CN112379087B - Lysate applied to novel coronavirus inactivated vaccine and antigen dissociation method - Google Patents
Lysate applied to novel coronavirus inactivated vaccine and antigen dissociation method Download PDFInfo
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Abstract
The invention relates to a lysate applied to a novel coronavirus inactivated vaccine and an antigen dissociation method, wherein the raw materials of the lysate comprise diethanolamine, Triton X-100, sodium citrate, urea and EDTA, and PBS buffer solution is used for preparing the lysate; the dissociation of the aluminum adsorption novel coronavirus inactivated vaccine antigen by using the lysate comprises the following steps: preparing a lysate, performing antigen lysis and measuring the content of the antigen. Experiments prove that when the cracking temperature is 4 ℃ and the cracking time is 90min, the cracking effect of the lysate on the new corona inactivated vaccine is good, and the recovery rate and linearity (R) are good2) And the coefficient of variation (CV%) is respectively 104%, 0.99 and 1%, the lysate has stable and reliable lysis effect on the novel coronavirus inactivated vaccine, and the novel coronavirus inactivated vaccine antigen can be efficiently recovered, so that the content of the antigen adsorbed on aluminum in the vaccine can be accurately determined.
Description
Technical Field
The invention relates to a lysate applied to a novel coronavirus inactivated vaccine and an antigen dissociation method, belonging to the technical field of vaccine detection and evaluation.
Background
With the global outbreak of new coronavirus pneumonia, the world enters an emergency state. So far, patients infected with new coronary pneumonia have broken through millions of customs, which has great influence on China and even the world and seriously hinders the development of the global economy and society.
Research has shown that new coronary pneumonia is caused by a novel coronavirus (SARS-Cov-2), a positive-strand RNA virus with a diameter of about 80-120nm, belonging to the family Coronaviridae (Coronaviridae) the subfamily orthocoronaviridae (Orthoconaviridae) which comprises 4 genera (α, β, γ and δ), of which β is the causative agent of new coronary pneumonia. The viral genome consists of 29891 nucleotides and can encode 9860 amino acids. The whole virus is mainly composed of 4 proteins, namely S protein (Spike protein), E protein (Envelope protein), M protein (Membrane protein) and N protein (nucleomapped protein). Most of the existing researches mainly aim at S antigen and N antigen, and the researches show that S protein has rich B cell and T cell epitopes, can induce and generate effective neutralizing antibody to neutralize virus, and has protective effect in animal experiments. In addition, the N antigen also has stronger antigenicity and can induce the generation of neutralizing antibodies.
The use of aluminum hydroxide adjuvant in inactivated vaccine has been known for many years, and the vaccine using aluminum hydroxide as adjuvant can effectively enhance the immune effect of the vaccine, and research shows that the aluminum hydroxide can retain antigen at the injection site, present the antigen to APC cell, induce inflammatory reaction, promote the release of cytokine and other media which activate dendritic cell, thereby enhancing immune response, and is safe and reliable, so that the SARS-Cov-2 inactivated vaccine also uses aluminum hydroxide as adjuvant, animal experiments show that SARS-Cov-2 virus is inactivated treated, S, N antigen can induce high-level neutralizing antibody in rhesus monkey after being adsorbed by aluminum, and the antibody titer is positively correlated with the content of S, N antigen, therefore how to accurately evaluate the contents of S antigen and N antigen adsorbed on aluminum in the vaccine is particularly important, and is an important index for ensuring the safety and effectiveness of the vaccine, however, S antigen and N antigen adsorbed on aluminum cannot be directly detected by ELISA, and it is necessary to cleave the antigen from aluminum in advance with a cleavage agent and then to perform ELISA detection and quantification. The principle of the adsorption of the aluminum hydroxide to the antigen is complex, and the aluminum hydroxide is considered to be adsorbed to the antigen through the physical or chemical actions such as electrostatic interaction, hydrophobic interaction, surface hydroxyl group and phosphate group on the antigen by the current theory.
