CN115219726B - Method for detecting serum neutralizing antibody titer by SARS-CoV-2 pseudovirus - Google Patents
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Abstract
The invention belongs to a detection method of new coronavirus antibody titer, in particular to a method for detecting neutralizing antibody titer in serum by using SARS-CoV-2 pseudovirus, comprising the following steps: 1) Setting a blank control group, a virus control group and a sample group on the detection plate, and setting parallel holes; 2) Culturing the blank control group and the virus control group and the sample group, and then respectively carrying out luminescence detection to obtain the average value of the respective luminescence intensities; 3) Calculating the inhibition rate of the neutralizing antibody; 4) Calculating the neutralizing antibody titer by adopting statistical software according to the neutralizing antibody inhibition rate calculated in the last step; compared with the prior art, the method provided by the invention overcomes the defect that the quantitative detection of the neutralizing antibody titer in serum at the cell level cannot be realized in the existing method, has high sensitivity, can be used for experimental animals of different species, and has higher practical value in the safety and effectiveness evaluation of new crown vaccines.
Description
Technical Field
The invention belongs to a method for detecting the antibody titer of a novel coronavirus, in particular to a method for detecting the neutralizing antibody titer of serum by SARS-CoV-2 pseudovirus.
Background
The development of related researches is greatly limited by the new higher protection standard, and a pseudo virus neutralizing antibody detection method (PBNAs) is used as an alternative and conceptual verification detection means, so that the method has higher practical value in the safety and effectiveness evaluation of vaccines.
SARS-CoV-2 pseudoviruses generally use replication-defective HIV viruses as a backbone or replication-defective VSV-G viruses as a backbone, and a capsid coats the surface Spike glycoprotein (spiken) of SARS-CoV-2, which binds to the human angiotensin converting enzyme2 (ACE 2) Receptor glycoprotein complex through a Spike protein Receptor Binding Domain (RBD), infecting cells overexpressing human ACE2, and simulating viral invasion processes. Since pseudoviruses can only be infected once and cannot be replicated, they can be handled and used in biosafety class 2 (BSL-2) laboratories.
The SARS-CoV-2 pseudovirus disclosed by Beijing Vetong biological technology Co., ltd takes HIV virus as a framework, and is coated with Spike protein of SARS-CoV-2, so that cells which over express human ACE2 can be infected, and simultaneously, the cells are provided with FireflyLuc and EGFP marks, and the infection efficiency is detected through FireflyLuc and EGFP. However, the pseudovirus in this technique is only used for detecting whether a cell line (recommended to infect hACE2-Cos7 cells) is infected with the pseudovirus, and is not a detection method, and cannot be used for characterizing the activity of neutralizing antibodies in serum after vaccine, and quantitative detection of the titer of neutralizing antibodies in serum at the cell level cannot be achieved.
Disclosure of Invention
In order to solve the technical problems, the invention discloses a method for detecting the titer of neutralizing antibodies in serum by SARS-CoV-2 pseudovirus.
The technical scheme of the invention is as follows:
a method for detecting neutralizing antibody titer in serum by SARS-CoV-2 pseudovirus, comprising the steps of:
1) Setting a blank control group, a virus control group and a sample group on the detection plate, and setting parallel holes;
2) Culturing the blank control group, the virus control group and the sample group, and respectively performing fluorescence detection to obtain a luminous intensity average value of the blank control group, a luminous intensity average value of the virus control group and a luminous intensity average value of the sample group;
3) Calculating the inhibition rate of the neutralizing antibody = [1- (the average value of the luminous intensity of the sample group-the average value of the luminous intensity of the blank group)/(the average value of the luminous intensity of the virus control group-the average value of the luminous intensity of the blank group) ]x100%;
4) Inputting the neutralizing antibody inhibition rate calculated in the previous step into statistical software for nonlinear fitting, and calculating the neutralizing antibody titer;
the blank control contains hACE2-Cos7 cells;
the virus control contains SARS-CoV-2 pseudovirus and hACE2-Cos7 cells;
the sample group contains SARS-CoV-2 pseudovirus, hACE2-Cos7 cells and serum to be tested;
the luminous intensity average value is determined by the following steps:
31 Cell plating: inoculating 5000 hACE2-Cos7 cells per well in 96-well plate on day D1, and culturing overnight at 37deg.C;
32 Sample dilution: inactivating the serum to be tested at 56 ℃ for 30min on day D1, and standing overnight at 4 ℃ with 40 mu L of each serum; carrying out gradient dilution on serum to be tested in D2 days, adding 570 mu L of diluent into 30 mu L of serum, adding 600 mu L of diluent into the 1 st row of 24-hole plates, uniformly mixing the two rows of holes, adding 450 mu L of diluent into each hole of the five rows of holes, uniformly mixing the first row of holes, adding 150 mu L of diluent into the second row of holes, and sequentially diluting until the sixth row of holes is discarded to 150 mu L; preferably, the gradient dilution ratio is selected as follows: 1: 20. 1:80, 1:320, 1:1280, 1: 5120. 1:20480.
33 Neutralization detection of viral infection): d2, taking out SARS-CoV-2 pseudovirus from a refrigerator at the temperature of minus 80 ℃ to melt on ice, mixing the pseudovirus and diluted serum according to the infection complex (MOI) of the current virus to target cells in a sample group, mixing the pseudovirus with a complete culture medium in a virus control group, and incubating for 1h at the temperature of 37 ℃ to obtain a mixed solution; sucking original cell culture solution, and adding 100 μl of the prepared mixed solution into each well; 100 mu L of complete culture medium is added into the blank control group, and the blank control group is gently shaken; centrifuging at 4deg.C for 2 hr (centrifuging at the same time), discarding the culture medium containing pseudovirus, washing with PBS, adding 30 μl of 0.25% pancreatin into each well, digesting at 37deg.C for 5min, adding 100 μl complete culture medium, lightly blowing with a row gun for 4-5 times, re-suspending uniformly, and culturing at 37deg.C overnight;
34 Cell exchange fluid: d3, D4, changing the culture medium every day, and continuously culturing at 37 ℃;
35 Protein expression detection: and (5) carrying out fluorescence detection on D5 days, and calculating the average value of the luminous intensity.
Furthermore, in the method for detecting the titer of neutralizing antibodies in serum by using SARS-CoV-2 pseudovirus, the fluorescence detection in the step 35) is a Luciferase quantitative determination.
Further, the method for detecting the neutralizing antibody titer in serum by using the SARS-CoV-2 pseudovirus comprises the following steps:
1) Sample processing: PBS was washed twice, the supernatant was discarded, 40. Mu.L of cell lysate was added to each well, lysed on ice for 15min, intermittently blow-suspended during lysis to allow complete lysis, transferred to a 1.5mLEP tube, centrifuged at 10000g for 3min, and the supernatant was used for the assay.
2) The detection method comprises the following steps: 20. Mu.L of the sample was added to the assay plate, and 100. Mu.L of firefly luciferase assay reagent dissolved and equilibrated to room temperature was added and mixed well. Incubating for 5min at room temperature after shaking to stabilize the luminescence signal. The Luciferase signal value at a wavelength of 520nm was detected using a chemiluminescent detector.
Preferably, the chemiluminescent detection instrument model used: spectraMaxL.
Preferably, the following kit is used: bright-Lumi ™ firefly luciferase reporter gene detection kit, with the product number of RG051S; firefly luciferase reporter cell lysate, accession No. RG126S.
Further, the method for detecting the neutralizing antibody titer of serum by using the SARS-CoV-2 pseudovirus is obtained from Beijing Vetong Biotechnology Co., ltd., product No. VS-SC-0001.
Furthermore, in the method for detecting the neutralizing antibody titer of serum by using the SARS-CoV-2 pseudovirus, the SARS-CoV-2 pseudovirus takes HIV virus as a framework, and is coated with Spike protein of SARS-CoV-2, so that cells which over-express human ACE2 can be infected, and simultaneously, the cells are provided with FireflyLuc and EGFP marks, and the infection efficiency is detected by the FireflyLuc and EGFP.
Furthermore, in the method for detecting the neutralizing antibody titer of the serum by using the SARS-CoV-2 pseudovirus, the serum to be detected is from positive serum after being immunized by SARS-CoV-2 vaccine, and preferably, rat positive serum collected at different time points after three-needle intramuscular injection is selected.
Furthermore, the statistical software is log (agonist) vs. response-Variableslope in GraphPadprism.
Furthermore, the detection method is applied to the safety/effectiveness evaluation of the novel crown vaccine.
Comparison of the description before the optimization of the invention:
the original procedure for the use of pseudoviruses (extracted from the official Web View http:// www.vitalstar.com.cn/Index/lists/catid/342. Html) included, as a comparison with the procedure of the present invention, the following steps:
1. the first day, 96-well plates were seeded with 5000 hACE2-COS7 cells per well.
2. The next day, the medium in the plates was removed, fresh medium (100. Mu.L/well) and pseudovirus 2X 10 3 TU and Polybrene were mixed (5 ug/ml final concentration), added to the culture wells and centrifuged (1200 g x2h,4 ℃).
3. Centrifuging the infected cells, sucking up the medium, and washing once with PBS; mu.L of 0.25% pancreatin was added to each well, incubated at 37℃for 5min, 100. Mu.L of complete medium was added, and the culture was continued.
4. Culture was continued for 3 days after infection (medium change every day), and luc detection was performed, and eGFP fluorescence was also observed (medium change as the case may be). The invention has the following beneficial effects:
the protocol of the original pseudovirus instructions only shows that infection can be detected by FireflyLuc and eGFP, and the invention is based on the development of antibodies that can quantitatively detect serum of vaccinated animals. The method overcomes the defect that the quantitative detection of the neutralizing antibody titer in serum at the cell level cannot be realized in the existing method, has high sensitivity, can be used for experimental animals of different species, and has higher practical value in the safety and effectiveness evaluation of the novel crown vaccine.
Drawings
FIG. 1 shows the inhibition of neutralizing antibodies after serum gradient dilution of different dose groups of rats immunized with SARS-CoV-2 vaccine; and (3) injection: results have subtracted the blank background RLU, fitted by graphpadprism5.0 log (agonist) vs. response-Variableslope nonlinear; the ordinate is the inhibition rate of the neutralizing antibody, and the abscissa is the dilution multiple after Log conversion;
FIG. 2 shows the inhibition of neutralizing antibodies after serum gradient dilution at D28 after immunization of rats with SARS-CoV-2 vaccine in different dose groups;
FIG. 3 shows the inhibition of neutralizing antibodies after serum gradient dilution of D15 and D28 following immunization of high dose group 1 rats with SARS-CoV-2 vaccine;
FIG. 4 shows the inhibition of neutralizing antibodies after serum gradient dilution of D15 and D28 following immunization of high dose group No. 2 rats with SARS-CoV-2 vaccine;
FIG. 5 is a graph showing detection of EC by serum neutralizing antibodies after immunization of rats with SARS-CoV-2 vaccine 50 Results;
FIG. 6 shows the inhibition of neutralizing antibodies after serum gradient dilution of the Beijing Tiantan pseudovirus system, different species;
FIG. 7 shows the inhibition of neutralizing antibodies by Vildahl pseudovirus system after serum gradient dilution of different species.
Detailed Description
The method of the present invention will be further described with reference to examples, and the experimental methods without specific reference to the examples may be generally performed under conventional conditions, such as those described in molecular cloning Experimental guidelines written by J.Sambucus et al, or according to the manufacturer's recommendations. The present invention may be better understood and appreciated by those skilled in the art by reference to the examples. However, the method of implementing the present invention should not be limited to the specific method steps described in the embodiments of the present invention.
Example 1
Detection of neutralizing antibody titer in serum after immunization of rats with SARS-CoV-2 vaccine
1 detection results of pseudo-virus neutralizing antibodies
Three mice were given serum at different time points (D15 after immunization with vaccine 2, D28 days before end of recovery period) after three intramuscular injections (D1, D7, D15) of the SARS-CoV-2 vaccine, 6 samples were taken, the serum-derived rats were divided into 3 groups, 1-vehicle control group (1 sample of D28), 2-vaccine low dose group (1 sample of D28), 3-vaccine high dose group (4 samples of No. 1 and No. 2 rats at D15 and D28) by the SARS-CoV-2 pseudovirus neutralizing antibody detection method (pbnasseudovirus-basedneutracentionassays), 1: 20. 1:80, 1:320, 1:1280, 1: 5120. gradient dilution of 20480, and detection of neutralizing antibody level of SARS-CoV-2 in rat serum at different time points.
The inhibition curve is shown in figure 1, the inhibition rate of each dilution multiple of the solvent control group is between-6% and 6% at the time of D28, the inhibition rate of the serum of the low-dose group is 53% at the time of dilution 1280 times, the inhibition rate of the serum of two animals of the high-dose group is 84% and 96% respectively at the time of dilution 1280 times, and the result shows that the neutralizing antibody is positive, and the result is shown in figures 3 and 4; EC (EC) 50 As a result, as shown in FIG. 5, the vehicle control group failed to calculate EC at D28 50 Vaccine high dose group EC 50 Average value 3893.5, vaccine low dose group EC 50 1374.0. Both neutralizing antibody inhibition and neutralizing antibody titer results are time-dose dependent. The statistics are shown in tables 1-3 below and the individual data are shown in Table 4.
2. Experimental method
The same batch of assay plates was run with a blank (no virus, no serum, only cell culture medium) and virus control (virus, no serum), test serum groups (co-incubation of virus and post-immunization serum), and plates were run in the following table with 2 parallel wells per group except for the low dose group. Neutralizing antibody inhibition = 1-luminescence intensity of experimental group/luminescence intensity of virus control group.
The experimental groupings and loading were as shown in table 5 below:
note that: x represents that the component is not added to the cell culture well, and O represents that the component is added to the culture well.
The 96-well plate rows are shown in tables 6 and 7 below:
the specific experimental operation is as follows:
1: cell plating: d1 was seeded with 5000 hACE2-Cos7 cells per well in 96-well plates and incubated overnight at 37 ℃.
2: sample dilution: d1 serum to be tested was inactivated at 56 ℃ for 30min, left overnight at 4 ℃ (40 μl each), and 6 serum portions were serially diluted 1: 20. 1:80, 1:320, 1:1280, 1: 5120. gradient dilution was performed at a ratio of 1:20480, 30. Mu.L of serum was diluted with 570. Mu.L of the diluted solution, and the mixture was added to column 1 of a 24-well plate and mixed well at 600. Mu.L per well. After adding 450 mu L of diluent to each hole of the last five columns, a row gun uniformly mixes the first column, then adding 150 mu L of diluent to the second column, and sequentially diluting until the sixth column is discarded to 150 mu L.
3: neutralization detection of viral infection: d2 SARS-CoV-2 pseudovirus was thawed on ice from a-80 ℃ refrigerator and 2 μl of pseudovirus per well was recommended based on the MOI of the pseudoviraled cells of the batch, 2 μl of pseudovirus was mixed with 98 μl of diluted serum in the sample group, pseudovirus was mixed with complete medium in the virus control group, and incubated at 37 ℃ for 1h (formulation 1.1 times the volume used, i.e. 2.2 μl of pseudovirus was mixed with 107.8 μl of diluted serum). Sucking the original culture solution of the cells, adding 100 mu L of the prepared mixed solution into each hole, adding 100 mu L of complete culture medium into a blank control group, gently shaking, centrifugally infecting for 2 hours at 4 ℃, removing the culture medium containing pseudoviruses by 1200g, washing with PBS once, adding 30 mu L of 0.25% pancreatin into each hole, digesting for 5 minutes at 37 ℃, adding 100 mu L of complete culture medium, gently blowing for 4-5 times by a gun, re-suspending uniformly, and culturing overnight at 37 ℃.
4: cell exchange liquid: d3, D4 medium was changed daily and culture was continued at 37 ℃.
5: protein expression detection: d5 Luciferase assay was performed.
Quantitative determination of luciferases:
sample processing: PBS was washed twice, the supernatant was discarded, 40. Mu.L of cell lysate was added to each well, lysed on ice for 15min, intermittently blow-suspended during lysis to allow complete lysis, transferred to a 1.5mLEP tube, centrifuged at 10000g for 3min, and the supernatant was used for the assay.
