Therefore, there is a large unmet medical need to develop a simple and accurate assay that can overcome these limitations and provide clinicians with valuable quantitative measurements that they can then use to optimize the management of patients on biologic therapies. Here, we have developed and validated a novel homogenous mobility shift assay (HMSA) using
size-exclusion high-performance liquid chromatography (SE-HPLC) to quantitatively measure both induced antibodies-to-infliximab (ATI) levels and IFX levels in serum samples collected from IBD patients being treated with IFX. Individual serum samples from healthy controls were obtained from anti-PD-1 monoclonal antibody blood bank donors (Golden West Biologics, Temecula, CA). Sera from IBD patients treated with IFX were obtained from residual samples leftover after testing for ATI and IFX levels in our laboratories and the patient information was de-identified. Unless
otherwise noted, all reagents and chemicals were obtained from either Thermo Fisher Scientific (Waltham, INK 128 price MA) or Sigma Aldrich Corporation (St. Louis, MO). Commercially-available infliximab (RemicadeTM, Janssen Biotech, Inc., Horsham, PA) was buffer exchanged with phosphate buffered saline (PBS, pH 7.3) and labeled with AlexaFluor 488 (Life Technology, Carlsbad, CA) following the manufacturer’s instructions. Briefly, a reaction mixture consisting of 10 mg of IFX, 154 μg of AlexaFluor 488 dye, and 1 mL 1 × PBS (pH 8.0) was incubated in the dark at room temperature (RT) for 1 h with constant stirring. A desalting column was then used to remove free AlexaFluor 488, and the infliximab-AlexaFluor Montelukast Sodium 488 conjugate (IFX-488) was collected. The protein concentration and labeling efficiency of the conjugate was measured using a NanoDrop Spectrophotometer (Thermo Fisher Scientific, Waltham, MA). The NanoDrop spectrophotometer measures the A280 value for the protein concentration and the A494 value for AlexaFluor 488 concentration. The approximate molar extinction coefficient of the AlexFluor 488 dye at 494 nm is 71,000 cm− 1 M− 1 and the labeling efficiency
is calculated as follows: molesdyepermoleprotein=A494×dilutionfactor71,000×proteinconcentrationM Only those conjugates containing 2 to 3 fluorescent dyes per antibody qualified for the ATI-HMSA. The procedure for the labeling of recombinant TNF-α (RayBiotech, Inc, Norcross, GA) with AlexaFluor 488 was identical to that used for the labeling of IFX. The molar ratio of TNF-α to fluorescent dye in the reaction mixture was 1:6 and the resulting TNF-α-AlexaFluor 488 conjugate (TNF-488) contained 1–2 dye molecules per TNF-α. Activated AlexaFluor 488 (1 mg) and 4 mL 1 M Tris buffer (pH 8.0) were mixed for 1 h on a magnetic stirrer at RT to block the active site on the dye. The resulting solution was buffer-exchanged with 1 × PBS. The blocked AlexaFluor 488 was used as the IC and combined with either IFX-488 or TNF-488 at a molar ratio of 1:1.