CN114441754A

CN114441754A - Use of antibodies to annexin for detecting the severity of SARS-CoV-2 and/or COVID-19

Info

Publication number: CN114441754A
Application number: CN202210107547.6A
Authority: CN
Inventors: 余枝广; 黄涛; 芦亚飞; 崔易航; 吴晓丽; 汪梅花; 柴凡; 肖良品
Original assignee: Medcaptain Medical Technology Co Ltd
Current assignee: Medcaptain Medical Technology Co Ltd
Priority date: 2022-01-28
Filing date: 2022-01-28
Publication date: 2022-05-06

Abstract

The application provides the application of an antibody of annexin as a detection target and/or a reagent for detecting the antibody generation level of the annexin in the preparation of a product for detecting SARS-CoV-2; through detecting the antibody of the annexin, whether the SARS-CoV-2 is infected or not can be known, which is beneficial to individual early warning and disease prevention and control. The application also provides the application of the antibody of the annexin as a detection target and/or a reagent for detecting the antibody generation level of the annexin in preparing a product for detecting the severity of the COVID-19, and a product for detecting SARS-CoV-2 and/or detecting the severity of the COVID-19.

Description

Use of antibodies to annexin for detecting the severity of SARS-CoV-2 and/or COVID-19

Technical Field

The present application relates to the field of biomedicine, and in particular to the use of antibodies to annexin for the detection of the severity of SARS-CoV-2 and/or COVID-19.

Background

SARS-CoV-2 is a new strain of beta coronavirus, which can be transmitted by close contact, respiratory droplets and high concentration aerosol to cause lung Disease-dominated new coronavirus pneumonia (Corona Virus Disease 2019, COVID-19). The new coronavirus pneumonia becomes a global pandemic, so that the detection of the new coronavirus and the severity of the new coronavirus pneumonia is particularly important.

Disclosure of Invention

In view of the above, the application of the reagent for detecting the antibody of the annexin as the detection target and/or detecting the antibody generation level of the annexin in the preparation of products for detecting SARS-CoV-2 and for detecting the severity of COVID-19; whether the individual is infected or not is judged by detecting SARS-CoV-2, and the disease risk level of the individual is judged by detecting the severity of COVID-19, so that the individual early warning, prevention and control are enhanced, and the spread of diseases is favorably controlled.

In a first aspect, the present application provides the use of an antibody to annexin as a detection target and/or a reagent for detecting the level of antibody production by said annexin in the manufacture of a product for the detection of SARS-CoV-2.

Optionally, the annexin comprises at least one of annexin a1, annexin a2, annexin A3, annexin a4, annexin a5 and annexin A8.

Optionally, the antibody comprises at least one of an IgG antibody and an IgM antibody.

Optionally, the product comprises at least one of a kit and a diagnostic system.

Optionally, the detecting comprises detecting the level of production of antibodies to the annexin by at least one of chemiluminescence, protein chip assay and enzyme linked immunosorbent assay.

Optionally, the reagent comprises at least one of a chemiluminescent detection reagent, a protein chip detection reagent and an enzyme-linked immunoassay detection reagent.

Further, the chemiluminescent detection reagent comprises an annexin-coated magnetic bead solution, a chemiluminescent group-labeled secondary antibody solution and a chemiluminescent substrate solution.

Still further, the concentration of the magnetic beads in the annexin-coated magnetic bead solution is 0.15mg/mL-0.75 mg/mL.

Still further, the concentration of the antibody in the chemiluminescent group labeled secondary antibody solution is 0.01ng/mL-10000 ng/mL.

Still further, the secondary antibody solution labeled with a chemiluminescent group comprises at least one of a mouse anti-human IgG antibody labeled with a chemiluminescent group, a mouse anti-human IgM antibody labeled with a chemiluminescent group, a rabbit anti-human IgG antibody labeled with a chemiluminescent group, a rabbit anti-human IgM antibody labeled with a chemiluminescent group, a sheep anti-human IgG antibody labeled with a chemiluminescent group, and a sheep anti-human IgM antibody labeled with a chemiluminescent group.

In a second aspect, the present application provides the use of an antibody to annexin as a detection target and/or an agent for detecting the level of antibody production by said annexin in the manufacture of a product for use in the detection of the severity of COVID-19.

Further, the chemiluminescence detection reagent comprises a magnetic bead solution coated with annexin, a secondary antibody solution labeled with a chemiluminescence group and a chemiluminescence substrate solution.

In a third aspect, the present application provides a product for detecting SARS-CoV-2 and/or for detecting the severity of COVID-19, comprising an agent that detects the antibody-producing level of an annexin protein by at least one of chemiluminescence, protein chip assay, and enzyme linked immunosorbent assay, using an antibody to the annexin protein as a detection target.

Optionally, the chemiluminescent detection reagent comprises an annexin-coated magnetic bead solution, a chemiluminescent group-labeled secondary antibody solution and a chemiluminescent substrate solution.

Furthermore, the concentration of the magnetic beads in the annexin-coated magnetic bead solution is 0.15mg/mL-0.75 mg/mL.

Further, the concentration of the antibody in the secondary antibody solution marked by the chemiluminescence group is 0.01ng/mL-10000 ng/mL.

Further, the secondary antibody solution labeled with the chemiluminescent group comprises at least one of a mouse anti-human IgG antibody labeled with the chemiluminescent group, a mouse anti-human IgM antibody labeled with the chemiluminescent group, a rabbit anti-human IgG antibody labeled with the chemiluminescent group, a rabbit anti-human IgM antibody labeled with the chemiluminescent group, a sheep anti-human IgG antibody labeled with the chemiluminescent group and a sheep anti-human IgM antibody labeled with the chemiluminescent group.

The application provides the application of an antibody of annexin as a detection target and/or a reagent for detecting the antibody generation level of the annexin in the preparation of a product for detecting SARS-CoV-2 and for detecting the severity of COVID-19; whether the individual is infected or not is judged by detecting SARS-CoV-2, and the disease risk level of the individual is judged by detecting the severity of COVID-19, so that the individual early warning, prevention and control are enhanced, and the spread of diseases is favorably controlled.

Drawings

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present application, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a graph showing the results of measurement of a sample of a subject in the examples.

Detailed Description

The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

The inventors of the present application have found that the level of annexin antibody production is related to the severity of SARS-CoV-2 infection, as well as COVID-19 infection. Accordingly, the present application provides the use of antibodies to annexin as detection targets and/or reagents for detecting antibody production levels of annexin in the preparation of products for the detection of SARS-CoV-2. That is, the use of an antibody to annexin as a detection target in the preparation of a product for the detection of SARS-CoV-2, and/or the use of a reagent for detecting the antibody production level of annexin in the preparation of a product for the detection of SARS-CoV-2; therefore, whether the subject is infected with SARS-CoV-2 is further judged according to the generation level of the annexin antibody, thereby achieving the purposes of individual early warning, prevention and control and disease spread control.

The application also provides the application of the antibody of the annexin as a detection target and/or a reagent for detecting the antibody generation level of the annexin in the preparation of a product for detecting the severity of COVID-19. That is, the use of an antibody to annexin as a detection target in the manufacture of a product for detecting the severity of COVID-19, and/or the use of an agent that detects the antibody-producing level of annexin in the manufacture of a product for detecting the severity of COVID-19; thus, the severity of the patient suffering from the COVID-19 can be judged according to the generation level of the annexin antibody, and the medicine can be specifically administered according to the severity of the patient suffering from the disease, so that the cure rate and the survival rate of the patient are improved.

The present application also provides a product for detecting SARS-CoV-2 and/or for detecting the severity of COVID-19, comprising an agent for detecting the antibody-producing level of annexin by at least one of chemiluminescence, protein chip method and enzyme linked immunosorbent assay, using an antibody of annexin as a detection target. The antibody of the annexin is used as a detection target, and the generation level of the antibody of the annexin is detected by adopting a reagent, so that whether a subject is infected with SARS-CoV-2 or not is evaluated, the severity of the patient with COVID-19 is evaluated, and the prevention and control of diseases are facilitated.

In an embodiment of the present application, the annexin comprises at least one of annexin a1, annexin a2, annexin A3, annexin a4, annexin a5 and annexin A8. In one embodiment of the present application, the annexin is annexin a 2.

In embodiments herein, the antibody comprises at least one of an IgG antibody and an IgM antibody. Specifically, at least one of an annexin a 1-specific IgG antibody, an annexin a 1-specific IgM antibody, an annexin a 2-specific IgG antibody, an annexin a 2-specific IgM antibody, an annexin A3-specific IgG antibody, an annexin A3-specific IgM antibody, an annexin a 4-specific IgG antibody, an annexin a 4-specific IgM antibody, an annexin a 5-specific IgG antibody, an annexin a 5-specific IgM antibody, an annexin A8-specific IgG antibody, and an annexin A8-specific IgM antibody may be used as the detection target.

In an embodiment of the present application, the detecting comprises detecting the level of production of antibodies to annexin by at least one of a chemiluminescence method, a protein chip method, and an enzyme-linked immunosorbent assay. In embodiments of the present application, the reagents include at least one of a chemiluminescent detection reagent, a protein chip detection reagent, and an enzyme-linked immunoassay detection reagent.

In the embodiment, the chemiluminescent detection reagent comprises an annexin-coated magnetic bead solution, a chemiluminescent group labeled secondary antibody solution and a chemiluminescent substrate solution. In the present application, a sample of an annexin-coated magnetic bead subject is incubated, and when an annexin antibody is present in the sample of the subject, the annexin-coated magnetic bead can be combined with the annexin antibody in the sample of the subject to form a complex, the complex is combined with a chemiluminescent-labeled secondary antibody, and a chemiluminescent substrate acts on the chemiluminescent group to generate luminescence, so that the annexin antibody is detected. In one embodiment, the annexin coated magnetic beads comprise annexin coated superparamagnetic magnetic beads. In another embodiment, the magnetic beads have a micron-sized particle size, which may be, but not limited to, 0.8 μm, 1.0 μm, 1.2 μm, 1.5 μm, 2.0 μm, 2.5 μm, 2.8 μm, 3.0 μm, 3.5 μm, and the like.

In the embodiment of the application, the concentration of the magnetic beads in the annexin-coated magnetic bead solution is 0.15mg/mL-0.75 mg/mL. Specifically, the concentration of the magnetic beads in the annexin-coated magnetic bead solution can be, but is not limited to, 0.2mg/mL, 0.25mg/mL, 0.3mg/mL, 0.4mg/mL, 0.5mg/mL, 0.6mg/mL, 0.7mg/mL, 0.75mg/mL, or the like. In one embodiment, the pH of the annexin-coated magnetic bead solution is 3-10.5. Specifically, the pH of the annexin-coated magnetic bead solution can be, but is not limited to, 4, 5, 6, 7, 8, 9, 10, or the like. In one embodiment, the concentration of the magnetic beads in the annexin-coated magnetic bead solution is 0.5mg/mL, and the pH is 8.0.

In an embodiment of the application, the magnetic beads are coated with a full-length or partial fragment of at least one of annexin a1, annexin a2, annexin A3, annexin a4, annexin a5 and annexin A8. In one embodiment, annexin a2 is exemplified, and in one embodiment, annexin a2 has an amino acid sequence (N-terminal to C-terminal) of MSTVHEILCKLSLEGDHSTPPSAYGSVKAYTNFDAERDALNIETAIKTKGVDEVTIVNILTNRSNAQRQDIAFAYQRRTKKELASALKSALSGHLETVILGLLKTPAQYDASELKASMKGLGTDEDSLIEIICSRTNQELQEINRVYKEMYKTDLEKDIISDTSGDFRKLMVALAKGRRAEDGSVIDYELIDQDARDLYDAGVKRKGTDVPKWISIMTERSVPHLQKVFDRYKSYSPYDMLESIRKEVKGDLENAFLNLVQCIQNKPLYFADRLYDSMKGKGTRDKVLIRIMVSRSEVDMLKIRSEFKRKYGKSLYYYIQQDTKGDYQKALLYLCGGDD; the magnetic beads may be coated with at least one of the full-length fragment and the partial fragment of annexin a 2. In one embodiment, the magnetic beads are coated with a C-terminal polypeptide fragment of annexin a 2. In another embodiment, the magnetic beads are coated with an N-terminal polypeptide fragment of annexin a 2. In yet another embodiment, the magnetic beads are coated with an intermediate polypeptide fragment of annexin a 2. Wherein, the intermediate polypeptide fragment can be selected from the full-length fragment of annexin A2. Specifically, when the magnetic beads are coated with a partial fragment of annexin, the partial fragment contains at least ten amino acids.

In an embodiment of the present application, a method for preparing an annexin-coated magnetic bead solution includes: placing the aqueous solution of the Tosyl activated ester magnetic beads on a mixing instrument for mixing uniformly, and removing a supernatant after adsorbing the magnetic beads; adding phosphate buffer solution and annexin, uniformly mixing, performing ultrasonic incubation and rotary incubation, adsorbing magnetic beads, and removing supernatant; adding a Tris buffer solution containing bovine serum albumin, uniformly mixing, performing rotary incubation, adsorbing magnetic beads, removing supernatant, and adding the magnetic bead buffer solution to obtain a magnetic bead solution coated with annexin. Thus, the magnetic bead solution with good dispersibility, high stability, good sensitivity and good specificity can be prepared. In the application, a magnetic frame can be adopted to adsorb the magnetic beads, the pH of a phosphate buffer solution can be 6.5-8.5, the pH of a Tris buffer solution can be 7.5-10, the rotary incubation can be incubation for 20-30 h at the rotating speed of 15-20 rpm, and the adding amount of the magnetic bead buffer solution is selected according to the concentration of the magnetic bead solution for coating the annexin.