The detection method comprises the following steps: 20. Mu.L of the sample was added to the assay plate, and 100. Mu.L of firefly luciferase assay reagent dissolved and equilibrated to room temperature was added and mixed well. Incubating for 5min at room temperature after shaking to stabilize the luminescence signal. The Luciferase signal value (instrument model: spectraMaxL) at a wavelength of 520nm was detected using a chemiluminescent detector.
The kit comprises: bright-Lumi ™ firefly luciferase reporter gene detection kit, with the product number of RG051S; firefly luciferase reporter cell lysate, accession No. RG126S.
Protective measures are as follows:
the virus used in this experiment was a replication-defective pseudovirus and was not harmful to humans and the environment.
The contacting of the virus-containing specimen is performed in a negative pressure environment (negative pressure laboratory, biosafety cabinet).
The virus-contacted specimens and consumables were sterilized by immersing them in 10% 84 sterilizing solution at high temperature and high pressure before being discarded. The suspected contaminated specimen or area (e.g., the surface of a centrifuge tube for storing the sample) is sterilized with medical alcohol.
Example 2
Compared with the detection method of pseudo virus neutralizing antibodies of other systems, positive antibodies are added:
the same 5 samples and positive antibodies were tested for comparison using our method and the method provided by Beijing Tiantan pharmaceutical biotechnology development company. While detecting the presence or absence of nonspecific binding of other species (rhesus) to pseudoviruses.
Sample information: blank serum of 2 rhesus monkeys, serum after 3 SD rats were immunized (3 rats were derived from adjuvant control group, vaccine low dose group, vaccine high dose group, respectively), positive antibody information: novel coronavirus neutralizing antibody, lot number: 20200615, (give) providing units: a sho derivative; the original concentration is 0.92mg/mL, and the dilution is 0.046mg/mL by 20 times.
1. Detection result of neutralizing antibody from Beijing Tiantan pharmaceutical biotechnology development company
The cells were recommended and pseudovirus was added at the optimal infectious titer (cat# 800, lot # 20200904, titer 95000TCID 50 Per mL, recommended dilution factor: 7) The negative background of 2 monkeys and 1 mouse infected with hACE2-Cos7 was nearly 0%, the titer of the neutralizing antibody in the serum of the low-dose rats was 3579, the titer of the neutralizing antibody in the serum of the high-dose rats was 6514, and the inhibition rate of the neutralizing antibody after gradient dilution of the positive antibody with the final concentration of 0.046mg/mL was 100%. The experiment CC, VC had three complex holes, and others had no complex holes. The results of the detection are shown in FIG. 6 and tables 8-9.
2. Vito-derived pseudovirus neutralizing antibody detection results
The addition was performed according to the recommended cell number and pseudovirus optimal infection titer (lot number: 20200904, titer: 1.6X10) 6 TU/mL, dilution fold: 38, about 2000TU per well), the negative background of infected hACE2-Cos7,2 monkeys and 1 mouse was nearly 0%, no nonspecific binding to pseudoviruses was seen, the titer of the serum neutralizing antibody in the low dose group was 1233, the titer of the serum neutralizing antibody in the high dose group was 1665, the detection results were dose-effect dependent, the inhibition rate of the neutralizing antibody after gradient dilution of the positive antibody at a final concentration of 0.046mg/mL was 100%, six multiplex wells were found in the experiment CC, VC, and the other no multiplex wells were found in the results shown in FIG. 7 and tables 10 and 11.
In conclusion, the detection method of the neutralizing antibody of the pseudovirus has comparability with the detection method of other systems, blank serum of the monkey and the rat is not combined in a non-specific way, the inhibition rate of the neutralizing antibody of the positive antibody is 100% in the 20-20480-fold dilution process, and the sensitivity is 0.045 mug/mL after the system verification.
The foregoing is only illustrative of the present invention and is not to be construed as limiting the scope of the invention, which is defined by the appended claims and their equivalents.
