P092 Development and evaluation of i-Tracker Ustekinumab and i-Tracker Anti-ustekinumab kits: fast and innovative chemiluminescent assays for the monitoring of patients treated with ustekinumab

Background Ustekinumab, a monoclonal antibody directed against IL12/23, is a drug widely used for the treatment of Chronic Inflammatory Diseases (Psoriasis, Crohn’s disease, etc.). Therapeutic Drug Monitoring is currently proposed to provide useful information to clinicians to improve the efficacy of the treatment. Theradiag has just developed the innovative i-Tracker ustekinumab and i-Tracker Anti-ustekinumab kits: fast quantification of ustekinumab and Anti-ustekinumab antibodies fully automated on the random access i-Track10 chemiluminescent analyzer. Methods Analytical performances were assessed using 2 types of serum samples: human serum spiked with ustekinumab or Anti-ustekinumab antibodies, and samples from Inflammatory Bowel Diseases patients treated with ustekinumab (n=32). For drug measurement, ustekinumab from serum sample was captured by anti-idiotypic antibody coupled magnetic microparticles and anti-ustekinumab polyclonal antibodies conjugated to acridinium ester were used for the detection of ustekinumab. For anti-drug antibodies measurement, Anti-ustekinumab antibodies were captured according to Ustekinumab coupled magnetic microparticles and detected with the use of ustekinumab conjugated to acridinium ester. Light emission was linked to the quantity of ustekinumab, or anti-ustekinumab antibodies presents in the sample. Results Ustekinumab measurement showed high accuracy (recovery was comprised between 98% and 120%). High precision weas reached for both assays (intra-precision CV were below 9.1% and 5.3% for ustekinumab and Anti-ustekinumab assays; inter-precision CV were below 4.6% and 10.6% for ustekinumab and Anti-ustekinumab assays) and no interference was seen with biologic agents (bilirubin, hemoglobin, lipids, biotin and rheumatoid factors). The dynamic ranges of the assays were 100ng/ml to 10 000ng/ml for ustekinumab quantification and 1 AU/ml to 250 AU/ml for anti-ustekinumab antibodies quantification. i-Tracker ustekinumab and i-Tracker anti-ustekinumab assays were compared to respective ELISA based LISA-TRACKER assays and showed excellent correlation (R² = 0.96, Slope = 0.93 for ustekinumab assay; Spearman’s coefficient correlation was 0.95 for Anti-ustekinumab assay). Conclusion i-Tracker kits are innovative assays which exhibit fast (time to results < 40min), accurate and reproducible results for the quantification of ustekinumab and Anti-ustekinumab antibodies. Excellent agreements were observed with respective LISA-TRACKER assays. i-Tracker kits are valuable tools for the monitoring of patients treated with ustekinumab.

Development of a PTHrP Chemiluminescent Immunoassay to Assess Humoral Hypercalcemia of Malignancy

Background: Measurement of parathyroid hormone related peptide (PTHrP) is helpful in the diagnosis and clinical management of patients suspected of humoral hypercalcemia of malignancy (HHM). In these patients uncontrolled release of PTHrP by tumor cells is responsible for the hypercalcemia and PTH concentrations are typically suppressed. Objective: Develop a sensitive and specific assay for quantitation of PTHrP in plasma. Method: Calibrators (PTHrP 1-86) and samples (50uL) were incubated with an anti-PTHrP goat polyclonal acridinium ester labeled antibody. Complexes were transferred and incubated in a microplate coated with an anti-PTHrP polyclonal rabbit antibody. After washing, the acridinium ester generated signal, which is directly proportional to the amount of PTHrP in sample, was quantified. Results: In this assay PTHrp was stable for 24 hours ambient, 3 days refrigerated, 34 days frozen and through 3 freeze/thaws. Intra and inter-assay imprecision in EDTA plasma (~0.16-35.0 pmol/L) ranged from 2.2-8.6% and 5-15%, respectively. The limit of detection was 0.04 pmol/L and the limit of quantitation was 0.16 pmol/L (15% CV). The analytical measuring range was 0.39-50.5 pmol/L (slope of 1.07 and r2 of 0.99). Average spike recovery was 98% (range 85-108%). The assay was not affected by hemoglobin of ≤500 mg/dL, triglycerides of ≤2000 mg/dL, or bilirubin of ≤50mg/dL. No hook effect was noted up to 500 pmol/L. PTH (1-84) did not cross-react in the assay. C-terminal PTHrP(107-139), and N-terminal PTHrP(1-36) had no significant cross-reactivity (≤1.1%). Mid-PTHrP(38-94) had 8.3% cross-reactivity. Comparison with an in-house PTHrP assay (n=267) showed an r2 of 0.96, and slope of 2.25 by Passing-Bablok regression fit. The 97.5% reference interval for PTHrP (n=114) was ≤0.7 pmol/L, however a higher concentration (≤4.2 pmol/L) was identified as a more specific clinical cut-off. A retrospective clinical validation study showed that using ≤4.2 pmol/L resulted in a 91% clinical sensitivity and a 98% clinical specificity. Conclusion: We have developed an analytically and clinically sensitive and specific PTHrP immunoassay. A cutoff of ≤4.2 pmol/L is clinically useful in the evaluation of patients suspected of hypercalcemia of malignancy.

