CN117517653A - FRNT (FRNT-FRNT) detection method for neutralizing activity of Mpox virus antibody for non-diagnostic purposes - Google Patents
FRNT (FRNT-FRNT) detection method for neutralizing activity of Mpox virus antibody for non-diagnostic purposes Download PDFInfo
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Abstract
The invention belongs to the technical field of molecular biology detection, and discloses a method for detecting neutralizing activity FRNT of a Mpox virus antibody for non-diagnosis purpose, which comprises the steps of plating, diluting a sample to be detected, preparing a sample-virus mixture, infecting, fixing, breaking a membrane, adding a detection antibody, developing, scanning and counting, and calculating the neutralizing activity. Compared with the existing PRNT and CPE methods, the FRNT detection method has the advantages of time saving, high efficiency and less required sample to be detected. The detection method provided by the invention can evaluate the immune effect of the Mpox vaccine by detecting the level of the neutralizing antibody of the Mpox virus in serum, and is expected to be applied to the research and development of the monkey pox vaccine. The invention can also evaluate the neutralizing activity of the Mpox virus monoclonal antibody, can be applied to the research and development of monkey pox monoclonal neutralizing antibody medicaments, and provides methodological support for Mpox patient specific humoral immune response, preventive vaccine evaluation and neutralizing antibody activity evaluation.
Description
Technical Field
The invention belongs to the technical field of molecular biology detection, and particularly relates to a method for detecting neutralizing activity FRNT of a Mpox virus antibody for non-diagnosis.
Background
Monkey pox (also called M pox) is a viral zoonosis caused by infection of monkey pox virus (MPXV, also called Mpox virus), and the clinical manifestations are mainly fever, rash, lymphadenectasis and other symptoms. The Mpoxvirus is a double-stranded DNA virus having a genome of about 197kb in length, and belongs to the genus orthopoxvirus (poxvirus) of the family Poxviridae, which also includes 3 viruses pathogenic to humans, such as variola virus (variola), vaccinia virus (vaccinia virus), and the like. Monkey pox has become the most important orthopoxvirus in the public health field since humans have eliminated smallpox in 1980 and subsequently stopped vaccinating with smallpox vaccine.
The form of the Mpoxvirus is consistent with that of other orthopoxviruses, the shape of the Mpoxvirus is round brick or oval, the size of the Mpoxvirus is 200nm multiplied by 250nm, the outer membrane of the Mpoxvirus is 30nm on the periphery, and the Mpoxvirus surrounds a homogeneous core body. They all contain soluble antigens, nucleoprotein antigens and hemagglutinin, and the antigenic properties are essentially identical, with cross-immunity to each other. The national health commission release bulletin of 2023, 9 and 15: from day 20, 9 of 2023, the monkey pox is taken into the infectious disease of class B for management, and preventive and control measures of the infectious disease of class B are taken. The monkey pox neutralizing antibody and the method for detecting the neutralizing activity thereof are important tools for developing monkey pox vaccines and medicaments, and have important significance for controlling the epidemic situation of the monkey pox.
The neutralizing antibody (neutralizing antibody) can be combined with surface proteins or other structural substances of pathogens (such as viruses, bacteria and the like) so as to prevent the pathogens from invading cells and replicating and reproducing, thereby playing an anti-infection role and being an important target for vaccine development and antiviral drug development. The virus neutralization test is a method of incubating a mixture of a virus and a specific antibody under appropriate conditions in vitro to allow the virus and the antibody to react with each other, infecting sensitive cells with the mixture, and then determining the infectivity of the remaining virus. The method can be used for measuring the neutralizing activity of the antibody, is commonly used for evaluating the effects of vaccines and neutralizing antibody medicines, and has important clinical significance.
