Method for evaluating neutralizing antibody by using virus pseudovirus particles

Abstract

The invention provides a method for evaluating a neutralizing antibody by using virus pseudovirus particles, which comprises the following steps: respectively inoculating rabies virus, HIV (Human Immunodeficiency Virus) and novel coronavirus pseudovirus in different cell lines such as 293T, 293T-CD4, 293T-ACE2 and the like, carrying out gradient dilution test, calculating the PD50 value of the neutralizing antibody by combining with an improved Reed-Muench method, and finally quantitatively evaluating the inactivated virus amount and titer of the neutralizing antibody. According to the method, cell preparation, virus preparation, mixed incubation and detection, result analysis and other links are all optimized, for example, the natural death rate of cells is detected in advance, the non-specific influence is deducted, and the experiment accuracy and the data credibility are improved. Through parallel testing of multiple pseudoviruses, the safety and the detection efficiency are effectively improved, and the method is suitable for high-throughput screening and refined research and has innovativeness and practical value.

Description

Technical Field

[0001] The present invention relates to the technical field of methods for evaluating virus pseudovirion particles, and in particular to a method for evaluating neutralizing antibodies using virus pseudovirion particles. Background Art

[0002] Pseudovirus particles (PVPs) are widely used in virology research because they are highly similar in appearance to real viruses but do not contain pathogenic genetic material of the virus and are therefore non-infectious. Compared with live viruses, the use of pseudovirus particles can significantly reduce the biosafety risk of experiments and have the advantages of simple preparation, easy labeling, and flexible operation. Therefore, the neutralizing antibody evaluation method based on pseudovirus particles has become a safe and efficient alternative, especially in the research of new viruses and related vaccine development. It has significant application potential.

[0003] Neutralizing antibodies are a type of antibody that can prevent the virus from entering host cells by specifically binding to key proteins on the surface of the virus (such as spike proteins), thereby neutralizing the virus's ability to infect. They mainly inhibit the virus's infection and replication by binding to key proteins on the surface of the virus and blocking the interaction between the virus and the host cells.

[0004] The titer of neutralizing antibodies refers to the strength or ability of neutralizing antibodies to neutralize viral infection, that is, under certain conditions, the efficiency of neutralizing antibodies in preventing or inhibiting viral infection of host cells. The titer is usually determined by measuring the lowest effective concentration of antibodies to neutralize viruses in in vitro experiments and is expressed in the form of titers or neutralization indexes. A high titer means that the antibody can effectively neutralize the virus at a lower concentration, whereas a low titer requires a higher concentration of antibodies to achieve the same neutralizing effect. Titer testing is an important indicator for evaluating antibody quality and efficacy, and is widely used in vaccine development, antibody drug development, and clinical treatment evaluation.

[0005] The current methods for evaluating neutralizing antibodies using pseudovirus particles mainly include the preparation of pseudovirus particles, cell infection experiments, and the detection of reporter genes such as fluorescence or luciferase. Pseudovirus particles are generated by introducing the genes of key structural proteins (such as envelope proteins or spike proteins) of the target virus into host cells, and the host cells are infected with pseudovirus particles carrying reporter genes to observe the expression level of the reporter gene, thereby indirectly reflecting the ability of neutralizing antibodies to inhibit viral infection. However, these methods still have certain technical bottlenecks, such as insufficient stability of pseudovirus particles, insufficient detection sensitivity and specificity, and low standardization of test results.

