title: Comprehensive characterization and optimization of anti-LRRK2 (leucine-rich repeat kinase 2) monoclonal antibodies.
abstract: Missense mutations in LRRK2 (leucine-rich repeat kinase 2) are a major cause of PD (Parkinson's disease). Several antibodies against LRRK2 have been developed, but results using these polyclonal antibodies have varied widely leading to conflicting conclusions. To address this challenge, the Michael J. Fox Foundation for Parkinson's Research generated a number of monoclonal antibodies targeting epitopes across the LRRK2 protein. In the present paper, we report optimized protocols and results for ten monoclonal antibodies for immunoblotting, immunohistochemistry, immunoprecipitation and kinase activity assays, in rat, mouse and human brain tissue. Several efficacious antibodies were identified, but results demonstrate that the mouse monoclonal N241A/34 is suitable for most applications, with the best overall rabbit monoclonal antibody being c41-2. These antibodies produced a dominant band of the expected size via immunoblotting and a lack of labelling in tissue derived from LRRK2-knockout animals under optimized conditions. A significant proportion of LRRK2 protein localizes to insoluble fractions and no evidence of truncated LRRK2 protein was detected in any fraction from rodent or human tissues. An assay was developed for the robust detection of LRRK2 kinase activity directly from frozen mouse and human brain tissue, but precipitous declines in activity were observed that corresponded to increasing post-mortem intervals and processing times. Finally, we demonstrate the highest levels of brain-localized LRRK2 in the striatum, but note differential expression patterns between rat and mouse in both striatum and cortex. Anti-LRRK2 monoclonal antibodies that are unlimited in availability together with the proposed standardized protocols should aid in the definition of LRRK2 function in both health and disease.
authors.affiliation: MRC Protein Phosphorylation Unit, University of Dundee, Nethergate, Dundee DD1 4HN, Scotland, UK.
authors.forename: Paul
authors.initials: P
authors.lastname: Davies
authors.forename: Kelly M
authors.initials: KM
authors.lastname: Hinkle
authors.forename: Nour N
authors.initials: NN
authors.lastname: Sukar
authors.forename: Bryan
authors.initials: B
authors.lastname: Sepulveda
authors.forename: Roxana
authors.initials: R
authors.lastname: Mesias
authors.forename: Geidy
authors.initials: G
authors.lastname: Serrano
authors.forename: Dario R
authors.initials: DR
authors.lastname: Alessi
authors.forename: Thomas G
authors.initials: TG
authors.lastname: Beach
authors.forename: Deanna L
authors.initials: DL
authors.lastname: Benson
authors.forename: Charles L
authors.initials: CL
authors.lastname: White
authors.forename: Rita M
authors.initials: RM
authors.lastname: Cowell
authors.forename: Sonal S
authors.initials: SS
authors.lastname: Das
authors.forename: Andrew B
authors.initials: AB
authors.lastname: West
authors.forename: Heather L
authors.initials: HL
authors.lastname: Melrose
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D001921:Brain / Q000201:enzymology; D002460:Cell Line; D018604:Epitope Mapping; D006801:Humans; D015151:Immunoblotting; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D010300:Parkinson Disease / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000276:immunology*; D011817:Rabbits; D051381:Rats
chemical_list: D000911:Antibodies, Monoclonal; C495280:LRRK2 protein, human; C547428:LRRK2 protein, rat; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; C495279:Lrrk2 protein, mouse; D017346:Protein Serine-Threonine Kinases
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