title: A quantitative TR-FRET plate reader immunoassay for measuring autophagy.
abstract: Autophagy involves the isolation and targeting of unwanted cellular components to lysosomes for their digestion and reuse. Autophagic dysregulation has recently been implicated in a wide range of disease processes, yet facile methods for quantifying autophagy have been lacking in the field. Here we describe the generation of a quantitative plate reader assay for measuring the autophagic activity of cells. One of the best characterized autophagy markers is the protein LC3B, which normally resides in the cytosol (LC3B-I) but upon induction of autophagy becomes lipidated and embedded in autophagosomal membranes (LC3B-II). To quantify autophagy, we reasoned that GFP-tagged LC3B could serve as a time-resolved fluorescence resonance energy transfer (TR-FRET) acceptor upon cell lysis in the presence of terbium-labeled LC3B antibodies. Using this TR-FRET immunoassay approach, we screened a panel of LC3B antibodies and identified an antibody that exhibits strong preferential affinity toward autophagosome-associated LC3B-II and thereby facilitates specific detection of autophagic activity. The plate reader format provides both a quantitative and an objective result, thus overcoming some of the key limitations of the traditional immunoblotting and imaging approaches used to monitor autophagy. Moreover, since the assay step requires only a single addition of cell lysis buffer containing the detection antibody its simple workflow is both automation-friendly and scalable, which renders it suitable for high-throughput screening. We demonstrate how this TR-FRET immunoassay for GFP-tagged LC3B-II can be applied to quantitatively detect changes in the autophagy activity of cells, including estimating effects on autophagic flux.
authors.affiliation: Discovery & ADMET-TOX Systems--Life Technologies Corporation, Madison, WI, USA. michael.hancock@lifetech.com
authors.forename: Michael K
authors.initials: MK
authors.lastname: Hancock
authors.forename: Spencer B
authors.initials: SB
authors.lastname: Hermanson
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Dolman
mesh_terms: D001343:Autophagy; D045744:Cell Line, Tumor; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation* / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007118:Immunoassay / Q000295:instrumentation* / Q000379:methods*; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D010588:Phagosomes / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors
chemical_list: D008869:Microtubule-Associated Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins