CN112945919B - Method and system for detecting virus neutralizing antibody and application thereof - Google Patents
Method and system for detecting virus neutralizing antibody and application thereof Download PDFInfo
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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Abstract
The invention provides a method and a system for detecting virus neutralizing antibodies and application thereof. The detection method comprises the steps of obtaining microscopic images of experimental group samples in an orifice plate, analyzing the microscopic images of the experimental group samples, and obtaining the infection degree of the host cells and the virus neutralization capacity of the to-be-detected object based on the image analysis result of the experimental group samples. The detection method is used for imaging and analyzing the fluorescent marked sample in the pore plate, so that experimental data of the virus neutralizing antibody capacity can be obtained quickly, the interference of the cell lysis or pancreatin digestion process on the natural state of the cell is avoided, and the method is simple in operation flow and does not need special materials or time-consuming maintenance.
Description
Technical Field
The invention belongs to the technical field of biological medicines, in particular relates to development and screening of medicines, and particularly relates to a detection method and system of virus neutralizing antibodies and application of the detection method and system.
Background
Functional studies of virus-neutralizing antibodies mainly involve studies of virus-neutralizing ability and antibody binding ability. Antibody binding capacity is typically studied using methods such as Enzyme-linked immunosorbent assay (Enzyme-Linked ImmunoSorbent Assay, ELISA), biofilm interference techniques (Biolayer Interferometry, BLI) and flow cytometry fluorescence sorting techniques (Fluorescence activated Cell Sorting, FACS). Antibody neutralization capacity is generally assessed by a pseudoviral reporter or viral plaque reduction neutralization assay (PRNT). However, the above methods have respective drawbacks and limitations, and are difficult to meet the urgent requirements of rapid high-throughput screening.
The core of the enzyme-linked immunosorbent assay is to combine an antibody with an enzyme complex and then detect the combined antibody by color development; the method cannot accurately evaluate the combination of the antibody and the antigen protein in the natural conformation, and false positive data is easy to generate. The biological membrane interference technology is based on the displacement change of interference spectrogram to detect the interaction between biological molecules, and the method needs the protein to be detected to have a label so as to be fixed on the sensor in an affinity mode.
The working principle of FACS is that cells to be detected are put into a sample tube after being dyed by specific fluorescent dye, enter a flow chamber filled with sheath liquid under the pressure of gas, are arranged into a single row under the constraint of the sheath liquid and are sprayed out by a nozzle of the flow chamber to form a cell column, the cell column vertically intersects with an incident laser beam, and cells in the liquid column are excited by the laser to generate fluorescence; the method can rapidly and objectively detect multiple characteristics of single cells, but the required instruments are relatively expensive, the operation is complex, the maintenance cost is high, and the management of special personnel is required.
Plaque reduction neutralization assays are a more sensitive method of detecting serum neutralizing antibodies, with the dilution of serum that reduces plaque number by 50% as the titer therein. The experiment uses quantitative virus and equal serum with different dilutions to mix and then drive, inoculate a prepared monolayer of cells, cover nutrient agar, place the monolayer of cells in a carbon dioxide incubator at 37 ℃ for culture, count the number of plaques after several days, and calculate the plaque neutralization titer of the serum. The operation principle is approximately the same as that of the traditional serum neutralization experiment; the method has long experimental period, very relies on manual operation and has low automation degree.
Thus, developing a method to rapidly and efficiently detect antibody binding and function would be helpful in evaluating and screening antibodies targeting viruses.
Disclosure of Invention
In view of the problems existing in the prior art, the invention aims to provide a detection method and a detection system for virus neutralizing antibodies and application thereof. The method for measuring the virus neutralizing antibody test by high flux has the advantages of high detection speed and high flux, and has positive influence on the evaluation and screening of the targeted virus antibody.
To achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a method for detecting a virus neutralizing antibody, comprising the steps of:
acquiring microscopic images of experimental group samples in an orifice plate, wherein the experimental group samples are obtained by mixing at least two concentrations of to-be-detected substances with virus particles respectively and then mixing the to-be-detected substances with host cells, wherein the virus particles carry fluorescent markers A or are combined with the markers carrying the fluorescent markers A, and microscopic imaging is carried out on the experimental group samples under the excitation of a bright field and/or fluorescence excitation light matched with the fluorescent markers A to obtain microscopic images of the experimental group samples;
analyzing microscopic images of the experimental group samples to obtain image analysis results of the experimental group samples;
and obtaining the infection degree of the host cells and the virus neutralization capacity of the test object based on the image analysis result of the experimental group sample.
According to the method, the fluorescent marked sample in the micro-pore plate is rapidly imaged and analyzed through the high-flux image analysis equipment, so that experimental data of virus neutralizing antibody capacity is rapidly obtained, and the method is not only suitable for detection analysis of the 96-pore plate, but also suitable for detection analysis of other 384-pore plates, and has the advantages of high flux and high detection efficiency; the method provided by the invention does not belong to a liquid flow analysis system, can be combined with fluorescence microscopic imaging, image recognition and image superposition synthetic analysis technology, can directly image an orifice plate, can classify cells combined with and not combined with an object to be detected at a cell level, and can carry out various statistical analyses according to photographed pictures to obtain various parameters including average fluorescence intensity.
Therefore, the high-flux detection method is based on the cell level, results are directly obtained from images, the detection process is visual and convenient to verify, the detection speed is high, the flux is high, and the method has important significance for preparation of antibodies or medicines and high-flux screening.
Meanwhile, the method provided by the invention is carried out by using a high-flux cell analysis platform, cells can be suspension cells or adherent cells, the interference of the cell lysis or pancreatin digestion process on the natural state of the cells is avoided, the operation flow is simple, and no special materials or time-consuming maintenance are needed.
As a preferred technical solution of the present invention, the detection method further includes:
obtaining a microscopic image of a positive control group and a microscopic image of a negative control group, wherein the samples in the positive control group are obtained by mixing positive samples with virus particles and then mixing the positive samples with host cells, the samples in the negative control group are obtained by mixing negative samples with the virus particles and then mixing the negative samples with the host cells,
analyzing the microscopic images of the positive control group and the microscopic images of the negative control group to obtain image analysis results, wherein the image analysis results comprise any one or a combination of at least two of positive control average fluorescence intensity, negative control average fluorescence intensity, total number of cells and cell infection number.
Further, the degree of infection of the host cell is expressed as the infection rate or binding inhibition rate;
the infection rate is calculated using the following formula:
infection rate = number of cell infections/total number of cells;
the binding inhibition rate is calculated using the following formula:
binding inhibition ratio = (average fluorescence intensity of experimental group-average fluorescence intensity of negative control)/(average fluorescence intensity of positive control-average fluorescence intensity of negative control)
Further, the method further comprises constructing a fitted curve to determine the virus neutralization capacity of the test object;
the fitting method comprises the following steps: and taking the concentration of the object to be detected, the average fluorescence intensity of the experimental group, the number of cell infections, the infection rate and the binding inhibition rate as variables to obtain a dose-dependent graph reflecting the virus neutralization capacity.
Further, a dose-dependent curve reflecting virus neutralization capacity is fitted based on a dose-effect relationship equation:
Y＝Bottom+(Top-Bottom)/(1+10^((Log IC50-X)*Hill Slope)
x represents the concentration of the object to be measured;
y represents average fluorescence intensity of an experimental group, cell infection number, infection rate and binding inhibition rate of an object to be detected under the concentration of X;
bottom represents the average fluorescence intensity/cell infection number/infection rate/binding inhibition rate of the negative control;
top represents the mean fluorescence intensity of positive control/number of cell infections/infection rate/binding inhibition rate;
neutralization titer IC50 represents the concentration of test agent that is effective in causing 50% of individuals;
hill Slope is a Slope, and is obtained by solving at least two groups of XY data.