Methods currently in common use to detect neutralizing antibody activity include plaque reduction neutralization assays (plaque reductionneutralizationtest, PRNT) and cytopathic effects (Cytopathic effect, CPE) methods. The PRNT method is generally completed in a 12-hole plate, and needs 2 12-hole plates to detect 1 sample, so that the efficiency is low; the volume of the sample required by the 12-hole plate is increased by about 10 times compared with that required by the 96-hole plate, and the cost is obviously increased; in addition, it takes about 3 days to form macroscopic plaques after the cells are infected, which takes a long time. CPE method is generally completed in 96 well plates, 1 sample is detected under the optimal condition of one 96 well plate, compared with PRNT method, efficiency is improved, cost is reduced, but CPE needs more time than plaque formation, detection is generally needed 4-5 days after infection, and time is longer.
Disclosure of Invention
Aiming at the defects of low flux and long detection period in the prior art, the invention aims to provide a method for detecting the neutralizing activity FRNT of a Mpox virus antibody with a non-diagnostic purpose.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a method for detecting the neutralizing activity FRNT of an Mpox virus antibody of non-diagnostic interest comprising the steps of:
(1) And (3) paving: inoculating the susceptible cells into a 96-well plate according to the inoculum size of 1.0-3.0 ten thousand cells/well, and culturing in an incubator for 16-24 hours;
(2) Diluting a sample to be tested: in a U-shaped bottom 96-well plate, diluting a sample to be tested by a ratio of 2-5 times by using a maintenance culture medium containing complement, and carrying out 6-10 dilutions, wherein each dilution is carried out by 2-3 duplicate wells;
(3) Preparation of sample-virus mixtures: diluting the Mpoxvirus to 2000-6000FFU/ml by using a maintenance culture medium, adding the Mpoxvirus after equal volume dilution into the pore plate in the step (2), uniformly mixing, and placing the mixture into an incubator for culturing for 1-2 hours;
(4) Infection: discarding the medium in the 96-well plate in the step (1), adding 100 mu L of the sample-virus mixture prepared in the step (3) into the medium, and then placing the medium in an incubator for continuous culture for 16-24 hours;
(5) Fixing: taking out the 96-well plate in the step (4), discarding the culture medium, soaking in a fixing solution, and fixing at room temperature for 30-60 minutes;
(6) Rupture of membranes: discarding the fixing solution in the 96-well plate in the step (5), adding 100-200 mu L of membrane rupture buffer solution into each well, and rupture the membrane at room temperature for 10-20 minutes;
(7) Adding a detection antibody: 50-100 mu L of diluted Mpox virus detection antibody is added into each hole, and the mixture is incubated for 2-4 hours at room temperature;
(8) Color development: adding 50-100 mu L of chromogenic substrate into each well, and developing at room temperature for 5-10 minutes;
(9) Scanning and counting: scanning and counting by an ELISPot plate reader;
(10) Calculating neutralization activity: according to the counting result of the step (9), the neutralization activity of each well is calculated according to a formula (100-100 times of the test well reading value/virus control well average reading value), a neutralization curve is fitted by GraphPad Prism software, and half inhibition concentration is calculated.
Preferably, the susceptible cells in step (1) are selected from one of Vero E6, vero, BHK-21, BS-C-1, BSC-40, heLa cells.
Preferably, the Vero E6 cells in step (1) are pre-digested and centrifuged to form a cell suspension and then inoculated.
Preferably, the sample to be tested in the step (2) is selected from one of monoclonal antibodies, polyclonal antibodies, plasma, proteins, polypeptides or small molecule viral infection inhibitors.
Preferably, the maintenance medium in the step (2) is DMEM medium containing 2% fetal bovine serum and 1% penicillin, and the content of complement in the maintenance medium is 2%.
Preferably, steps (3), (4), (5) are carried out in a biosafety tertiary laboratory.
Preferably, the fixing solution in the step (5) is 4% paraformaldehyde.
Preferably, in the step (7), the dilution is specifically dilution of the Mpox virus detection antibody and the antibody diluent according to a volume ratio of 1 (500-1000).
Preferably, the Mpox virus detection antibody in step (7) is selected from HRP-labeled VACV polyclonal or fluorescence-labeled VACV polyclonal. It should be noted that other polyclonal antibodies or monoclonal antibodies that bind to the Mpox virus can be used as the Mpox virus detection antibodies in the present invention.
Preferably, when the Mpox virus detection antibody is a fluorescent labelled Mpox virus detection antibody, the method of FRNT detection of the neutralizing activity of the Mpox virus antibody of non-diagnostic interest does not comprise step (8).
Preferably, the steps (7), (8) and (9) are preceded by washing the 96-well plate with PBS buffer.
Due to the adoption of the technical scheme, the invention has the beneficial effects that:
(1) The method for detecting the neutralizing activity FRNT of the Mpox virus antibody disclosed by the invention utilizes the principles of interaction and enzymatic reaction of virus antigen and antibody, combines a cell imaging technology, can detect a virus protein signal at early stage of virus infection (the detection result can be obtained after 18 hours after infection), and obviously shortens the detection time compared with the existing PRNT and CPE methods (which need 3-5 days).
(2) The method for detecting the neutralizing activity of the Mpox virus antibody can detect 3 samples by one pore plate, and is more efficient compared with the existing PRNT (detecting 0.5 samples) and CPE (detecting 1 sample under the optimal condition).
(3) The method for detecting the neutralizing activity FRNT of the Mpox virus antibody disclosed by the invention has the advantages that the amount of a sample to be detected is reduced: the FRNT method requires about 1/10 of the sample to be measured by the PRNT method and 1/2 of the sample to be measured by the CPE method.
(4) The detection method provided by the invention can evaluate the immune effect of the Mpox vaccine by detecting the level of the neutralizing antibody of the Mpox virus in serum, and is expected to be applied to the research and development of the monkey pox vaccine. The invention can also evaluate the neutralizing activity of the Mpox virus monoclonal antibody, can be applied to the research and development of monkey pox monoclonal neutralizing antibody medicaments, and provides methodological support for Mpox patient specific humoral immune response, preventive vaccine evaluation and neutralizing antibody activity evaluation.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the result of observing cytopathic effect under a microscope in example 1.
FIG. 2 shows the results of nucleic acid detection in the test wells and control wells of example 1.
FIG. 3 is the results of scanning and counting with an ELISPot plate reader in example 3.
FIG. 4 is the results of scanning and counting with an ELISPot plate reader in example 4.
FIG. 5 shows the results of the measurement of the neutralizing activity of the antibody in example 4.
Detailed Description
Embodiments of the present invention will now be described more fully hereinafter with reference to the accompanying examples, in which some, but not all embodiments of the invention are shown. The following description of at least one exemplary embodiment is merely exemplary in nature and is in no way intended to limit the invention, its application, or uses. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Unless otherwise indicated, the instruments and materials used in the examples below are those conventionally used in laboratories, and the technical solutions are conventional in the art.
The study was approved by the ethics Committee of the third people Hospital in Shenzhen (approval number: 2021-030), and participants provided written informed consent for sample collection and subsequent analysis.
Example 1: mpoxvirus isolation and identification
The present example isolated the Mpoxvirus from clinical samples of monkey pox patients and identified by qPCR, comprising the steps of:
and (1) under the condition of informed consent, collecting a clinical diagnosis monkey pox patient pox liquid sample, and sending the sample to a biosafety third-level laboratory for storage.
Step (2), digesting Vero E6 cells with pancreatin, centrifuging at 1000rpm, removing supernatant, resuspending cells in complete medium, counting, and adjusting cell concentration to 2×10 5 Per mL, 6-well plates were plated, 2mL of cell suspension was added to each well, and cultured overnight.
Step (3), in a biosafety tertiary laboratory, re-suspending a monkey pox patient pox fluid sample with 2mL of maintenance medium and filtering the diluted sample with a 0.45 μm filter; the Vero E6 cell culture medium in the 6-well plate of step (2) was aspirated, the filtered samples (test wells) were added, and a control group was set with corresponding addition of maintenance medium (control wells). The 6-well plate was placed at 37℃with 5% CO 2 The cells were infected in the incubator for 1 hour.
And (4) sucking and discarding the sample in the 6-hole plate in the step (3), adding 2.5mL of maintenance medium, placing the mixture in a cell culture box for continuous culture, and observing cytopathic condition every day. Typical cytopathy is observed under a microscope on day 3 of culture, as shown in FIG. 1, in which white arrows show viral plaques, suggesting that cells may be infected with the Mpoxvirus and proliferation of the virus occurs.
And (5) after culturing until the 5 th day, repeatedly freezing and thawing for 3 times, transferring the liquid in the 6-hole plate to a 15mL centrifuge tube, centrifuging at 3000rpm for 10 minutes, respectively taking 200 mu L of supernatant from a test hole and a control hole, extracting viral nucleic acid according to a specification of a magnetic bead method virus genome DNA/RNA extraction kit, and subpackaging the rest supernatant for later use in a refrigerator at minus 80 ℃.
And (6) detecting the nucleic acid of the test hole and the control hole extracted in the step (5) by a qPCR method according to the specification of the monkey pox virus nucleic acid detection kit. The detection results are shown in fig. 2, the characteristic amplification curve is detected in the nucleic acid sample of the experimental hole, and the characteristic amplification curve is not found in the sample of the control hole, which indicates that the monkey pox virus in the sample of the experimental hole is positive, and the monkey pox virus is successfully separated.
The source information for the reagents and materials used in this example are shown below:
complete medium: DMEM contains 10% fetal bovine serum and 1% penicillin;
maintenance medium: DMEM contains 2% fetal bovine serum and 1% penicillin;
DMEM (Gibco, cat No. 11965092), fetal bovine serum (Gibco, cat No. 10091148), green chain mycin (Gibco, cat No. 15070063), vero E6 cells (ATCC, cat No. CRL-1586), pancreatin (Gibco, cat No. 25200056), a magnetic bead method viral genomic DNA/RNA extraction kit (tengyne constant peak, cat No. MP 301S), a monkey poxvirus nucleic acid assay kit (fluorescent PCR method) (river organism, cat No. ZD-076-02).
Example 2: mpoxvirus amplification culture
The embodiment uses Vero E6 cells to amplify and culture the Mpox virus, and specifically comprises the following steps:
step (1), vero E6 cells were cultured according to a 1X 10 protocol 7 Inoculating the inoculum size of each bottle into T175 cell culture flask, placing at 37deg.C, 5% CO 2 The cells were cultured overnight in a cell incubator.
In the step (2), in a biosafety tertiary laboratory, 1mL of the Mpox virus for freezing and storing in the example 1 is taken and melted in a biosafety cabinet, and the virus is diluted to 5mL by a maintenance medium.
Step (3), discarding the cell culture medium in the T175 cell culture flask, adding 5mL of diluted virus, and infecting in a cell culture box for 1 hour.
Step (4), virus is absorbed and removed, 30mL of maintenance medium is added into each bottle, and the culture is continued for 3 days.
And (5) taking out the T175 cell culture flask from the incubator, and freezing and thawing 3 times in the environment of minus 80 ℃ and 37 ℃.
And (6) transferring the liquid to a 50mL centrifuge tube, centrifuging at 3000rpm for 10 minutes, and taking the supernatant to be split-packed and frozen in a refrigerator at minus 80 ℃ for later use.
This example amplified cultures of the Mpox virus isolated and identified in example 1 were used in subsequent experiments.
The Vero E6 cells used in this example and the maintenance medium source were the same as in example 1.
Example 3: mpoxvirus titration
In this embodiment, the titration of the Mpox virus is performed by using a focal forming unit (Focus formingunit, FFU) assay, which specifically includes the following steps:
step (1), inoculating Vero E6 cells into a 96-well plate according to an inoculum size of 1.5 ten thousand cells/well, and placing the cells at 37 ℃ and 5% CO 2 The cells were cultured overnight in a cell incubator.
Step (2), taking out the Mpox virus for freezing and storing in the biological safety tertiary laboratory, and diluting the Mpox virus by 10 times with a maintenance medium for 5 dilutions.
And (3) discarding the culture medium in the 96-well plates of the Vero E6 cells, adding the virus diluents with different concentrations prepared in the step (2) into the 96-well plates of the Vero E6 cells (100 mu L is added to each well, 3 compound wells are arranged for each diluent), and placing the 96-well plates in a cell culture box at 37 ℃ for culturing for 18 hours.
And (4) taking out the 96-well plate from the incubator, discarding the culture medium, soaking the 96-well plate in 4% paraformaldehyde, and fixing the 96-well plate at room temperature for 30 minutes.
Step (5), discarding 4% paraformaldehyde fixed solution in the 96-well plate, adding 200 mu L of membrane rupture buffer solution into each well, and rupture the membrane at room temperature for 10 minutes.
Step (6), washing the 96-well plate 2 times by adding 200 μl of PBS buffer to each well, adding 100 μl of HRP-labeled VACV polyclonal antibody diluted with antibody diluent to each well (volume ratio of HRP-labeled VACV polyclonal antibody to antibody diluent is 1:800), and incubating at room temperature for 2 hours.
Step (7), washing the 96-well plate 3 times by using PBS buffer solution, adding 50 mu L of chromogenic substrate into each well, and developing at room temperature for 5 minutes.
Step (8), washing the 96-well plate 2 times with PBS buffer, scanning with ELISPot plate reader and counting.
Step (9), according to the counting result of the step (8), according to the formula: the average read x 10 x dilution was used to calculate the viral titer. As shown in the figure 3 of the drawings,the batch of Mpox virus titres were: (80+96+77)/3×10×100=8.43×10 4 FFU/mL。
In this example, the titer calculation was performed on the frozen Mpox virus after the amplification culture in example 2, and the dilution basis of the Mpox virus titer was provided for the subsequent experiments.
The source information for the reagents and materials used in this example are shown below:
vero E6 cells, maintenance medium source as in example 1;
membrane rupture buffer solution: 10 XPerm/Wash buffer (BD, cat. No. 554723) was diluted ten times with 0.15% Triton X-100;
antibody dilution: ten-fold dilution of deionized water with 10 XPerm/Wash buffer (BD, cat. No. 554723);
PBS buffer (Bio, cat# E607008-3000), 4% paraformaldehyde (bio, cat# E672002-0500), HRP-labeled VACV polyclonal antibody (Invitrogen, cat# PA 1-73192), chromogenic substrate (SeraCare, cat# 5510-0030).
Example 4: mpoxvirus neutralization assay
The present example uses a focus-reduced neutralization assay (Focus reduction neutralization test, FRNT) to detect the neutralizing activity of VACV polyclonal antibodies against the Mpox virus, and specifically comprises the following steps:
(1) And (3) paving: the Vero E6 cells were digested with pancreatin and centrifuged, and the cells were resuspended in complete medium to give a fraction of 1.5X10 5 cell/mL of cell suspension was inoculated in 96-well plates at an inoculum size of 1.5 ten thousand cells/well, placed at 37℃and 5% CO 2 The cells were cultured overnight in a cell incubator.
(2) Diluting a sample to be tested: a maintenance medium containing 2% complement was prepared and 3-fold specific dilutions of VACV polyclonal antibodies (VACV pAb) were made in U-bottom 96-well plates at an initial concentration of 200. Mu.g/mL, 8 dilutions were made, 3 multiplex wells were made for each dilution, and the volume of each well was 60. Mu.L after dilution. Meanwhile, a Rabbit antibody control (Rabbit IgG) group and a virus control (without a sample to be tested) group are additionally arranged.
(3) Preparation of sample-virus mixtures: in a biosafety tertiary laboratory, the Mpox virus for frozen stock of example 2 was diluted with maintenance mediumTo 4000FFU/mL, adding 60 mu L of the solution into the U-shaped bottom 96-well plate in the step (2) to be mixed with the sample to be tested in an equal volume, blowing and sucking the mixture uniformly, and then placing the mixture at 37 ℃ and 5% CO 2 Incubate in cell incubator for 1 hour.
(4) Infection: the medium in the 96-well plates of the Vero E6 cells in the step (1) is discarded, and 100 mu L (200 FFU containing virus) of the sample-virus mixture prepared in the step (3) is added into the 96-well plates of the Vero E6 cells per well, and then the mixture is placed in a cell incubator at 37 ℃ for continuous culture for 18 hours.