In one embodiment, the method for preparing the annexin-coated magnetic bead solution comprises the following steps: and (3) balancing the aqueous solution of the Tosyl activated ester magnetic beads at room temperature, then placing the aqueous solution on a mixing instrument, and slowly mixing the mixture for 5-20 min until the mixture is completely dispersed. Sucking a certain amount of uniformly mixed magnetic beads into a centrifugal tube, placing the centrifugal tube on a magnetic frame, standing for at least 1min, completely adsorbing the magnetic beads, and removing the supernatant of the magnetic beads; adding phosphate buffer solution (pH 6.5-8.5) into a centrifuge tube, adding annexin, fully mixing, and performing ultrasonic treatment on the suspension in the centrifuge tube by using an ultrasonic cell crusher under the ice bath condition (the parameter setting is 10%, the total working time is 1min, the ultrasonic on-time is 30s, the ultrasonic off-time is 30s, and the alarm temperature is 37 ℃); placing on a rotary blending machine in a constant temperature and humidity box at 37 ℃, rotating at 15-20 rpm, and performing rotary incubation for about 24 h; placing the centrifugal tube in a magnetic frame, standing for at least 1min, and removing the supernatant of the magnetic beads after the magnetic beads are completely adsorbed; adding Tris buffer solution (pH 7.5-10) containing bovine serum albumin, and mixing well; covering a centrifuge tube, placing on a rotary blending machine in a constant temperature and humidity box at 37 ℃, rotating at 15-20 rpm, and performing rotary incubation for about 24 h; taking out the centrifugal tube from the constant temperature and humidity box, placing the centrifugal tube on a magnetic frame, standing for at least 1min, and removing the supernatant of the magnetic beads. Adding the magnetic bead buffer solution to prepare the annexin-coated magnetic bead solution. It can be understood that the annexin-coated magnetic bead mother solution can be prepared, and the annexin-coated magnetic bead mother solution is diluted into the annexin-coated magnetic bead working solution with the use concentration when the annexin-coated magnetic bead mother solution is used.

In the embodiment of the present application, the concentration of the antibody in the chemiluminescent-group-labeled secondary antibody solution is 0.01ng/mL to 10000 ng/mL. Specifically, the concentration of the antibody in the chemiluminescent-labeled secondary antibody solution may be, but is not limited to, 0.1ng/mL, 1ng/mL, 20ng/mL, 100ng/mL, 300ng/mL, 1000ng/mL, 5000ng/mL, 8000mg/mL, or the like. In one embodiment, the pH of the chemiluminescent-labeled secondary antibody solution is 3 to 10.5. Specifically, the pH of the chemiluminescent-group-labeled secondary antibody solution may be, but is not limited to, 4, 5, 7, 9, 10, or the like. In one embodiment, the concentration of antibody in the chemiluminescent-labeled secondary antibody solution is 300ng/mL and the pH is 7. In an embodiment of the present application, the secondary chemiluminescent group-labeled antibody solution includes at least one of a chemiluminescent group-labeled mouse anti-human IgG antibody, a chemiluminescent group-labeled mouse anti-human IgM antibody, a chemiluminescent group-labeled rabbit anti-human IgG antibody, a chemiluminescent group-labeled rabbit anti-human IgM antibody, a chemiluminescent group-labeled sheep anti-human IgG antibody, and a chemiluminescent group-labeled sheep anti-human IgM antibody.

In embodiments of the present application, the chemiluminescent group comprises at least one of acridinium ester, alkaline phosphatase, luminol, and ruthenium terpyridyl. In one embodiment, the chemiluminescent detection reagent comprises an acridinium ester labeled secondary antibody solution. In an embodiment of the present application, a method for preparing an acridinium ester-labeled secondary antibody solution includes: and adding a secondary antibody to be marked and the activated acridinium ester mother solution into the acridinium ester marking buffer solution, oscillating, uniformly mixing and reacting in a dark place to obtain the acridinium ester marker. The acridinium ester marker is dialyzed by acridinium ester marker buffer solution to obtain a secondary antibody solution marked by acridinium ester. In the present application, the acridinium ester labeling buffer may be a 2- (N-morpholino) ethanesulfonic acid buffer, with a pH of 5.0-7.5; the secondary antibody can be a mouse anti-human IgG or anti-human IgM monoclonal secondary antibody; the activated acridinium ester mother liquor can be an N, N-dimethylformamide solution dissolved with acridinium ester; after the dialysis of the acridinium ester marker, the concentration of the acridinium ester marker can be adjusted as required to obtain a secondary antibody solution labeled by the acridinium ester. In one embodiment, the acridinium ester labeled secondary antibody solution is prepared by a method comprising: the acridinium ester labeling buffer was mixed with the secondary antibody to prepare a 1mg/mL secondary antibody solution. Adding an activated acridinium ester mother solution (1mg/mL acridinium ester is dissolved in a DMF organic solvent) into the prepared secondary antibody solution, shaking and uniformly mixing for about 5s, wrapping a centrifuge tube with tin foil paper, and reacting for at least 1h in a dark place to obtain the acridinium ester marker. Transferring the acridinium ester marker into a dialysis tube, putting the tube into a beaker filled with acridinium ester marker buffer solution, and stirring and dialyzing for about 1h in a dark place; replacing the acridinium ester labeling buffer solution, and dialyzing for about 2 hours; finally, the acridinium ester labeling buffer solution is replaced with fresh one, and dialysis is carried out for at least 12 h. The dialyzed acridinium ester labeled secondary antibody was removed, zeroed with acridinium ester labeled buffer, and the protein absorbance peak a280 and the acridinium ester absorbance peak a370 were measured using an ultraviolet-visible spectrophotometer to calculate the acridinium ester/antibody ratio (IR) and the acridinium ester labeled secondary antibody concentration.

