Serum samples were diluted 1:200 (human) or 1:100 (P. leucopus) in Protein Array Blocking (PAB) buffer (Whatman Inc, Sanford, ME) supplemented with 10% (vol/vol) DH5alpha E. coli lysate (MCLAB, San Francisco, CA). This suspension was incubated for 30 min at 22°C, with gentle constant shaking. Arrays were re-hydrated with PAB buffer for 30 min at 22°C. Primary antibody was placed on re-hydrated array pads and incubated for 12 h at 4°C on a rocker platform (Bellco, Vineland, NJ). Pads were rinsed 6 times, followed by three 5-minute washes in Tween-TBS. Cy3-conjugated secondary antibody, goat anti-human IgG heavy and light chain or goat anti-Peromyscus leucopus IgG heavy and light chain (KPL, Gaithersburg, MD), were diluted 1:200 in PAB buffer, and incubated with pads in the dark for 1 h at 22°C on a rocking platform. Arrays were washed in Tween-TBS as described above and this process repeated with TBS alone.
Scan protocol
Slides were dismounted from their frames, rinsed in double-distilled H2O and centrifuged 20 min at 500 x g to dry. Probed array slides were scanned in a Perkin Elmer ScanArray Express HT at a wavelength of 670 nm, at 95% laser power and 55% PMT. The output RGB TIFF files generated by the scanner were quantitated using ProScanArray Express software (Perkin Elmer, Waltham, MA) with spot-specific background correction.
Data processing
For analysis of antibody binding to OspC proteins on the microarray the following steps were taken: (i) raw values of antibody binding measured as the mean pixel intensity of spots of printed protein were log10-transformed; raw values less than 1.0 were set to 0; (ii) the mean, standard deviation, 95% confidence intervals and z-scores of antibody binding intensity to each OspC type were calculated for the human LD patient, P. leucopus and respective control sera groups. Inclusion criterion for cross-reactivity analysis was a minimum reactivity corresponding to a z score of 2 to at least one OspC protein.
Inferring Epitopes of a Polymorphic Antigen Amidst Broadly Cross-Reactive Antibodies Using Protein Microarrays: a Study of OspC Proteins of Borrelia burgdorferi
Data table header descriptions
ID_REF
VALUE
Array signal intensity after log10-transformation.