[0006] Therefore, developing an efficient, stable, and sensitive neutralizing antibody evaluation method based on pseudovirus particles can not only improve the reliability and efficiency of the experiment, but also provide key technical support for vaccine development and antibody drug screening. This method requires technical breakthroughs in pseudovirus particle preparation, detection system optimization, and evaluation index standardization to ensure the accuracy, repeatability, and applicability of the test results, thereby promoting the widespread application and development of neutralizing antibody evaluation technology. Summary of the invention

[0007] In view of the above problems, the object of the present invention is to provide: a method for evaluating neutralizing antibodies using viral pseudovirions, comprising the following steps: S1. Cell preparation and inoculation: The cultured cells inoculated with three kinds of pseudoviruses were inoculated in 96-well plates, with about 1×10 3 cells and add 50 μL of DMEM medium containing 2% FBS; S2. Confirmation of cell status: After inoculation, the cells were cultured for 24 hours, and the cell morphology and growth status were observed under a microscope to ensure that the cells were full, evenly distributed, and free of contamination. The cells in the three wells of the blank control wells were digested with trypsin and counted, and the average value was taken. The result was 1×10 4 Pieces / hole; S3. Preparation of neutralizing antibodies and viruses; specifically including: S3.1. Preparation of neutralizing antibody gradients: dilute the neutralizing antibody into at least 4 gradients using DMEM medium containing 2% FBS, with 50 μL prepared for each gradient; S3.2. Virus preparation: Take the pseudovirus solution with calibrated activity and prepare it into a virus working solution with a concentration of 2 times the TCID50 value; S4, mixed incubation: Take 50 μL of the above virus working solution and mix it with equal amounts of each gradient of neutralizing antibody solution, and incubate at 37°C for 2 hours to allow the neutralizing antibody to fully bind to the virus; S5. Virus inoculation: 100 μL of the mixed solution after incubation was slowly added to the 96-well plates with each cell line, corresponding to different gradients; and culture was continued for 48 hours; S6. Result detection and recording: Observe and photograph the cells under an inverted fluorescence microscope; record each well based on whether the cell fluorescence marker CPE appears to determine the degree of virus infection in each well; S7. Calculation of the PD50 value of neutralizing antibodies: The PD50 value of neutralizing antibodies was calculated based on the improved Reed-Muench method.

[0008] S8. Neutralizing antibody titer expression: The obtained PD50 value is converted into the dilution factor of neutralizing antibody required to protect half of the cells in the culture well from pathology or death, and based on the virus titer or virus copy number corresponding to the TCID50 value, the actual amount of virus inactivated by the neutralizing antibody and its titer are quantitatively evaluated.

[0009] Furthermore, the cells inoculated with virus pseudovirus include 293T cells inoculated with rabies virus pseudovirus, 293T-CD4 cells inoculated with human immunodeficiency virus pseudovirus, and 293T-ACE2 cells inoculated with new coronavirus pseudovirus; at least 23 wells are inoculated for each cell type, of which 20 wells are used for subsequent inoculation of gradient diluted virus solution; and the remaining 3 wells are used as blank control wells for cell counting before virus inoculation.

[0010] Furthermore, in step S1, before inoculating the cells into a 96-well plate, 293T cells, 293T-CD4 cells and 293T-ACE2 cells were passaged for at least 2 generations respectively to make them stably sensitive to the respective inoculated viruses, and the culture medium containing 2% FBS was replaced within 48 hours before inoculation.

[0011] Furthermore, in step S3.1, the neutralizing antibodies include neutralizing antibodies against the novel coronavirus Spike Protein S1, neutralizing antibodies against HIV virus gp120, and neutralizing antibodies against rabies virus G protein.

[0012] Furthermore, in step S3.1, the four gradients of neutralizing antibody dilution were 500-fold, 1000-fold, 5000-fold, and 10000-fold, respectively.

