Multiplex human papillomavirus serology based on in situ-purified glutathione s-transferase fusion proteins.
BACKGROUND
More than 100 different human papillomaviruses (HPVs) can cause proliferative diseases, many of which are malignant, such as cervical cancer. HPV serology is complex because infection and disease lead to distinct type-specific antibody responses. Using bead-based technology, we have developed an assay platform that allows the simultaneous detection of antibodies against up to 100 in situ affinity-purified recombinant HPV proteins.


METHODS
Twenty-seven HPV proteins were expressed as glutathione S-transferase fusion proteins and affinity-purified in one step by incubation of glutathione-displaying beads in bacterial lysate. Spectrally distinct bead sets, each carrying one particular antigen, were mixed, incubated with serum, and differentiated in a flow cytometer-like analyzer (xMAP; Luminex Corp). Antibodies bound to the antigens were detected via fluorescent secondary reagents. We studied 756 sera from 2 case-control studies of cervical cancer.


RESULTS
Glutathione S-transferase fusion proteins bound with high affinity to glutathione-displaying beads (Kd = 6.9 x 10(-9) mol/L). The dynamic range of multiplex serology covered 1.5 orders of magnitude, and antibodies were detected at serum dilutions >1:1,000,000. Imprecision (median CV) was < or = 5.4%, and assay reproducibility was high (R2 = 0.97). Results on clinical samples showed high concordance with ELISA (kappa = 0.846), but multiplex serology exhibited increased detection of weak antibody responses. Antibodies to the E6 oncoproteins of the rare HPV types 52 and 58 were associated with cervical cancer (P < 0.001).


CONCLUSIONS
Multiplex serology enables antibody analyses of large numbers of sera against up to 100 antigens in parallel and has the potential to replace ELISA technology.
Waterboer
Tim
T
Infection and Cancer Program, German Cancer Research Center (DKFZ), Heidelberg, Germany. t.waterboer@dkfz.de
Sehr
Peter
P
Michael
Kristina M
KM
Franceschi
Silvia
S
Nieland
John D
JD
Joos
Thomas O
TO
Templin
Markus F
MF
Pawlita
Michael
M
D000914:Antibodies, Viral / Q000097:blood*; D000956:Antigens, Viral / Q000737:chemistry / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005982:Glutathione Transferase / Q000737:chemistry*; D006801:Humans; D027383:Papillomaviridae / Q000276:immunology; D030361:Papillomavirus Infections / Q000097:blood / Q000175:diagnosis*; D040081:Protein Array Analysis / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000302:isolation & purification; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012698:Serologic Tests / Q000379:methods*; D012984:Software; D002583:Uterine Cervical Neoplasms / Q000097:blood / Q000175:diagnosis* / Q000821:virology
D000914:Antibodies, Viral; D000956:Antigens, Viral; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase