CN111122878A

CN111122878A - Method for detecting biological activity of adjuvant

Info

Publication number: CN111122878A
Application number: CN202010115594.6A
Authority: CN
Inventors: 赵干; 何忠淮; 俞庆龄
Original assignee: Advaccine Suzhou Biopharmaceutical Co ltd
Current assignee: Advaccine Suzhou Biopharmaceutical Co ltd
Priority date: 2019-02-28
Filing date: 2020-02-25
Publication date: 2020-05-08
Anticipated expiration: 2040-02-25
Also published as: WO2020173511A2; WO2020173511A3; CN111122878B; CN109752560A

Abstract

The invention provides a method for detecting the biological activity of an adjuvant. Which comprises the following steps: mixing an adjuvant with a co-stimulant to obtain an adjuvant/co-stimulant mixture; applying the adjuvant/co-stimulant mixture to a cell model; and detecting the amount of the protein factors secreted by the cell model to characterize the biological activity of the adjuvant. The detection method and the detection reagent for the adjuvant biological activity can accurately identify the biological activity of different adjuvants, thereby simplifying the evaluation means of the adjuvant and being applicable to the rapid evaluation of the quality of the adjuvant-containing vaccine. The detection method provided by the invention is easy to operate, strong in practicability and wide in application range, and is a great innovation in adjuvant quality control and evaluation.

Description

Method for detecting biological activity of adjuvant

Technical Field

The invention belongs to the field of adjuvant detection, and particularly relates to a method for detecting the bioactivity of an adjuvant.

Background

Adjuvants are a class of substances that nonspecifically alter or enhance the immune response of the body to an antigen, activating NLRP3 (nuclear binding oligomerization domain like receptor 3, NLRP3) inflammasome NLRP3, which is a member of the nod (nuclear binding oligomerization domain) like receptor family, can oligomerize via Caspase Activation and Recruitment Domains (CARD), which interact with aspartic protease 1 to form inflammasome, and modify pro-inflammatory proteins IL-1 β and IL-18 precursor molecules to mature IL-1 β and IL-18.

Experiments on in vitro cell lines show that the adjuvant can activate the secretion of Caspase-1 and its downstream protein factors such as IL-1, IL-18, IL-33, etc. in cells. The lysosomal membrane also suffers some damage during the process as reactive oxygen species are generated after the adjuvant particles enter the cell in an endocytosed form. Both reactive oxygen species and lysosomal damage are upstream activation signals of the NLRP3 inflammasome, promoting activation of the inflammasome; besides, the adjuvant can further induce apoptosis of cells at a small-range injection site and release uric acid. Local high-concentration urate crystal in tissue space forms endogenous danger signals and Damage-related molecular patterns (DAMPs) indirectly activate formation of NLRP3 inflammasome. The NLRP3 inflammasome formed by the adjuvant directly or indirectly can stimulate inflammatory dendritic cells, thereby promoting the uptake, processing and presentation of antigens, and regulating innate immune response and acquired immune response so as to play a nonspecific activation activity.

Therefore, the adjuvant can not only induce the organism to generate long-term and efficient immune response, but also reduce the using amount of antigen, the production cost and the immunization times. The quality evaluation of the adjuvant is an important index in the quality standard of the vaccine. At present, the detection items of the adjuvant in the national quality standard comprise chemical components, physicochemical properties, biochemical properties and purity detection, wherein the biochemical property detection refers to the inspection of the adjuvant from physical properties such as identification, adsorption rate and the like, and the detection process uses more equipment and has more complex steps, which is not beneficial to rapidly knowing the result so as to evaluate the quality of the adjuvant-containing vaccine. And since the adjuvant acts as a non-specific immunostimulant, it is necessary to develop a method for detecting the biological activity of the adjuvant.

Based on the enhancement effect of the adjuvant on the immune response of the antigen, the biological activity of the adjuvant is usually detected by combining the adjuvant with the antigen to immunize a mouse in the traditional method, the biological activity of the adjuvant is evaluated by detecting the secretion amount of an antibody in the serum of the mouse, although the investigation result is visual, the time is long, the result is unstable, and the accurate and rapid evaluation on the biological activity of the adjuvant is not facilitated.

According to systematic research and scientific practice of earlier work, various mammal primary cells and cell line systems, co-stimulators, target detection objects and the like are systematically screened, and the problems that the primary cells, the cell line in-vitro detection systems and the target detection objects are unstable, the response value is low, the batch-to-batch difference is large and the like can be solved. By optimizing a cell system, exploring a target detection object with proper co-stimulus concentration and high responsiveness, and the like, an in-vitro accurate and rapid evaluation method of the adjuvant can be established. The invention improves the quality control and evaluation method and standard of the existing adjuvant, provides a new technical approach for the evaluation of the bioactivity of the adjuvant, has easy operation, strong practicability and wide application range, and is a great innovation in the quality control and evaluation of the adjuvant.

Disclosure of Invention

The invention provides a method for evaluating the biological activity of an adjuvant by using a cell model, aiming at the problems in the prior art. The detection method and the detection reagent provided by the invention can accurately identify the biological activities of different adjuvants, thereby simplifying the evaluation means of the adjuvants and being applicable to the rapid evaluation of the quality of the vaccine containing the adjuvants.

Term(s) for

Co-stimulants: means a substance capable of exerting an assisting or synergistic effect.

LPS: a lipopolysaccharide.

Poly I: C: polyinosinic acid-polycytidylic acid.

Lipid A: lipid A.

MPL: monophosphoryl lipid a.

PAM 3: triacylglycerols are synthesized.

R848: and (3) the resiquimod.

CpG: oligo cytosine-phosphate-guanine.

DC cell: a dendritic cell.

In one aspect, the present invention provides a method for detecting the biological activity of an adjuvant, wherein the method comprises the following steps:

step 1, acting an adjuvant on a cell model;

and 2, detecting the amount of the protein factors secreted by the cell model in the step 1 to characterize the biological activity of the adjuvant.

In some embodiments, the greater the amount of the same protein factor secreted, the greater the biological activity of the representative adjuvant in the same cell model experiment;

further, the adjuvant in the step 1 is selected from one or more of aluminum hydroxide, aluminum sulfate, aluminum phosphate, ammonium alum, potassium alum, TLRs ligand, ATP and inorganic mineral salt;

alternatively, the adjuvant in step 1 can be mixed with co-stimulant to act on the cell model.

Further, the co-stimulant is selected from microbial extracts, chemically synthesized molecules or naturally extracted or recombined expression products by utilizing a genetic engineering technology and the like;

preferably, the microbial extract is LPS;

preferably, the chemically synthesized molecule is selected from one or more of Poly I C, Lipid A, MPL, PAM3, R848, and CpG;

preferably, the naturally extracted or recombinant expression product by using a genetic engineering technology is flagellin;

further, the co-stimulant is selected from one or more of the microbial extracts, chemically synthesized molecules and naturally extracted or recombinant expression products by using genetic engineering technology;

preferably, the adjuvant/co-stimulant mixture of step 1 comprises 0.24-240 μ g/mL adjuvant and 0.3-300ng/mL co-stimulant;

the adjuvant/co-stimulant mixture further preferably contains 40-240 μ g/mL of adjuvant; more preferably 60-220. mu.g/mL; more preferably 100-;

the co-stimulant content of the adjuvant/co-stimulant mixture is further preferably 40-240 ng/mL; more preferably 60-220 ng/mL; more preferably 100 and 200 ng/mL;

further, the cells in the cell model in the step 1 are derived from mammals;

preferably, the mammal may be a human, mouse, rabbit, dog, or the like;

preferably, the cell is a mammalian antigen presenting cell;

further, the antigen presenting cell is derived from a human or a mouse;

preferably, the cell is selected from a macrophage, a DC cell or a monocyte of a mammal;

preferably, the cell may be derived from human peripheral blood, mouse peritoneal cavity, mouse spleen, mouse lymph node, mouse peripheral blood or mouse bone marrow;

preferably, the cell may be a human THP or a human CAL-1 monocyte;

preferably, the cell may be the mouse DC cell line JAWSII;

preferably, the cell may be the mouse macrophage cell line RAW 264.7;

preferably, the cell may be a DC cell line DC 2.4;

preferably, the cell may be a mammalian reporter cell line;

preferably, the action time in the step 1 is 0.5-72 h; further preferably 20-26 h; more preferably 21-25 h;

further, the protein factors in the step 2 are selected from one or more of cytokine class (IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-22, IL-23, IL-27, IL-31, IL-33, TNF α, IFN α, IFN β, IFN gamma) and chemokine class (MCP-1/CCL2, MIP-1 α/CCL3, MIP-1 β/CCL4, RANTES/CCL5, CXCL9, CXCL10 and CXCL 12);

preferably, the method for detecting the amount of the protein factors secreted by the biological model in the step 2 is an ELISA detection method;

in another aspect, the invention provides the use of the aforementioned detection method in the quality assessment of an adjuvanted vaccine.

Compared with the prior art, the invention has the beneficial effects that:

(1) compared with the prior art for inspecting the adjuvant from the aspects of physical properties such as adsorption rate, purity and the like, the method disclosed by the invention can be used for inspecting the biological properties of the adjuvant more directly, objectively, quickly and accurately, thereby simplifying the evaluation means of the adjuvant and being applied to quick evaluation of the quality of the adjuvant-containing vaccine.

(2) Compared with the traditional method in which the adjuvant is combined with the antigen to immunize the mouse, the evaluation method for deducing the biological activity of the adjuvant by detecting the secretion amount of the antibody in the serum of the mouse, the cell model detection method provided by the invention reduces the time consumption, and the presented result can quantitatively detect the biological activity of the adjuvant by using accurate numbers and ranges, so that the results of different batches can be longitudinally compared and the result is more accurate.

Detailed Description

The following non-limiting examples are presented to enable those of ordinary skill in the art to more fully understand the present invention and are not intended to limit the invention in any way. The following is merely an exemplary illustration of the scope of the invention as claimed, and various changes and modifications of the invention of the present application may be made by those skilled in the art based on the disclosure, which also fall within the scope of the invention as claimed.

The present invention will be further described below by way of specific examples. The various chemicals used in the examples of the present invention were obtained by conventional commercial routes unless otherwise specified.

The invention provides a method for detecting the biological activity of an adjuvant, which can be used for evaluating the biological activity of the adjuvant by detecting the secretion amount of a protein factor in a cell model, namely: under the same experimental conditions, the more the same protein factor is secreted by the same cell model, the stronger the biological activity of the adjuvant acting on the cell model.

The detection of the biological activity of the adjuvant by adopting the traditional method and the method provided by the invention is compared, and the result of a comparison experiment shows that the detection method provided by the invention can be used for quickly and accurately detecting the biological activity of the adjuvant, and has obvious advantages compared with the traditional method.

The terms:

MEM medium: low minimal medium.

FBS: fetal bovine serum.

rmGM-CSF: recombinant mouse granulocyte-macrophage colony stimulating factor.

TMB: 3,3',5,5' -tetramethylbenzidine.

In the embodiment of the invention, "Alum" refers to aluminum hydroxide adjuvant, Alum1, Alum 2 and Alum3 are different aluminum hydroxide adjuvants, and the performances of each aspect of Alum1 are better than those of Alum 2, and the performances of each aspect of Alum 2 are better than those of Alum 3.

Alum1 was purchased from Invivogen under the VAC-ALU-250 designation.

Alum 2 was purchased from ThermoFisher as cat # 77161.

Alum3 was formulated according to standard methods.

Poly I: C was purchased from Invivogen under the designation tlrl-picw.

R848 is available from Invivogen under the designation tlrl-R848.

CpG was purchased from Invivogen under the designation tlrl-1826.

Example 1 biological Activity of aluminum adjuvants Using conventional methods

(1) Preparation of Experimental materials

6-8 week old female C57BL/C mice, purchased from Beijing Huafukang, 6 mice per group. Purified water and food are used for feeding during the experiment, and the illumination period is 12 h. The stimulators are mutually matched according to the amount of ovalbumin OVA (10 mu g), aluminum adjuvant (200 mu g) and LPS (100ng) used by each mouse, and are uniformly mixed at room temperature for 30min, then the mice (200 mu L/mouse) are immunized by an intraperitoneal injection mode on the 1 st day, the boosting immunity is carried out 14 days after the primary immunization, and the mouse serum is respectively collected for standby 3h and 7d after the boosting immunity; experimental groups and specific stimulus addition levels are shown in the following table:

TABLE 1 vaccine antigens with aluminum adjuvant, LPS grouping and addition

(2) Specific antibody detection:

standing the mouse serum at room temperature for 2h, and centrifuging at 4000rpm and 4 ℃ for 30min to separate the mouse serum; the content of ovalbumin OVA specific antibody in serum is determined by a quantitative ELISA method, and the specific detection steps are as follows:

antigen coating: diluting the standard capture antibody to 0.5. mu.g/mL with ELISA coating solution; OVA antigen is 2 mug/mL, a 96-well plate is coated with the diluted antigen, 100 mug/well, and the temperature is 4 ℃ overnight;

and (3) sealing: PBST (0.05% Tween20 in PBS) was washed 3 times for 5min each, 5% skim milk powder, 100. mu.L/well, blocked at 37 ℃ for 1 h;

IgG standards and serum incubation: PBST is washed for 3 times, each time is 5min, IgG standard substance is diluted by 2 times of skimmed milk powder in a gradient way, mouse serum is diluted by 200 times of skimmed milk powder in 2%, each hole is 100 mu L, and incubation is carried out for 1h at 37 ℃;

and (3) secondary antibody incubation: PBST was washed 5 times for 5min each, added with goat anti-mouse IgG labeled with HRP (1:4000) at 100. mu.L/well, incubated at 37 ℃ for 1 h; wherein the goat anti-mouse IgG labeled with HRP is purchased from Kinsley, and has the product number of A00160.

Color development: PBST is washed for 5 times, each time is 5min, substrate TMB is added for color development, 100 mu L/hole is added, and color development is carried out for 15min in a dark place at 37 ℃;

and (4) terminating: adding 2mol/L sulfuric acid to stop color development, wherein the color development is 50 mu L/hole;

reading: the optical density values were measured at OD 450nm/620 nm. And (5) making a standard curve according to the content of the standard substance and the OD value, and calculating the content of the antibody in the serum sample.

As shown in Table 2, after mice were immunized with the mixture of aluminum adjuvant/LPS in combination with OVA antigen, OVA-Alum 1+ LPS, OVA-Alum 2+ LPS and OVA-Alum 3+ LPS activated the body to produce more antigen-specific antibodies than the control group, while aluminum adjuvants with different activities were significantly different in promoting antibody production.

The greater the amount of specific antibody produced, the greater the biological activity of the aluminum adjuvant. Therefore, according to the experimental results, the strong and weak relationship of the biological activity of the 3 aluminum adjuvants is as follows: the experimental results are in accordance with the theoretical range, and the biological activity of the aluminum adjuvant can be visually inspected by adopting a traditional method.

However, the time for stimulating and culturing the mice is long, and due to the fact that the mice are living systems, the system deviation is large, and the like, in the experiment, the deviation of an OVA-Alum 1+ LPS experimental group is 60%, the deviation of an OVA-Alum 2+ LPS experimental group is 82%, the deviation of an OVA-Alum 2+ LPS experimental group is 77%, and the deviation of each experimental group is large, so that the quick and accurate evaluation on the bioactivity of the aluminum adjuvant is not facilitated.

TABLE 2 amount of antigen-specific antibody production in mouse model

Example 2 detection of the biological Activity of an aluminum adjuvant Using the method of the present invention

In this embodiment, a mouse DC cell detection system is used as a biological model for detecting the biological activity of the aluminum adjuvant, and the specific operation steps are as follows:

(1) cell resuscitation

Frozen mouse DC cell JAWSII cell line (from department of molecular virology education in medicine of university of Bekken/professor of Wangbin university of Weijian, key laboratory) was taken out from liquid nitrogen at-196 deg.C, quickly placed in a 37 deg.C water bath, quickly thawed, washed 2 times with medium (MEM medium), resuspended in 10mL of MEM complete medium (MEM medium + 20% FBS +5ng/mLrmGM-CSF), and then suspended at 1X 10⁶And (3) spreading the cells/dish in a 10cm cell culture dish, culturing for 5-7 days, after the cells are spread to the bottom of the dish, digesting with 0.25% trypsin, collecting the cells, carrying out passage, and continuing the subsequent test after 1-2 passages.

(2) Cell stimulation

Adjusting the concentration of the collected JAWSII cells to 5X 10⁵After the cells/wells are laid on a 24-well plate and cultured for 24h, different stimulators are added at 37 ℃ and 5% CO₂Culturing for 24h under the condition. The stimuli were LPS (3ng/mL), Alum (200. mu.g/mL), Alum + LPS mixture (3ng/mL LPS + 200. mu.g/mL Alum), LPS, Alum as positive controls, Medium as negative control.

(3) Protein factor detection

After the culture, cell supernatants were collected, centrifuged at 12000rpm at 4 ℃ for 5min to remove cell debris and aluminum adjuvant particles, and IL-1 β content in the cell supernatants was measured using IL-1 β ELISA kit (purchased from Union, cat # 70-EK201B 4/2).

The detection procedure of the protein factor IL-1 β ELISA kit is as follows according to the specification of the kit, all reagents and samples are balanced to room temperature, 300 mu L of 1 Xwashing liquor is added into a pre-coated plate, the pre-coated plate is kept still and soaked for 30s, after the washing liquor is discarded, a microporous plate is dried on absorbent paper, 100 mu L of standard product diluted by 2 times is added into a repeated hole, 100 mu L of standard product diluent is added into a repeated hole of a blank hole, 100 mu L of cell culture supernatant sample is added into a sample hole, 50 mu L of diluted detection antibody is added into each hole, the sample adding process is completed within 15min, a sealing plate membrane is used for sealing, the oscillation is carried out at 300r/min, the liquid is discarded after the room temperature incubation is carried out for 1.5h, 300 mu L of washing liquor washing plate is added into each hole, the washing liquor is washed for 6 times, the plate is dried after the patting is carried out on the absorbent paper, 100 mu L of horseradish peroxidase-labeled streptavidin is added into each hole, a new sealing plate is used for sealing, the sealing membrane is carried out at 300r/min, the oscillation is discarded, the liquid is added into each hole, the washing liquor is washed for 6 times, the washing liquor for incubation is added, the washing liquor for each hole, the detection is carried out at room temperature, the color of the yellow detection, the color of the detection is changed from the detection, the detection of the detection.

