We're working on mucosal vaccine that produces an immune response as a set of infection, which has the potential to prevent infection and interrupt transmission. We have developed a cationic nanoemulsion-encapsulated with retinoic acid delivery system. Immunization with ovalbumin by intramuscular injection which triggers both systemic and mucosal immune responses.
In future studies, animal models of intestinal related diseases can be used to further evaluate the effect and the mechanism of cationic nanoemulsion-encapsulated retinoic acid in enhancing mixing protection. Begin aqueous phase preparation by dissolving 0.15 grams of polysorbate 80 in 28.2 milliliters of PBS while stirring at 40 degrees Celsius. To prepare the oil phase formulation, dissolve sorbitan trioleate 85, DOTAP and retinoic acid in squalene while stirring at 40 degrees Celsius.
Next dropwise, add the aqueous phase at one milliliter per minute to the oil phase stirring at 1400 RPM for primary oil and water emulsion preparation. Pre emulsify the mixture using a high shear emulsifier at 30, 000 RPM for six minutes. Treat the primary oil and water emulsion with a high pressure homogenizer for 12 cycles at 900 bar to obtain a homogenous nanoemulsion.
Collect the emulsion in a 50 milliliter flask. And, filter sterilize it through a syringe with a 0.2 micron PES filter. Connect the flask to the Schlenk line through the catheter, rotating the three-way stopcock to connect the system to the vacuum tube.
Turn on the vacuum pump to create a vacuum in the flask. Then, rotate the three-way stopcock to connect the system to the Argon tube and fill it with Argon. Replace the flasks cork and seal it with a paraffin film.
Wrap the flask in tinfoil and store it at four degrees Celsius. Switch on the dynamic light scattering instrument and wait for 30 minutes for the laser light source to stabilize. Dilute the nanoemulsion 50 fold with ultrapure water and add one milliliter of sample to the corresponding sample cell.
On the instrument, set the temperature to 25 degrees Celsius. Choose water as the dispersing medium and disposable plastic dishes as the measuring cell. Load the samples and measure the particle size.
Report the mean value of three repeated measurements for each sample as the result. To begin antigen coating, dilute ovalbumin to five micrograms per milliliter with a coating buffer. Coat in 96-well ELISA plate with 100 microliters of ovalbumin solution per well overnight at four degrees Celsius.
The next day, wash the wells five times with PBST. Block the wells with 250 microliters of 1%BSAPBST at 37 degrees Celsius for two hours. To prepare a 1000x serum dilution, first dilute 20 microliters of serum to one milliliter using 0.5%BSAPBST.
Then, remove 25 microliters of serum aliquot and dilute it to 500 microliters using 0.5%BSAPBST. Add 200 microliters of serum diluted to 1000x to the first row of the coated plate. Then add 100 microliters of 0.5%BSAPBST to all wells except the wells in the first row.
Transfer 100 microliters from the first row to the second row and mix 10 times. To create varying concentrations of serum for IgG detection, discard 100 microliters of liquid from the last row. Next dilute mice vaginal lavage, bronchi alveolar lavage, nasal lavage fluids with 0.5%BSAPBST, and small intestinal lavage fluid with 20%fetal calf serum PBST.
Incubate the plate at 37 degrees Celsius for one hour. Wash the wells five times with PBST. Add 100 microliters of HRP conjugated goat anti-mouse antibody to the appropriate wells and incubate at 37 degrees Celsius for 40 minutes.
After washing the wells five times with PBST, add 100 microliters of TMB Eliza substrate and incubate in a light protected area for five minutes. Add 100 microliters of 450 nanometer stop solution to terminate the reaction. Measure the absorbance at 450 nanometers on a microplate reader.
Calculate the antibody titer defining a positive response value as 2.1 times the serum value of the control mouse group. The nanoemulsion adjuvant vaccine significantly increase the ovalbumin specific IgG, IgG1 and IgG2a antibody titers in serum. The levels of specific IgA in the vaginal lavage fluid and small intestine lavage fluid were significantly enhanced when ovalbumin was adjuvanted with cationic nanoemulsion-encapsulated retinoic acid.