Further, the host cells in the experimental group sample carry a fluorescent marker B, and the fluorescent marker A and the fluorescent marker B are matched with each other to obtain fluorescence excitation light, and the detection method further comprises the step of obtaining microscopic images of the experimental group sample obtained after the fluorescence excitation light matched with the fluorescent marker B is excited.
Further, the detection method further comprises: comparing the first microscopic image and the second microscopic image with respect to the first microscopic image respectively photographed under the fluorescent channel matched with the fluorescent mark A and the second microscopic image photographed under the fluorescent channel matched with the fluorescent mark B, and determining whether the position information of the cells in each pore plate in the first microscopic image and the second microscopic image is consistent according to the comparison result.
In a second aspect, the present invention also provides a system for detecting virus-neutralizing antibodies, the system comprising the following modules:
the microscopic imaging module is used for acquiring microscopic images of samples in the pore plate, wherein the samples are obtained by mixing and incubating an object to be detected and/or a standard sample with a virus sample and then mixing the virus sample with host cells, the virus particles carry fluorescent markers A or are combined with the markers carrying the fluorescent markers A, and the samples are subjected to microscopic imaging under the excitation of a bright field and/or fluorescence excitation light matched with the fluorescent markers A to obtain microscopic images of the samples;
and the analysis processing module is used for analyzing the microscopic image of the sample to obtain an image analysis result of the sample and determining the virus neutralization capacity of the to-be-detected object based on the image analysis result of the to-be-detected sample.
Further, the system further comprises: the sample platform module is used for bearing the pore plate, the pore plate is provided with a plurality of sample holes and is used for bearing at least two types of objects to be detected, the objects to be detected of the same type in the plurality of sample holes at least comprise two concentrations, and the microscopic imaging module is used for respectively shooting samples to be detected in all holes on the pore plate to obtain microscopic images of all the samples to be detected.
Further, the system further comprises: and the sample automatic replacement module is used for updating the pore plate, and the microscopic imaging module shoots the sample on the updated pore plate to obtain a microscopic image of the sample on the updated pore plate.
In a third aspect, the present invention provides the use of a method as described in the first aspect or a system as described in the second aspect in the screening of a neutralising viral antibody drug, mRNA, DNA vaccine, a pharmaceutically acceptable adjuvant or a chemically synthesized drug.
The virus neutralization experiment not only refers to the neutralization effect of the antibody, but also can be widely understood to be the capability of inhibiting virus infection cells of non-antibody virus drugs, vaccines and the like, so that the application has good application potential in the evaluation of mRNA, DNA vaccines, adjuvants, chemical synthesis drugs and the like.
The numerical ranges recited herein include not only the recited point values, but also any point values between the recited numerical ranges that are not recited, and are limited to, and for the sake of brevity, the invention is not intended to be exhaustive of the specific point values that the recited range includes.
Compared with the prior art, the invention has at least the following beneficial effects:
(1) The invention develops a test method for measuring virus neutralizing antibodies in high flux, which rapidly images and analyzes fluorescent marked samples in a microplate by high flux image analysis equipment to rapidly obtain experimental data of virus neutralizing antibodies; the method not only can detect suspended cells, but also can analyze adherent cells, avoids the interference of processes such as cell lysis or pancreatin digestion on the natural state of the cells, has simple operation flow, and does not need special materials or time-consuming maintenance;
(2) The high-flux detection method provided by the invention combines the image recognition and image superposition synthetic analysis technology, directly images the pore plate, distinguishes and classifies cells in different fluorescent states under the bright field and the fluorescent channel, and realizes the analysis of the capacity of inhibiting virus protein by virus neutralizing antibodies at the cell level;
(3) The method can directly judge the binding capacity of the antibody and the virus protein through image and data analysis, and the method does not use a liquid flow analysis system, so that the method can directly image the pore plate, combines fluorescence microscopic imaging and image synthesis analysis methods, is quick, visual and accurate, is not only suitable for detection analysis of 96 pore plates, but also suitable for detection analysis of other pore plates such as 384, and has the advantages of high flux, high detection efficiency, visual and accurate detection results and the like.