As shown by the results in Table 3, when JAWSII cells were stimulated with the aluminum adjuvant/LPS mixture, Alum1+ LPS, Alum 2+ LPS and Alum 3+ LPS were all able to stimulate secretion of more IL-1 β, with a typical synergistic effect compared to Alum, LPS stimulation alone, while different aluminum adjuvants stimulated cell lines with significantly different abilities to secrete IL-1 β.

Theoretically, the experimental results in example 1 show that the biological activity of each aluminum adjuvant is in a relationship of Alum 1> Alum 2> Alum 3. in the results of this example, the relationship of the amount of the protein factor IL-1 β stimulated and secreted by each experimental group is Alum1+ LPS > Alum 2+ LPS > Alum 3+ LPS, which conforms to the theory and the trend described in example 1, so that the more the amount of the protein factor is secreted, the stronger the biological activity of the corresponding adjuvant is, and the method provided by the invention can be used for describing the biological activity of the aluminum adjuvant.

TABLE 3 amount of IL-1 β produced in cell model

Example 3 verification of the accuracy of the detection method of the invention

The procedure was followed 3 consecutive times as described in example 2, where the stimulus was selected only for the compatibility of the Alum + LPS mixture (3ng/mL LPS + 200. mu.g/mL Alum), and the suitability of the procedure for the detection of the biological activity of the aluminum adjuvant was examined. Acceptance criteria: the deviation between the detection results of 3 times is less than or equal to 5 percent, which both meet the theoretical range and accord with the bioactivity trend of different aluminum adjuvants obtained by the traditional detection method in the embodiment 1. The experimental results are shown in table 4, the 3 detection results are all within the theoretical range, and accord with the bioactivity trends of different aluminum adjuvants obtained by the conventional detection method described in example 1, and meanwhile, the deviation of the 3 detection results is not more than 5%, which indicates that the detection method of the invention has better accuracy.

TABLE 4 accuracy of IL-1 β production by cell models in experiments

Example 4 verification of the reproducibility of the method of the invention

The method for detecting the IL-1 β in the supernatant comprises the steps of continuously detecting Alum in the same batch for 6 times according to the detection method described in example 2, wherein the stimulus only selects the compatibility of an Alum + LPS mixture (3ng/mL LPS +200 mug/mL Alum), the batch numbers are respectively Alum 1: Alum 1-1, Alum1-2 and Alum 1-3, Alum 2: Alum 2-1, Alum 2-2 and Alum 2-3 and Alum 3: Alum 3-1, Alum3-2 and Alum 3-3, stimulating cells after being combined with the LPS respectively, and detecting the IL-1 β production amount in the supernatant, wherein the acceptable standard is that the deviation between 6 detection results is less than or equal to 5% and the detection results between the same batches are less than or equal to 5%.

TABLE 5 production of IL-1 β from cell models in repeated experiments

Comparing the detection results of examples 1, 2, 3 and 4, it can be seen that the method for detecting the biological activity of the adjuvant provided by the invention has high accuracy, good repeatability and short time, and can be quickly and accurately applied to the evaluation of the biological activity of the adjuvant product.

Example 5 detection of the biological Activity of an aluminum adjuvant by IL-6 biomarkers Using the method of the invention (TLR4 Ligand System)

(1) cell recovery reference example 2.

(2) Cell stimulation

Adjusting the concentration of the collected JAWSII cells to 5X 10⁵After the cells/wells are laid on a 24-well plate and cultured for 24h, different stimulators are added at 37 ℃ and 5% CO₂Culturing for 24h under the condition. The stimulus isLPS (30ng/mL), Alum1 (60. mu.g/mL), Alum1+ LPS mixture (30ng/mL LPS + 60. mu.g/mL Alum 1), LPS, Alum1 as positive control, Medium as negative control.

(3) Protein factor detection

Collecting cell supernatant after culturing, centrifuging at 12000rpm at 4 ℃ for 5min to remove cell debris and aluminum adjuvant particles, and detecting the content of IL-6 in the cell supernatant by using an IL-1 β ELISA kit.

Protein factor IL-6ELISA kit detection procedures according to the kit instructions refer to example 2.

As shown by the results in Table 6, when JAWSII cells were stimulated with the aluminum adjuvant/LPS mixture, Alum1+ LPS was able to stimulate the secretion of more IL-6, with a typical synergistic effect of Alum1+ LPS compared to Alum1, LPS stimulation alone.

