Quantitation of Trastuzumab and an Antibody to SARS-CoV-2 in Minutes Using Affinity Membranes in 96-Well Plates
Quantitation of Trastuzumab and an Antibody to SARS-CoV-2 in Minutes Using Affinity Membranes in 96-Well Plates
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DOI：
10.1021/acs.analchem.1c03654
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发表时间：
2022-01
影响因子：
7.4
通讯作者：
H. Tan;Junyan Yang;J. Linnes;C. Welch;M. Bruening
中科院分区：
文献类型：
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作者：
H. Tan;Junyan Yang;J. Linnes;C. Welch;M. Bruening
人血清中治疗性单克隆抗体（mAb）的定量可确保患者具有足够水平的mAb进行有效治疗。本研究描述了在96孔板格式中使用亲和玻璃纤维膜快速（<5 min）定量治疗性mAb曲妥珠单抗和抗SARS-CoV-2刺突蛋白的mAb。含聚丙烯酸的膜在膜孔中的吸附和膜中−COOH基团的活化使亲和肽或蛋白质与膜共价连接。含mAb的血清通过亲和膜可在1 min内捕获mAb。随后冲洗、与荧光团结合的二抗结合和第二次冲洗可在威尔斯孔中产生mAb浓度依赖性荧光强度。根据不同日期的分析建立的校准曲线具有低变异性，并且允许在单独制备的样品中测定mAb水平，平均误差<10%，尽管单次重复测量的误差可能达到40%。该试验可在具有生理相关mAb浓度的稀释血清中进行，也可在未稀释血清中进行。因此，含有亲和膜的96孔板、酶标仪和简单的真空歧管的组合在<5分钟内提供方便的mAb定量。
Quantitation of therapeutic monoclonal antibodies (mAbs) in human serum could ensure that patients have adequate levels of mAbs for effective treatment. This research describes the use of affinity, glass-fiber membranes in a 96-well-plate format for rapid (<5 min) quantitation of the therapeutic mAb trastuzumab and a mAb against the SARS-CoV-2 spike protein. Adsorption of a poly(acrylic acid)-containing film in membrane pores and activation of the −COOH groups in the film enable covalent-linking of affinity peptides or proteins to the membrane. Passage of mAb-containing serum through the affinity membrane results in mAb capture within 1 min. Subsequent rinsing, binding of a secondary antibody conjugated to a fluorophore, and a second rinse yield mAb-concentration-dependent fluorescence intensities in the wells. Calibration curves established from analyses on different days have low variability and allow determination of mAb levels in separately prepared samples with an average error <10%, although errors in single-replicate measurements may reach 40%. The assays can occur in diluted serum with physiologically relevant mAb concentrations, as well as in undiluted serum. Thus, the combination of 96-well plates containing affinity membranes, a microplate reader, and a simple vacuum manifold affords convenient mAb quantitation in <5 min.