A method for detecting the antigen content of a novel coronavirus nanoparticle recombinant protein vaccine and application thereof

Abstract

The application belongs to the technical field of biological detection, and particularly relates to a novel coronavirus nanoparticle recombinant protein vaccine antigen content detection method and application thereof. In order to solve the problems of qualitative and quantitative determination of novel coronavirus nanoparticle vaccine antigens, in-vitro titer evaluation and adjuvant adsorption efficiency determination, the application establishes a new detection method. According to the physical characteristics of protein nanoparticle macromolecules, two antibodies are set for different proteins of VLP nanoparticles, i.e. a rabbit anti Anti-HPF antibody designed for VLP ferritin inner core and a HRP-labeled rabbit anti Anti-RBD antibody recognizing the VLP surface coupled RBD antigen, so as to accurately detect the antigen content of the nanoparticle recombinant protein vaccine. The detection method has the advantages of convenient operation, low cost, low laboratory requirement, high safety, etc. The method can be implemented in ordinary laboratories and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a method for detecting the antigen content of a novel coronavirus nanoparticle recombinant protein vaccine and its application. Background Technology

[0002] The novel coronavirus (SARS-CoV-2) is a highly contagious and insidious virus, clinically manifesting as viral pneumonia / lung infection. People of all ages can be infected with the novel coronavirus, but adults are the most susceptible, with the elderly and those with weakened immune systems being particularly vulnerable, making prevention and control challenging. Therefore, the research and development and dissemination of a novel coronavirus vaccine are of paramount importance.

[0003] According to incomplete statistics, there are currently more than 200 research and development projects on novel coronavirus vaccines being carried out globally. Among them, virus-like particles (VLPs) are nanoparticles assembled from multiple protein subunits, which are expected to be developed into various vaccine antigens, adjuvants or drug carriers, and have become one of the hot topics in the field of biopharmaceutical engineering in recent years. Studies have shown that by utilizing the surface of the Helicobacter pylori nonheme ferritin (H. pylori feritin, HPF) backbone, through the autocatalytic binding of histidine (Lys) and aspartic acid (Asp) to form heteropeptide bonds, and equipping it with the receptor-binding domain (RBD) from the Spike protein of SARS-CoV-2, nanoparticles (NPs) based on RBD subunits can be generated to produce vaccines. This allows for the simultaneous display of 24 identical or different novel coronavirus antigens on the surface of a single nanovaccine. [Ma, X. et al. Nanoparticle Vaccines Based on the Receptor Binding Domain (RBD) and Heptad Repeat (HR) of SARS-CoV-2 Elicit Robust Protective Immune Responses. Immunity 53, 1315-1330.e1319 (2020). Zhang, X. et al. Improvement of a SARS-CoV-2 vaccine by enhancing the conjugation efficiency of the immunogen to self-assembled nanoparticles. Cellular & Molecular Immunology 18,] [2042-2044 (2021).] This means that the RBD protein of different mutant strains of the novel coronavirus can be displayed on the surface of nanoparticle vaccines.Meanwhile, studies in three animal models (BALB / c mice, hACE2 mice, and rhesus monkeys) revealed that the nanoparticle vaccine significantly induced the production of high-titer neutralizing antibodies and demonstrated cross-protection against different strains of the novel coronavirus [Yuan, Y. et al. A bivalent nanoparticle vaccine exhibits potent cross-protection against the variants of SARS-CoV-2. Cell reports 38, 110256 (2022). Chen, R. et al. Development of Receptor Binding Domain (RBD)-Conjugated Nanoparticle Vaccines with Broad Neutralization against SARS-CoV-2 Delta and Other Variants. Advanced science (Weinheim, Baden-Wurttemberg, Germany) 9, e2105378 (2022).]. In terms of safety, the nanoparticle vaccine also exhibits relatively high safety.

[0004] However, there is currently no method for detecting antigen content in RBD-based NPs vaccines, making their development challenging. Therefore, given the self-assembly characteristics of novel coronavirus nanoparticle vaccines (with an internal core of HPF self-assembled nanoparticles and RBD loading on the surface), corresponding detection methods need to be developed to address issues such as qualitative and quantitative analysis of nanoparticle antigens, in vitro potency assessment, and adjuvant adsorption efficiency determination, thereby reducing the difficulty of novel coronavirus vaccine development. Summary of the Invention

[0005] To overcome the shortcomings of the existing technology, this invention establishes a new detection method for the detection of recombinant protein vaccine antigens from novel coronavirus nanoparticles. This detection method is convenient to operate, low in cost, has low laboratory requirements, high safety, and can be implemented in ordinary laboratories, showing good application prospects.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] The first aspect of this invention provides a method for detecting the antigen content of a novel coronavirus nanoparticle recombinant protein vaccine. Specifically, the method involves using an ELISA double-antibody sandwich method, pre-coating a rabbit anti-Anti-HPF antibody against the core of a novel coronavirus nanoparticle recombinant protein vaccine (HPF-RBD type VLP vaccine), then adding the novel coronavirus nanoparticle recombinant protein vaccine to be tested, a standard, and an HRP-labeled rabbit anti-Anti-RBD antibody that recognizes the antigen conjugated on the surface of the novel coronavirus nanoparticle recombinant protein vaccine. After incubation and color development, the OD value at a wavelength of 450 nm is read, and a standard curve is plotted based on the standard. Finally, the antigen content in the vaccine is calculated based on the standard curve.

[0008] This invention establishes a novel detection method based on the physical properties of protein nanoparticles (HPF-RBD type VLP vaccines). Two types of antibodies are designed to target different proteins that make up VLP nanoparticles: a rabbit anti-Anti-HPF antibody targeting the VLP ferritin core and an HRP-labeled rabbit anti-Anti-RBD antibody recognizing the RBD antigen conjugated on the VLP surface. This allows for the accurate detection of the antigen content in the recombinant protein nanoparticle vaccine. This detection method has the advantages of convenient operation, low cost, low laboratory requirements, and high safety. It can be implemented in ordinary laboratories and has good application prospects.

[0009] Preferably, the novel coronavirus nanoparticle recombinant protein vaccine to be tested includes vaccine stock solution and semi-finished or finished formulation prepared by adding adjuvants.

[0010] Preferably, the method for pre-coating rabbit anti-Anti-HPF antibody is as follows: the rabbit anti-Anti-HPF antibody is diluted with coating buffer and added to a high-adsorption 96-well plate, incubated, and then blocked with 5% skim milk blocking solution.

[0011] Preferably, the concentration range of the added standard is 25-500 ng / mL.

[0012] Preferably, in the novel coronavirus nanoparticle recombinant protein vaccine to be tested, the limit of quantification of the antigen is 80.786 ng / mL.

[0013] The second aspect of this invention provides the application of the detection method described in the first aspect in the detection of antigen content and in vitro efficacy of novel coronavirus nanoparticle recombinant protein vaccines.

[0014] It should be noted that the antigen content detection method of the present invention may also be applicable to enzyme immunoassay, immunofluorescence assay, indirect hemagglutination assay, radioimmunoassay, etc.

[0015] Compared with the prior art, the beneficial effects of the present invention are:

[0016] This invention establishes a novel detection method for the antigen of recombinant protein vaccine made from nanoparticles for the novel coronavirus. To prevent the steric hindrance of VLP nanoparticles from affecting the binding of surface antibodies and to avoid interference between antibodies against the same antigen, this invention, based on the physical properties of the protein nanoparticle macromolecules, sets up two types of antibodies targeting different proteins that make up the VLP nanoparticles. Specifically, a rabbit anti-Anti-HPF antibody is designed targeting the VLP ferritin core, and a detection antibody is designed to recognize the RBD antigen coupled to the VLP surface. This allows for the accurate detection of the antigen content of the recombinant protein vaccine made from nanoparticles. This detection method has the advantages of convenient operation, low cost, low laboratory requirements, and high safety. It can be implemented in ordinary laboratories and has good application prospects. Attached Figure Description

[0017] Figure 1 This represents the results of a standard curve reliability test. Detailed Implementation

[0018] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0019] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0020] Example 1: A method for detecting the antigen content of a novel coronavirus nanoparticle recombinant protein vaccine.

[0021] Detection Principle: For HPF-RBD type VLP vaccines, this method uses a modified one-step double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) (i.e., ELISA double-antibody sandwich method) to determine antigen content. Due to the large molecular size and steric hindrance of virus-like nanoparticles (VLPs), conventional double-antibody sandwich ELISA detection can produce deviations in quantification. Therefore, considering the physical characteristics of protein VLPs and to prevent interference between antibodies targeting the same antigen, rabbit anti-Anti-HPF antibodies targeting the VLP core (internal core is HPF self-assembled nanoparticles with RBD loaded on the surface) are pre-coated. Then, sample / standard / control and HRP-labeled rabbit anti-Anti-RBD antibody recognizing the antigen conjugated on the VLP surface are added sequentially. Because the detection antibody is conjugated to HRP molecules, the colorimetric operation can be performed directly without incubating the second antibody, significantly reducing the operation time. Furthermore, the pre-coating with rabbit anti-Anti-HPF antibody avoids cross-reaction between antibodies. Simultaneously, the substrate TMB is used for color development. TMB is converted to blue under the catalysis of peroxidase, and then to the final yellow under the action of acid. The intensity of the color is linearly positively correlated with the content of RBD protein in the original sample solution. The concentration of the sample can be accurately calculated by measuring the absorbance (OD value) at a wavelength of 450 nm using an ELISA reader.

[0022] 1. Preparation before the experiment

[0023] (1) Information on reagents and consumables

[0024] As shown in the table below:

[0025]

[0026] (2) Preparation of relevant solutions

[0027] As shown in the table below:

[0028]

[0029]

[0030] (3) Pre-coating of rabbit anti-Anti-HPF antibody

[0031] 1) Wrapping board

[0032] Dilute the Rabbit-Anti-SD-HPF antibody to 2.5 μg / mL with Coating buffer, add 50 μL to each well of a high-adsorption 96-well plate, seal the reaction wells with sealing film, and incubate at room temperature for 4 h or at 4°C overnight.

[0033] 2) Closed

[0034] Prepare a 5% skim milk blocking solution using washing buffer. Add 50 μL to each well of a high-adsorption 96-well plate. Seal the reaction wells with a sealing membrane and incubate at room temperature for 1-2 hours or at 4°C for 10-12 hours.

[0035] 2. Detection of antigen content in RBD-ferritin nanoparticle stock solution:

[0036] (1) Standard dilution: RBD-ferritin nanoparticle stock solution standard was diluted with PBS to 500, 300, 200, 150, 100 and 50 ng / mL;

[0037] (2) Washing the plate: Take out the plate pre-coated with antibody, discard the liquid, add 300 μL of washing buffer to each well, gently tap and shake to remove the washing buffer, repeat this washing process 3 times, and pat dry on absorbent paper for the last time;

[0038] (3) Sample incubation: Add vaccine sample (novel coronavirus HPF-RBD nanoparticle vaccine stock solution SS220502A, 500 times dilution) and diluted standard to each well at a rate of 100 μL / well, set up two replicates for each well, and add two blank control wells (PBS dilution) at the same time. Incubate at 37°C for 1 h.

[0039] The information on the vaccine stock solution is shown in the table below:

[0040]

[0041]

[0042] (4) Washing the plate: Take out the high adsorption 96-well plate, discard the liquid, add 300 μL of washing buffer to each well, gently tap and shake to remove the washing liquid, repeat this washing process 3 times, and pat dry on absorbent paper for the last time;

[0043] (5) Antibody incubation: Add 100 μL of Anti-RBD-HRP antibody diluted with PBS (1:3000 dilution) to each well and incubate at 37°C for 1 h;

[0044] (6) Washing the plate: Take out the high adsorption 96-well plate, discard the liquid, add 300 μL of washing buffer to each well, gently tap and shake to remove the washing liquid, repeat this washing process 3 times, and pat dry on absorbent paper for the last time;

[0045] (7) Color development: Add 50 μL of TMB substrate to each well and incubate at room temperature in the dark for 12 min;

[0046] (8) Termination and reading: Add 50 μL of ELISA Stop Solution to each well and measure the OD value of each well at a wavelength of 450 nm;

[0047] (9) Calculation: In the Excel worksheet, linear regression analysis is used. The standard curve is plotted with the concentration value of the standard on the X-axis and its OD value on the Y-axis. The linear equation is obtained as: Y = bX + a. Then, a, b, and R are obtained. 2 Value. Where b is the slope, a is the y-intercept, and R is the value. 2 The coefficient of variation (CV) is the determination coefficient for the goodness of fit. The coefficient of variation (CV) is calculated as: (Standard deviation of detected values ​​ / Mean) × 100%. Where R0... 2 The trend line's estimated value reflects the degree of fit between the estimated value and the corresponding actual data; the higher the degree of fit, the more reliable the trend line. The coefficient of variation (CV) reflects the degree of deviation of each sample's data from the mean. Finally, the concentration value of each sample is calculated according to the curve equation, and then multiplied by the dilution factor to obtain the actual concentration of the sample.

[0048] The test results of the stock solution standard are shown in Table 1. 2 =0.9925, indicating high reliability of the test results. The test results for vaccine stock solution SS220502A, at a 500-fold dilution, are shown in Table 2. After 500-fold dilution, the antigen content of the vaccine was 126 ng / mL, while the antigen concentration in the vaccine stock solution was 63 μg / mL. There was no difference in antigen content, and the results were not significantly different from those obtained with BCA, indicating that the test results are accurate and reliable.

[0049] Table 1. Detection results of vaccine stock solution SS220502A

[0050]

[0051]

[0052] Note: 450OD-1 is the OD value of the first hole, 450OD-2 is the OD value of the second duplicate hole, and 450OD mean is the average OD value of the two duplicate holes.

[0053] Table 2. Detection results of vaccine stock solution SS220502A and its 500-fold dilution

[0054]

[0055] Note: 450OD-1 is the OD value of the first hole, 450OD-2 is the OD value of the second duplicate hole, and 450OD mean is the average OD value of the two duplicate holes.

[0056] Example 2: Feasibility Verification of a Method for Detecting Antigen Content in Novel Coronavirus Nanoparticle Recombinant Protein Vaccine

[0057] (1) Reliability test:

[0058] Following the detection method of Example 1, within a linear range of 25-500 ng / mL, each concentration point (0, 50, 100, 150, 200, 300, 500 ng / mL) was measured three times. When R... 2 A value ≥0.98, and the backcalculated concentration at each concentration point is within ±20% of the theoretical value, indicates that the standard curve reliability test has been passed.

[0059] The experimental results are shown in Table 3 and Figure 1 As shown, with the concentration value of the standard substance X and its OD value Y, when the standard curve R... 2 When the coefficient of performance (CV) is 0.9931, the CV at each concentration point is ≤20%, and the relative deviation is ≤20%, indicating that the plotted standard curve is reliable.

[0060] Table 3. Reliability test results of the standard curve

[0061]

[0062] (2) Accuracy test:

[0063] According to the detection method in Example 1, 100 μL of each of the six vaccine stock solutions (Shenzhen Regenerative City Biomedical Technology Co., Ltd., batch number: 20220802) of the same concentration were taken as test samples, and 10 μL of stock solution standard (500 ng / mL) was added to each as a recovery sample group. Each sample was measured three times.

[0064] The experimental results are shown in Tables 4 and 5. When the standard curve R... 2 When the coefficient of performance (COP) is 0.982, the sample recovery rate is within the range of 80% to 120%, and the CV is ≤20%, indicating that the recovery rate is good and meets the detection requirements.

[0065] Table 4 Test results of sample groups

[0066]

[0067] Table 5 Test results of the recovered sample group

[0068]

[0069] (3) Repeatability test:

[0070] Following the detection method of Example 1, high, medium, and low concentrations (50, 150, and 300 ng / mL) were measured 10 times within the linear range, and the CV was calculated for each concentration point.

[0071] The experimental results are shown in Table 6. When the standard curve R2 When the coefficient of variation (CV) is 0.9978, the sample was measured 10 times, and the CV was ≤20%, indicating that the repeatability was good and met the detection requirements.