Drawings
The structure of the high throughput cell analysis system used in the example of FIG. 1 is schematically shown.
FIG. 2 is a schematic flow chart of a method for detecting virus-neutralizing antibodies according to the present invention.
FIG. 3 is a micrograph (scale 0.4 mm) of the experimental group cells of example 1 under a 4 Xobjective of a high throughput cell analyzer.
FIG. 4 is a micrograph (scale 1 mm) of the experimental group cells of example 1 under a high throughput cell analyzer 10X objective lens.
Detailed Description
The following embodiments are further described with reference to the accompanying drawings, but the following examples are merely simple examples of the present invention and do not represent or limit the scope of the invention, which is defined by the claims.
In the present invention, the structure of the high-throughput cell analysis system is shown in fig. 1, and specifically includes: the device comprises a light source module, a microscopic imaging module, a sample table module, a control module, an analysis processing module and a sample automatic replacement module;
(1) The analysis processing module is connected with the microscopic imaging module and is used for analyzing and processing microscopic images acquired by the microscopic imaging device to acquire image analysis results, wherein the image analysis results comprise but are not limited to average fluorescence intensity of an experimental group, total number of cells, number of cell infection and the like;
(2) The control module is respectively connected with the light source module, the sample table module, the microscopic imaging module and the automatic sample replacing module and respectively controls the light source channel, the sample position movement, the microscopic imaging setting and the automatic sample replacement;
(3) The light source module comprises a bright field light source and/or a fluorescent light source, and provides fluorescent excitation light to form image information through a sample in the sample stage module;
specifically, the light source module comprises a fluorescent light source assembly and a fluorescent light source seat for supporting and moving a fluorescent light source; the fluorescent light source component comprises a filtering cube and a fluorescent generating unit; the light source module further comprises a fluorescent light source motor which is connected with the fluorescent light source and used for adjusting the position of the fluorescent light source; the fluorescent light source motor is connected with the control module;
(4) The microscopic imaging module acquires optical information of a sample in the sample stage module to form a microscopic image; the microscopic imaging module comprises an objective lens, a tube lens and a camera;
(5) The automatic sample replacing module can automatically replace the sample carrier plates, carry out batch detection on samples on a plurality of sample carrier plates, can work continuously for 24 hours, and efficiently realize high-flux detection on the samples.
Unless specified, the reagents and experimental consumables used in the invention are available from manufacturers routine in the art; similarly, unless otherwise indicated, all methods and techniques used are those well known to those skilled in the art.
The flow chart of the method for detecting virus neutralizing antibodies provided by the invention is shown in figure 2, and is specifically as follows:
s1, acquiring microscopic images of samples in an orifice plate
The high throughput cell analysis system obtains microscopic images of the sample in a cell culture plate on a sample stage module, which is used to carry co-cultured samples, which may be single well or multi-well (e.g., 96-well, 384-well, etc.). The sample is obtained by mixing at least two concentrations of an analyte and virus particles respectively and then mixing the mixture with host cells, wherein the analyte comprises but is not limited to a virus protein blocking antibody, serum, vaccine or the like, and the analyte is subjected to gradient dilution before detection; the host cells comprise suspension cells and/or adherent cells, so that the interference of processes such as cell lysis or pancreatin digestion on the natural state of the cells is avoided.
