Extraction And Purification Of F1 Capsule Antigen From Y. Pestis

In this chapter, I describe a simple and efficient strategy to obtain purified F1 antigen from Yersinia pestis. Yersinia pestis strain EV76 is inoculated into brain-heart infusion medium containing sterile glass beads and then incubated at 37 degrees C for 72 h with shaking at 175 rpm. The supernatant is collected by centrifugation and then precipitated with 40% (w/v) saturated ammonium sulfate. The protein pellet is collected by centrifugation, redissolved in 0.01 M phosphate-buffered saline (pH 7.2), and then precipitated with 25% (w/v) saturated ammonium sulfate. The supernatant is precipitated with 5% saturated ammonium sulfate. Finally, highly purified F1 protein is obtained using a Sephacryl S-200HR column. The F1 protein is quantified based on the area and the intensity of its band in a sodium dodecyl sulfate-polyacrylamide gel stained with Coomassie Brilliant Blue. The F1 protein content is determined using a bicinchoninic acid assay. The purity of the F1 antigen is determined by high-performance liquid chromatography, andWestern blotting is used to confirm the specificity of the F1 antigen using a specific monoclonal antibody against the F1 antigen.