(5) Fixing: taking out the 96-well plate in the step (4), discarding the culture medium, soaking in 4% paraformaldehyde, and fixing at room temperature for 30 minutes.
(6) Rupture of membranes: the 4% paraformaldehyde fixed solution in the 96-well plate is discarded, 200 mu L of membrane rupture buffer solution is added to each well, and membrane rupture is carried out at room temperature for 10 minutes.
(7) Adding a detection antibody: the 96-well plate was washed 2 times with 200. Mu.L of PBS buffer added to each well, and 100. Mu.L of HRP-labeled VACV polyclonal antibody diluted with antibody diluent (the volume ratio of HRP-labeled VACV polyclonal antibody to antibody diluent was 1:800) was added to each well and incubated at room temperature for 2 hours.
(8) Color development: the 96-well plate was washed 3 times with PBS buffer, 50. Mu.L of chromogenic substrate was added to each well, and the plate was developed at room temperature for 5 minutes.
(9) Scanning and counting: the 96-well plate was washed 2 times with PBS buffer, scanned and counted with an ELISpot reader, and the count results are shown in fig. 4.
(10) Calculating neutralization activity: according to the counting result of the step (9), the neutralization activity of each well is calculated according to a formula (100-100 times of the test well reading value/virus control well average reading value), a neutralization curve is fitted by GraphPad Prism software, and half inhibition concentration (IC 50) is calculated. The results of the calculation are shown in FIG. 5, in which the rabbit IgG control antibody showed no neutralizing activity against the Mpox virus infection of Vero E6 cells, whereas the rabbit-derived VACV polyclonal antibody had a neutralizing effect with an IC50 of 73.87. Mu.g/mL. The result shows that the FNRT method is suitable for detecting the neutralizing activity of the Mpox, and has application potential for evaluating the activity of monoclonal antibodies, polyclonal antibodies, blood plasma, blood serum and other proteins, polypeptides and small molecule virus infection inhibitors with neutralizing effect.
The source information for the reagents and materials used in this example are shown below:
vero E6 cells, pancreatin, complete medium, maintenance medium, 4% paraformaldehyde, membrane rupture buffer, PBS buffer, antibody dilution, HRP-labeled VACV polyclonal antibody, chromogenic substrate source as in previous examples;
VACV polyclonal antibody (Invitrogen, cat No. PA 1-7258), rabbit antibody rabit IgG (cat No. CR1, shimeji).
The invention utilizes the basic principles of virus antigen and antibody specific combination and enzymatic reaction to combine with cell imaging technology to develop a non-diagnostic Mpox virus antibody neutralization activity FRNT detection method, which has the advantages of time saving, high efficiency and less required sample to be detected compared with the existing PRNT and CPE methods. Experiments prove that the method can rapidly and efficiently detect the neutralizing activity of the Mpox virus antibody, and has important application value in the development and evaluation of monkey pox vaccine and antibody medicaments.
The foregoing is a further detailed description of the present application in connection with the specific embodiments, and it is not intended that the practice of the present application be limited to such descriptions. It will be apparent to those skilled in the art to which the present application pertains that several simple deductions or substitutions may be made without departing from the spirit of the present application.
