The effect of changing single radial hemolysis assay method when quantifying influenza A antibodies in serum

Single radial hemolysis (SRH) is a simple method for measuring concentrations of antibody specific for influenza A viruses. There are many differences in reported assay methods and these changes may significantly affect results. This study evaluated the effect of changing erythrocyte concentrations, influenza virus concentrations used to sensitize erythrocytes, incubation times, and incubation temperatures. These factors were shown to significantly affect results of the SRH assay. A method for standardizing the assay and expressing results as relative antibody concentrations is described. Standardized relative antibody concentrations show significantly less variability than do hemolysis zone measurements when the SRH assay method is changed. This paper demonstrates that use of this method would allow for more consistent results to be obtained within laboratories. Use of this method with quantified, internationally standardized sera would allow results of the single radial hemolysis assay to be directly compared between laboratories.