Comparison of immunologic amplification vs enzymatic deposition of fluorochrome-conjugated tyramide as detection...

Using various sizes and dilutions of hapten-conjugated DNA probes, we compared catalyzed reporter deposition (CARD) to fluorochrome-conjugated antibody layering (immunological method) for amplifying FISH signals. Cosmid and phage probes that contained human DNA inserts of 40 KB and 15 KB, respectively, and were mapped to chromosome 15q11.2 were used to evaluate these amplification methods. The probes were used either at standard concentrations (10 ng/microliter) or at dilutions up to 1:40 (0.25 ng/microliter). Detection of FISH signals using either immunological (three antibody layers) or CARD methods were comparable when the undiluted (10 ng/microliter) or 1:4 dilution (2.5 ng/microliter) of the cosmid probe was used. Use of a single fluorochrome-conjugated antibody layer produced very weak FISH signals. However, addition of an unlabeled secondary antibody followed by a third antibody conjugated to the same fluorochrome (i.e., two rounds of amplification) produced a strong signal that was detected at a 1:4 probe dilution but was not successfully detected at probe dilutions of 1:10 or greater. In contrast, intense probe signals were produced with the CARD method at all probe dilutions, particularly when coupled to extended hapten-antibody incubation times. The 15-KB phage probe was difficult to detect at a 1:4 dilution with the standard immunologic amplification methods but was readily detected with the CARD method. These data suggest that CARD may be useful for FISH in that (a) less probe may be needed and therefore valuable probe reagents may be conserved, and (b) smaller targets may be detected, thus extending the range of this technique.