[0072] Table 6. Results of Repeatability Tests on Test Samples

[0073]

[0074] (4) Specificity test: Following the detection method in Example 1, 100 μL of the original standard solution (300 ng / mL) and negative matrix (PBS) were taken, and 4 μL of HPF interference protein (300 ng / mL) was added to each. Each sample was measured three times. The information on HPF interference protein is shown in the table below:

[0075]

[0076]

[0077] The experimental results are shown in Table 7. When the standard curve R 2 When the concentration was 0.9939, the sample matrix and its interference groups were lower than those of the standard (300 ng / mL), and the recovery rate of the standard was 80-120%, indicating that it had good specificity and met the detection requirements.

[0078] Table 7 Specificity Test Results

[0079]

[0080] (5) Limit of Quantitation Test: The limit of quantitation refers to the lowest amount of analyte that can be quantitatively determined in the test. A commonly used method is based on the standard deviation of the response value and the slope of the standard curve, calculated using the formula LOQ = 10σ / S. Where LOQ is the limit of quantitation; σ is the deviation of the response value; and S is the slope of the standard curve. σ can be obtained by measuring the standard deviation of the blank value. Based on the above principle, following the detection method of Example 1, the blank sample (0 ng / mL) was measured 20 times, and the standard relative deviation was calculated.

[0081] The experimental results are shown in Table 8, indicating that when the standard curve R... 2 When the value is 0.9818, the limit of quantitation of the detection method described in Example 1 is 80.786 ng / mL.

[0082] Table 8 Results of Limit of Quantitation Test

[0083]

[0084] In summary, this invention establishes a novel detection method for the detection of recombinant protein vaccine antigens from novel coronavirus nanoparticles. This method is convenient to operate, low in cost, requires minimal laboratory expertise, and is highly safe; it can be implemented in ordinary laboratories.

[0085] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. A method for detecting the antigen content of a novel coronavirus nanoparticle recombinant protein vaccine, characterized in that, The ELISA double-antibody sandwich method was used. Rabbit anti-Anti-HPF antibody targeting the core of the novel coronavirus nanoparticle recombinant protein vaccine was pre-coated. Then, the novel coronavirus nanoparticle recombinant protein vaccine to be tested, the standard, and the HRP-labeled rabbit anti-Anti-RBD antibody that recognizes the RBD antigen conjugated on the surface of the novel coronavirus nanoparticle recombinant protein vaccine were added. After incubation and color development, the OD value at 450 nm wavelength of the ELISA reader was read. A standard curve was plotted based on the standard. Finally, the antigen content in the vaccine was calculated based on the standard curve. The novel coronavirus nanoparticle recombinant protein vaccine is the novel coronavirus HPF-RBD nanoparticle vaccine.

2. The method for detecting the antigen content of a novel coronavirus nanoparticle recombinant protein vaccine according to claim 1, characterized in that, The novel coronavirus nanoparticle recombinant protein vaccine to be tested includes vaccine stock solution and semi-finished or finished products made by adding adjuvants.

3. The method for detecting the antigen content of a novel coronavirus nanoparticle recombinant protein vaccine according to claim 1, characterized in that, The method for pre-coating rabbit anti-Anti-HPF antibody is as follows: the rabbit anti-Anti-HPF antibody is diluted with coating buffer and added to a high-adsorption 96-well plate, incubated, and then blocked with 5% skim milk blocking solution.

4. The method for detecting the antigen content of a novel coronavirus nanoparticle recombinant protein vaccine according to claim 1, characterized in that, The concentration range for adding the standard is 25-500 ng / mL.

5. The method for detecting the antigen content of a novel coronavirus nanoparticle recombinant protein vaccine according to claim 1, characterized in that, In the novel coronavirus nanoparticle recombinant protein vaccine to be tested, the limit of quantification of the antigen is 80.786 ng / mL.

6. The application of the detection method according to any one of claims 1-5 in the detection of antigen content and in vitro efficacy of novel coronavirus nanoparticle recombinant protein vaccines.

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