Partial Purification of the Virus of Epidemic Influenza by Adsorption on Calcium Phosphate. ∗

Abstract

The purpose of the present communication is to summarize certain of a series of experiments on the partial purification of influenza virus using calcium phosphate as the adsorbing agent. The procedure is simple and the amount of material that can be handled is limited only by the volume of infected lung suspension or chick embryonic tissue suspension available.

The procedure herein employed is a modification of a previously reported method 1 , † for the removal of bacteria from large quantities of broth culture. By the addition of calcium chloride and phosphate buffer, at pH 7.5, a precipitate of calcium phosphate is formed in the broth culture. The precipitate adsorbs the bacteria, settles quickly, and can readily be separated from the supernatant fluid. The bacteria are then “eluted’ from the calcium phosphate by adjusting the hydrogen ion concentration of the resuspended precipitate to about pH 4.5. This causes the calcium phosphate to go into solution, leaving the bacteria suspended in a small volume of fluid from which they can be separated by centrifugation. Because of the instability of epidemic influenza virus when exposed to an acidity as high as pH 4.5, 2 this method for the removal of calcium phosphate could not be employed. Accordingly, the modification introduced was one in which the calcium phosphate was dissolved at neutrality by the use of the sodium salt of citric acid.

In this report, a typical experiment will be described in which a comparison is made between the infectivity, the immunogenicity, and the nitrogen content in the untreated and the partially purified preparation of a suspension of infected mouse lung. The amount of residual infective and immunogenic material which remained in the supernatant fluid after adsorption was also tested.

2.8 grams of the lung tissue of mice infected with the PR-8 strain of influenza virus were ground with alundum and diluted with 11.2 cc of physiological salt solution and 16.8 cc of 0.05 M phosphate buffer (pH 7.8) to form a 10% suspension. This was centrifuged for 25 minutes in the Swedish Angle Centrifuge to remove gross particles of tissue. One portion was partially purified in the following manner involving 3 successive adsorptions, although it has since been found that only 2 are necessary. To 15 cc of the suspension in a centrifuge tube, 4.5 cc of 0.1 M calcium chloride and 4.9 cc of 0.1 M sodium hydroxide were added.