A quantitative complement fixation test with influenza immune sera and virus antigens obtained from allantoic fluid is described. The method utilizes a photoelectric densitometer which provides a simple, objective, and accurate determination of the hemolytic reaction. The enhancement of the hemolytic activity of complement in the presence of serum or allantoic fluid necessitates a preliminary titration of complement in the presence of these agents. An accurate appraisal of the activity of the complement under the conditions of the actual test permits the selection of an optimal amount of complement and greatly increases the sensitivity of the test. The substance (or substances) responsible for the enhanced hemolytic activity of complement has been found in human and many animal sera and in allantoic fluids obtained from the developing chick embryo. It requires the presence of both complement and hemolysin, resists heating at 100°C. for 2 hours, and is dialyzable.
Allantoic fluid or mouse lung preparations of influenza virus contain a complement-fixing antigen which is intimately associated with the virus particle. It sediments in the high speed centrifuge at the same rate as the hemagglutinin and infective particle and, like the latter, is adsorbed by fowl red blood cells and eluted from the cells on standing at room temperature or 37°C. It cannot be separated from the virus particle by repeated washings in the centrifuge or repeated adsorptions with red blood cells; the hemagglutinin and complement-fixing antigen titers remain roughly proportional. This antigen shows a high degree of strain specificity in cross complement fixation tests with PR8, W.S., and swine ferret antisera, and, as found with the neutralization test, it shows little or no strain specificity with human sera. A soluble antigen is also present in influenza virus preparations which can be readily separated from the virus particle by centrifugation. It is not adsorbed by red blood cells. Furthermore, it reacts in lower titer with ferret antisera and usually shows less strain specificity in cross complement fixation tests. In general, allantoic fluid virus preparations contain much less of the soluble antigen than mouse lung extracts.