least one of a chemiluminescence method, a protein chip method, and an enzyme-linked immunoassay method. When the detection result of the specific IgA is higher than the IgA cut-off value, the specific Sexual IgA is positive; when the detection result of the specific IgM is lower than the IgM cut-off value, the specific IgM is negative.
The application according to claim 3, wherein the evaluation comprises:

The individual specific IgA and IgM are detected by a colloidal gold method, and when the specific IgA test is positive and the specific IgM test is negative, the individual is evaluated as having a risk of COVID-19.
The application according to claim 1, wherein the early stage is 3 to 5 days after the individual has fever, respiratory symptoms or imaging features of pneumonia.
A kit for early assessment of the risk of individuals suffering from COVID-19, characterized in that specific IgA and IgM are used as detection targets, and the early stage is 2-7 days after the individual has fever, respiratory symptoms or imaging features of pneumonia.
The kit according to claim 7, wherein the early stage is 3 to 5 days after the individual has fever, respiratory symptoms or imaging features of pneumonia.
The application of reagents for detecting specific IgA and IgM in the preparation of a kit for early assessment of the risk of individuals suffering from COVID-19, characterized in that, the early stage is 2~ after the individual has fever, respiratory symptoms or imaging features of pneumonia 7 days.
The application according to claim 9, wherein the early stage is 3 to 5 days after the individual has fever, respiratory symptoms or imaging features of pneumonia.