Meanwhile, the samples can be divided into an experimental group sample, a negative control group sample, a positive control group sample, a blank cell sample and a blank virus sample; wherein, only host cells exist in the blank cell sample, the blank virus sample only comprises virus particles and host cells, no to-be-detected object is added, and the blank cell sample and the blank virus sample are used for proving the fluorescence intensity of virus positive, so that the host cells can be infected; the negative control group samples replace the to-be-detected object with a negative standard substance, and the positive control group samples replace the to-be-detected object with a positive standard substance, so that the specificity of the antibody is proved, and the antigen can be specifically identified.
In some embodiments, the virus particles carry fluorescent markers A, the virus particles carrying fluorescent markers A are mixed with an analyte, incubated, then mixed with host cells to serve as an experimental group, and the culture is continued, and a negative control group and a positive control group are simultaneously arranged; the fluorescent label A can be various fluorescent labels (such as FITC, PE, APC, etc.), can be HRP peroxide labels (if HRP peroxide is adopted, bright field shooting is needed), and can also be a direct-connection-resistant label; in other embodiments, the viral particles do not themselves carry a fluorescent label, and it is desirable to bind to the substance carrying the fluorescent label. Mixing virus particles which do not carry fluorescent markers with an object to be detected, incubating, mixing with host cells to serve as an experimental group, continuing culturing, and simultaneously setting a negative control group and a positive control group; and after the continuous culture is finished, adding a first antibody of the viral protein into the mixed solution, incubating, and then adding a second antibody carrying a fluorescent label A, and continuously incubating to enable the fluorescent label A to be indirectly combined with the viral protein.
In some embodiments, the host cell also carries a fluorescent label, fluorescent label B is a different label than fluorescent label a, which may also be a nuclear dye (such as DAPI, PI, hoechst, etc.), and the fluorescent label a and fluorescent label B match with each other to give off fluorescence; the method that the host cells also carry fluorescent markers is a double-dyeing method, and compared with a single-dyeing method that the host cells do not carry fluorescent markers, the accuracy of experimental results can be further improved.
In a specific embodiment, the standard virus (100 CCID) is expressed as an equal volume of VSVG-GFP 50 0.1 mL) and the diluted Gibco serum are used as virus particles and an object to be detected, the Gibco serum is diluted by a DMEM cell culture solution in a continuous multiple ratio to obtain a plurality of concentration gradients, and the mixture of the two is placed in a 37 ℃ incubator for incubation; then taking the obtainedAdding the mixture of the cells into a 96-well cell culture plate in which 293T cells grow into a single layer, and connecting a plurality of well cells at each dilution; meanwhile, a control group is set, comprising: (1) normal cells (without serum and standard virus added) served as blank control; (2) VSVG-GFP standard virus and 293T cells (without serum addition) were used as virus controls; (3) an antibody positive serum control (replacement of test serum with antibody positive serum); (4) an antibody negative serum control (replacement of the test serum with an antibody negative serum); 96-well cell culture plates were placed at 37℃in 5% CO 2 Incubating in an incubator, and acquiring a bright field image of the cell culture plate and a microscopic image under a green fluorescent channel by using a high-flux cell analysis system;
in another specific example, PSPAX2-RFP standard virus is used as virus particles, mixed with Gibco serum, 293T cells are used as host cells, and further AO dye is used to stain the 293T cells, and then a high throughput cell analysis system is used to obtain bright field images of the cell culture plate, microscopic images under the red fluorescent channel and under the green fluorescent channel.
In another specific embodiment, a plurality of virus protein blocking antibodies diluted in a continuous multiple ratio concentration gradient are used as a to-be-detected object, are mixed with the virus protein of lymphocytic choriomeningitis (Lymphocytic Choriomeningitis Virus, LCMV) to be detected, and are added into a cell culture plate of Diffuse Large B Cell Lymphoma (DLBCL) cells with cells growing into a single layer, and a plurality of holes are inoculated at each dilution; and adding LCMV virus protein primary antibody into the cell culture plate, and adding FITC labeled secondary antibody after incubation to obtain an experimental group sample.
