B-321 Development of Chemiluminescent Enzyme Immunoassay (CLEIA) for Detecting Methotrexate in Blood

Abstract

Abstract Background Methotrexate (MTX) is a folate metabolism antagonist used in the treatment of rheumatoid arthritis, psoriasis, and antineoplastic agents. Therapeutic drug monitoring (TDM) is required to avoid serious adverse reactions of high-dose MTX/leucovorin rescue therapy or in patients with possible delayed excretion due to renal dysfunction or other factors. Although there are many reagents for MTX measurement based on the immunoassay method, they have a narrow measurement range to 2 µmol/L. Therefore, high concentration samples often make users conduct dilution and retest. In addition, most reagents have a cross-reactivity rate of over 40% with 2,4-diamino-N10-methylpteroic acid (DAMPA), one of the metabolites of MTX, so users must take note in the interpretation of measurement results. We have established an antibody with high specificity against MTX, and have developed a fully-automated, chemiluminescence immunoassay for LUMIPULSE® L2400 system for detecting MTX in blood, Lumipulse Presto® Methotrexate, which can significantly improve the issues described above. Methods MTX assay is a one-step competitive immunoassay on the LUMIPULSE® L2400 System (FUJIREBIO INC.), which requires 10 µL of sample. The resulting reaction signals are derived within 30 min/sample, and are proportional to the amount of MTX in the sample allowing quantitative determination of MTX in plasma or serum. Results The detection limit of the assay was 0.019 µmol/L, and the limit of quantitation was 0.030 µmol/L. A 20-day precision study was performed during a 31-day period using two controls and three panel specimens, and within-laboratory precision showed CV ≤ 3.1 %. Linearity was demonstrated over the range 0.017–28.821 µmol/L. No interference was observed with unconjugated (≤20.1 mg/dL) or conjugated bilirubin (≤20.2 mg/dL), chyle (≤1770 FTU), hemoglobin (≤450 mg/dL), and RF (rheumatoid factor, ≤5522 IU/mL). The correlation coefficient and the regression slope of this assay and LC-MS/MS were 1.00 and 1.0, respectively (N = 171). Compared to existing immunoassay reagents, the assay showed much lower cross-reactivity to DAMPA. Conclusion The performance of our novel MTX assay using LUMIPULSE® L2400 system was satisfactory, suggesting it is useful for routine analysis. In particular, the measurement range is wide, 0.030 to 25.000 µmol/L, which greatly reduces the frequency of dilution and retest of high concentration samples. Used with the auto dilution system in LUMIPULSE® L2400, it is possible to measure almost all clinical specimen automatically. Because of the low cross-reactivity with DAMPA, it is expected to be utilized in patient receiving glucarpidase. Acknowledgment: Some of the samples were provided in accordance with “Guidelines for clinical measurement and diagnosis technology improvement project”. The guidelines are promoted by Tsukuba Medical Laboratory of Education and Research Center, and University of Tsukuba Hospital, Japan.