title: Adaptive Focused Acoustics (AFA) Improves the Performance of Microtiter Plate ELISAs.
abstract: We investigated the use of Adaptive Focused Acoustics (AFA) technology to improve the performance of microtiter plate enzyme-linked immunosorbent assays (ELISAs). Experiments were performed with commercially available AFA instrumentation and off-the-shelf 96-well microtiter plate sandwich ELISAs. AFA was applied over a range of acoustic energies, temperatures, and durations to the antigen/antibody binding step of an ELISA for measuring HIV-1 p24 in tissue culture samples. AFA-mediated antigen/antibody binding was enhanced up to 2-fold over passive binding at comparable temperatures and was superior or comparable at low temperature (8-10 °C) to passive binding at 37 °C. Lower nonspecific binding (NSB), lower inter- and intra-assay coefficients of variation (CVs), higher Z' factors, and lower limits of detection (LODs) were measured in AFA-mediated assays compared with conventional passive binding. In a more limited study, AFA enhancement of antigen/antibody binding and lower NSB was measured in an ELISA for measuring IGFBP-3 in human plasma. We conclude from this study that application of AFA to antigen/antibody binding steps in microtiter plate ELISAs can enhance key assay performance parameters, particularly Z' factors and LODs. These features render AFA-mediated binding assays potentially more useful in applications such as high-throughput screening and in vitro diagnostics than assays processed with conventional passive antigen/antibody binding steps.
authors.affiliation: Biotechnology Consulting Associates, Fitzwilliam, NH, USA davidgreen@myfairpoint.net.
authors.forename: David J
authors.initials: DJ
authors.lastname: Green
authors.affiliation: Covaris, Inc., Woburn, MA, USA.
authors.forename: Edwin A
authors.initials: EA
authors.lastname: Rudd
authors.affiliation: Covaris, Inc., Woburn, MA, USA.
authors.forename: James A
authors.initials: JA
authors.lastname: Laugharn
mesh_terms: D000162:Acoustics; D000906:Antibodies / Q000737:chemistry; D000937:Antigen-Antibody Reactions; D004797:Enzyme-Linked Immunosorbent Assay / Q000295:instrumentation* / Q000379:methods*; D016655:HIV Core Protein p24 / Q000737:chemistry*; D006801:Humans; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000097:blood*; D007700:Kinetics; D057230:Limit of Detection; D009693:Nucleic Acid Hybridization; D011485:Protein Binding; D015203:Reproducibility of Results; D013696:Temperature
chemical_list: D000906:Antibodies; D016655:HIV Core Protein p24; C515497:IGFBP3 protein, human; D018972:Insulin-Like Growth Factor Binding Protein 3; C514799:p24 protein, Human Immunodeficiency Virus Type 1
keywords: Adaptive Focused Acoustics; LOD; NSB in ELISAs; Z′ factor; high-throughput screening