Lin Yong-Qing, Zhang Yilu, Li Connie, Li Louis, Zhang Kelley, Li Shawn
Alliance Pharma, Inc., 17 Lee Blvd. Malvern, PA 19355, USA.
J Pharmacol Toxicol Methods. 2012 Jan;65(1):44-8. doi: 10.1016/j.vascn.2011.11.003. Epub 2011 Dec 8.
To evaluate the dried blood spot (DBS) technique in ELISA quantification of larger biomolecular drugs, an anti-CD20 monoclonal antibody drug was used as an example. A method for the quantification of the anti-CD20 drug in human DBS was developed and validated.
The drug standard and quality control samples prepared in fresh human blood were spotted on DBS cards and then extracted. A luminescent ELISA was used for quantification of the drug from DBS samples.
The assay range of the anti-CD20 drug standards in DBS was 100-2500ng/mL. The intra-assay precision (%CV) ranged from 0.4% to 10.1%, and the accuracy (%Recovery) ranged from 77.9% to 113.9%. The inter assay precision (%CV) ranged from 5.9% to 17.4%, and the accuracy ranged from 81.5% to 110.5%. The DBS samples diluted 500 and 50-fold yielded recovery of 88.7% and 90.7%, respectively. The preparation of DBS in higher and lower hematocrit (53% and 35%) conditions did not affect the recovery of the drug. Furthermore, the storage stability of the anti-CD20 drug on DBS cards was tested at various conditions. It was found that the anti-CD20 drug was stable for one week in DBS stored at room temperature. However, it was determined that the stability was compro]mised in DBS stored at high humidity, high temperature (55°C), and exposed to direct daylight for a week, as well as for samples stored at room temperature and high humidity conditions for a month. Stability did not change significantly in samples that underwent 3 freeze/thaw cycles.
Our results demonstrated a successful use of DBS technique in ELISA quantification of an anti-CD20 monoclonal antibody drug in human blood. The stability data provides information regarding sample storage and shipping for future clinical studies. It is, therefore, concluded that the DBS technique is applicable in the quantification of other large biomolecule drugs or biomarkers.
为了评估干血斑（DBS）技术在较大生物分子药物酶联免疫吸附测定（ELISA）定量中的应用，以一种抗CD20单克隆抗体药物为例，开发并验证了一种在人DBS中定量抗CD20药物的方法。
将新鲜人血中制备的药物标准品和质量控制样品点样于DBS卡上，然后进行提取。采用发光ELISA法对DBS样品中的药物进行定量。
DBS中抗CD20药物标准品的测定范围为100 - 2500ng/mL。批内精密度（%CV）范围为0.4%至10.1%，准确度（%回收率）范围为77.9%至113.9%。批间精密度（%CV）范围为5.9%至17.4%，准确度范围为81.5%至110.5%。稀释500倍和50倍的DBS样品回收率分别为88.7%和90.7%。在较高和较低血细胞比容（53%和35%）条件下制备DBS不影响药物回收率。此外，在各种条件下测试了抗CD20药物在DBS卡上的储存稳定性。发现抗CD20药物在室温下储存于DBS中可稳定一周。然而，确定在高湿度、高温（55°C）下储存一周且暴露于直射日光下的DBS以及在室温及高湿度条件下储存一个月的样品中稳定性受损。经历3次冻融循环的样品稳定性无显著变化。
我们的结果表明DBS技术成功用于人血中抗CD20单克隆抗体药物的ELISA定量。稳定性数据为未来临床研究提供了有关样品储存和运输的信息。因此，得出结论，DBS技术适用于其他大生物分子药物或生物标志物的定量。
