Shi Xiaoping, Gao Hui, Li Zhong, Li Jinghua, Liu Yang, Li Lujuan, Zhang Qi
Pharmacy Department of Dalian Children's Hospital, No.154, Zhongshan Road, Dalian City, 116012, Xigang District, China.
Hematology Ward of Dalian Children's Hospital, No.154, Zhongshan Road, Dalian City, 116012, Xigang District, China.
BMC Pharmacol Toxicol. 2019 Jan 9;20(1):3. doi: 10.1186/s40360-018-0283-5.
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 (R = 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%.
甲氨蝶呤是许多化疗方案中的重要组成部分。甲氨蝶呤的血药浓度用于确定亚叶酸的给药方案。然而，在中国，西门子检测试剂盒的下限为0.30μmol/L。本研究通过优化酶放大免疫分析技术检测的参数，将下限从0.3μmol/L扩展至0.05μmol/L，并降低了检测成本。
修改酶放大免疫分析技术检测的参数以减少试剂A和试剂B的用量。然后制备一组新的定制校准品的标准曲线以检测低浓度。通过对照品和样本评估日内和日间精密度。通过分析一系列浓度已知为0.05至1.00μmol/L的质量控制品来验证修改后检测方法的线性。最后，分别用修改后的酶放大免疫分析技术检测和液相色谱 - 串联质谱检测对相同样本进行检测。进行简单线性回归以验证修改后的酶放大免疫分析技术检测的有效性。
日内和日间精密度在所有水平（0.05、0.12、0.43、0.82μmol/L）均显示出良好的重复性。修改后检测方法的线性方程为y = 0.9913x + 0.0046，其中y为测量浓度的均值，x为目标浓度（R = 0.9994）。在0.05 - 10.00μmol/L范围内，修改后的检测方法与液相色谱 - 串联质谱检测之间的相关性显著（r = 0.9968）。在0.30 - 10.00μmol/L范围内，修改后的检测方法与商业检测方法之间的相关性也显著（r = 0.9987）。
修改后的检测方法将西门子VIVA - E的灵敏度提高至0.05μmol/L。此外，试剂盒的检测次数从140次增加到210次。这意味着检测成本降低了约30%。
