Genepix pro 5.0 program

Recombinant human IgG1 mAbs were screened for binding on protein microarrays (ProtoArray) (catalog no. PAH0525101; Invitrogen) precoated with >9,400 human proteins in duplicate. The binding patterns of human anti-HA bnAbs were compared to the human myeloma protein 151 K in lot-matched arrays. Array-bound anti-human IgG served as the loading control for the detection Ab, and array-bound human IgG served as the loading control for the secondary reagent. Abs were screened for reactive antigens on protein microarrays following the manufacturer’s instructions and as described previously^{16 (link)}. The ProtoArray microarray (Invitrogen) was blocked and incubated on ice with 2 μg/ml of HA mAb or isotype control 151 K for 90 min. Ab binding to array protein was detected with 1 μg/ml of Alexa Fluor 647-labeled anti-human IgG secondary Ab (Invitrogen). The ProtoArray microarrays were scanned using a GenePix 4000B scanner (Molecular Devices) at 635 nm, with 10-μm resolution. Fluorescence intensities were quantified with GenePix Pro 5.0 program (Molecular Devices) using lot-specific protein location information provided by the microarray manufacturer.

Binding of recombinant CH103 Ab mutants to 9,400 human proteins was determined using a ProtoArray (Invitrogen, Waltham, MA) in duplicate, according to the manufacturer instructions and as described previously (13 (link), 23 (link), 50 (link), 51 (link)). In brief, protein microarrays were blocked with 2 µg/ml of recombinant Abs or 151K, an isotype-matched (IgG1, κ) human myeloma control Ab (Southern Biotech) for 1.5 hours at 4°C. Ab binding to array proteins was detected using 1 µg/ml of Alexa Fluor 647-conjugated anti-human IgG (Invitrogen), using mild agitation at 4°C for 1.5 hours. Microarrays were scanned using a GenePix 4000B scanner (Molecular Devices, San Jose, CA). Fluorescence intensities of Ab binding to each protein were quantified by aligning image data with the GenePix Pro 5.0 program (Molecular Devices) using lot-specific protein spot definitions provided by the manufacturer, and by comparing Ab-binding patterns to the 151K control.

MAbs were screened for binding on protein microarrays (ProtoArray) (PAH0525101; Invitrogen) pre-coated with 9,400 human proteins in duplicate and screened following manufacturer’s instructions and as previously described (Liu et al., 2015 (link)). Briefly, after blocking, the microarray was incubated on ice with 2 μg/ml of mAbs or isotype control 151K for 90 min. Ab binding to array protein was detected with 1 μg/ml of Alexa Fluor 647-labeled anti-human IgG (Invitrogen) secondary Ab. Microarrays were scanned using a GenePix 4000B scanner (Molecular Devices) at a wavelength of 635 nm, with 10-μm resolution, using 100% power and 600 gain. Fluorescence intensities were quantified with GenePix Pro 5.0 program (Molecular Devices) using lot-specific protein location information provided by the microarray manufacturer.