title: Direct detection of antibody concentration and affinity in human serum using microscale thermophoresis.
abstract: The direct quantification of both the binding affinity and absolute concentration of disease-related biomarkers in biological fluids is particularly beneficial for differential diagnosis and therapy monitoring. Here, we extend microscale thermophoresis to target immunological questions. Optically generated thermal gradients were used to deplete fluorescently marked antigens in 2- and 10-fold-diluted human serum. We devised and validated an autocompetitive strategy to independently fit the concentration and dissociation constant of autoimmune antibodies against the cardiac β1-adrenergic receptor related to dilated cardiomyopathy. As an artificial antigen, the peptide COR1 was designed to mimic the second extracellular receptor loop. Thermophoresis resolved antibody concentrations from 2 to 200 nM and measured the dissociation constant as 75 nM. The approach quantifies antibody binding in its native serum environment within microliter volumes and without any surface attachments. The simplicity of the mix and probe protocol minimizes systematic errors, making thermophoresis a promising detection method for personalized medicine.
authors.affiliation: Systems Biophysics, Center for Nanoscience, Physics Department, Ludwig Maximilians Universität München, München, Germany.
authors.forename: Svenja
authors.initials: S
authors.lastname: Lippok
authors.forename: Susanne A I
authors.initials: SA
authors.lastname: Seidel
authors.forename: Stefan
authors.initials: S
authors.lastname: Duhr
authors.forename: Kerstin
authors.initials: K
authors.lastname: Uhland
authors.forename: Hans-Peter
authors.initials: HP
authors.lastname: Holthoff
authors.forename: Dieter
authors.initials: D
authors.lastname: Jenne
authors.forename: Dieter
authors.initials: D
authors.lastname: Braun
mesh_terms: D001323:Autoantibodies / Q000097:blood*; D015415:Biomarkers / Q000097:blood*; D002311:Cardiomyopathy, Dilated / Q000175:diagnosis; D004586:Electrophoresis; D006801:Humans; D008832:Microchemistry; D013696:Temperature
chemical_list: D001323:Autoantibodies; D015415:Biomarkers