[Purification of coxiella burneti antigen by density gradient centrifugation (author's transl)]

Coxiella burneti, strain Henzerling, Phase II, was recovered from yolk sacs of infected chidken embryos by mechanical separation, treated with Freon and purified by density gradient centrifugation. A J-21 Beckman Centrifuge was applied and the JCF-Z Rotor with a zonal as well as a continuous flow core was used. The technique of producing different types of gradients with a wide range of density (1-60% Sucrose) without a gradient pump is described. With the zonal core a good separation of antigen from unspecific proteins was achieved with 60 Min. at 15000 rpm (Fig. 2-4). The antigens bandet in 1-4 characteristic peaks within the range of 45-49% Sucrose (1,2102-1,2532 g/ml), according to the gradient used. A certain amount of specific fluorescent antigen was also found within the peaks of unspecific protein at densities of 18 -- 20% Sucrose (1,0776-1,0820 g/ml). Centrifugation for more than 1 hour only caused a shift of the antigen peaks into a higher range of density. Using the continuous flow core 2 antigen peaks were found within a density of 43-50% Sucrose (1,1961-1,2341 g/ml). Unspecific protein banded with a peak at 15% Sucrose (1,0612 g/ml); small amounts of specific fluorescent antigen were also found within that density. Approaching equilibrium by centrifuging up to 44 hours at 19000 rpm caused the Coxiella antigens to band at 54% Sucrose (1,2544 g/ml).