Shanghai Genext Medical Technology Co., Ltd., Shanghai, 200433, China.
Department of Pharmacy, Shanghai Changhai Hospital, Naval Medical University, Shanghai, 200433, China.
J Pharm Biomed Anal. 2021 Oct 25;205:114290. doi: 10.1016/j.jpba.2021.114290. Epub 2021 Jul 29.
Accurate quantification of low level of blood drugs by Latex enhanced immunoturbidimetric assay (LEITA) remains a challenge due to its inherent limited sensitivity. To deal with this problem, we designed a new agglutination-enhancement strategy, and applied it for development of a highly sensitive and accurate tacrolimus LEITA. By this principle, a very small amount of biotin labeled anti-tacrolimus monoclonal antibodies (BLATMA) can arise agglutination strong enough for accurate reading of the increased absorbance since the BLATMA bears multiple biotin molecules, and the agglutination mediated by BLATMA can be inhibited by a similarly small amount of tacrolimus when the drug binds to BLATMA, giving rise to an improved sensitivity. The limit of detection (LOD) and functional sensitivity obtained by the proposed tacrolimus LEITA was 0.22 ng/mL and 0.59 ng/mL, respectively, 8-20 times more sensitive than the conventional drug-latex or antibody-latex based direct inhibition LEITA formats. Good precision was observed in the whole range of clinically significant tacrolimus concentration. The reliability of the tacrolimus LEITA was demonstrated by its strong correlation with both liquid chromatography-tandem mass spectrometry (LC-MS/MS) (R = 0.977, slope = 0.998) and the ABBOTT tacrolimus chemiluminescent magnetic immunoassay (CMIA) (R = 0.982, slope = 1.01) in the analysis of 119 clinical samples. It's concluded that the agglutination-enhancement strategy can be applied to construct highly sensitive LEITA for accurate tacrolimus analysis; owing to the improved sensitivity, this technique can be expected not only to improve the reliability of LEITA for low-level drug monitoring, but also to broaden the scope of analytes detectable by LEITA.
由于其固有的灵敏度限制，通过乳胶增强免疫比浊测定法（LEITA）准确量化低水平血液药物仍然是一个挑战。为了解决这个问题，我们设计了一种新的聚集增强策略，并将其应用于开发一种高度敏感和准确的他克莫司 LEITA。根据这一原理，由于 BLATMA 携带多个生物素分子，少量的生物素标记的抗他克莫司单克隆抗体（BLATMA）可以引起足够强的聚集，以便准确读取增加的吸光度，并且当药物与 BLATMA 结合时，BLATMA 介导的聚集可以被同样少量的他克莫司抑制，从而提高了灵敏度。所提出的他克莫司 LEITA 的检测限（LOD）和功能灵敏度分别为 0.22ng/mL 和 0.59ng/mL，比传统的药物-乳胶或抗体-乳胶直接抑制 LEITA 格式敏感 8-20 倍。在整个临床有意义的他克莫司浓度范围内观察到良好的精密度。通过与液相色谱-串联质谱（LC-MS/MS）（R = 0.977，斜率 = 0.998）和 ABBOTT 他克莫司化学发光磁免疫分析（CMIA）（R = 0.982，斜率 = 1.01）的强烈相关性，证明了他克莫司 LEITA 的可靠性，在分析 119 个临床样本时。结论是，聚集增强策略可应用于构建高度敏感的 LEITA 以准确分析他克莫司；由于灵敏度的提高，该技术不仅有望提高 LEITA 进行低水平药物监测的可靠性，而且还可以拓宽 LEITA 可检测分析物的范围。