In the application, a secondary antibody solution marked by a chemiluminescent group can be prepared into different concentrations, and is matched with a fixed-concentration annexin-coated magnetic bead solution to measure a relative luminescent value; the secondary antibody solution labeled with the chemiluminescent group is selected at an appropriate concentration according to the relative luminescence value. In one embodiment, the annexin coated magnetic bead solution and the chemiluminescent-group labeled secondary antibody solution may further comprise a preservative to prolong the useful life of the reagents.

In the present application, the chemiluminescent substrate solution is selected accordingly based on the chemiluminescent group. In one embodiment, where the chemiluminescent group comprises an acridinium ester, the chemiluminescent substrate solution comprises an acidic pre-excitation liquid and a basic excitation liquid. Specifically, the addition volume of the pre-excitation solution is 1. mu.L-10 mL (e.g., 20. mu.L, 100. mu.L, 300. mu.L, 1mL, 5mL, or 8 mL), and the addition volume of the alkaline excitation solution is 1. mu.L-10 mL (e.g., 20. mu.L, 100. mu.L, 300. mu.L, 1mL, 5mL, or 8 mL); the time interval between the addition of the acidic pre-excitation liquid and the addition of the alkaline excitation liquid is 0.001s-10 min. For example, the pre-excitation liquid may be H₂O₂The solution is NaOH solution. In this case, the chemiluminescence is flash chemiluminescence. In the present application, where the chemiluminescent group comprises alkaline phosphatase, the chemiluminescent substrate solution comprises a1, 2-dioxetane derivative (AMPPD) solution; when the chemiluminescent group comprises horseradish peroxidase, the chemiluminescent substrate solution comprises luminol solution; chemiluminescence when the chemiluminescent group comprises ruthenium terpyridylThe substrate solution comprises tripropylamine solution where chemiluminescence is electrochemiluminescence. In one embodiment, the detection is performed using a chemiluminescent detection reagent and a chemiluminescent detector, wherein the window time for the photomultiplier tube to acquire the signal in the chemiluminescent detector is 0.001s-100s, such as 0.001s, 0.01s, 0.1s, 5s, 20s, 50s, 75s, 100s, or the like. In the application, the chemiluminescence method is adopted for detection, so that the accuracy and the speed of detection can be improved.

In a specific embodiment, the use of an antibody against annexin as a detection target and/or a reagent for detecting the antibody production level of annexin in for the preparation of a product for detecting SARS-CoV-2, wherein the product is a kit comprising a solution of annexin-coated magnetic beads and a chemiluminescent-labeled secondary antibody solution. Specifically, the volume of the annexin-coated magnetic bead solution and the chemiluminescent group-labeled secondary antibody solution can be, but is not limited to, 6.6 mL.

In the embodiment of the application, the protein chip detection reagent comprises a protein chip, a secondary antibody and a color developing agent. Wherein, the protein chip is provided with a detection point and a quality control point coated with annexin, and the secondary antibody is an antibody marked by a marker; by incubating the protein chip and a sample of a subject, when the sample of the subject contains an annexin antibody, the annexin antibody is combined with the annexin at a detection point, after a second antibody is added, the second antibody is combined with the annexin antibody, and then a color developing agent is added for color development, so that the annexin antibody is detected. Specifically, the marker can be, but is not limited to, horseradish peroxidase, and the developer can be, but is not limited to, tetramethylbenzidine developing solution.

In the embodiment of the application, the enzyme-linked immunoassay reagent comprises an ELISA plate coated with annexin, a secondary antibody and a color developing agent. Through incubating the ELISA plate and a sample of a subject, when the sample of the subject has the annexin antibody, the annexin antibody is combined with the annexin on the ELISA plate, after the second antibody is added, the second antibody is combined with the annexin antibody, and then a color developing agent is added for color development, so that the annexin antibody is detected. The secondary antibody is an antibody marked by a marker, specifically, the secondary antibody can be an antibody marked by horseradish peroxidase or an antibody marked by biotin, and the developer can be a tetramethylbenzidine developing solution; when the secondary antibody is a biotin-labeled antibody, horseradish peroxidase-labeled avidin is also added before the color developing agent is added. Furthermore, the enzyme-linked immunoassay reagent also comprises a washing solution and a stop solution. Specifically, the washing solution can be phosphate buffer solution containing tween-20, and the stop solution can be sulfuric acid solution of 1mol/L-5 mol/L.

