Biotinylated anti-human IgG (Jackson) was used as secondary antibody followed after washing in T-TBS by streptavidin-PBXL3 conjugate (Martek Biosciences), both at 1/200 in blocking buffer.
Hybridization protocol
Human sera were used at 1/100 dilution in protein array blocking buffer (Whatman) containing E. coli lysate (Antigen Discovery Inc, Irvine CA) to a final concentration of 10mg/ml protein to block anti-E. coli antibodies. Sera were preincubated at RT for 30 minutes with constant mixing prior to applying to the arrays in triplicate and incubating overnight at 4oC with gentle agitation. After washing in TBS + 0.05% Tween 20% (T-TBS), biotinylated anti-human IgG (Jackson) was used as secondary antibody followed after washing in T-TBS by streptavidin-PBXL3 conjugate (Martek Biosciences), both at 1/200 in blocking buffer.
Scan protocol
After washing five times, slides were air dried by brief centrifugation and then scanned and analyzed using a Perkin Elmer ScanArray Express HT microarray scanner (PerkinElmer). Fluorescence intensities were quantified from tiff images by using ProScanArray Express software (PerkinElmer).
Description
References: Baldi, P., and Hatfield, G.W. (2002). DNA Microarrays and Gene Expression: From Experiments to Data Analysis and Modeling. Cambridge University Press Baldi, P., and Long, A.D. (2001). A Bayesian framework for the analysis of microarray expression data: regularized t -test and statistical inferences of gene changes. Bioinformatics 17, 509-519. Benjamini, Y., and Hochberg, Y. (1995). Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. Roy. Statist. Soc. Ser. B 57, 289--300. Hermanson, G., Chun, S., Felgner, J., Tan, X., Pablo, J., Nakajima-Sasaki, R., Molina, D.M., Felgner, P.L., Liang, X., and Davies, D.H. (2011). Measurement of antibody responses to Modified Vaccinia virus Ankara (MVA) and Dryvax((R)) using proteome microarrays and development of recombinant protein ELISAs. Vaccine 30, 614-625. Long, A.D., Mangalam, H.J., Chan, B.Y., Tolleri, L., Hatfield, G.W., and Baldi, P. (2001). Improved statistical inference from DNA microarray data using analysis of variance and a Bayesian statistical framework. Analysis of global gene expression in Escherichia coli K12. J Biol Chem 276, 19937-19944.
Data processing
Data analysis was performed using the R (http://www.r-project.org) statistical software. In order to stabilize the variance observed in microarray signals, the vsn normalization was implemented as part of the Bioconductor suite (www.bioconductor.org) to the quantified array intensities using the 'no DNA' spots (IVTT reactions lacking the template DNA) as normalization controls. Inter-group comparisons were performed using a Bayes regularized t-test adapted from Cyber-T for protein arrays (Baldi and Hatfield, 2002; Baldi and Long, 2001). To account for multiple comparison conditions, p-values were corrected by the Benjamini and Hochberg (pBH) method to control the false discovery rate (Benjamini and Hochberg, 1995). After correction, p-values < 0.05 were considered significant. An individual was defined as a responder if the ratio of retransformed signal intensities before and after DVX vaccination (d28/d0) was >2.0 for 2 or more of 6 signature antigens (WR101/H3L, WR113/D8L, WR148, WR150/A27L, WR070/I1L, and WR118/D13L). Seroprevalence rates on d0 and d28 were determined for each antigen based on a cut off defined as the averaged+2SD of the naïve take population (n=50). Individuals positive for one or more of the 6 signature antigens were considered seropositive.
