Profiling the Humoral Immune Response to Infection by Using Proteome Microarrays: High-Throughput Vaccine and Diagnostic Antigen Discovery
Despite the increasing availability of genome sequences from many human pathogens, the production of complete proteomes remains at a bottleneck. To address this need, a high-throughput PCR recombination cloning and expression platform has been developed that allows hundreds of genes to be batch-proc...
Saved in:
| Published in: | Proceedings of the National Academy of Sciences - PNAS Vol. 102; no. 3; pp. 547 - 552 |
|---|---|
| Main Authors: | , , , , , , , , , , , , , |
| Format: | Journal Article |
| Language: | English |
| Published: |
United States
National Academy of Sciences
18-01-2005
National Acad Sciences |
| Subjects: | |
| Online Access: | Get full text |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| Summary: | Despite the increasing availability of genome sequences from many human pathogens, the production of complete proteomes remains at a bottleneck. To address this need, a high-throughput PCR recombination cloning and expression platform has been developed that allows hundreds of genes to be batch-processed by using ordinary laboratory procedures without robotics. The method relies on high-throughput amplification of each predicted ORF by using gene specific primers, followed by in vivo homologous recombination into a T7 expression vector. The proteins are expressed in an Escherichia coli-based cell-free in vitro transcription/translation system, and the crude reactions containing expressed proteins are printed directly onto nitrocellulose microarrays without purification. The protein microarrays are useful for determining the complete antigen-specific humoral immune-response profile from vaccinated or infected humans and animals. The system was verified by cloning, expressing, and printing a vaccinia virus proteome consisting of 185 individual viral proteins. The chips were used to determine Ab profiles in serum from vaccinia virus-immunized humans, primates, and mice. Human serum has high titers of anti-E. coli Abs that require blocking to unmask vaccinia-specific responses. Naïve humans exhibit reactivity against a subset of 13 antigens that were not associated with vaccinia immunization. Naïve mice and primates lacked this background reactivity. The specific profiles between the three species differed, although a common subset of antigens was reactive after vaccinia immunization. These results verify this platform as a rapid way to comprehensively scan humoral immunity from vaccinated or infected humans and animals. |
|---|---|
| Bibliography: | ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 ObjectType-Article-1 ObjectType-Feature-2 To whom correspondence should be addressed at: Center for Virus Research, 3221 McGaugh Hall, University of California, Irvine, CA 92697. E-mail: pfelgner@uci.edu. Communicated by Masayasu Nomura, University of California, Irvine, CA, December 2, 2004 Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. AY871843–AY872187). Author contributions: D.H.D., X.L., P.B., L.P.V., and P.L.F. designed research; D.H.D., X.L., J.E.H., S.H., Y.M., K.M.R., T.T.N., M.K.-D., and S.C. performed research; A.R. and P.B. contributed new reagents/analytic tools; D.H.D., X.L., J.E.H., S.C., and P.L.F. analyzed data; and D.H.D., X.L., and P.L.F. wrote the paper. Abbreviations: HA, hemagglutinin; VIG, vaccinia immune globulin. |
| ISSN: | 0027-8424 1091-6490 |
| DOI: | 10.1073/pnas.0408782102 |