Detection of cystic fibrosis delta F508 mutation by anti-double-stranded DNA antibody.
This study evaluated an enzyme immunoassay (EIA) as a screening tool for detection of the most common mutation (delta F508) in cystic fibrosis (CF). Guthrie card bloodspots were extracted to remove whole blood polymerase chain reaction (PCR) inhibitors. The washed filter paper was directly amplified under standard (98 bp amplicons) or modified PCR conditions (491 bp amplicons) for the delta F508 mutation. Monoclonal anti-double stranded deoxyribonucleic acid antibody was used to detect competent hybrid formation between PCR products and normal (N) and mutant (M) cDNA (deoxyribonucleic acid) probes coated to microtiter plate wells. Under standard conditions, mean relative color production (N/M) via an enzyme-linked horseradish peroxidase secondary antibody was 8.8, 0.6 and 0.04 for individuals normal, heterozygous and homozygous for the CF delta F508 mutation, respectively (n = 27). Comparison of EIA results to those obtained by tris-borate-EDTA/8 percent polyacrylamide gel electrophoresis (TBE-PAGE) yielded excellent correlation (100 percent) for all three genotypes (n = 27). In comparison to TBE-PAGE, the EIA was 5 to 10 fold more sensitive when serially diluted PCR samples were evaluated. Larger PCR products (491 bp amplicons) for the CF delta F508 mutation obtained under modified conditions were not resolved by TBE-PAGE. The EIA, however, demonstrated equal sensitivity to the 98 bp and 491 bp amplicons. Performance time for TBE-PAGE analysis was substantially shorter (25 percent) than the EIA (3.5 to 4 h and 4.5 to 5 h, respectively) when small batches of samples (n = 5) were analyzed. The TBE-PAGE was not, however, convenient for screening large numbers of PCR-amplified samples (n > 15).
Hopfer
S M
SM
Department of Laboratory Medicine, University of Connecticut School of Medicine, Farmington 06030, USA.
Makowski
G S
GS
Davis
E L
EL
Aslanzadeh
J
J
D000974:Antibodies, Antinuclear; D001483:Base Sequence; D003550:Cystic Fibrosis / Q000235:genetics*; D004247:DNA / Q000097:blood*; D015342:DNA Probes; D004591:Electrophoresis, Polyacrylamide Gel; D005374:Filtration; D005838:Genotype; D006801:Humans; D007124:Immunoenzyme Techniques; D008969:Molecular Sequence Data; D009154:Mutation; D010209:Paper; D016133:Polymerase Chain Reaction
D000974:Antibodies, Antinuclear; D015342:DNA Probes; D004247:DNA