Detection
of serum Abs on NAPPA arrays was performed as described.16 (link) Plasmid DNA (1.5 μg/mL), capture antibody
(50 μg/mL anti-GST antibody, GE Healthcare Biosciences, Piscataway,
NJ) or anti-FLAG antibody (Sigma-Aldrich, St. Louis, MO), protein
cross-linker (2 mM, BS3, Pierce, Rockford, IL), and BSA (3 mg/mL,
Sigma-Aldrich) were co-printed onto the array surface. All samples
were printed using a Genetix QArray2 with 300 μm solid tungsten
pins on amine-treated glass slides. The printed DNA was transcribed
and translated in situ using reticulocyte lysate according to previously
published protocols.14 (link) Protein expression
was detected using anti-GST mAb (Cell Signaling, Danvers, MA) diluted
at 1:200. For detecting serum antibodies, the arrays were incubated
with serum diluted 1:250–1:600 in 5% PBS milk with 0.2% Tween
20 overnight and detected with anti-human IgG-HRP (Jackson ImmunoResearch
Laboratories, West Grove, PA) with Tyramide (PerkinElmer, Waltham,
MA). Slides were scanned with a PerkinElmer ProScanArray HT. The highly
immunogenic EBV-derived antigen, EBNA-1, was included as N- and C-terminal
fragments for positive control antigens. Negative controls included
empty vectors and no DNA controls. Registration spots for array alignment
were printed with purified human IgG proteins.
of serum Abs on NAPPA arrays was performed as described.16 (link) Plasmid DNA (1.5 μg/mL), capture antibody
(50 μg/mL anti-GST antibody, GE Healthcare Biosciences, Piscataway,
NJ) or anti-FLAG antibody (Sigma-Aldrich, St. Louis, MO), protein
cross-linker (2 mM, BS3, Pierce, Rockford, IL), and BSA (3 mg/mL,
Sigma-Aldrich) were co-printed onto the array surface. All samples
were printed using a Genetix QArray2 with 300 μm solid tungsten
pins on amine-treated glass slides. The printed DNA was transcribed
and translated in situ using reticulocyte lysate according to previously
published protocols.14 (link) Protein expression
was detected using anti-GST mAb (Cell Signaling, Danvers, MA) diluted
at 1:200. For detecting serum antibodies, the arrays were incubated
with serum diluted 1:250–1:600 in 5% PBS milk with 0.2% Tween
20 overnight and detected with anti-human IgG-HRP (Jackson ImmunoResearch
Laboratories, West Grove, PA) with Tyramide (PerkinElmer, Waltham,
MA). Slides were scanned with a PerkinElmer ProScanArray HT. The highly
immunogenic EBV-derived antigen, EBNA-1, was included as N- and C-terminal
fragments for positive control antigens. Negative controls included
empty vectors and no DNA controls. Registration spots for array alignment
were printed with purified human IgG proteins.