In Vitro ELISA and Cell-Based Assays Confirm the Low Immunogenicity of VNAR Therapeutic Constructs in a Mouse Model of Human RA: An Encouraging Milestone to Further Clinical Drug Development

Figure 4

Detection and quantification of serum D1-NDure™-C4 and drug-specific mouse ADA using direct-binding ELISA. (a) Detecting and quantifying serum D1-NDure™-C4 in 30 mg/kg D1-NDure™-C4-treated mice serum samples diluted at 1 : 500 in sterile PBS, pH 7.4 prior to adding to designated ELISA wells. Serum D1-NDure™-C4 was detected and quantified in a direct-binding ELISA with wells coated with 2 μg/mL hTNF-α, and secondary antibody detection was performed using an anti-poly-histidine-peroxidase antibody. Standard D1-NDure™-C4 (D1-NDure™-C4 Std) was used at a top known concentration of 1 μg/mL. (b) Direct-binding ELISA detection of mouse ADA to anti-hTNF-α drug in 30 mg/kg D1-NDure™-C4 serum samples diluted at 1 : 100 in sterile PBS, pH 7.4 prior to adding to designated ELISA wells. Detection of drug-specific mouse ADA was determined using wells coated with 2 μg/mL hTNF-α and an anti-mouse IgG antibody- (whole molecule) peroxidase as detection reagent. D1-NDure™-C4 Std (con 1) and D1-NDure™-C4 Std (con 2) are controls used at known concentrations, with secondary antibody detection using an anti-mouse IgG antibody- (whole molecule) peroxidase or an anti-poly-histidine-peroxidase antibody, respectively. The results shown are the ( with two replicates per experiment). (c) Detection of circulating serum mouse IgG in 30 mg/kg D1-NDure™-C4-treated mice serum samples diluted at 1 : 500 in sterile PBS, pH 7.4 prior to adding to designated ELISA wells. Indirect capture ELISA wells coated with an anti-mouse IgG (Fc specific) antibody and serum mouse IgG detected using an anti-mouse IgG antibody- (whole molecule) peroxidase were used. The result shown are the ( with two replicates per experiment).

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