A comparison of chemiluminescent and colorimetric substrates in a hepatitis B virus DNA hybridization assay.
We compared the sensitivity of a chemiluminescent substrate 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl- 1,2-dioxetane (AMPPD) and a chromogenic substrate 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT) for detection of an alkaline phosphatase label in a hepatitis B virus "core antigen" DNA (HBVc) probe hybridization assay. Chemiluminescent signal obtained from AMPPD hydrolysis by alkaline phosphatase was detected with Polaroid Instant Black and White Type 612 film. The chemiluminescent assay detected 1.18 x 10(6) copies of HBVc plasmid DNA in 30 min. By comparison, 9.8 x 10(7) copies of DNA could be measured using chromogenic BCIP/NBT substrate within the same incubation time. After further development, the chemiluminescent endpoint permitted detection of 4.39 x 10(4) copies of HBVc plasmid DNA in 2 h.
Bronstein
I
I
Tropix, Inc., Bedford, Massachusetts 01730.
Voyta
J C
JC
Edwards
B
B
D000218:Adamantane / Q000031:analogs & derivatives; D000469:Alkaline Phosphatase; D003124:Colorimetry; D015342:DNA Probes; D004279:DNA, Viral / Q000032:analysis*; D006512:Hepatitis B Core Antigens / Q000235:genetics*; D006515:Hepatitis B virus / Q000235:genetics* / Q000276:immunology; D007211:Indoles; D008163:Luminescent Measurements; D009580:Nitroblue Tetrazolium; D009693:Nucleic Acid Hybridization; D010957:Plasmids
D015342:DNA Probes; D004279:DNA, Viral; D006512:Hepatitis B Core Antigens; D007211:Indoles; C061523:3-(2'-spiroadamantane)-4-methoxy-4-(3''-phosphoryloxy)phenyl-1,2-dioxetane; D009580:Nitroblue Tetrazolium; C035455:5-bromo-4-chloro-3-indoxyl phosphate; D000469:Alkaline Phosphatase; D000218:Adamantane