Most of traditional lysis solutions are prepared by using some surfactants, such as diethanolamine, Triton X-100 and the like, 2010 version of pharmacopoeia of the people's republic of China discloses a lysis solution prepared from diethanolamine and Triton X-100, which is used for detecting the antigen content of aluminum-adsorbed hepatitis A inactivated vaccine and EV71 inactivated vaccine, and has good effect, but the lysis solution is not suitable for aluminum-adsorbed SARS-Cov-2 inactivated vaccine, has poor lysis effect and low antigen recovery rate, which is only 46%, so that the lysis solution for the aluminum-adsorbed SARS-Cov-2 inactivated vaccine is urgently needed to be found at present.
Disclosure of Invention
In order to overcome the problems of the prior art, the invention provides a lysate applied to a novel coronavirus inactivated vaccine and an antigen dissociation method.
The purpose of the invention is realized as follows:
the utility model provides a be applied to lysate of novel coronavirus inactivated vaccine, its raw materials include diethanolamine, Triton X-100, sodium citrate, urea and EDTA, utilize PBS buffer solution to dispose this lysate, the concentration of each raw materials is respectively:
further, the concentration of the PBS buffer solution is 0.01 mol/L-0.1 mol/L, and the preferable concentration is 0.01 mol/L.
Another aspect of the invention:
a method for dissociating the antigen of an aluminum-adsorbed novel inactivated coronavirus vaccine utilizes the lysate to dissociate the antigen of the novel inactivated coronavirus vaccine, and specifically comprises the following steps:
step 1, preparation of lysate: preparing mixed solution containing diethanolamine, Triton X-100, sodium citrate, urea and EDTA by using PBS buffer solution to obtain the lysate;
step 2, antigen lysis: adding the lysate prepared in the step 1 into a novel coronavirus inactivated vaccine, and performing lysis at the temperature of 4-37 ℃ for 30-90 min to obtain an antigen dissociation solution;
step 3, antigen content determination: and measuring the content of the antigen of the novel coronavirus inactivated vaccine in the antigen dissociation solution by using ELISA.
Further, the addition amount of the lysis solution in the step 2 is as follows: the lysate was added to the vaccine at a volume ratio of 1: 1.
Further, the preferred conditions for antigen lysis in step 2 are lysis at 4 ℃ for 90 min.
Further, the specific measurement steps of ELISA in step 3 are:
step 301, preparing an ELISA plate: diluting a human anti-S antigen monoclonal antibody with a coating solution, coating the diluted human anti-S antigen monoclonal antibody into a 96-well plate, placing each well with 100 mu l of the coating solution at 4 ℃ overnight, then discarding the solution in the wells and patting the solution dry, adding 1% BSA into each well, placing the wells at 4 ℃ and sealing the wells overnight, discarding the sealing solution and patting the plate dry, vacuumizing and sealing the plate for packaging, and storing the plate at-20 ℃ for later use;
step 302, antigen determination: taking out an ELISA detection plate, balancing at room temperature for 30min, diluting the antigen dissociation solution obtained in the step 2 and a standard substance in series by 2 times, adding the diluted solution into the plate, adding 100 mu l of each hole, incubating at 37 ℃ for 2h, washing the plate with 0.05% Tween20 for 4 times, drying, adding a rabbit anti-SARS-Cov-2 antibody, adding 100 mu l of each hole, incubating at 37 ℃ for 1h, washing the plate with 0.05% Tween20 for 4 times, drying, adding goat anti-rabbit HRP, incubating at 37 ℃ for 0.5h with 100 mu l of each hole, washing the plate with 0.05% Tween20 for 4 times, drying, adding a TMB color development solution, developing at room temperature for 8min, adding 50 mu l of sulfuric acid into each hole after the color development is finished, reading the OD value of each hole by taking 450nm as a detection wavelength and 630nm as a reference wavelength, and calculating the antigen content of each antigen dissociation solution by using a double parallel line method according to the standard content.
Compared with the prior art, the invention has the advantages and beneficial effects that:
1. the lysate is based on the existing lysate used for the inactivated hepatitis A vaccine, urea and EDTA with a certain concentration are added to prepare a new lysate consisting of diethanolamine, Triton X-100, sodium citrate, urea and EDTA, surfactants such as diethanolamine and Triton X-100 destroy the non-covalent bond combination between the antigen and aluminum hydroxide, the antigen is released by the dissolution of the citrate on the aluminum hydroxide, the urea causes the conformational change of the antigen, the EDTA used as a metal ion chelating agent makes the dissolution of aluminum ions difficult to precipitate, the interference of ELISA is reduced, the novel coronavirus inactivated vaccine antigen can be efficiently recovered, and the accurate determination of the contents of S antigen and N antigen adsorbed on aluminum in the vaccine is realized;
2. the invention discloses a method for dissociating the antigen of the novel aluminum-adsorbed coronavirus inactivated vaccine, which determines that the optimal lysis condition of the lysate on the SARS-Cov-2 inactivated vaccine is lysis temperature through the research of the lysate at different lysis temperatures and times: 4 ℃, cracking time: 90min, the lysis effect of the lysate on the new crown inactivated vaccine is good under the condition, and the recovery rate and linearity (R) are good2) And coefficient of variation (CV%) of 104%, 0.99, 1%, respectively. The repeatability and stability results show that after repeated experiments are carried out on the lysate, the recovery rate is over 95%, and the variation coefficient is less than 15%, so that the lysate has stable and reliable lysis effect on the new crown inactivated vaccine, the lysis effect of the lysate on the new crown inactivated vaccine with high and low antigen content is evaluated, and the results show that the lysate has good lysis effect on the new crown inactivated vaccine with different antigen concentrations, the recovery rates are 100% and 104%, and the lysis effect is not different.
Drawings
The invention is further illustrated by the following figures and examples.
FIG. 1 is a graph showing the results of recovery under different cleavage time (A) and cleavage temperature (B) in the antigen dissociation method described in example 3 of the present invention.
Detailed Description
Example 1:
the embodiment provides a lysate applied to a novel inactivated coronavirus vaccine, wherein the raw materials of the lysate comprise diethanolamine, Triton X-100, sodium citrate, urea and EDTA, the lysate is prepared by using 0.01mol/L PBS buffer solution, and the concentrations of the raw materials are respectively as follows:
example 2:
this example provides a method for dissociating the antigen of the novel aluminum-adsorbed inactivated coronavirus vaccine, wherein the lysate described in example 1 is used to dissociate the antigen of the novel inactivated coronavirus vaccine.
The preparation method of the SARS-Cov-2 inactivated vaccine comprises the following steps: SARS-Cov-2 virus seed (prepared by applicant) is cultured, the culture fluid is collected and inactivated, and after subsequent purification, aluminium hydroxide is added for adsorption, and prepared into inactivated vaccine with S antigen content of 150U/ml, 202U/ml and 250U/ml respectively for standby.
