Clinical Chemistry
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Clinical Chemistry 48: 1654-1660, 2002;
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(Clinical Chemistry. 2002;48:1654-1660.)
© 2002 American Association for Clinical Chemistry, Inc.

Microtiter Format for Simultaneous Multianalyte Detection and Development of a PCR-Chemiluminescent Enzyme Immunoassay for Typing Human Papillomavirus DNAs

Aldo Roda1a, Mara Mirasoli1, Simona Venturoli2, Monica Cricca2, Francesca Bonvicini2, Mario Baraldini3, Patrizia Pasini1, Marialuisa Zerbini2 and Monica Musiani2

1 Department of Pharmaceutical Sciences;
2 Department of Clinical and Experimental Medicine, Division of Microbiology; and
3 Institute of Chemical Sciences, University of Bologna, 40126 Bologna, Italy.

aAddress correspondence to this author at: Department of Pharmaceutical Sciences, University of Bologna, via Belmeloro 6, 40126 Bologna, Italy. Fax 39-051-343398; e-mail roda{at}alma.unibo.it.

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.




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An innovative, flow-assisted, noncompetitive chemiluminescent immunoassay for the detection of pathogenic bacteria.
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J. Wallace, B. A. Woda, and G. Pihan
Facile, Comprehensive, High-Throughput Genotyping of Human Genital Papillomaviruses Using Spectrally Addressable Liquid Bead Microarrays
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