title: Development and validation of a cell-based fluorescent method for measuring antibody affinity.
abstract: Monoclonal antibodies have become essential tools for diagnostic and therapeutic purposes. Antibody affinity is one of the critical factors influencing the therapeutic success of tumor-targeting antibodies. Therefore, developing an accurate and reliable method for determining antibody affinity is crucial. In this study, we describe a fluorescent cell-based immunosorbent assay that can accurately measure antibody affinity (KD) in the nanomolar range. This method involves the addition of fluorescently labeled antibodies to antigen-positive and antigen-negative cell lines fixed on 96-well plates. The fluorescent signals from nonspecific binding to negative control cell lines is subtracted from the specific binding to the antigen-positive cell lines. The KDvalues obtained by this method were comparable with values obtained by the flow cytometry and radioactive (I125) scatchard assay. Our results demonstrate that this modified cell-based fluorescent method allows for a convenient and efficient identification of therapeutically relevant leads.
authors.affiliation: Department of Pathology, Duke University Medical Center, Durham, NC, USA. Electronic address: xin.yu@duke.edu.
authors.forename: Xin
authors.initials: X
authors.lastname: Yu
authors.affiliation: Department of Pathology, Duke University Medical Center, Durham, NC, USA. Electronic address: cpegram@outlook.com.
authors.forename: Charles N
authors.initials: CN
authors.lastname: Pegram
authors.affiliation: Department of Pathology, Duke University Medical Center, Durham, NC, USA. Electronic address: darell.bigner@duke.edu.
authors.forename: Darell D
authors.initials: DD
authors.lastname: Bigner
authors.affiliation: Department of Pathology, Duke University Medical Center, Durham, NC, USA. Electronic address: vidyalakshmi.chandramohan@duke.edu.
authors.forename: Vidyalakshmi
authors.initials: V
authors.lastname: Chandramohan
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism; D000915:Antibody Affinity; D000941:Antigens / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D001681:Biological Assay / Q000379:methods*; D045744:Cell Line, Tumor; D011508:Chondroitin Sulfate Proteoglycans / Q000276:immunology / Q000378:metabolism; D066246:ErbB Receptors / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D005434:Flow Cytometry; D057809:HEK293 Cells; D006801:Humans; D008562:Membrane Glycoproteins / Q000276:immunology / Q000378:metabolism; D008565:Membrane Proteins / Q000276:immunology / Q000378:metabolism; D051379:Mice; D009154:Mutation; D011485:Protein Binding; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence; D041701:Swiss 3T3 Cells; D014162:Transfection; D014407:Tumor Cells, Cultured; D000099216:Chondroitin Sulfate Proteoglycan 4; D000098853:Podoplanin
chemical_list: D000911:Antibodies, Monoclonal; D000941:Antigens; D011508:Chondroitin Sulfate Proteoglycans; D066246:ErbB Receptors; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; C101390:CSPG4 protein, human; D000099216:Chondroitin Sulfate Proteoglycan 4; C117468:PDPN protein, human; D000098853:Podoplanin; C512478:EGFR protein, human
keywords: Antibody affinity; Flow cytometry; Fluorescent cell-based assay; Scatchard