In some embodiments, the high-throughput cell analysis system can sequentially and automatically acquire microscopic images of samples to be tested in multiple holes on the hole plate, and can also quickly replace a new hole plate for shooting through a sample automatic replacement module (such as a full-automatic mechanical arm) after shooting of one hole plate. In the application scene, for the same type of to-be-detected object, at least two to-be-detected objects with different concentrations can be prepared and placed in different sample holes. And the high-throughput cell analysis system sequentially and automatically shoots samples to be detected in different sample holes to obtain microscopic images of the samples to be detected under different concentrations of the objects to be detected.
When one pore plate is insufficient to meet detection requirements, if more than ten kinds of affinities of objects to be detected are required to be detected, samples to be detected can be placed in the plurality of pore plates, and the high-throughput cell analysis system can also rapidly replace a new pore plate to shoot through a sample automatic replacement module (such as a full-automatic mechanical arm) after shooting one pore plate. Therefore, microscopic images of a plurality of samples can be efficiently and accurately shot, and the interference of complex operations such as manual replacement of an orifice plate, focusing, moving of a sample stage module and the like on the shooting of the microscopic images is avoided.
S2, analyzing microscopic images of the sample
The analysis processing module of the high-flux cell analysis system analyzes microscopic images of the sample to be tested.
The image analysis results are information related to evaluating virus neutralization ability of the test object, and the obtained image analysis results include, but are not limited to, any one or a combination of at least two of an experimental group average fluorescence intensity, a positive control average fluorescence intensity, a negative control average fluorescence intensity, a total number of cells, and a cell infection number. Cell infection numbers can be counted by fluorescent cell numbers. In the single fluorescence method, the number of infected cells is the number of fluorescent cells, and the total number of cells can be determined by the number of fluorescent cells and the number of non-fluorescent cells. In the double fluorescence method, the number of cells infected is the number of double fluorescent cells, and the total number of cells can be determined by the number of double fluorescent cells and the number of single fluorescent cells.
In some embodiments, from the obtained microscopic pictures under bright field and fluorescent channels, the number of cell infections, total number of cells, and average fluorescent intensity in the experimental group and each control group can be obtained; the bright field image of the cells of the experimental group obtained is shown in FIG. 3 and FIG. 4, wherein FIG. 3 is a micrograph (scale 0.4 mm) obtained under a 4X objective lens, and FIG. 4 is a micrograph (scale 1 mm) obtained under a 10X objective lens. For the average fluorescence intensity of the image, the analysis processing module may first determine the fluorescence intensity after the viral particles bind to the host cells, and then calculate the sum of the fluorescence intensities and divide by the total number of cells to obtain the average fluorescence intensity. The present invention can clearly distinguish host cells to which viral particles are bound from cells to which viral particles are not bound based on the image, thereby determining the average fluorescence intensity.
The two microscopic images are obtained by the double fluorescence method, and the image analysis result, such as any one or a combination of at least two of the average fluorescence intensity, the total number of cells, or the number of virus-infected cells (i.e., the number of cell infections) in percentage of the total number of cells, can be more accurately obtained than by the single fluorescence method. Meanwhile, aiming at a first microscopic image shot under a fluorescent channel matched with the fluorescent marker A and a second microscopic image shot under a fluorescent channel matched with the fluorescent marker B, the two microscopic images are compared, so that cells in the microscopic images shot under the same visual field can be verified to be at the same position, and the accurate determination of an analysis result can be further verified.
S3, determining virus neutralization capacity of the object to be tested
The analysis processing module of the high throughput cell analysis system may determine virus neutralization capacity based on the image analysis results.