[0013] Furthermore, before the mixed incubation in S4, both the neutralizing antibody solution and the virus solution are subjected to microfiltration or sterile filtration; wherein the pH of the neutralizing antibody solution is between 7.2 and 7.4, and the pH of the virus solution is controlled between 7.0 and 7.2 to reduce nonspecific protein aggregation. Furthermore, the improved Reed-Muench method in step S7 specifically includes: S7.1. Conduct a baseline test on the natural mortality rate of the same batch of cells without adding virus and antibody in advance; correct the natural mortality rate to obtain a corrected baseline mortality rate; S7.2, and correct the observed CPE rate by the following formula: in, To correct the cytopathic rate; is the measured cytopathic rate; To correct for baseline mortality; S7.3. After obtaining the corrected cytopathic effect rate, the Reed-Muench formula is used for conversion. According to the CPE rate of each well, the equilibrium distance P value between the two dilutions that lead to 50% CPE of the cells is found. S7.4. Add the logarithm of the dilution factor less than 50% CPE to the P value to obtain the titration endpoint; S7.5, the antilogarithm of the titration endpoint is the PD50 sample value, which is then multiplied by the initial dilution multiple to obtain the PD50 value of the neutralizing antibody. Further, the natural mortality rate is corrected in step S7.1 to obtain a corrected baseline mortality rate including: S7.1.1. Multi-point measurement: Record the total number of cells in the blank control wells under the same conditions. The number of dead cells , calculate the mortality rate ,satisfy: S7.1.2. Take the average of the mortality rates of all blank control wells to obtain the initial baseline mortality rate ,satisfy: Where n represents the number of blank control wells; S7.1.3. Calculate the standard deviation of the initial baseline mortality rate ,satisfy: S7.1.4. Determine the revised baseline mortality rate through confidence test: Based on the pre-specified confidence level Estimate the confidence interval to satisfy: in, To calculate the confidence interval; is the confidence interval threshold; represents the critical value of the two-sided test; To correct for baseline mortality; Furthermore, in step S7, pseudoviruses with fluorescent markers such as GFP or mCherry are used for detection by fluorescence microscopy or whole-well fluorescence scanning imaging system, and image analysis software is used to count the number of fluorescence-positive cells in each well, so as to more accurately distinguish between infection-positive and infection-negative cells.

[0014] The beneficial effects of the present invention are: 1. Improved safety: This method is based on virus pseudovirus particles (such as pseudoviruses of rabies virus, HIV virus, new coronavirus, etc.) for neutralizing antibody evaluation, which is safer than real viruses and avoids the potential risks of operating highly pathogenic or high-risk viruses under conventional laboratory conditions.

[0015] 2. Simultaneous detection of virus spectrum: By inoculating pseudoviruses against multiple viruses in different cell lines such as 293T, 293T-CD4, and 293T-ACE2, the activity of neutralizing antibodies against multiple targets can be evaluated in parallel in one experiment. Compared with traditional single virus determination, the experimental cycle is greatly shortened and the experimental efficiency is improved.

[0016] 3. Improved Reed-Muench correction: In step S7, the concept of "corrected baseline mortality" is introduced. The natural mortality rate of cells (D_baseline) is accurately determined through multi-point measurement, variance calculation and confidence interval estimation, and this part is deducted in the CPE rate calculation to eliminate the interference of non-specific cell death; this improved Reed-Muench method can more accurately determine the dilution factor that leads to 50% CPE, making the calculation of PD50 more scientific and reliable.

[0017] 4. Automated and visualized detection: Use an inverted fluorescence microscope or a full-well fluorescence scanning imaging system to observe the results, and combine with image analysis software to obtain the number of fluorescence-positive cells in each well; fluorescent labeling can significantly improve the discrimination of positive / negative cells, reduce subjective judgment errors, and facilitate later image archiving and re-analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a schematic diagram of the method flow of the present invention; Figure 2 This is the result of the Spike Protein S1 neutralizing antibody PD50 value test; Figure 3 This is the gp120 neutralizing antibody PD50 value detection result diagram; Figure 4 This is a graph showing the test results of the PD50 value of the G protein neutralizing antibody. DETAILED DESCRIPTION

[0019] In order to deepen the understanding of the present invention, the present invention will be further described in detail below in conjunction with examples. The examples are only used to explain the present invention and do not constitute a limitation on the protection scope of the present invention.

