High-throughput polymerase chain reaction chemiluminescent enzyme immunoassay for typing and quantifying human papillomavirus DNAs Simone Ambretti a , Mara Mirasoli b , Simona Venturoli a , Marialuisa Zerbini a , Mario Baraldini c , Monica Musiani a , Aldo Roda b, * a Department of Clinical and Experimental Medicine, Division of Microbiology, University of Bologna, 40138 Bologna, Italy b Department of Pharmaceutical Sciences, University of Bologna, 40125 Bologna, Italy c Institute of Chemical Sciences, University of Bologna, 40127 Bologna, Italy Received 18 March 2004 Available online 21 July 2004 Abstract A miniaturized polymerase chain reaction (PCR) chemiluminescent enzyme immunoassay (CLEIA) based on a 384-well microt- iter plate for detection and typing of oncogenic high- and low-risk human papillomavirus (HPV) in genital lesions is described. The assay relies on PCR consensus amplification, hybridization of the digoxigenin-labeled product by means of type-specific biotin-la- beled oligoprobes immobilized on the streptavidin-coated wells of a 384-well microtiter plate, and quantification by means of a horseradish peroxidase-labeled antidigoxigenin antibody and chemiluminescence detection. The method provides semiquantitative information on the viral load, with a limit of detection of 10–50 DNA copies for HPV 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 54, 58, and 59 and high reproducibility (intraassay CV 7.5%, interassay CV 9.5%). Results obtained on 60 clinical samples were con- cordant with those obtained with a conventional PCR-enzyme-linked immunosorbent assay colorimetric assay. The 384 PCR- CLEIA method, which is amenable to automation, represents a fast and high-throughput method for detecting and typing HPV DNAs in screening programs and evaluating the viral load. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Human papillomavirus; PCR CLEIA; Chemiluminescence; Viral load; 384-Well microtiter plates Epidemiologic and molecular biological studies have shown that human papillomavirus (HPV) 1 infection is associated with the development of cervical carcinoma, with different degrees of risk. Based on these studies, various HPV genotypes have been classified as high or low oncogenic risk [1,2]. Moreover, various studies sug- gest that a high viral load, especially of high-risk geno- types, is a predictor for development of high-grade cervical lesions [3–5]. The involvement of HPVs in cervi- cal and many other types of carcinoma stress the need of rapid and miniaturized tests to be employed in screening programs. Since HPVs cannot be propagated in cell cultures and only very small amounts of viral antigens are present in body fluids [6], the diagnosis of HPV infection mainly re- lies on HPV nucleic acid detection [7]. The polymerase chain reaction (PCR) represents a valid tool for the diag- nosis of HPV-related diseases [8] and several hybrid cap- ture-immunoassays have been developed for the detection and typing of PCR products [9–11]. In the present study we have used a 384-well microtiter plate format for the 0003-2697/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2004.06.004 * Corresponding author. Fax: +39-051-343398. E-mail address: aldo.roda@unibo.it (A. Roda). 1 Abbreviation used: HPV, human papillomavirus; 384 PCR- CLEIA, 384-well polymerase chain reaction chemiluminescent enzyme immunoassay; HRP, horseradish peroxidase; ELISA, enzyme-linked immunosorbent assay; LSIL, low-grade intraepithelial lesion; HSIL, high-grade intraepithelial lesion; CL, chemiluminescence. www.elsevier.com/locate/yabio Analytical Biochemistry 332 (2004) 349–357 ANALYTICAL BIOCHEMISTRY