Microtiter format for simultaneous multianalyte detection and development of a PCR-chemiluminescent enzyme immunoassay for typing human papillomavirus DNAs.
BACKGROUND
To allow multianalyte binding assays, we have developed a novel polystyrene microtiter plate containing 24 main wells, each divided into 7 subwells. We explored its clinical potential by developing a PCR-chemiluminescent immunoassay (PCR-CLEIA) for simultaneous detection and typing of seven high oncogenic risk human papillomavirus (HPV) DNAs in one well.


METHODS
Seven different oligonucleotide probes, each specific for a high-risk HPV genotype, were separately immobilized in the subwells. Subsequently, a digoxigenin-labeled consensus PCR amplification product was added to the main well. The PCR product hybridized to the immobilized probe corresponding to its genotype and was subsequently detected by use of a peroxidase-labeled anti-digoxigenin antibody and chemiluminescence imaging with an ultrasensitive charge-coupled device camera. Results obtained for 50 cytologic samples were compared with those obtained with a conventional colorimetric PCR-ELISA.


RESULTS
The method was specific and allowed detection of 50 genome copies of HPV 16, 18, 33, and 58, and 100 genome copies of HPV 31, 35, and 45. Intra- and interassay CVs for the method were 5.6% and 7.9%, respectively. All results obtained for clinical samples were confirmed by the conventional PCR-ELISA.


CONCLUSIONS
PCR-CLEIA allows rapid, single-tube simultaneous detection and typing of seven high-risk HPV DNAs with small reagent volumes. The principle appears applicable to the development of other single-tube panels of tests.
D004279:DNA, Viral / Q000032:analysis*; D005260:Female; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods; D008163:Luminescent Measurements; D027383:Papillomaviridae / Q000235:genetics*; D030361:Papillomavirus Infections / Q000175:diagnosis; D016133:Polymerase Chain Reaction; D011137:Polystyrenes; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured; D014412:Tumor Virus Infections / Q000175:diagnosis; D014773:Virology / Q000379:methods
D004279:DNA, Viral; D011137:Polystyrenes