[0020] Embodiment 1

[0021] like Figure 1 As shown, this embodiment provides a method for evaluating neutralizing antibodies using viral pseudovirus particles, comprising the following steps: S1. Cell preparation and inoculation: The cultured cells inoculated with three kinds of pseudoviruses were inoculated in 96-well plates, with each well inoculated with approximately cells and add 50 μL of DMEM medium containing 2% FBS; S2. Confirmation of cell status: After the cells are inoculated, they are cultured for 24 hours, and the cell morphology and growth status are observed under a microscope to ensure that the cells are full, evenly distributed and free of contamination; the cells in the three wells of the blank control wells are digested with trypsin and counted, and the average value is taken. Pieces / hole; S3. Preparation of neutralizing antibodies and viruses; specifically including: S3.1. Preparation of neutralizing antibody gradients: dilute the neutralizing antibody into at least 4 gradients using DMEM medium containing 2% FBS, with 50 μL prepared for each gradient; S3.2. Virus preparation: Take the pseudovirus solution with calibrated activity and prepare it into a virus working solution with a concentration of 2 times the TCID50 value; S4, mixed incubation: Take 50 μL of the above virus working solution and mix it with equal amounts of each gradient of neutralizing antibody solution, and incubate at 37°C for 2 hours to allow the neutralizing antibody to fully bind to the virus; S5. Virus inoculation: 100 μL of the mixed solution after incubation was slowly added to the 96-well plates with each cell line, corresponding to different gradients; and culture was continued for 48 hours; S6. Result detection and recording: Observe and photograph the cells under an inverted fluorescence microscope; record each well based on whether the cell fluorescence marker CPE appears to determine the degree of virus infection in each well; S7. Calculation of the PD50 value of neutralizing antibodies: The PD50 value of neutralizing antibodies was calculated based on the improved Reed-Muench method.

[0022] S8. Neutralizing antibody titer expression: The obtained PD50 value is converted into the dilution factor of neutralizing antibody required to protect half of the cells in the culture well from pathology or death, and based on the virus titer or virus copy number corresponding to the TCID50 value, the actual amount of virus inactivated by the neutralizing antibody and its titer are quantitatively evaluated.

[0023] Furthermore, the cells inoculated with virus pseudovirus include 293T cells inoculated with rabies virus pseudovirus, 293T-CD4 cells inoculated with human immunodeficiency virus pseudovirus, and 293T-ACE2 cells inoculated with new coronavirus pseudovirus; at least 23 wells are inoculated for each cell type, of which 20 wells are used for subsequent inoculation of gradient diluted virus solution; and the remaining 3 wells are used as blank control wells for cell counting before virus inoculation.

[0024] Furthermore, in step S1, before inoculating the cells into a 96-well plate, 293T cells, 293T-CD4 cells and 293T-ACE2 cells were passaged for at least 2 generations respectively to make them stably sensitive to the respective inoculated viruses, and the culture medium containing 2% FBS was replaced within 48 hours before inoculation.

[0025] Furthermore, in step S3.1, the neutralizing antibodies include neutralizing antibodies against the novel coronavirus Spike Protein S1, neutralizing antibodies against HIV virus gp120, and neutralizing antibodies against rabies virus G protein.

[0026] Furthermore, in step S3.1, the four gradients of neutralizing antibody dilution were 500-fold, 1000-fold, 5000-fold, and 10000-fold, respectively.

[0027] Furthermore, before the S4 mixed incubation, both the neutralizing antibody solution and the virus solution are subjected to microfiltration or sterile filtration; wherein the pH of the neutralizing antibody solution is between 7.2 and 7.4, and the pH of the virus solution is controlled between 7.0 and 7.2 to reduce nonspecific protein aggregation.

