P405 Development and validation of a rapid immunoassay for monitoring of ustekinumab concentrations in Inflammatory Bowel Disease patients

Abstract

Abstract Background Several studies have reported an association between ustekinumab serum concentrations and clinical outcomes, suggesting a potential role of therapeutic drug monitoring (TDM) for guiding clinical decision-making and treatment optimisation in ustekinumab-treated patients with inflammatory bowel diseases. Usually, ustekinumab concentrations are measured with an enzyme-linked immunosorbent assay (ELISA). Disadvantages of using ELISA for TDM are the rather long time-to-result and the necessity of collecting multiple samples in order to reduce the price per determination. The performance of TDM could be further enhanced by using a rapid point-of-care assay, which would allow immediate and individualised dose optimisation based on real-time pharmacokinetic information instead of awaiting dose adjustments at the subsequent administration. Methods A fiber-optic surface plasmon resonance (FO-SPR) assay was developed on a White FOx 1.0 device (FOx Biosystems), using a monoclonal antibody (MA)/MA sandwich approach. MA-UST56A2D11 was covalently immobilised to a gold-sputtered fiber surface to capture ustekinumab. After incubation with the serum sample, biotinylated MA-UST56C1H12 was used for detection of bound ustekinumab, and the signal was amplified using a polyclonal anti-biotin antibody conjugated to gold nanoparticles. A calibration curve was constructed by spiking ustekinumab in 1/1000 diluted human serum. Recovery and imprecision were assessed, and the performance of the FO-SPR assay was compared with that of our previously developed in-house ELISA using twelve serum samples of ustekinumab-treated patients. Results A dose-response curve ranging from 1.25 – 40 ng/mL was obtained, allowing quantification of ustekinumab concentrations from 0.31 µg/mL (using a 1/250 dilution) up to 80 µg/mL (using a 1/2000 dilution) in serum samples. Measurement of ustekinumab-spiked samples (1 – 25 µg/mL) showed an average intra-assay recovery of 111% (range: 95-124%) and imprecision of 10% (range: 8-15%), and an average inter-assay recovery of 109% (range: 100-122%) and imprecision of 5% (range: 1-8%). Comparison of measurements between FO-SPR and ELISA revealed a very good correlation (Pearson r coefficient of 0.980, 95% CI 0.928-0.995, p<0.001; Figure 1). Using a pre-functionalised fiber, the FO-SPR assay requires 55 min for measuring a single ustekinumab serum sample. Conclusion A rapid FO-SPR assay for quantification of ustekinumab concentrations in serum samples was established and showed a very good correlation with ELISA. The reduced assay time and the possibility of measuring a single sample are advantages compared to ELISA, and may allow implementation of FO-SPR as a point-of-care diagnostic tool.