Specifically, the analysis processing module may analyze the neutralization titer IC50 of the analyte based on the image analysis result; the IC50 (half maximal inhibitory concentration) represents the concentration of the test agent that is effective to cause half of the individual, i.e., half-inhibitory concentration of the test agent, indicating that a certain drug or substance (inhibitor) has reached half-inhibitory concentration in inhibiting viral infection. In the single fluorescence method, the infection degree of the host cells is expressed as an infection rate, which is expressed as the infection rate=the number of fluorescent cells (i.e., the number of infected cells)/the total number of cells; in the double fluorescence method, the infection rate can also be calculated by using the ratio of the number of double fluorescent cells (i.e., the number of infected cells) to the number of single fluorescent cells;
in addition, if a positive control group and a negative control group are provided, the infection degree of the host cell can also be expressed in terms of the binding inhibition ratio: the binding inhibition ratio = (experimental group average fluorescence intensity-negative control average fluorescence intensity)/(positive control average fluorescence intensity-negative control average fluorescence intensity).
Specifically, a fitting curve is constructed by taking the concentration of the object to be detected, the average fluorescence intensity of an experimental group, the number of infected cells, the infection rate and the binding inhibition rate as variables, so as to determine the virus neutralization capacity of the object to be detected. More specifically, IC50 values were determined using GraphPad Prism to fit a dose-dependent curve reflecting virus neutralization capacity based on a dose-effect relationship equation:
Y＝Bottom+(Top-Bottom)/(1+10^((Log IC50-X)*Hill Slope)
x represents the concentration of the object to be measured;
y represents average fluorescence intensity of an experimental group, cell infection number, infection rate and binding inhibition rate of an object to be detected under the concentration of X;
bottom represents the average fluorescence intensity/cell infection number/infection rate/binding inhibition rate of the negative control;
top represents the mean fluorescence intensity of positive control/number of cell infections/infection rate/binding inhibition rate;
neutralization titer IC50 represents the concentration of test agent that is effective in causing 50% of individuals;
hill Slope is a Slope, and is obtained by solving at least two groups of XY data.
In addition, the analysis processing module of the high-flux cell analysis system can output the dose-dependent curves of a plurality of objects to be detected on the same coordinate system to carry out high-flux and more visual comparison.
In summary, according to the method for testing the virus neutralizing antibody by high-throughput measurement, the fluorescent marked sample in the microplate is rapidly imaged and analyzed by the high-throughput image analysis equipment, and the method is simple in operation flow, visual and accurate in result and high in reliability.
The applicant declares that the above is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be apparent to those skilled in the art that any changes or substitutions that are easily conceivable within the technical scope of the present invention disclosed by the present invention fall within the scope of the present invention and the disclosure.
Claims (6)
1. A method for detecting a virus neutralizing antibody, comprising the steps of:
acquiring microscopic images of experimental group samples in an orifice plate, wherein the experimental group samples are obtained by mixing at least two concentrations of to-be-detected substances with virus particles respectively and then mixing the to-be-detected substances with host cells, wherein the virus particles carry fluorescent markers A or are combined with the markers carrying the fluorescent markers A, and microscopic imaging is carried out on the experimental group samples under the excitation of a bright field and/or fluorescence excitation light matched with the fluorescent markers A to obtain microscopic images of the experimental group samples;
analyzing microscopic images of the experimental group samples to obtain image analysis results of the experimental group samples;
obtaining the infection degree of the host cells and the virus neutralization capacity of the object to be tested based on the image analysis result of the experimental group sample;
the detection method further comprises the following steps:
obtaining microscopic images of a positive control group and microscopic images of a negative control group, wherein the samples in the positive control group are samples obtained by mixing positive sample host cells,
analyzing the microscopic images of the positive control group and the microscopic images of the negative control group to obtain image analysis results, wherein the image analysis results comprise any one or a combination of at least two of positive control average fluorescence intensity, negative control average fluorescence intensity, total number of cells and cell infection number;
