Amplified direct immunofluorescence (AMDI) for detection of Epstein-Barr virus nuclear antigen

Low antigen concentrations could be identified in human cells by sequential application of (a) FITC-labeled human antibodies at high dilution, (b) rabbit antiserum to the hapten fluorescein isothiocyanate (FITC), and (c) FITC-anti-rabbit globulin. The high specificity of direct immunofluorescence (a) was not affected by the amplifying steps (b) and (c). Using this AMDI technique Epstein-Barr (EB) virus nuclear antigen (EBNA) could be specifically stained with human sera up to a dilution of 1 : 4000. Owing to the high dilutions applied, unwanted antibody reactivity in the FITC-labeled serum could be blocked by preincubating with unlabeled undiluted human sera. Thus EBNA was selectively stained in EB virus producer cells. Moreover, EBNA was specifically detected in human tumor biopsy material by the use of AMDI.