Streptavidin r phycoerythrin sa pe

Recombinant display on Staphylococcus carnosus TM300 [20] (link) was performed as previously described [16] (link). The ADAPTs were subcloned to a bacterial display vector [16] (link) in fusion to a reporter tag with affinity for human IgG. 100 nM HSA-Alexa Fluor 488-conjugate (Invitrogen, labeled according to the supplier's recommendations) or 50 nM biotinylated human ERBB2-His₆ (Sino Biological) was used together with polyclonal human IgG-Alexa Fluor 647 conjugate (Invitrogen). The fluorescently labeled human IgG was used for monitoring the surface expression level of individual bacteria through binding to the reporter tag. The monitoring allowed normalization of the ERBB2-binding signals to minimize potential biases from differences in surface expression levels between bacteria as described previously [16] (link). Streptavidin-R-phycoerythrin (SA-PE) (Invitrogen) was used for detection of ERBB2. Cells were analyzed on a Gallios flow cytometer (Beckman Coulter, Brea, CA, USA). Staphylococci expressing the scaffold albumin-binding domain were used as a negative control and cells expressing ADAPT_ERBB2-1 or an ERBB2-binding Affibody molecule (Z02891; [21] (link)) as positive controls.

ADAs were analyzed using a previously described Luminex multiplexed assay^[21b] with minor modifications. Briefly, the plasma samples were diluted 500‐fold in PBS. Diluted plasma samples (50 µL) were transferred to a black 96‐well‐plate (Corning). Next, OVA‐, OVA‐PEG‐, and OVA‐POEGMA‐coupled magnetic beads were added to each well (50 µL; 2500 beads per set) and incubated for 1 h on a plate shaker. The magnetic beads were separated on a magnetic ring, and wells were washed with 0.2% w/v I‐Block (Thermo Scientific) in PBS (Hyclone)—termed the assay buffer. To analyze IgGs, R‐Phycoerythrin‐conjugated goat anti‐mouse IgG (Jackson Immunoresearch; #115‐115‐164) was transferred to the wells (5 µg mL⁻¹; 100 µL) and incubated for 1 h. To analyze IgMs, biotin‐conjugated goat anti‐mouse IgM (Jackson Immunoresearch; #115‐065‐075) was used at the same concentration and volume and for the same duration. Next, the wells were washed with the assay buffer. For the analysis of IgM‐class ADAs, streptavidin‐R‐phycoerythrin (SAPE; Invitrogen) was transferred into the wells (7.5 µg mL⁻¹; 100 µL) and incubated for 30 min, followed by washing the wells with the assay buffer. The beads were resuspended in 100 µL assay buffer, and their fluorescence signal was measured by MAGPIX (Luminex).

The binding between the purified G proteins and ephrinB2 and ephrinB3 was measured to determine the ligand cross-reactivity. G protein-coupled magnetic microspheres were mixed after sonication such that all assays were multiplexed. A total of 1500 mixed microspheres were added per well. The recombinant mouse ephrin-B2 Fc chimera biotinylated protein (R&D systems, Minneapolis, MN, USA) and recombinant human ephrin-B3 Fc chimera biotinylated protein (R&D systems) were diluted in PBSA and added into the microsphere-containing wells with two replicates per concentration. The 96-well assay plate (Corning Inc, Corning, NY, USA) was incubated for 60 min at 24 °C on a plate shaker at 800 rpm. Streptavidin-R-phycoerythrin (SAPE) (Invitrogen) was diluted to a concentration of 12 µg/mL. A total of 10 µL of diluted SAPE was added to each well. The assay plate was incubated for 30 min at 24 °C on a plate shaker at 800 rpm. The supernatant was removed using a magnetic plate separator (Luminex Corporation). The plate was washed three times with PBSA, and the binding was measured using Luminex MAGPIX instrument (Luminex Corporation). The net median fluorescence intensities (MFI) were recorded and used to draw the binding curve.