the degree of infection of the host cell is expressed as the infection rate or binding inhibition rate;
the infection rate is calculated using the following formula:
infection rate = number of cell infections/total number of cells;
the binding inhibition rate is calculated using the following formula:
binding inhibition = (experimental group average fluorescence intensity-negative control average fluorescence intensity)/(positive control average fluorescence intensity-negative control average fluorescence intensity);
the detection method further comprises the steps of constructing a fitting curve to determine the virus neutralization capacity of the object to be detected;
the fitting method comprises the following steps: taking the concentration of the object to be detected, the average fluorescence intensity of an experimental group, the number of cell infections, the infection rate and the binding inhibition rate as variables to obtain a dose-dependent graph reflecting the virus neutralization capacity;
fitting based on a quantity-effect relation equation to obtain a dose-dependent curve reflecting virus neutralization capacity:
Y＝Bottom+(Top-Bottom)/(1+10^((Log IC50-X)*Hill Slope)
x represents the concentration of the object to be measured;
y represents average fluorescence intensity of an experimental group, cell infection number, infection rate and binding inhibition rate of an object to be detected under the concentration of X;
bottom represents the average fluorescence intensity/cell infection number/infection rate/binding inhibition rate of the negative control;
top represents the mean fluorescence intensity of positive control/number of cell infections/infection rate/binding inhibition rate;
neutralization titer IC50 represents the concentration of test agent that is effective in causing 50% of individuals;
hill Slope is the Slope, and is obtained by solving at least two groups of XY data;
host cells in the experimental group sample carry fluorescent markers B, the fluorescent markers A and the fluorescent markers B are matched with different fluorescence excitation lights, and the detection method further comprises the steps of obtaining microscopic images of the experimental group sample obtained after the fluorescence excitation lights matched with the fluorescent markers B are excited;
the detection method further comprises the following steps:
comparing the first microscopic image and the second microscopic image with respect to the first microscopic image respectively photographed under the fluorescent channel matched with the fluorescent mark A and the second microscopic image photographed under the fluorescent channel matched with the fluorescent mark B, and determining whether the position information of the cells in each pore plate in the first microscopic image and the second microscopic image is consistent according to the comparison result.
2. The method according to claim 1, wherein the image analysis result includes any one or a combination of at least two of an average fluorescence intensity of an experimental group, a total number of cells, or a number of infected cells.
3. A system for performing the method of detection of a viral antibody according to any one of claims 1-2, characterized in that the system comprises the following modules:
the microscopic imaging module is used for acquiring microscopic images of samples in the pore plate, wherein the samples are obtained by mixing and incubating an object to be detected and/or a standard sample with virus particles and then mixing the virus particles with host cells, the virus particles carry fluorescent markers A or are combined with the markers carrying the fluorescent markers A, and the samples are subjected to microscopic imaging under the excitation of a bright field and/or fluorescence excitation light matched with the fluorescent markers A to obtain microscopic images of the samples;
and the analysis processing module is used for analyzing the microscopic image of the sample to obtain an image analysis result of the sample and determining the virus neutralization capacity of the object to be detected based on the image analysis result of the sample.
4. A system according to claim 3, wherein the system further comprises:
the sample platform module is used for bearing the pore plate, the pore plate is provided with a plurality of sample holes and is used for bearing at least two types of objects to be detected, the objects to be detected of the same type in the plurality of sample holes at least comprise two concentrations, and the microscopic imaging module is used for respectively shooting samples to be detected in all holes on the pore plate to obtain microscopic images of all the samples to be detected.
5. The system of claim 4, wherein the system further comprises:
and the sample automatic replacement module is used for updating the pore plate, and the microscopic imaging module shoots the sample on the updated pore plate to obtain a microscopic image of the sample on the updated pore plate.
6. Use of a method according to any one of claims 1 to 2 or a system according to any one of claims 3 to 5 in the screening of virus antibody neutralizing drugs, mRNA, DNA vaccines, pharmaceutical adjuvants or chemically synthesized drugs.
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