Fast solid support detection of human papillomavirus in in vitro amplified DNA using a DNP-anti DNP monoclonal antibody couple.
In some anogenital lesions the detection of certain types of human papilloma virus, especially oncogenic types, is of interest. In a first step during a prospective study, we compared two methods for the detection of human papillomavirus (HPV) DNA in clinical samples: Southern blotting followed by hybridization with a cloned radioactive genomic probe and a classical polymerase chain reaction (PCR) followed by hybridization with a 32P-labelled oligonucleotide probe. 118 biopsies and swabs were examined for HPV 6/11, 16, 18 and 33, 67 positive reactions were found by both methods, 5 positives only by PCR and 2 positives only by Southern blot for unidentified HPV. Patients with anogenital condylomas, dysplasias and carcinomas or asymptomatic patients were studied. Most high grade (II and III) dysplasias were associated with HPV 16 and HPV 18. Condylomata lesions and low grade dysplasia (grade I) were associated mostly with HPV 6/11, mixed type of HPV, less frequently with HPV 16 or HPV 18. As a second step a nested PCR coupled to solid support detection method was used as described by Sauvaigo et al. (1990) Nucleic Acids Res. 18, 3175-3183) to study a panel of 30 previously qualified different HPV DNA extracts. In this procedure the second round of PCR amplification involves biotinylated and dinitrophenylated labelled primers allowing the capture of PCR amplified HPV DNA sequences on streptavidin coated tubes and its revelation. We describe an improvement of HPV DNA detection by means of single-step immunoenzymatic revelation involving anti-DNP monoclonal antibodies conjugated to horseradish peroxidase enzyme. A perfect correlation with the previous results was obtained. This solid support method allows a faster and easier HPV typing compared to methods using membrane transfer.
Baccard-Longère
M
M
Service de Virologie, C.H.U. de Grenoble, France.
Alpha-Bazin
B
B
Chypre
C
C
Sauvaigo
S
S
Téoule
R
R
Bernard
P
P
Seigneurin
J M
JM
D000911:Antibodies, Monoclonal / Q000276:immunology*; D001005:Anus Neoplasms / Q000821:virology*; D001426:Bacterial Proteins; D001483:Base Sequence; D001710:Biotin; D015139:Blotting, Southern; D003218:Condylomata Acuminata / Q000821:virology*; D015344:DNA Probes, HPV; D004279:DNA, Viral / Q000032:analysis*; D004140:Dinitrophenols / Q000276:immunology*; D005260:Female; D005833:Genital Neoplasms, Female / Q000821:virology*; D006801:Humans; D007124:Immunoenzyme Techniques; D007163:Immunosorbent Techniques; D008297:Male; D008969:Molecular Sequence Data; D027383:Papillomaviridae / Q000145:classification / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity; D030361:Papillomavirus Infections / Q000821:virology*; D010412:Penile Neoplasms / Q000821:virology*; D016133:Polymerase Chain Reaction; D011446:Prospective Studies; D019809:Streptavidin; D014412:Tumor Virus Infections / Q000821:virology*; D002578:Uterine Cervical Dysplasia / Q000821:virology*
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D015344:DNA Probes, HPV; D004279:DNA, Viral; D004140:Dinitrophenols; D001710:Biotin; D019809:Streptavidin