Assay for circulin based on the release of cellular constituents.

Abstract

Circulin causes the release of cellular components from susceptible cells exposed to its action; within a given range of circulin concentrations this release is proportional to the amount of circulin present. This relationship can be used as a basis of a rapid and sensitive method for determining circulin. Inasmuch as cells on exposure to circulin release numerous compounds, various means can be used to measure this release, and the assay can be adapted to the equipment available. Two applications of this principle are described. They are based on the release from suspensions of Escherichia coli ATCC 26 of compounds that absorb radiations having a wave length of 260 mμ, or of P32-containing compounds from cells grown in a medium containing radioactive phosphate. Basically the same procedure is used for both assays: The cells are exposed to the action of various concentrations of circulin in centrifugation tubes. After two hours the cells are centrifuged down, and an aliquot of the supernatant liquid is examined either for absorption at 260 mμ, or for radioactivity. Unknown amounts can be determined by a comparison of their effectiveness in causing release of cellular materials to that of known amounts. By adjusting the initial concentrations of cells, one can vary the range of circulin concentrations over which the assay can be used. This method is more sensitive and more rapid than the filter disk – agar diffusion method of assay.