Tan, Hui YinYang, JunyanLinnes, Jacqueline C.Welch, Christopher J.Bruening, Merlin L.2023-04-252023-04-252022Tan HY, Yang J, Linnes JC, Welch CJ, Bruening ML. Quantitation of Trastuzumab and an Antibody to SARS-CoV-2 in Minutes Using Affinity Membranes in 96-Well Plates. Anal Chem. 2022;94(2):884-891. doi:10.1021/acs.analchem.1c03654https://hdl.handle.net/1805/32576This article is made available for unrestricted research re-use and secondary analysis in any form or be any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.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.en-USPublic Health EmergencyBiopolymersImmunologyMembranesPeptidesProteinsSerumArticle