Journal: Bio-protocol

Article Title: Assessments of HLA-I Specificities of Anti-HLA-I Monoclonal Antibodies Using Solid Phase Bead Arrays

doi: 10.21769/BioProtoc.3655

Figure Lengend Snippet: Labscreen Single Antigen HLA class-I Combination bead (each labeled with identifiable fluorescent dye and HLA-I) suspension array is successively incubated with biotinylated antibody, washed, incubated with streptavidin-PE, washed, and finally measured on the Labscreen flow analyzer.

Article Snippet: 1.5 ml centrifuge tubes (Thermo Fisher Scientific, catalog number: 05-408-129) 30 K MWCO centricon filters (MilliporeSigma, catalog number: UFC903008) Biotinylated anti-Bw6 (Thermo Fisher Scientific, One Lambda Inc., catalog number: BiH0038, store at 4 °C) Biotinylated anti-Bw4 (Thermo Fisher Scientific, One Lambda Inc., catalog number: BiH0007, store at 4 °C) W6/32 purified from supernatants or ascites derived from hybridoma line ATCC HB-95 using a protein G column (GE Lifesciences, catalog number: 17040401, store at 4 °C in 20% ethanol) ( Springer, 2001 ) or procured as purified protein from a commercial source (for example, Bio X Cell, catalog number: BE0079) EZ-Link NHS-PEG 4 -Biotin (Thermo Fisher Scientific, catalog number: 21330, store at -20 °C) Labscreen Single Antigen HLA class-I Combination bead suspension array, which contains 97 common HLA-A, -B, and -C bound beads derived from individual cell lines, a positive control bead sample, and a negative control bead sample, where each bead is distinguished based on the fluorescence intensity readout (Thermo Fisher Scientific, One Lambda Inc., Labscreen ® , catalog number: Class-I-LS1A04NC, store at 4 °C) Note: For flow cytometry-based experiments, FlowPRA Single Antigen Beads, which contain 32 total beads (8 HLA-I and one negative control bead sample in 4 groups, where the 9 beads are labeled with differing amounts of fluorophore that can be measured in the PE channel of a flow cytometer) can be used and supplemented with additional kits if necessary (Thermo Fisher Scientific, One Lambda Inc., catalog number: FL1HD store at 4 °C) 10x LabScreen Wash buffer (Thermo Fisher Scientific, One Lambda Inc., Labscreen ® , catalog number: LSPWABUF, store at -65 °C or lower until open, then store at 4 °C for up to three months) PE Conjugated Streptavidin (Thermo Fisher Scientific Inc., One Lambda Inc., Labscreen ® , catalog number: LT-SAPE, store at 4 °C) FITC Conjugated Streptavidin (Biolegend, catalog number: 405201, store at 4 °C) 1x PBS buffer (Life Technologies Corporation, Gibco, catalog number: 10010-023)

Techniques: Labeling, Suspension, Incubation

Journal: Bio-protocol

Article Title: Assessments of HLA-I Specificities of Anti-HLA-I Monoclonal Antibodies Using Solid Phase Bead Arrays

doi: 10.21769/BioProtoc.3655

Figure Lengend Snippet: FlowPRA single antigen beads come as four groups, each containing 8 HLA-I beads and one negative control bead (without HLA-I labeled 8, and colored teal in dot plots) that are individually identifiable in the PE channel. The beads are successively incubated and washed: biotinylated antibody, buffer, streptavidin-FITC, buffer. They are then measured on a flow cytometer. The dot plots are from a representative experiment, where isotype control, IgG2a (left panel), or W6/32 (right panel) were incubated with Group 1 of FlowPRA single antigen beads.

Article Snippet: 1.5 ml centrifuge tubes (Thermo Fisher Scientific, catalog number: 05-408-129) 30 K MWCO centricon filters (MilliporeSigma, catalog number: UFC903008) Biotinylated anti-Bw6 (Thermo Fisher Scientific, One Lambda Inc., catalog number: BiH0038, store at 4 °C) Biotinylated anti-Bw4 (Thermo Fisher Scientific, One Lambda Inc., catalog number: BiH0007, store at 4 °C) W6/32 purified from supernatants or ascites derived from hybridoma line ATCC HB-95 using a protein G column (GE Lifesciences, catalog number: 17040401, store at 4 °C in 20% ethanol) ( Springer, 2001 ) or procured as purified protein from a commercial source (for example, Bio X Cell, catalog number: BE0079) EZ-Link NHS-PEG 4 -Biotin (Thermo Fisher Scientific, catalog number: 21330, store at -20 °C) Labscreen Single Antigen HLA class-I Combination bead suspension array, which contains 97 common HLA-A, -B, and -C bound beads derived from individual cell lines, a positive control bead sample, and a negative control bead sample, where each bead is distinguished based on the fluorescence intensity readout (Thermo Fisher Scientific, One Lambda Inc., Labscreen ® , catalog number: Class-I-LS1A04NC, store at 4 °C) Note: For flow cytometry-based experiments, FlowPRA Single Antigen Beads, which contain 32 total beads (8 HLA-I and one negative control bead sample in 4 groups, where the 9 beads are labeled with differing amounts of fluorophore that can be measured in the PE channel of a flow cytometer) can be used and supplemented with additional kits if necessary (Thermo Fisher Scientific, One Lambda Inc., catalog number: FL1HD store at 4 °C) 10x LabScreen Wash buffer (Thermo Fisher Scientific, One Lambda Inc., Labscreen ® , catalog number: LSPWABUF, store at -65 °C or lower until open, then store at 4 °C for up to three months) PE Conjugated Streptavidin (Thermo Fisher Scientific Inc., One Lambda Inc., Labscreen ® , catalog number: LT-SAPE, store at 4 °C) FITC Conjugated Streptavidin (Biolegend, catalog number: 405201, store at 4 °C) 1x PBS buffer (Life Technologies Corporation, Gibco, catalog number: 10010-023)

Techniques: Negative Control, Labeling, Incubation, Flow Cytometry, Control