In embodiments of the present application, the antibody to annexin is derived from a blood sample of the subject. Specifically, the blood sample may be, but is not limited to, at least one of serum, plasma, and whole blood; the blood sample may be undiluted or diluted. In one embodiment, the reagent is used to detect the level of production of antibodies to annexin in a blood sample of a subject. In another embodiment, the blood sample is mixed with a diluent to obtain a diluted blood sample. Further, the pH of the diluent is 3 to 10.5. Specifically, the diluent may be, but is not limited to, a phosphate buffer, and the pH of the phosphate buffer may be, but is not limited to, 7.2. Further, the volume ratio of the blood sample to the diluent is 1: (1-1000). Specifically, the volume ratio of the blood sample to the diluent may be, but not limited to, 1:1, 1:100, 1:400, 1:500, 1:750, 1:900, or 1: 1000. In the present application, the subject may be a suspected case of a new corona, so that whether the subject is infected with the new corona can be more accurately confirmed; the subject can also determine the case of the new crown, so as to judge the severity of the illness state of the case of the determination, which has important significance for guiding medication and is also helpful for improving the survival rate of the subject. It can be understood that means such as lung CT detection, nucleic acid detection and the like can be carried out to jointly confirm whether the subject has the COVID-19 and the severity of the disease, and further, the targeted prevention and treatment can be carried out on cases with different disease risk levels, so that the cure rate is improved.

In one embodiment of the present application, a blood sample of a subject is pretreated and incubated with a solution of annexin-coated magnetic beads; after washing, incubating with a chemiluminescent group-labeled secondary antibody solution; then adding a chemiluminescence substrate solution for color development, and reading the luminescence intensity under a specific wavelength. If IgG antibody or IgM antibody of annexin exists in a blood sample of the subject, a magnetic bead coated with annexin, IgG antibody or IgM antibody of annexin, and a secondary antibody-chemiluminescence substrate complex marked by a chemiluminescence group can be formed, and the luminous intensity of the chemiluminescence group is in a positive correlation with the content of IgG antibody or IgM antibody of annexin. Optionally, pretreatment includes, but is not limited to, inactivation, dilution, and the like.

In embodiments of the present application, the reagent further comprises a negative control sample. By detecting the negative control sample, a Cut-off value (Cut-off) is determined based on the detection results of the subject sample and the negative control sample. In the present application, the sum of the detection average of the negative control sample and three times of the standard deviation is used as a cut-off value to judge whether the sample of the subject is negative or positive. In one embodiment, the test result is negative if the ratio of the test result to the cutoff value of the subject sample is less than 0.9; if the ratio of the detection result of the sample of the subject to the cutoff value is more than 1.1, the sample is positive; if the ratio of the detection result of the sample of the subject to the cutoff value is 0.9-1.1, the gray area is determined, and further judgment is needed. In the application, the detection sensitivity and specificity are improved by setting the cutoff value, so that the negative and positive of the sample of the subject can be better judged.

In embodiments of the present application, the product comprises at least one of a kit and a diagnostic system. In one embodiment of the present application, where the product comprises a kit, the reagents of any of the above embodiments are included in the kit. In one embodiment, the article of manufacture comprises a kit comprising a chemiluminescent detection reagent by which an IgG antibody and/or an IgM antibody of annexin is detected. In another embodiment of the present application, where the article of manufacture comprises a diagnostic system, the diagnostic system comprises a detection module for detecting the amount of annexin antibodies in a sample from the subject and an output module for outputting whether the subject is infected with SARS-CoV-2 and/or the severity of the subject's COVID-19 based on the detection results from the detection module. In one embodiment, the detection module comprises a reagent for detecting the antibody production level of annexin, and particularly can comprise the reagent in any one of the above embodiments. In another embodiment, the output module includes an input unit, an analysis unit and an output unit, the input unit is configured to receive a detection result of the detection module, the analysis unit is configured to analyze the detection result of the detection module according to a preset cutoff value and obtain an analysis result, and the output unit is configured to output the analysis result of the analysis unit. As will be appreciated, the result of the analysis is whether the subject is infected with SARS-CoV-2 and/or the severity of the subject's COVID-19.

Examples

And (3) balancing the aqueous solution of the Tosyl activated ester magnetic beads at room temperature, then placing the aqueous solution on a mixing instrument, and slowly mixing the mixture for 5-20 min until the mixture is completely dispersed. Sucking a certain amount of uniformly mixed magnetic beads into a centrifugal tube, placing the centrifugal tube on a magnetic frame, standing for at least 1min, completely adsorbing the magnetic beads, and removing the supernatant of the magnetic beads; adding a magnetic bead coating solution A (phosphate buffer solution with the pH of 6.5-8.5) into a centrifuge tube, adding recombinant human annexin A2 (with the amino acid sequence as described above) to be coated, fully mixing, and performing ultrasonic treatment on the suspension in the centrifuge tube by using an ultrasonic cell crusher under the ice bath condition (the parameters are set to be 10%, the total working time is 1min, the ultrasonic on-time is 30s, the ultrasonic off-time is 30s, and the alarm temperature is 37 ℃); covering a centrifuge tube, placing on a rotary blending machine in a constant temperature and humidity box at 37 ℃, rotating at 15-20 rpm, and performing rotary incubation for about 24 h; placing the centrifugal tube on a magnetic frame, standing for at least 1min, and removing the supernatant of the magnetic beads after the magnetic beads are completely adsorbed; adding magnetic bead coating solution C (Tris buffer solution containing bovine serum albumin, pH7.5-10), and mixing completely; covering a centrifuge tube, placing on a rotary blending machine in a constant temperature and humidity box at 37 ℃, rotating at 15-20 rpm, and performing rotary incubation for about 24 h; taking out the centrifugal tube from the constant temperature and humidity box, placing the centrifugal tube on a magnetic frame, standing for at least 1min, and removing the supernatant of the magnetic beads. The mother solution of magnetic beads coated with annexin A2 was prepared by adding a magnetic bead buffer (containing 50mM Tris, 150mM sodium chloride and 0.1% Tween 20, pH 8.0).

Taking acridinium ester labeled buffer solution (phosphate buffer solution, pH 6.5-8.5) in a centrifuge tube, taking mouse anti-human IgG or anti-human IgM monoclonal secondary antibody to be labeled, adding the monoclonal secondary antibody into the acridinium ester labeled buffer solution, and preparing into 1mg/mL secondary antibody solution. Adding activated acridine ester mother liquor (1mg/mL acridine ester is dissolved in DMF organic solvent) into the prepared mouse anti-human IgG or IgM solution, shaking and uniformly mixing for about 5s, wrapping a centrifuge tube with tin foil paper, and carrying out a light-shielding reaction for at least 1h to obtain the acridine ester marker. Taking a new dialysis tube, transferring the acridinium ester marker into the cleaned dialysis tube, and screwing down the cap of the dialysis tube; putting into a 5L beaker filled with 4L acridinium ester labeling buffer solution, and stirring and dialyzing for about 1h in a dark place; replacing 4L acridinium ester labeled buffer solution, and dialyzing for about 2 h; finally, 4L of fresh acridinium ester labeled buffer solution is changed, and dialysis is carried out for at least 12h to obtain acridinium ester labeled mouse anti-human IgG or IgM second antibody mother solution.

Preparing a magnetic bead mother solution coated with the catenin A2 into a magnetic bead working solution (R1) of 0.25mg/mL by using a magnetic bead buffer solution; preparing the dialyzed and purified acridinium ester labeled mouse anti-human IgG or IgM secondary antibody mother liquor into 3-5 secondary antibody label working solutions (R2) with different concentrations (0.001ng/mL to 10mg/mL) by using an acridinium ester label buffer solution (phosphate buffer solution, pH 6.5-8.5); the subject sample was tested by using a magnetic bead working solution (R1) and a secondary antibody marker working solution (R2), and an appropriate concentration was selected based on the intensity of the luminescence value (RLU) measured. Determining the dilution ratio, and preparing a working solution of acridinium ester labeled mouse anti-human IgG or IgM second antibody; matching the magnetic beads with the acridinium ester marker working solution to form a finished product kit;

according to the standard operation procedures in the industry and the instructions of blood collection tube manufacturers, an EDTA anticoagulation tube or an SST blood collection tube is adopted to extract a certain amount of blood samples of the testee; centrifuging the blood sample in the blood collecting tube at 2000rpm for not less than 10 min; sucking the upper layer serum or plasma, and adding the upper layer serum or plasma into the sample cup; or directly placing the blood sampling tube on the instrument sample rack.

Respectively placing the magnetic beads and the acridinium ester marker working solution at specified positions of an instrument, and placing a quality control product and a sample to be detected on a sample rack; firstly, starting a quality control test; the instrument automatically absorbs a certain amount of magnetic beads (50 mu L) and quality control substances (100 mu L), then the magnetic beads are mixed with acridinium ester marker working solution (containing nonspecific antigens, 100 mu L), after reaction lasts for 10min, the magnetic beads are attracted by a magnet, and after the magnetic beads are uniformly mixed and cleaned by cleaning solution (phosphate buffer solution, pH7-8, 100 mu L), the cleaning solution is removed by suction. Adding acidic pre-excitation solution (100 μ L), and adding alkaline excitation solution (100 μ L); the software calculates the concentration value of the quality control product according to the luminous value and judges whether the concentration value is in the designated range. After the quality control test is passed, starting the test of the blood sample of the subject; the procedure is as above, with a sample volume of 100 μ L; the software adopts the mean value of the luminescence values of the negative samples and the standard deviation of 3 times as the Cut-off value (Cut-off) set by each batch of reagent according to the luminescence values and the standard test deviations of a group of negative samples, and then calculates the ratio of the test luminescence values and the Cut-off values of the IgG or IgM antibodies according to the Cut-off value, thereby judging whether the result is negative or positive. If the ratio is greater than 1.1, judging the test result to be positive; if the ratio is less than 0.9, judging the test result to be negative; the ratio is between 0.9 and 1.1, which is a gray area, and the test is repeated or other methods are adopted to further screen the negative and positive.

