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A detailed study has been made of the nature of the variables inherent in the chicken red cell agglutination test for influenza virus in an effort to obtain a method of measurement of biological activity of sufficient accuracy that it might be employed as a reliable index of chemical purity of preparations of the virus. It was found that the temperature at which the test is conducted has a marked effect on the titer, whereas within the range of pH 6–8 the pH has a negligible effect. It was also found that a variation in results may be encountered due to a variation in the specific behavior of red cells from different chickens and to an instability of the red cells themselves. Preparations of purified influenza virus held at 4°C., on the other hand, were found to be stable with respect to chicken red cell agglutinating activity for several months. This fact, together with the fact that in duplicate measurements upon different samples the accuracy was such that the chances were 19 out of 20 that differences of 8.4 per cent in the mean end points were significant, made it possible to establish a reproducible standard of CCA activity based on a unit weight of purified virus material. As a result, it was possible to devise a standardized procedure for carrying out with high accuracy quantitative measurements of influenza virus.
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