Open AccessCandidate Antigens for Q Fever Serodiagnosis Revealed by Immunoscreening of a Coxiella burnetii Protein Microarray
Author(s) -
Paul A. Beare,
Chen Chen,
Timo Bouman,
Jozelyn Pablo,
Berkay Unal,
Diane C. Cockrell,
Wendy C. Brown,
Kent Barbian,
Stephen F. Porcella,
James E. Samuel,
Philip L. Felgner,
Robert A. Heinzen
Publication year - 2008
Publication title -
clinical and vaccine immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.649
H-Index - 77
eISSN - 1556-6811
pISSN - 1556-679X
DOI - 10.1128/cvi.00300-08
Subject(s) - immunoscreening , coxiella burnetii , q fever , antigen , virology , biology , protein microarray , microarray , microbiology and biotechnology , immunology , gene , genetics , peptide sequence , gene expression , cdna library
Q fever is a widespread zoonosis caused byCoxiella burnetii . Diagnosis of Q fever is usually based on serological testing of patient serum. The diagnostic antigen of test kits is formalin-fixed phase I and phase II organisms of the Nine Mile reference strain. Deficiencies of this antigen include (i) potential for cross-reactivity with other pathogens; (ii) an inability to distinguish betweenC. burnetii strains; and (iii) a need to propagate and purifyC. burnetii , a difficult and potentially hazardous process. Consequently, there is a need for sensitive and specific serodiagnostic tests utilizing defined antigens, such as recombinantC. burnetii protein(s). Here we describe the use of aC. burnetii protein microarray to comprehensively identify immunodominant antigens recognized by antibody in the context of humanC. burnetii infection or vaccination. Transcriptionally active PCR products corresponding to 1,988C. burnetii open reading frames (ORFs) were generated. Full-length proteins were successfully synthesized from 75% of the ORFs by using anEscherichia coli -based in vitro transcription and translation system (IVTT). Nitrocellulose microarrays were spotted with crude IVTT lysates and probed with sera from acute Q fever patients and individuals vaccinated with Q-Vax. Immune sera strongly reacted with approximately 50C. burnetii proteins, including previously identified immunogens, an ankyrin repeat-domain containing protein, and multiple hypothetical proteins. Recombinant protein corresponding to selected array-reactive antigens was generated, and the immunoreactivity was confirmed by enzyme-linked immunosorbent assay. This sensitive and high-throughput method for identifying immunoreactiveC. burnetii proteins will aid in the development of Q fever serodiagnostic tests based on recombinant antigen.