[0028] Furthermore, the improved Reed-Muench method in step S7 specifically includes: S7.1. Conduct a baseline test on the natural mortality rate of the same batch of cells without adding virus and antibody in advance; correct the natural mortality rate to obtain a corrected baseline mortality rate; S7.2, and correct the observed CPE rate by the following formula: in, To correct the cytopathic rate; is the measured cytopathic rate; To correct for baseline mortality; S7.3. After obtaining the corrected cytopathic effect rate, the Reed-Muench formula is used for conversion. According to the CPE rate of each well, the equilibrium distance P value between the two dilutions that lead to 50% CPE of the cells is found. S7.4. Add the logarithm of the dilution factor less than 50% CPE to the P value to obtain the titration endpoint; S7.5. The antilogarithm of the titration endpoint is the PD50 sample value, which is then multiplied by the initial dilution factor to obtain the PD50 value of the neutralizing antibody. The test results are as follows: Figure 2-Figure 4 shown.

[0029] Furthermore, the corrected baseline mortality rate obtained by correcting the natural mortality rate in step S7.1 includes: S7.1.1. Multi-point measurement: Record the total number of cells in the blank control wells under the same conditions. The number of dead cells , calculate the mortality rate ,satisfy: S7.1.2. Take the average of the mortality rates of all blank control wells to obtain the initial baseline mortality rate ,satisfy: Where n represents the number of blank control wells; S7.1.3. Calculate the standard deviation of the initial baseline mortality rate ,satisfy: S7.1.4. Determine the revised baseline mortality rate through confidence test: Based on the pre-specified confidence level Estimate the confidence interval to satisfy: in, To calculate the confidence interval; is the confidence interval threshold; represents the critical value of the two-sided test; Corrected for baseline mortality.

[0030] Furthermore, in step S7, pseudoviruses with fluorescent markers such as GFP or mCherry are used for detection by fluorescence microscopy or whole-well fluorescence scanning imaging system, and image analysis software is used to count the number of fluorescence-positive cells in each well, so as to more accurately distinguish between infection-positive and infection-negative cells.

[0031] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for evaluating neutralizing antibodies using viral pseudovirions, characterized in that: The following steps are involved: S1. Cell preparation and inoculation: The cultured cells inoculated with three kinds of pseudoviruses were inoculated in 96-well plates, with each well inoculated with approximately cells and add 50 μL of DMEM medium containing 2% FBS; S2. Confirmation of cell status: After the cells are inoculated, they are cultured for 24 hours, and the cell morphology and growth status are observed under a microscope to ensure that the cells are full, evenly distributed and free of contamination; the cells in the three wells of the blank control wells are digested with trypsin and counted, and the average value is taken. Pieces / hole; S3. Preparation of neutralizing antibodies and viruses; specifically including: S3.

1. Preparation of neutralizing antibody gradients: dilute the neutralizing antibody into at least 4 gradients using DMEM medium containing 2% FBS, with 50 μL prepared for each gradient; S3.

2. Virus preparation: Take the pseudovirus solution with calibrated activity and prepare it into a virus working solution with a concentration of 2 times the TCID50 value; S4, mixed incubation: Take 50 μL of the above virus working solution and mix it with equal amounts of each gradient of neutralizing antibody solution, and incubate at 37°C for 2 hours to allow the neutralizing antibody to fully bind to the virus; S5. Virus inoculation: 100 μL of the mixed solution after incubation was slowly added to the 96-well plates with each cell line, corresponding to different gradients; and culture was continued for 48 hours; S6. Result detection and recording: Observe and photograph the cells under an inverted fluorescence microscope; record each well based on whether the cell fluorescence marker CPE appears to determine the degree of virus infection in each well; S7. Calculation of neutralizing antibody PD50 value: The PD50 value of neutralizing antibody was calculated based on the improved Reed-Muench method; S8. Neutralizing antibody titer expression: The obtained PD50 value is converted into the dilution factor of neutralizing antibody required to protect half of the cells in the culture well from pathology or death, and based on the virus titer or virus copy number corresponding to the TCID50 value, the actual amount of virus inactivated by the neutralizing antibody and its titer are quantitatively evaluated.

2. A method for evaluating neutralizing antibodies using viral pseudovirions according to claim 1, characterized in that: The cells inoculated with virus pseudoviruses include 293T cells inoculated with rabies virus pseudoviruses, 293T-CD4 cells inoculated with human immunodeficiency virus pseudoviruses, and 293T-ACE2 cells inoculated with new coronavirus pseudoviruses; at least 23 wells are inoculated for each cell type, of which 20 wells are used for subsequent inoculation of gradient diluted virus solutions; and the remaining 3 wells are used as blank control wells for cell counting before virus inoculation.

3. A method for evaluating neutralizing antibodies using viral pseudovirus particles according to claim 2, characterized in that: In step S1, before inoculating the cells into 96-well plates, 293T cells, 293T-CD4 cells and 293T-ACE2 cells were passaged for at least 2 generations respectively to make them stably sensitive to the viruses they were inoculated with, and the culture medium containing 2% FBS was replaced within 48 hours before inoculation.

4. The method for evaluating neutralizing antibodies using viral pseudovirus particles according to claim 2, characterized in that: In step S3.1, the neutralizing antibodies include neutralizing antibodies against the novel coronavirus Spike Protein S1, neutralizing antibodies against HIV virus gp120, and neutralizing antibodies against rabies virus G protein.

5. The method for evaluating neutralizing antibodies using viral pseudovirions according to claim 1, characterized in that: In step S3.1, the four dilution gradients of neutralizing antibodies were 500-fold, 1000-fold, 5000-fold, and 10000-fold, respectively.

6. The method for evaluating neutralizing antibodies using viral pseudovirus particles according to claim 1, characterized in that: Before the S4 mixed incubation, both the neutralizing antibody solution and the virus solution are subjected to microfiltration or sterile filtration treatment; wherein the pH of the neutralizing antibody solution is between 7.2 and 7.4, and the pH of the virus solution is controlled between 7.0 and 7.2 to reduce nonspecific protein aggregation.

7. The method for evaluating neutralizing antibodies using viral pseudovirions according to claim 1, characterized in that: The improved Reed-Muench method in step S7 specifically includes: S7.

1. Conduct a baseline test on the natural mortality rate of the same batch of cells without adding virus and antibody in advance; correct the natural mortality rate to obtain a corrected baseline mortality rate; S7.2, and correct the observed CPE rate by the following formula: in, To correct the cytopathic rate; is the measured cytopathic rate; To correct for baseline mortality; S7.

3. After obtaining the corrected cytopathic effect rate, the Reed-Muench formula is used for conversion. According to the CPE rate of each well, the equilibrium distance P value between the two dilutions that lead to 50% CPE of the cells is found. S7.

4. Add the logarithm of the dilution factor less than 50% CPE to the P value to obtain the titration endpoint; S7.

5. The antilogarithm of the titration endpoint is the PD50 sample value, which is then multiplied by the initial dilution factor to obtain the PD50 value of the neutralizing antibody.

8. The method for evaluating neutralizing antibodies using viral pseudovirions according to claim 1, characterized in that: In step S7.1, the baseline mortality rate corrected for the natural mortality rate is obtained by: S7.1.

1. Multi-point measurement: Record the total number of cells in the blank control wells under the same conditions. The number of dead cells , calculate the mortality rate ,satisfy: S7.1.

2. Take the average of the mortality rates of all blank control wells to obtain the initial baseline mortality rate ,satisfy: Where n represents the number of blank control wells; S7.1.

3. Calculate the standard deviation of the initial baseline mortality rate ,satisfy: S7.1.

4. Determine the revised baseline mortality rate through confidence test: Based on the pre-specified confidence level Estimate the confidence interval to satisfy: in, To calculate the confidence interval; is the confidence interval threshold; represents the critical value of the two-sided test; Corrected for baseline mortality.

9. The method for evaluating neutralizing antibodies using viral pseudovirions according to claim 1, characterized in that: In step S7, pseudoviruses with fluorescent markers such as GFP or mCherry are used for detection by fluorescence microscopy or whole-well fluorescence scanning imaging system, and image analysis software is used to count the number of fluorescence-positive cells in each well, so as to more accurately distinguish infection-positive and infection-negative cells.

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