The antigen dissociation of the inactivated vaccine specifically comprises the following steps:
step 1, preparation of lysate: preparing a mixed solution containing diethanolamine, Triton X-100, sodium citrate, urea and EDTA by using 0.01mol/L PBS buffer solution as described in example 1, wherein the concentrations of the diethanolamine, Triton X-100, sodium citrate, urea and EDTA are respectively 0.3% (V/V), 1% (V/V), 10% (w/V), 1mol/L and 0.05mol/L, and the lysate is obtained;
step 2, antigen lysis: adding 0.2ml of vaccine with antigen concentration of 202U/ml into 1.5ml of EP tube, adding 0.2ml of lysate prepared in the step 1, and performing lysis at 4 ℃ for 90min to obtain antigen dissociation solution;
step 3, antigen content determination: measuring the content of the antigen of the novel coronavirus inactivated vaccine in the antigen dissociation solution by using ELISA, and specifically operating as follows:
step 301, preparing an ELISA plate: diluting a human anti-S antigen monoclonal antibody with a coating solution, coating the diluted human anti-S antigen monoclonal antibody into a 96-well plate, placing each well with 100 mu l of the coating solution at 4 ℃ overnight, then discarding the solution in the wells and patting the solution dry, adding 1% BSA into each well, placing the wells at 4 ℃ and sealing the wells overnight, discarding the sealing solution and patting the plate dry, vacuumizing and sealing the plate for packaging, and storing the plate at-20 ℃ for later use;
step 302, antigen determination: taking out an ELISA detection plate, balancing at room temperature for 30min, diluting the antigen dissociation solution obtained in the step 2 and a standard substance in series by 2 times, adding the diluted solution into the plate, adding 100 mu l of each hole, incubating at 37 ℃ for 2h, washing the plate with 0.05% Tween20 for 4 times, drying, adding a rabbit anti-SARS-Cov-2 antibody, adding 100 mu l of each hole, incubating at 37 ℃ for 1h, washing the plate with 0.05% Tween20 for 4 times, drying, adding goat anti-rabbit HRP, incubating at 37 ℃ for 0.5h with 100 mu l of each hole, washing the plate with 0.05% Tween20 for 4 times, drying, adding a TMB color development solution, developing at room temperature for 8min, adding 50 mu l of sulfuric acid into each hole after the color development is finished, reading the OD value of each hole by taking 450nm as a detection wavelength and 630nm as a reference wavelength, and calculating the antigen content of each antigen dissociation solution by using a double parallel line method according to the standard content.
Reagents used in this example: rabbit anti-SRAS-Cov-2 antibody, goat anti-rabbit HRP, 2mol/L sulfuric acid and S antigen standard are all prepared by the applicant; the one-component TMB color former was purchased from Sollarbio.
Example 3:
in order to explore the lysis effect of the new crown inactivated vaccine under different lysis conditions, the experiment of lysing the vaccine at different times and temperatures was performed in this example.
Taking 6 EP tubes with the volume of 1.5ml, adding 0.2ml of SARS-Cov-2 inactivated vaccine with the antigen concentration of 202U/ml into each EP tube, adding 0.2ml of the lysate described in example 1, and randomly extracting two tubes, respectively placing the two tubes at 4 ℃, 25 ℃ and 37 ℃ for cracking for 30 min; the above operations are repeated to crack for 60min and 90min respectively.
The sample recovery rate is calculated by dividing the cracked antigen content by the theoretical antigen content, the recovery rate is subjected to t test by SPSS17.0, the P <0.05 is taken as a statistical difference, and the correlation analysis is performed among the recovery rate, the cracking temperature and the cracking time.
The lysis solution of example 1 is used for lysing the aluminum-adsorbed SARS-Cov-2 inactivated vaccine under different temperature and time conditions, wherein the average recovery rate and the average R are obtained when the lysis temperature is 37 ℃ and the lysis time is 30min, 60min and 90min2And coefficient of variation (CV%) of 80%, 93%, 94%, 0.98, 0.99, 0.98, 10%, 1%, 7%, respectively; average recovery, average R at each cracking time when the cracking temperature is 25 deg.C2And coefficient of variation (CV%) of 80%, 88%, 106%, 0.99, 0.97, 0.98, 1%, 2%, respectively; when in useAverage recovery, average R at 4 ℃ cracking temperature2And coefficient of variation (CV%) of 81%, 96%, 104%, 0.91, 1.00, 0.99, 13%, 1%, respectively, the results are shown in table 1. Under each temperature condition, the recovery rate is increased along with the prolonging of the cracking time, the recovery rate is positively correlated with the cracking time, and the correlation coefficients are respectively 0.985, 0.976 and 0.896, but the cracking effect is gradually reduced along with the increasing of the temperature, the recovery rate is negatively correlated with the cracking temperature, and the correlation coefficients are respectively-0.933, -0.511 and-0.670, and the results are shown in figure 1. When the cracking temperature is 4 ℃ and the cracking time is 90min, the cracking effect is best, and the cracking condition is determined to be the proper cracking condition for the cracking solution.
