Triplicate protein samples (40?L) were loaded onto the sample plate for each measurement. polyspecificity and low risk for rapid clearance.35 The other two lead variants (VH Y30H and VL Y94K) showed slightly elevated baculovirus ELISA compared to the parent, suggesting similar or slightly increased polyspecificity. The number of predicted T cell epitopes was identical to the parent (measured at different concentrations was then used to extrapolate the diffusion interaction parameter (among measurements at different concentrations Cefodizime sodium The masses of the solution before and after dilution were measured to calculate the dilution factor. The density of the diluted sample was assumed to be 0.999?g/mL, while the density of the concentrated solution was calculated based on predicted osmolality.69 The concentration of the diluted solution was measured using 8453 spectrometer (Agilent) in a 10?mm quartz absorption cuvette (Hellma). The extinction coefficient for each variant was calculated based on the corresponding amino acid sequences (Table S4). The computed extinction coefficients were used to calculate the concentration from the absorbance at 280?nm (A280). Viscosity measurements were performed on a Discovery HR-30 rheometer (TA Instruments) equipped with a cone-and-plate geometry with 20?mm diameter and 1.01306 angle. Triplicate protein samples (40?L) were loaded onto the sample plate for each measurement. After incubation at 25C for 0.5?min, the viscosity was recorded 10 times during a 2?min time frame using a constant sheer rate of 1 1,000 s?1. The final viscosity was reported as the mean values of the total of 30 reads. Surface plasmon resonance The binding affinity of all generated variants was determined by SPR on a Biacore 8K+ (Cytiva). 3?g/mL of antibody samples were flowed through at a flow rate of 10?l/min for 1?min to be captured on a series S sensor chip Protein A (Cytiva). Injection of the antigen at a range of concentrations (0.2, 1, 2.5, and 5?nM) in HBS-EP buffer (100?mM HEPES, pH 7.4, 150?mM NaCl, 3?mM EDTA, 0.05% (v/v) polysorbate 20) was then conducted at a flow rate of 100?L/min at 37C. The Cefodizime sodium association was monitored for 3?min and the dissociation was monitored for 10?min. The Cefodizime sodium sensorgrams were analyzed by reference and blank subtraction, followed by fitting to a one-to-one Langmuir binding model supplied by the Biacore insight evaluation software (Version 4.0.8) to determine the kinetic constants and dissociation constant. Baculovirus ELISA The polyspecificity of lead anti-GCGR variants was assessed using a baculovirus ELISA as previously described.35 Computation of pI and TAP descriptor values and flags The pI values for all anti-GCGR variants as Fv fragments and also IgG1 were calculated from their amino acid sequences70,71 using the IsoelectricPoint class from Biopython. The five individual TAP scores72 were computed for all Cefodizime sodium anti-GCGR variants using a containerized version of the software licensed from OxPig (Table S3). In silico prediction of immunogenic T cell Cefodizime sodium epitopes The presence of immunogenic T cell epitopes in the parent and lead anti-GCGR variants was assessed computationally using NetMHCIIpan-4.0 EL36 for nine common HLA-DR alleles (HLA-DRB1??01:01, HLA-DRB1??03:01, HLA-DRB1??04:01, HLA-DRB1??07:01, HLA-DRB1??08:01, HLA-DRB1??09:01, HLA-DRB1??11:01, HLA-DRB1??13:01, and HLA-DRB1??15:01). 12C20-mer peptides were considered to be strongly presented on HLA-DR if the relative HLA-II presentation ranking was within the top 10%. Epitopes predicted to be presented on more than one HLA-DR molecule were defined as potential promiscuous epitopes. To identify potential immunogenic T cell epitopes in the Fab region for each molecule, we searched the natural antibody repertoire (Observed Antibody Space (OAS))73 for similar nine-mer peptides using the BioPhi humanness analysis tool.37 Epitopes Mouse monoclonal to CD48.COB48 reacts with blast-1, a 45 kDa GPI linked cell surface molecule. CD48 is expressed on peripheral blood lymphocytes, monocytes, or macrophages, but not on granulocytes and platelets nor on non-hematopoietic cells. CD48 binds to CD2 and plays a role as an accessory molecule in g/d T cell recognition and a/b T cell antigen recognition found in over 93% of OAS human subjects were removed from further analysis. Epitopes were excluded if their sequence was identically aligned to nine-mer peptides from the human proteome as identified using PEPMatch.74 Of the remaining epitopes, only those predicted to bind.