TABLE 6 amount of IL-6 production in cell models

Example 6 detection of the biological Activity of an aluminum adjuvant by IL-18 biomarkers Using the method of the invention (TLR4 Ligand System)

(1) cell recovery reference example 2.

(2) Cell stimulation reference example 5.

(3) Protein factor detection

Collecting cell supernatant after culturing, centrifuging at 12000rpm at 4 ℃ for 5min to remove cell debris and aluminum adjuvant particles, and detecting the content of IL-18 in the cell supernatant by using an IL-1 β ELISA kit.

Protein factor IL-18ELISA kit detection procedures reference example 2 according to the kit instructions.

As shown by the results in table 7, when JAWSII cells were stimulated with the aluminum adjuvant/LPS mixture, Alum1+ LPS stimulated secretion of more IL-18, with a typical synergistic effect for Alum1+ LPS compared to Alum1, LPS stimulation alone.

TABLE 7 amount of IL-18 production in cell models

Example 7 detection of the biological Activity of aluminum adjuvant by TNF- α biomarkers Using the method of the invention (TLR4 Ligand System)

(1) cell recovery reference example 2.

(2) Cell stimulation reference example 5.

(3) Protein factor detection

Collecting cell supernatant after culture, centrifuging at 12000rpm and 4 ℃ for 5min to remove cell debris and aluminum adjuvant particles, and detecting the content of TNF- α in the cell supernatant by using an IL-1 β ELISA kit.

The detection procedure of the protein factor TNF- α ELISA kit was as described in example 2 with reference to the kit instructions.

As shown by the results in table 8, when JAWSII cells were stimulated with the aluminum adjuvant/LPS mixture, Alum1+ LPS stimulated more TNF- α, with a typical synergistic effect compared to Alum1, LPS stimulation alone.

TABLE 8 amount of TNF- α production in cell model

Example 8 detection of the biological Activity of an aluminum adjuvant by IL-1 β biomarkers Using the method of the invention (TLR3 Ligand System)

(1) cell recovery reference example 2.

(2) Cell stimulation

Adjusting the concentration of the collected JAWSII cells to 5X 10⁵After the cells/wells are laid on a 24-well plate and cultured for 24h, different stimulators are added at 37 ℃ and 5% CO₂Culturing for 24h under the condition. Stimuli were Poly I: C (0.3, 3, 30ng/mL), Alum1 (60. mu.g/mL), Alum1+ Poly I: C mix (0.3, 3, 30ng/mL Poly I: C + 60. mu.g/mL Alum 1), Poly I: C, Alum1 was a positive control, and Medium (Medium) was a negative control.

(3) Protein factor detection

Collecting cell supernatant after culture, centrifuging at 12000rpm at 4 ℃ for 5min to remove cell debris and aluminum adjuvant particles, and detecting the content of IL-1 β in the cell supernatant by using an IL-1 β ELISA kit.

The detection procedure of the protein factor IL-1 β ELISA kit was as described in example 2 with reference to the kit instructions.

As shown by the results in Table 9, Alum1+ Poly I: C was able to stimulate secretion of more IL-1 β when JAWSII cells were stimulated with the aluminum adjuvant/Poly I: C mixture, and Alum1+ Poly I: C had a typical synergistic effect compared to Alum1, Poly I: C stimulation alone, and there was a dose dependence of this synergy.

TABLE 9 amount of IL-1 β production in cell models

Example 9 detection of the biological Activity of an aluminum adjuvant by IL-6 biomarkers Using the method of the invention (TLR3 Ligand System)

(1) cell recovery reference example 2.

(2) Cell stimulation

Adjusting the concentration of the collected JAWSII cells to 5X 10⁵After the cells/holes are laid on a 24-hole plate for adherent culture for 24 hours, the cells/holes are addedThe same irritant, 5% CO at 37 deg.C₂Culturing for 24h under the condition. Stimuli were Poly I: C (30ng/mL), Alum1 (60. mu.g/mL), Alum1+ Poly I: C mix (30ng/mL Poly I: C + 60. mu.g/mL Alum 1), Poly I: C, Alum1 as positive control, and Medium (Medium) as negative control.

(3) Protein factor detection

As shown by the results in Table 10, Alum1+ Poly I: C was able to stimulate secretion of more IL-6 when JAWSII cells were stimulated with the aluminum adjuvant/Poly I: C mixture, with a typical synergistic effect for Alum1+ Poly I: C compared to Alum1, Poly I: C stimulation alone.

TABLE 10 amount of IL-6 production in cell models

Example 10 detection of the biological Activity of an aluminum adjuvant by IL-18 biomarkers Using the method of the invention (TLR3 Ligand System)

(1) cell recovery reference example 2.

