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SinaryA novel quantitative ruminometric immunoassay (LIA)

Conclusion:The TK1 enzyme-labeled immunoassay uses a stable substrate, is precise, appears to be accurate, and

Methotrexate (MTX) is a cancer therapeutic drug for leukemia, osteosarcoma, non-Hodgkin's lymphoma and others. MTX levels in blood are monitored in patients to avoid toxic effects caused by the excessive dosage of this drug, and to determine when to intervene with the counter-acting ‘rescue’ therapy. An assay for serum and plasma MTX measurement on ARCHITECT i System (ARCHITECT Methotrexate) is being developed and the analytical feasibility of this assay is presented. METHODS: The ARCHITECT Methotrexate is a competitive immunoassay. First, the instrument mixes and incubates a sample (calibrators, controls, sera or plasma) with anti-methotrexate antibody-coated magnetic microparticles, biotinylated anti-methotrexate antibody and acridinium-conjugated MTX. Then, the instrument washes the microparticles and adds pre-trigger and trigger solutions to initiate the chemiluminescence reaction. Signals obtained as relative luminescent units are inversely proportional to the amount of MTX in the sample. RESULTS: The limit of quantitation was ≤ 0.040 µmol/L MTX. The 20-day imprecision study showed a total CV ≤ 7.1% within the range of 0.056 to 1705.433 µmol/L MTX with 6 controls and 5 panels on 4 instruments using 3 lots of reagents (n = 80). Dilution linearity showed the deviations from linearity were -4.1% to 7.2% within the range of 1.881 to 0.012 µmol/L MTX. Spike recovery revealed a mean recovery of 92% - 94% for samples at 0.045, 0.909 and 9.090 µmol/L of MTX. In the interference studies, the average levels of MTX in the individual interfering substance-spiked samples (10 endogenous substances including human anti-mouse antibody and rheumatoid factor, and 21 therapeutic drugs) were within 90% - 110% of that in the unspiked control samples. Method comparison of ARCHITECT Methotrexate to TDx/TDxFLx Methotrexate II (TDx) generated a Passing Bablok correlation as [ARCHITECT] = 0.00 + 1.01 [TDx] for samples within the range of 0.040 to 1.415 µmol/L (n = 92) and [ARCHITECT] = 0.00 + 1.04 [TDx] for samples within the range of 0.040 to 1624.760 µmol/L MTX , n = 142). Specimen storage study denoted that specimens could be stored at room temperature for 24 hours or at 2-8oC for 48 hours since the MTX values of the stored specimens described above were within 10% deviation from that of the baseline control specimens. Specimen Tube type study demonstrated that specimens collected in K2-EDTA, sodium heparin, and lithium heparin tubes was within ± 10% deviation from that collected in serum tubes across the range of 0.040 to 10.00 μmol/L MTX. Reagent on-board stability showed that the ARCHITECT Methotrexate reagents could remain on the analyzer for a minimum of 30 days with no more than 10% shift from baseline. CONCLUSION: The ARCHITECT Methotrexate under development was demonstrated to be an accurate, precise, sensitive and robust assay for the measurement of Methotrexate in human serum and plasma. Citation Format: Robert J. Smalley, Raymond E. Picard, Beth A. Burkhart, Robert L. Frescatore, Curtis L. Glover, Lisa C. Zhu, Elizabeth A. Roessner, Karin MajnesjÖ, Anders Öhrvik, Kuanglin He, Zhong Qian Li, Timothy R. Kettlety. An immunoassay for measurement of methotrexate on ARCHITECT i system. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3678. doi:10.1158/1538-7445.AM2014-3678

Human epididymis protein 4 (HE4), a member of Whey acidic four-disulfide core protein (WFDC) family, was demonstrated as one of the most useful biomarkers for ovarian cancer (HellstrÖm I, et al. 2003; Drapkin R, et al. 2005; Moore RG, et al. 2007).This study was to evaluate the use of Lumipulse G HE4 assay with patient serum for monitoring recurrence and progression of epithelial ovarian cancer. Recently, Lumipulse G HE4 assay was cleared by US FDA as an aid in monitoring recurrence or progressive disease in patients with epithelial ovarian cancer. Methods: Lumipulse G HE4 is a Chemiluminescent Enzyme Immunoassay (CLEIA) for the quantitative determination of HE4 in human serum and plasma on the Lumipulse G System by a two-step sandwich immunoassay method. In the assay, serum was added to and incubated with an anti-HE4 monoclonal antibody (MAb)-linked magnetic particles. The particles were then washed and rinsed to remove unbound materials. Alkaline phosphatase-labeled 2nd anti-HE4 MAb was added to and incubated with the HE4-bound particles. The particles were then washed and rinsed again to remove unbound materials. Substrate 3-(2’-spiroadamantane)-4-methoxy-4-(3”-phosphoryloxy) phenyl-1, 2-dioxetane disodium salt (AMPPD) solution was then added to and mixed with the particles. Luminescence signals were thus generated by the cleavage of dephosphorylated AMPPD and converted into the amount of HE4 in the serum. Results: In the monitoring study, changes in HE4 levels in serial serum samples collected in SST tubes from 72 subjects with epithelial ovarian cancer were compared to changes in disease status, that is, progression or no progression. A total of 330 observations were undertaken with an average number of 5.6 observations per subject. A positive change in the HE4 value was defined as an increase in the observation value that was at least 18% greater than the previous observation value. Of the 61 samples with a positive change, 49% of them correlated with the progression of epithelial ovarian cancer while 80% of the 269 subject serial samples with no significant change in the HE4 value correlated with no progression. The total concordance was 74%, positive predictive values (PPV) 35%, and negative predictive value (NPV) 87%. In addition, a comparison of Lumipulse G HE4 with the predicate device, HE4 EIA, was carried out using specimens consistent with CLSI Protocol EP09-A3 and weighted Deming regression analysis. The slope and correlation coefficient (r) obtained were 1.03 and 0.9891, respectively, for the tested specimens (n = 143) which ranged from 33.4 - 969.5 pmol/L, and slope and r of 1.03 and 0.9917, respectively, for the tested specimens (n = 168) ranged from 33.4 - 4602.0 pmol/L. Conclusion: The Lumipulse G HE4 assay has demonstrated to be useful in monitoring the course of disease in women with epithelial ovarian cancer and well correlated with the predicate device HE4 EIA. Citation Format: Rachel Radwan, Anders Öhrvik, Katherine Falcone, Sara Gannon, Candice Felman, Natalya Benina, Natascha Svensson, Savitha Raju, Sharee Jones, Catherine Peacock, Julianna Young, Zhong-Qian Li, Christian Fermer, Diana Dickson. Lumipulse G HE4 assay for monitoring of ovarian cancer recurrence and progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 437.