Claims (2)
1. A method for detecting neutralizing antibody titer in serum by SARS-CoV-2 pseudovirus, comprising the steps of:
1) Setting a blank control group, a virus control group and a sample group on the detection plate, and setting parallel holes;
2) Culturing the blank control group, the virus control group and the sample group, and respectively performing fluorescence detection to obtain a luminous intensity average value of the blank control group, a luminous intensity average value of the virus control group and a luminous intensity average value of the sample group;
3) Calculating the inhibition rate of the neutralizing antibody = [1- (the average value of the luminous intensity of the sample group-the average value of the luminous intensity of the blank group)/(the average value of the luminous intensity of the virus control group-the average value of the luminous intensity of the blank group) ]x100%;
4) Inputting the neutralizing antibody inhibition rate calculated in the previous step into statistical software for nonlinear fitting, and calculating the neutralizing antibody titer;
the blank group contains hACE2-Cos7 cells;
the virus control group contains SARS-CoV-2 pseudovirus and hACE2-Cos7 cells;
the sample group contains SARS-CoV-2 pseudovirus, hACE2-Cos7 cells and serum to be tested;
the luminous intensity average value is determined by the following steps:
31 Cell plating: inoculating 5000 hACE2-Cos7 cells per well in 96-well plate on day D1, and culturing overnight at 37deg.C;
32 Sample dilution: inactivating the serum to be tested at 56 ℃ for 30min on day D1, and standing overnight at 4 ℃ with 40 mu L of each serum; carrying out gradient dilution on serum to be tested in D2 days, adding 570 mu L of diluent into 30 mu L of serum, adding 600 mu L of diluent into the 1 st row of 24-hole plates, uniformly mixing the two rows of holes, adding 450 mu L of diluent into each hole of the five rows of holes, uniformly mixing the first row of holes, adding 150 mu L of diluent into the second row of holes, and sequentially diluting until the sixth row of holes is discarded to 150 mu L;
33 Neutralization detection of viral infection): d2, taking out the SARS-CoV-2 pseudovirus from a refrigerator at the temperature of minus 80 ℃ to melt on ice, mixing the pseudovirus and diluted serum according to the infection complex number of the pseudovirus to target cells in a sample group, mixing the pseudovirus with a complete culture medium in a virus control group, and incubating for 1h at the temperature of 37 ℃ to obtain a mixed solution; sucking original cell culture solution, adding 100 μl of the prepared mixed solution into each well, and adding 100 μl of complete culture medium into blank control group; gently shaking, centrifuging at 4deg.C for 2 hr, removing culture medium containing pseudovirus, washing with PBS, adding 30 μl of 0.25% pancreatin into each well, digesting at 37deg.C for 5min, adding 100 μl complete culture medium, lightly blowing with a row gun for 4-5 min, re-suspending, and culturing at 37deg.C overnight;
34 Cell exchange fluid: d3, D4, changing the culture medium every day, and continuously culturing at 37 ℃;
35 Protein expression detection: carrying out fluorescence detection in D5 days, and calculating the average value of luminous intensity;
the fluorescence detection of the step 35) is a Luciferase quantitative determination;
the Luciferase quantitative determination comprises the following steps:
1) Sample processing: washing twice with PBS, discarding supernatant, adding 40 μl of cell lysate into each well, performing lysis on ice for 15min, intermittently blowing suspension during lysis to completely lyse, transferring into 1.5mLEP tube, centrifuging 10000g for 3min, and collecting supernatant for measurement;
2) The detection method comprises the following steps: adding 20 mu L of sample into a detection plate, adding 100 mu L of firefly luciferase detection reagent which is dissolved and balanced to room temperature, and mixing uniformly; incubating for 5min at room temperature after oscillation to stabilize the luminous signals; detecting the Luciferase signal value at the wavelength of 520nm by using a chemiluminescent detector;
the SARS-CoV-2 pseudovirus takes HIV virus as a framework, is coated with Spike protein of SARS-CoV-2, can infect cells which over-express human ACE2, has FireflyLuc and EGFP marks, and detects infection efficiency through FireflyLuc and EGFP;
the serum to be tested is from positive serum after being immunized by SARS-CoV-2 vaccine;
the statistical software is log agonist vs. response-Variableslope in GraphPadPrism.
2. Use of the detection method according to claim 1 for the safety/effectiveness evaluation of a novel coronal vaccine.
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