A set of normal human serum samples (negative) and a set of serum samples (positive) from new coronary hospitalized patients were collected, each sample was tested using the annexin A2 chemiluminescence immunoassay kit, and the test was repeated 2 times in parallel to obtain the relative luminescence value (RLU) of the subject sample, the results are shown in FIG. 1, and the mean, standard deviation (STDEV) and Cut-off value (Cut-off) were obtained. Of these, 17 negative normal human serum samples had an average relative luminescence value of 68814.7RLU, a standard deviation STDEV of 27071.7, and a cutoff value (Cut-off) of 150030RLU, so that a luminescence value/cutoff value (S/CO) ratio of each sample was calculated, wherein samples having a ratio of 0.9 to 1.1 were considered as gray area samples, and only sample No. 4 among the negative samples was 1.02 and was in gray area, and the results are shown in table 1; a total of 25 serum samples from hospitalized patients with new crown infection were collected, and the relative luminescence values tested and the luminescence/cutoff (S/CO) ratios calculated are shown in Table 2, and all patients were judged to be positive if the luminescence/cutoff (S/CO) ratios were greater than 1.1. It can be seen that the detection of the generation level of the antibody of annexin can be used for detecting SARS-CoV-2, which is beneficial to individual early warning and disease prevention and control.

TABLE 1 test results of normal human serum samples

TABLE 2 detection results of serum samples from Xinguan-positive patients

Positive sample numbering	Relative luminous value (RLU)	Luminescence/cutoff (S/CO)
			1	453718.5	3.02
2	470507.0	3.14
			3	266672.5	1.78
4	266899.5	1.78
			5	234149.5	1.56
6	228459.5	1.52
			7	223856.5	1.49
8	269093.5	1.79
			9	235033.0	1.57
10	503972.0	3.36
			11	352753.0	2.35
12	320456.5	2.14
			13	430490.0	2.87
14	1097276.0	7.31
			15	485945.0	3.24
16	1237953.0	8.25
			17	1249784.0	8.33
18	889377.5	5.93
			19	211715.5	1.41
20	174648.0	1.16
			21	197004.5	1.31
22	239665.0	1.60
			23	220478.5	1.47
24	223466.0	1.49
			25	174102.5	1.16

While the preferred embodiments of the present invention have been described in detail, it will be understood that those skilled in the art will be able to make various changes, substitutions and alterations without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. Use of an antibody to annexin as a detection target and/or a reagent for detecting the antibody production level of the annexin in the preparation of a product for the detection of SARS-CoV-2.

2. The use of claim 1, wherein the annexin comprises at least one of annexin A1, annexin A2, annexin A3, annexin A4, annexin A5 and annexin A8.

3. The use of claim 1, wherein said antibody comprises at least one of an IgG antibody and an IgM antibody.

4. The use of claim 1, wherein the product comprises at least one of a kit and a diagnostic system.

5. The use of claim 1, wherein said detecting comprises detecting the level of production of antibodies to said annexin by at least one of chemiluminescence, protein chip assay and enzyme linked immunosorbent assay.

6. The use of claim 1, wherein the reagent comprises at least one of a chemiluminescent detection reagent, a protein chip detection reagent, and an enzyme-linked immunoassay detection reagent.

7. The use of claim 6, wherein the chemiluminescent detection reagent comprises a solution of annexin-coated magnetic beads, a solution of a chemiluminescent-group-labeled secondary antibody and a solution of a chemiluminescent substrate.

8. The use of claim 7, wherein the concentration of magnetic beads in the annexin-coated magnetic bead solution is 0.15mg/mL to 0.75 mg/mL;

the concentration of the antibody in the secondary antibody solution marked by the chemiluminescence groups is 0.01ng/mL-10000 ng/mL;

the secondary antibody solution marked by the chemiluminescent groups comprises at least one of a mouse anti-human IgG antibody marked by the chemiluminescent groups, a mouse anti-human IgM antibody marked by the chemiluminescent groups, a rabbit anti-human IgG antibody marked by the chemiluminescent groups, a rabbit anti-human IgM antibody marked by the chemiluminescent groups, a sheep anti-human IgG antibody marked by the chemiluminescent groups and a sheep anti-human IgM antibody marked by the chemiluminescent groups.

9. Use of an antibody to annexin as a detection target and/or an agent for detecting the level of antibody production by said annexin in the manufacture of a product for detecting the severity of COVID-19.

10. A product for detecting SARS-CoV-2 and/or for detecting the severity of COVID-19, comprising an antibody to annexin as a detection target, comprising a reagent for detecting the antibody production level of said annexin by at least one of chemiluminescence, protein chip, and enzyme linked immunosorbent assays.