Extraction modified protocols(1) antigens in vaccines formulated in oily adjuvants (OA)
Antigens for vaccine use can be put in solution with oily adjuvants such as squalene or oil components that a long storage they allow and same time assures that vaccine remains for many months in inoculation site. To extract the antigen from the solution vaccinate in oily adjuvants to verify quality and typology of it, benzyl alcohol is used for breaks the emulsion and the antigens is released and recovered in the resultant aqueous layer. For procedure in preheated (37 °C) or pre-cooled (4 °C) test-tubes, 9 ml of formulated vaccine in mixture water
il (30:70 or 50:50) with coinciding temperature is distributed and add 1 ml of benzyl alcohol, per 10% final concentration. Vortex for 20 mins at maximum speed using a bench-top vortex and then centrifuge at 16100 g for 10 mins or at 10900 rpm for 20 mins to form three layers. Protein is recovered in the aqueous (middle) layer and they concentration measured with BCA method and therefore dilute in coating buffer and to proceed at coated microplate step.
(1)Aron P. Miles, Allan Saul : Extraction and Characterization of Vaccine Antigens from Water-in-Oil Adjuvant Formulations. Methods in Molecular Biology – Vol. 308 – Therapeutic Proteins, 2005, pp. 293-300