Chemiluminescent immunoperoxidase assay for the dot blot hybridization detection of Parvovirus B19 DNA using a low light imaging device.
A new synthesized stable trifluoro-substituted acridan (2',3',6'-trifluorophenyl 10-methylacridan-9-carboxylate known as Lumigen PS-3) has been applied as chemiluminescent substrate of the horseradish peroxidase (HRP) enzyme (neutral isoenzyme C) in a dot blot hybridization assay for the detection of B19 Parvovirus DNA. The dot blot hybridization assay uses digoxigenin-labeled DNA probes which are immunoenzymatically revealed by anti-digoxigenin Fab fragments conjugated with HRP. The results obtained using PS-3 reagent or the luminol-based enhanced chemiluminescence detection system (ECL Amersham and Renaissance DuPont NEN kits) were compared. A high-performance, low-intensity-light imaging luminograph apparatus to collect light emission was used. The detection systems using the different chemiluminescent substrates gave sensitive and reproducible results for calibration graphs, with high precision (relative standard deviation 5-18%). With the chemiluminescent assay it was possible to detect 0.5, 1, or 2 pg of target homologous DNA, using PS-3, ECL, or Renaissance (RE) reagents, respectively, while colorimetry had a detection limit of 5 pg. When clinical samples were analyzed the positive reference sera and the PCR-positive products gave light emissions with values higher than background at 2 sigma level, while the negative samples gave a signal comparable to the background noise for all chemiluminescent reagents. The PS-3 reagent detected one more dilution (1/256) than ECL and RE (1/128) of positive reference sera.
Girotti
S
S
Institute of Chemical Sciences, University of Bologna, Italy.
Musiani
M
M
Ferri
E
E
Gallinella
G
G
Zerbini
M
M
Roda
A
A
Gentilomi
G
G
Venturoli
S
S
D004279:DNA, Viral / Q000032:analysis*; D006735:Horseradish Peroxidase; D006801:Humans; D007124:Immunoenzyme Techniques; D008163:Luminescent Measurements; D009693:Nucleic Acid Hybridization; D016732:Parvovirus B19, Human / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D004279:DNA, Viral; D006735:Horseradish Peroxidase