Ultrasensitive chemiluminescence immunoassay with enhanced precision for the detection of cTnI amplified by acridinium ester-loaded microspheres and internally calibrated by magnetic fluorescent nanoparticles

Hydrogel microspheres sensitive to temperature as new potential signal enhancers and magnetic fluorescent nanoparticles as internal standards were used to establish a new CLIA method for the accurate diagnosis of cTnI in the human body.

P693 Development and evaluation of i-TRACKER infliximab and i-TRACKER anti-Infliximab kits: fast and innovative chemiluminescent assays for the monitoring of patients treated with infliximab

Background Infliximab, a monoclonal antibody directed against TNFα, is a drug widely used for the treatment of inflammatory diseases (rheumatoid arthritis, Crohn’s disease, etc.). Therapeutic drug monitoring is currently proposed to provide useful information to clinicians to improve the efficacy of the treatment. Theradiag has just developed the innovative i-TRACKER Infliximab and i-TRACKER Anti-Infliximab kits: fast quantifications of Infliximab and Anti-Infliximab antibodies fully automated on the random access i-TRACK10 chemiluminescent analyser. Methods Analytical performances were assessed using two types of serum samples: human serum spiked with Infliximab or Anti-Infliximab antibodies, and samples from inflammatory bowel disease patients treated with Infliximab (n = 41). On one hand, Infliximab from serum sample was captured by TNFα coupled magnetic microparticles and Anti-Infliximab polyclonal antibodies conjugated to acridinium ester were used for the detection of Infliximab. On the other hand, Anti-Infliximab antibodies were captured according to Infliximab coupled magnetic microparticles and detected with the use of Infliximab conjugated to acridinium ester. Light emission was linked to the quantity of Infliximab, or anti-Infliximab antibodies, presents in the sample. Results Infliximab measurement showed high accuracy (recovery was comprised between 80% and 107%). High precisions were reached for both assays (intra-precision CV were below 8.1% and 2.7% for Infliximab and Anti-Infliximab assays; inter-precision CV were below 11.7% and 4.8% for Infliximab and Anti-Infliximab assays) and no interferences were seen with biologic agents (bilirubin, haemoglobin, lipids, biotin and rheumatoid factors). The dynamic ranges of the assays were 0.3–24 µg/ml for Infliximab quantification and 10–2000 ng/ml for Anti-Infliximab antibodies quantification. i-TRACKER Infliximab and i-TRACKER anti-Infliximab assays were compared with respective LISA-TRACKER assays and showed excellent correlation (R² = 0.94, Slope = 1.04 for Infliximab assay; Spearman’s coefficient correlation was 0.98 for Anti-Infliximab assay). Conclusion i-TRACKER kits are innovative assays which exhibit fast (time to results <40 min), accurate (standardised with NIBSC/WHO international standard Infliximab) and reproducible results for the quantification of princeps and biosimilar molecules (CT-P13, SB2). Excellent agreements were observed with respective LISA-TRACKER assays. i-TRACKER kits are valuable tools for the monitoring of patients treated with Infliximab.

Flow Injection Analysis with Microdialysis Probes Enable Minimally Invasive and Dynamic H2O2 Measurements

This paper describes the optimization of a published flow injection analysis system coupled with microdialysis probes (MDP-FIA) for in-situ sampling and online measurements of hydrogen peroxide (H2O2). By modifying the commonly used Na2CO3 buffer by addition of EDTA and a changed order in reagent injection, interfering transition metals such as Fe(II) and Fe(III) are complexed and removed from the system without interfering with the chemiluminescent reaction of the used acridinium ester and H2O2. The system was then used to monitor changes in H2O2 concentration upon microwaving seawater and filtered seawater in the presence and absence of agar.