Modified enzyme multiplied immunoassay technique of methotrexate assay to improve sensitivity and reduce cost

Abstract

BackgroundMethotrexate is an important component in many chemotherapy protocols. The blood concentration of Methotrexate is used to determine the regimen of folinic acid. However, the lower limit of Siemens assay kit is 0.30 μmol/L in China. This study extended the limit from 0.3 to 0.05 μmol/L and reduced the test cost by optimizing the parameters of Enzyme Multiplied Immunoassay Technique assay.MethodsParameters of Enzyme Multiplied Immunoassay Technique assay were modified to decrease the volume of reagents A and B. Then a standard curve with a new custom set of calibrators was prepared to detect low concentration. Intra-day and inter-day imprecision were assessed by control material and samples. The linearity of the modified assay was verified by analyzing a range of quality controls with known concentration from 0.05 to 1.00 μmol/L. At last, the same samples were tested by modified Enzyme Multiplied Immunoassay Technique assay and Liquid Chromatography-tandem Mass Spectrometry assay respectively. A simple linear regression was performed to verify the validity of the modified Enzyme Multiplied Immunoassay Technique assay.ResultsIntra-day and inter-day imprecision show good reproducibility at all levels (0.05, 0.12, 0.43, 0.82 μmol/L). The linearity equation of modified assay was y = 0.9913x + 0.0046, in which y was the mean of measured concentration and x was the target concentration (R2 = 0.9994). In the range of 0.05–10.00 μmol/L, correlation between the Modified assay and Liquid Chromatography-tandem Mass Spectrometry assay was significant (r = 0.9968). In the range of 0.30–10.00 μmol/L, the correlation between modified and commercial assays was significant (r = 0.9987) as well.ConclusionsThe modified assay enhanced the sensitivity of Siemens VIVA-E to 0.05 μmol/L. In addition, the test number of a reagent Kit increased from 140 to 210. This means the cost of detection was reduced about 30%.

Creation of a Nanobody-Based Fluorescent Immunosensor Mini Q-body for Rapid Signal-On Detection of Small Hapten Methotrexate

“…Also, EC50 values were calculated as 37.6 ± 11.0, 34.6 ± 13.8, and 20.4 ± 9.0 nM for the Q-body with No linker, GGGS 2 , and GGGS 4 , respectively, which were slightly higher but similar in value to the K d obtained in the previous study (5 ± 2 nM) . Interestingly, TAMRA C6-labeled linker-less Q-body exhibited a lower LOD value (0.56 nM) compared with previous studies. − Since the goodness of linear fitting at the low concentration range of MTX ( R 2 = 0.997) was better than that of 4PL fitting ( R 2 = 0.988) (Figure S10), the value from the fitted straight line (0.56 nM) was adopted as the LOD. This value is less than 1/50 of the EC50 value, and it might not be generally observed in competitive ELISAs.…”

“…This has attracted much attention for the development of an immunoassay method that will quantify MTX in human blood. Several human MTX ELISA kits have already been commercialized, and other immunoassay methods such as fluorescence polarization immunoassay (FPIA), chemiluminescent microparticle immunoassay, and enzyme multiplied immunoassay technique were established to meet the practical demand for therapeutic drug monitoring (TDM). Although low limits of quantification (∼50 nM) were achieved by these methods, the demand for further sensitivity and usability would remain.…”

Processing Validation Metrics of Syva Enzyme-Multiplied Immunoassay Technique (EMIT) Methotrexate Assay for Beckman Coulter System

“…Shi et al studied the Syva EMIT MTX assay in Siemens Viva-E autoanalyzer and had a bias ranging from -1.60% to -5.67% [ 21 ]. Our study, however, had a higher range of bias ranging from -3.54% to -7.21%.…”

Potential of Raman spectroscopy for the analysis of plasma/serum in the liquid state: recent advances

“…Human blood plasma or serum are the most commonly studied bodily fluids for disease diagnosis, biomarker discovery and therapeutic drug monitoring [51][52][53][54][55][56][57]. While plasma and serum are both cell-free fluids obtained from blood samples by centrifugation, they differ on the basis of whether clotting has been allowed or not.…”