TABLE 1 lysis effect of lysate on SARS-Cov-2 inactivated vaccine under different conditions
Example 4:
to explore the lytic effect of vaccines with different antigen content, the following experiments were performed in this example:
taking 6 1.5ml EP tubes, adding 0.2ml of vaccine with the antigen content of 250U/ml into each tube, adding 0.2ml of lysate, and performing pyrolysis at 4 ℃ for 90 min; taking another 6 tubes of 1.5ml EP, adding 0.2ml of vaccine with antigen content of 150U/ml into each tube, adding 0.2ml of lysate, and splitting at 4 deg.C for 90 min.
The lysis effect of the lysate on vaccines with different antigen contents as described in example 1 is as follows:
the vaccine with high content and low content is split for 60min at 4 ℃, the average recovery rate is 100% and 104%, respectively, the statistical analysis shows that the average R is not statistically different (t is 1.363, P is 0.203), and the average R is20.98 and 0.99, respectively, and coefficients of variation of 6% and 9%, respectively, both less than 15%, the results are shown in table 2.
TABLE 2 lysis effect of lysate on vaccines with different antigen content at 4 deg.C
Example 5:
this example evaluated the reproducibility and precision of the lysate described in example 1, and the specific method was: performing lysis experiments on different days, taking 6 mEP tubes of 1.5ml each day, adding 0.2ml of vaccine with the antigen content of 250U/ml into each tube, adding 0.2ml of lysis solution described in example 1, and performing lysis at 4 ℃ for 90 min. The above operation was repeated 3 times, 1 time per day.
The lysis of the vaccine with the S antigen content of 250U/ml by the lysate in example 1 is repeated for a plurality of times within 3 days, the average recovery rate of the repeated tests in each day is 99%, 95% and 108%, and the average R20.98, 0.99 and 0.97 respectively, and coefficients of variation (CV%) 6%, 6% and 4%, respectively. The total recovery rate and coefficient of variation (CV%) were 101% and 7% in 3 days, respectively, and the lysate had good reproducibility and precision, and the results are shown in Table 3.
TABLE 3 reproducibility and precision results of lysates
Comparative example:
the comparative example provides a hepatitis A inactivated vaccine lysate, which comprises the raw materials of diethanolamine and TritonX-100, wherein the lysate is prepared by using 0.01mol/L PBS buffer solution, and the concentrations of the raw materials are respectively as follows:
diethanolamine 2.5% (V/V)
TritonX-100 0.2％(v/V)。
Comparing the lysis effect of the lysate applied to the novel inactivated coronavirus vaccine in example 1 with that of the inactivated hepatitis A vaccine lysate in the present comparative example, the specific operation is as follows:
12 pieces of 1.5ml EP tubes are taken, 0.2ml of SARS-Cov-2 inactivated vaccine with the antigen content of 250U/ml is added into each tube, the tubes are randomly divided into two groups, 6 tubes are respectively arranged, wherein, 0.2ml of lysate of the hepatitis A inactivated vaccine in the example 1 is added into each tube of one group, 0.2ml of lysate of the hepatitis A inactivated vaccine in the comparative example is added into each tube of the other group, and all samples are placed at 4 ℃ for 90min for lysis.
The S antigen content of each sample was determined by referring to the antigen content determination method described in step 3 of example 2. The lysate (1) and the lysate (2) for the lysis of the inactivated hepatitis a vaccine in example 1 lyse the same new corona inactivated vaccine, the recovery rates of the two lysates are 86% and 46%, respectively, the lysis effect (recovery rate) of the lysate in example 1 is significantly higher than that of the lysate for the lysis of the inactivated hepatitis a vaccine, and the lysate has significant statistical difference (t-9.312, P-0.000), and the average R of the two lysates is equal to-9.3122And the coefficients of variation were 0.97, 0.96, 7%, 11%, respectively, and the results are shown in Table 4.