(2) Cell stimulation reference example 9.

(3) Protein factor detection

As shown by the results in Table 11, Alum1+ Poly I: C was able to stimulate secretion of more IL-18 when JAWSII cells were stimulated with the aluminum adjuvant/Poly I: C mixture, with a typical synergistic effect for Alum1+ Poly I: C compared to Alum1, Poly I: C stimulation alone.

TABLE 11 amount of IL-18 production in cell models

Example 11 testing of the biological Activity of aluminum adjuvants with the method of the invention (TLR3 Ligand System) by TNF- α biomarkers

(1) cell recovery reference example 2.

(2) Cell stimulation reference example 9.

(3) Protein factor detection

As shown by the results in Table 12, Alum1+ Poly I: C stimulated secretion of more TNF- α when JAWSII cells were stimulated with the aluminum adjuvant/Poly I: C mixture, with a typical synergistic effect of Alum1+ Poly I: C compared to Alum1, Poly I: C stimulation alone.

TABLE 12 amount of TNF- α production in cell model

Example 12 detection of the biological Activity of an aluminum adjuvant by IL-1 β biomarkers Using the method of the invention (TLR7/8Ligand System)

(1) cell recovery reference example 2.

(2) Cell stimulation

Adjusting the concentration of the collected JAWSII cells to 5X 10⁵After the cells/wells are laid on a 24-well plate and cultured for 24h, different stimulators are added at 37 ℃ and 5% CO₂Culturing for 24h under the condition. The stimuli were R848(0.3, 3, 30ng/mL), Alum1 (60. mu.g/mL), Alum1+ R848 mixture (0.3, 3, 30ng/mL R848+ 60. mu.g/mL Alum 1), R848, Alum1 were positive controls, and Medium (Medium) was negative control.

(3) Protein factor detection

As shown by the results in table 13, when JAWSII cells were stimulated with the aluminum adjuvant/R848 mixture, Alum1+ R848 stimulated secretion of more IL-1 β, with a typical synergistic effect of Alum1+ R848 compared to Alum1, R848 stimulation alone, and with dose-dependence of this synergy.

TABLE 13 amount of IL-1 β production in cell models

Example 13 detection of the biological Activity of an aluminum adjuvant by IL-6 biomarkers Using the method of the invention (TLR7/8Ligand System)

(1) cell recovery reference example 2.

(2) Cell stimulation

Adjusting the concentration of the collected JAWSII cells to 5X 10⁵After the cells/well are spread on a 24-well plate for adherent culture for 24h, different stimulators are added, the temperature is 37 ℃, and the content is 5%CO₂Culturing for 24h under the condition. The stimuli were R848(30ng/mL), Alum1 (60. mu.g/mL), Alum1+ R848 mixture (30ng/mL R848+ 60. mu.g/mL Alum 1), R848, Alum1 were positive controls, and Medium (Medium) was negative control.

(3) Protein factor detection

As shown in the results in table 14, when JAWSII cells were stimulated with the aluminum adjuvant/R848 mixture, Alum1+ R848 stimulated secretion of more IL-6, with a typical synergistic effect of Alum1+ R848 compared to Alum1, R848 stimulation alone.

TABLE 14 amount of IL-6 production in cell models

Example 14 testing of the bioactivity of aluminium adjuvants with the method of the invention (TLR7/8Ligand System) by IL-18 biomarkers

(1) cell recovery reference example 2.

(2) Cell stimulation reference example 13.

(3) Protein factor detection

As shown by the results in table 15, when JAWSII cells were stimulated with the aluminum adjuvant/R848 mixture, Alum1+ R848 stimulated secretion of more IL-18 with a typical synergistic effect compared to Alum1, R848 stimulation alone.

TABLE 15 amount of IL-18 production in cell models

Example 15 testing of the biological Activity of aluminum adjuvants with the method of the invention (TLR7/8Ligand System) by TNF- α biomarkers

(1) cell recovery reference example 2.

(2) Cell stimulation reference example 13.

(3) Protein factor detection

As shown in the results in table 16, when JAWSII cells were stimulated with the aluminum adjuvant/R848 mixture, Alum1+ R848 stimulated secretion of more TNF- α with a typical synergistic effect compared to Alum1, R848 stimulation alone.

TABLE 16 amount of TNF- α production in cell model

Example 16 testing of the bioactivity of aluminum adjuvants with the method of the invention (TLR9 Ligand system) by IL-1 β biomarkers

(1) cell recovery reference example 2.

