Imagequant software version 8

For peptide ELISPOT the following peptides were designed:
peptide #1 - RVLAPALDSWGTGGGDYKDDD{LYS(BIOTIN)} (Genescript)
peptide #2 - LPKFSAPSASGPGGGDYKDDD{LYS(BIOTIN)} (Genescript)
peptide #3 - ESTRYQLWLPHQGGGDYKDDD{LYS(BIOTIN)} (Genescript)
control peptide - AVLAAALASWGTGGGDYKDDD{LYS(BIOTIN)} (Genescript)
In brief, 110 pg biotin-conjugated peptides were printed on nitrocelluose coated slides (10485323, Whatman) by SpotBot® 4 (Arrayit). For primary antibody human precleared serum (1:100) was used, for secondary antibody rabbit anti-human IgG (H&L) (HRP) (Abcam) was used. All incubations were done for 1 h at room temperature. Results were scanned using Ettan DigeImager (GE Healthcare Life Sciences) and images calculated using ImageQuant software version 8.1 (GE Healthcare Life Sciences).

M13 phage particles displaying the relevant peptides were printed onto nitrocellulose coated glass slides (10,485,323, Whatman) using SpotBot® 4 arrayer (Arrayit) in four dilutions (1:2, 1:10, 1:100, 1:1000). 1xPBS-20% glycerol and mQ were used as negative controls and human IgG serial dilutions (12–50 ng/µl) as positive controls. Human serum or plasma (1:50), previously precleared to plastic and E. coli/wild type M13 phage antigens, was used as primary antibody while rabbit anti-human IgG H&L (HRP) (#ab6759, Abcam, RRID: AB_955,434) (1:1000) was used as a secondary antibody. Both antibody incubations were conducted at room temperature for 1 h with agitation. The presence of bound human IgG antibodies was detected using reaction with catalyzed Signal Amplification (CSA) System II, Biotin-Free, HRP, DAB+ (Dako, Agilent, Cat#: K1497), diluted in the substrate buffer (1:100). Results were visualised using Ettan DigeImager (GE Healthcare Life Sciences) and signals (image colour-intensities) quantified using ImageQuant software version 8.1 (GE Healthcare Life Sciences).