Claims (10)
1. A method for detecting the neutralizing activity FRNT of an antibody against a non-diagnostic target Mpox virus, comprising the steps of:
(1) And (3) paving: inoculating the susceptible cells into a 96-well plate according to the inoculum size of 1.0-3.0 ten thousand cells/well, and culturing in an incubator for 16-24 hours;
(2) Diluting a sample to be tested: in a U-shaped bottom 96-well plate, diluting a sample to be tested by a ratio of 2-5 times by using a maintenance culture medium containing complement, and carrying out 6-10 dilutions, wherein each dilution is carried out by 2-3 duplicate wells;
(3) Preparation of sample-virus mixtures: diluting the Mpoxvirus to 2000-6000FFU/ml by using a maintenance culture medium, adding the Mpoxvirus after equal volume dilution into the pore plate in the step (2), uniformly mixing, and placing the mixture into an incubator for culturing for 1-2 hours;
(4) Infection: discarding the medium in the 96-well plate in the step (1), adding 100 mu L of the sample-virus mixture prepared in the step (3) into the medium, and then placing the medium in an incubator for continuous culture for 16-24 hours;
(5) Fixing: taking out the 96-well plate in the step (4), discarding the culture medium, soaking in a fixing solution, and fixing at room temperature for 30-60 minutes;
(6) Rupture of membranes: discarding the fixing solution in the 96-well plate in the step (5), adding 100-200 mu L of membrane rupture buffer solution into each well, and rupture the membrane at room temperature for 10-20 minutes;
(7) Adding a detection antibody: 50-100 mu L of diluted Mpox virus detection antibody is added into each hole, and the mixture is incubated for 2-4 hours at room temperature;
(8) Color development: adding 50-100 mu L of chromogenic substrate into each well, and developing at room temperature for 5-10 minutes;
(9) Scanning and counting: scanning and counting by an ELISPot plate reader;
(10) Calculating neutralization activity: according to the counting result of the step (9), the neutralization activity of each well is calculated according to a formula (100-100 times of the test well reading value/virus control well average reading value), a neutralization curve is fitted by GraphPad Prism software, and half inhibition concentration is calculated.
2. The method for detecting the neutralizing activity of a Mpox virus antibody for non-diagnostic purposes according to claim 1, wherein the susceptible cells in the step (1) are selected from one of Vero E6, vero, BHK-21, BS-C-1, BSC-40 and HeLa cells.
3. The method for detecting the neutralizing activity of a Mpox virus antibody for non-diagnostic purposes according to claim 1, wherein the Vero E6 cells in step (1) are digested and centrifuged in advance to prepare a cell suspension and inoculated.
4. The method for detecting the neutralizing activity FRNT of an Mpox virus antibody of non-diagnostic interest according to claim 1, wherein the sample to be tested in step (2) is one selected from the group consisting of monoclonal antibodies, polyclonal antibodies, plasma, proteins, polypeptides and inhibitors of small-molecule viral infection;
the maintenance medium in the step (2) is a DMEM medium containing 2% of fetal bovine serum and 1% of green streptomycin, and the content of complement in the maintenance medium is 2%.
5. The method for detecting the neutralizing activity of an Mpox virus antibody for non-diagnostic purposes according to claim 1, wherein the steps (3), (4) and (5) are carried out in a biosafety tertiary laboratory.
6. The method for detecting the neutralizing activity of a Mpox virus antibody for non-diagnostic purposes according to claim 1, wherein the fixative solution in step (5) is 4% paraformaldehyde.
7. The method for detecting the neutralizing activity FRNT of a Mpox virus antibody for non-diagnostic purposes according to claim 1, where the dilution in step (7) is specifically a dilution of the Mpox virus detection antibody with an antibody diluent in a volume ratio of 1 (500-1000).
8. The method for detecting the neutralizing activity FRNT of an Mpox virus antibody of non-diagnostic interest according to claim 1, wherein the Mpox virus detection antibody in step (7) is selected from HRP-labeled VACV polyclonal antibody or fluorescent-labeled VACV polyclonal antibody.
9. The method for detecting the neutralizing activity FRNT of an mx virus antibody of non-diagnostic purpose according to claim 1, characterized in that when the mx virus detection antibody in step (7) is a fluorescent-labeled mx virus detection antibody, the method for detecting the neutralizing activity FRNT of an mx virus antibody of non-diagnostic purpose does not include step (8).
10. The method for detecting the neutralizing activity of an Mpox virus antibody for non-diagnostic purposes according to claim 1, wherein the steps (7), (8) and (9) are further performed by washing the 96-well plate with PBS buffer.
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| CN106706582A (en) * | 2016-12-09 | 2017-05-24 | 中国人民解放军第四军医大学 | High-flux method for quickly detecting hantaan virus neutralizing antibody titer |
| CN112379105A (en) * | 2020-11-09 | 2021-02-19 | 新乡学院 | Method for detecting IPMA (ionic polymer antigen) neutralizing antibody of PRRSV (porcine reproductive and respiratory syndrome Virus) |
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