TABLE 4 comparison of the lysis effects of the two lysates on the inactivated vaccine
The research can determine that the lysate in example 1 can effectively lyse the S antigen from the aluminum hydroxide under the condition of 4-90 min, the recovery rate is within 80-120%, the linearity is good, the coefficient of variation is less than 15%, and the lysate can be used for lysing new crown inactivated vaccines.
Finally, it should be noted that the above only illustrates the technical solution of the present invention, but not limited thereto, and although the present invention has been described in detail with reference to the preferred arrangement, it will be understood by those skilled in the art that modifications or equivalent substitutions may be made thereto without departing from the spirit and scope of the technical solution of the present invention.
Claims (3)
1. The lysate applied to the novel inactivated coronavirus vaccine is characterized in that the raw materials of the lysate are diethanolamine, Triton X-100, sodium citrate, urea and EDTA, PBS buffer solution is used for preparing the lysate, and the concentrations of the raw materials are respectively as follows:
0.3 percent of diethanolamine
Triton X-100 1%
Sodium citrate 10%
1mol/L of urea
EDTA 0.05mol/L；
The concentration of the PBS buffer solution is 0.01-0.1 mol/L.
2. A method for dissociating the antigen of a novel aluminum-adsorbed inactivated coronavirus vaccine, which is characterized in that the lysate of claim 1 is used for dissociating the antigen of the novel inactivated coronavirus vaccine, and the method comprises the following steps:
step 1, preparation of lysate: preparing mixed solution containing diethanolamine, Triton X-100, sodium citrate, urea and EDTA by using PBS buffer solution to obtain the lysate;
step 2, antigen lysis: adding the lysate prepared in the step 1 into a novel coronavirus inactivated vaccine, and performing lysis at 4 ℃ for 90min to obtain an antigen dissociation solution;
step 3, antigen content determination: measuring the content of the antigen of the novel coronavirus inactivated vaccine in the antigen dissociation solution by using ELISA;
the specific measurement steps of ELISA are as follows:
step 301, preparing an ELISA plate: diluting a human anti-S antigen monoclonal antibody with a coating solution, coating the diluted human anti-S antigen monoclonal antibody into a 96-well plate, placing each well with 100 mu l of the coating solution at 4 ℃ overnight, then discarding the solution in the wells and patting the solution dry, adding 1% BSA into each well, placing the wells at 4 ℃ and sealing the wells overnight, discarding the sealing solution and patting the plate dry, vacuumizing and sealing the plate for packaging, and storing the plate at-20 ℃ for later use;
step 302, antigen determination: taking out an ELISA detection plate, balancing at room temperature for 30min, diluting the antigen dissociation solution obtained in the step 2 and a standard substance in series by 2 times, adding the diluted solution into the plate, adding 100 mu l of each hole, incubating at 37 ℃ for 2h, washing the plate with 0.05% Tween20 for 4 times, drying, adding a rabbit anti-SARS-Cov-2 antibody, adding 100 mu l of each hole, incubating at 37 ℃ for 1h, washing the plate with 0.05% Tween20 for 4 times, drying, adding goat anti-rabbit HRP, incubating at 37 ℃ for 0.5h with 100 mu l of each hole, washing the plate with 0.05% Tween20 for 4 times, drying, adding a TMB color development solution, developing at room temperature for 8min, adding 50 mu l of sulfuric acid into each hole after the color development is finished, reading the OD value of each hole by taking 450nm as a detection wavelength and 630nm as a reference wavelength, and calculating the antigen content of each antigen dissociation solution by using a double parallel line method according to the standard content.
3. The method according to claim 2, wherein the amount of the lysis solution added in step 2 is: the lysate was added to the vaccine at a volume ratio of 1: 1.
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