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

Methotrexate 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.

Parameters 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.

Intra-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.

The 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%.