Optimal fluorescein-to-protein ratios of bacterial direct fluorescent-antibody reagents

A number of bacterial systems were studied with specific direct fluorescent-antibody reagents prepared from rabbit antiserum fractions and having a wide range of fluorescein-to-protein ratios. These systems included Bacteroides, Bordetella, Clostridium, Escherichia, Legionella, Listeria, Salmonella, Shigella, and Streptococcus. For all systems studied, a fluorescein-to-protein ratio of 30 was optimal for conjugates prepared from ammonium sulfate fractions (greater than 75% gamma globulin) and pure immunoglobulin G desorbed from the Sepharose-bound protein A of Staphylococcus aureus. A pepsin digestion procedure is described that yielded the F(ab')2 piece of pure immunoglobulin G; this was labeled and studied at two fluorescein-to-protein ratios.