(2) Cell stimulation

Adjusting the concentration of the collected JAWSII cells to 5X 10⁵After the cells/wells are laid on a 24-well plate and cultured for 24h, different stimulators are added at 37 ℃ and 5% CO₂Culturing for 24h under the condition. The stimulators are CpG (0.3, 3, 30ng/mL), Alum 1(60 mu g/mL), Alum1+ CpG mixture (0.3, 3, 30ng/mL CpG +60 mu g/mL Alum 1), CpG and Alum1 are positive controls, and culture Medium (Medium) is negative control.

(3) Protein factor detection

As shown by the results in table 17, when JAWSII cells were stimulated with the aluminum adjuvant/CpG mixture, Alum1+ CpG stimulated secretion of more IL-1 β, with a typical synergistic effect of Alum1+ CpG compared to Alum1, CpG stimulation alone, and with a dose-dependent relationship.

TABLE 17 amount of IL-1 β production in cell model

Example 17 detection of the biological Activity of an aluminum adjuvant by IL-6 biomarkers Using the method of the invention (TLR9 Ligand System)

(1) cell recovery reference example 2.

(2) Cell stimulation

Adjusting the concentration of the collected JAWSII cells to 5X 10⁵After the cells/wells are laid on a 24-well plate and cultured for 24h, different stimulators are added at 37 ℃ and 5% CO₂Culturing for 24h under the condition. The stimulators are CpG (30ng/mL), Alum 1(60 μ g/mL), Alum1+ CpG mixture(30ng/mL CpG + 60. mu.g/mL Alum 1), CpG and Alum1 are positive controls, and the culture Medium (Medium) is negative control.

(3) Protein factor detection

As shown by the results in Table 18, Alum1+ CpG stimulates secretion of more IL-6 when JAWSII cells are stimulated with an aluminum adjuvant/CpG mixture, with a typical synergistic effect of Alum1+ CpG compared to Alum1, CpG alone.

TABLE 18 amount of IL-6 production in cell models

Example 18 detection of the biological Activity of an aluminum adjuvant by IL-18 biomarkers Using the method of the invention (TLR9 Ligand System)

(1) cell recovery reference example 2.

(2) Cell stimulation reference example 17.

(3) Protein factor detection

As shown by the results in Table 19, Alum1+ CpG stimulates secretion of more IL-18 when JAWSII cells are stimulated with an aluminum adjuvant/CpG mixture, with a typical synergistic effect of Alum1+ CpG compared to Alum1, CpG alone.

TABLE 19 amount of IL-18 production in cell models

Example 19 testing of the biological Activity of aluminum adjuvants with the method of the invention (TLR9 Ligand System) by TNF- α biomarkers

(1) cell recovery reference example 2.

(2) Cell stimulation reference example 17.

(3) Protein factor detection

As shown by the results in Table 20, Alum1+ CpG stimulated secretion of more TNF- α when JAWSII cells were stimulated with an aluminum adjuvant/CpG mixture, with a typical synergistic effect of Alum1+ CpG compared to Alum1, CpG alone.

TABLE 20 amount of TNF- α production in cell model

The above embodiments are merely preferred embodiments of the present invention, and the present invention is not limited thereto, and the experimental effects are equivalent to those of the above embodiments within the scope of the claims of the present invention. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for detecting the biological activity of an adjuvant is characterized by comprising the following steps:

step 1, acting an adjuvant on a cell model;

2. The detection method according to claim 1, wherein the adjuvant in step 1 is selected from one or more of aluminum hydroxide, aluminum sulfate, aluminum phosphate, ammonium alum, potassium alum, TLRs ligands, ATP and inorganic mineral salts.

3. The detection method according to claim 1, wherein the adjuvant in step 1 can be mixed with a co-stimulant and then co-acted on a cell model; wherein the co-stimulant is selected from one or more of LPS, Poly I: C, Lipid A, MPL, PAM3, R848, CpG, Flagellin.

4. The detection method according to claim 1, wherein the action time in step 1 is 0.5 to 72 hours.

5. The assay of claim 1, wherein the cells in the cell model of step 1 are derived from a mammal.

6. The method for detecting according to claim 1, wherein the protein factor in step 2 is selected from one or more of IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-22, IL-23, IL-27, IL-31, IL-33, MCP-1/CCL2, MIP-1 α/CCL3, MIP-1 β/CCL4, RANTES/CCL5, CXCL9, CXCL10, CXCL12, TNF α, IFN α, IFN β and IFN γ.

7. The assay of claim 3, wherein the adjuvant/co-stimulator mixture comprises 0.24-240 μ g/mL adjuvant and 0.3-300ng/mL co-stimulator.

8. The method of detecting according to claim 6, wherein the cell is an antigen presenting cell of a mammal.

9. The assay of claim 8, wherein the antigen presenting cell is derived from a human or mouse.

10. Use of the assay of any one of claims 1-9 in the quality assessment of an adjuvanted vaccine.