Conjugation of antibodies with fluorochromes: modifications to the standard methods

Abstract

A number of modification to the standard procedures for coupling of fluorochromes to antibodies are described. The suggested procedures result in economies of time, labour and materials, and allow the reliable production of high quality conjugates. The modifications include the use of staphylococcal protein A-Sepharose for (a) a simple one-step procedure for preparation of IgG from whole serum, (b) removal of undigested IgG after pepsin treatment, and (c) concentration of dilute solutions of IgG. Many other details of coupling procedures are discussed, and modifications suggested. Optimally coupled antibodies were separated by linear salt gradient elution from DEAESephadex, and the effects of fluorescein and tetramethyl rhodamine on the antibody isoelectric point studied.