The demonstrated protocol underlined the direct and effective experimental approach and the detailed steps to immune response effects of novel viral adjuvants. This protocol provides the research with not only a basically immune evaluation method for adjuvant vaccine but can also determines their mass stability. To begin, take the 10 milligrams per milliliter working solution of the MRSA HI antigen protein, and to it, sequentially add the three excipients, isopropyl myristate, polyoxyethylene castor oil, and propylene glycol, maintaining a mass ratio of one is to six is to three.
Stir the mixture well, then gradually add sterilized pure water to reach a system volume of 10 milliliters. To prepare the nanoemulsion vaccine by low energy emulsification method, stir it at 500 rotations per minute and 25 degrees Celsius for approximately 20 minutes. Subsequently, the nanoemulsion vaccine containing one milligram per milliliter protein is obtained as a clear transparent liquid.
For antigen coating of the enzyme linked immunosorbent assay or ELISA plate, dilute HI protein antigen to 10 micrograms per milliliter with coating solution, and add 100 microliters of the diluted antigen per well in the ELISA plate. Incubate the plate at four degrees Celsius overnight. After incubation, wash the plate with 300 microliters of PBST per well.
Seal the wells with 300 microliters of the sealing solution per well for two hours at 37 degrees Celsius, then dilute three microliters of the mice serum from each experimental group 100 times by adding 297 microliters of PBST, and vortex the diluted serum. Add 100 microliters of diluted serum per well in the coated ELISA plate for each serum sample. Similarly, dilute the positive and negative control serums 100 times with PBST by adding four microliters of positive or negative control serum to 396 microliters of PBST, followed by vortex mixing.
To perform double ratio dilution, add 200 microliters of the previously diluted experimental serum to the first row of the coated ELISA plate, then add 100 microliters of PBST to all wells, except those in rows one and 12. Next, adjust the pipette gun to 100 microliters and transfer 100 microliters of serum from the first row to the second row containing PBST. Mix the content in the second row 10 times using the pipette gun.
Similarly, perform one is to two successive dilutions of the serum til row 11. Once done, discard 100 microliters of liquid from row 11. Add 100 microliters of diluted negative and positive serum to each corresponding well in row 12 following the sample template.
Seal the ELISA plate with plastic wrap and incubate at 37 degrees Celsius for one hour. Meanwhile, dilute secondary antibody anti-mouse IgG HRP 10, 000 times by adding PBST. After the one-hour incubation, wash the ELISA plate with 300 microliters of PBST per well to clean the unbound antibodies, then add 100 microliters of the diluted secondary antibody to each well and incubate the plate at 37 degrees Celsius for 40 minutes.
Wash the plate with 300 microliters of PBST per well, then add 100 microliters of TMB color development solution to each well. Place the plates at 37 degrees Celsius and away from light for 10 minutes. After which, terminate the reaction immediately with 50 microliters of two molar sulfuric acid.
Detect the optical density or OD values at 415 nanometers using an enzyme marker within 15 minutes after termination. The physical characteristics of novel nanoemulsion vaccine were analyzed using transmission electron microscopy and atomic force microscopy. Dynamic light scattering was used to determine the particle size distribution and the zeta potential of the sample.
SDS-PAGE and western blotting showed that the amount of antigen in the precipitate and supernatant did not significantly defer after centrifugation, indicating the vaccine was structurally intact, specific, and immunogenic. The nanoemulsion adjuvant vaccine significantly increased the total IgG, IgG1, and IgG2a antibody titers of the mice. The immunogenicity of the protein vaccine was significantly improved.
The serum IgG2b OD values at 450 nanometers were highest when PBS was used at a one is to 5, 000 dilution. The lethal model of MRSA 252 mice reflected that the nanoemulsion vaccine showed a good protective effect and effectively inhibited bacterial infection with MRS A 252, improving the survival rate of infected mice. The preparation of nanoemulssion and the addition of serum to the plant are the most important.
Also, where detection the antibody titer, one should pay attention to correct to a wider and liquid-based out. Besides adjuvants, this method can also be used for the immune evolution of other math arrays, such as plastics or delivery systems.
