title: Rapid multiplexed flow cytometric assay for botulinum neurotoxin detection using an automated fluidic microbead-trapping flow cell for enhanced sensitivity.
abstract: A bead-based sandwich immunoassay for botulinum neurotoxin serotype A (BoNT/A) has been developed and demonstrated using a recombinant 50 kDa fragment (BoNT/A-HC-fragment) of the BoNT/A heavy chain (BoNT/A-HC) as a structurally valid simulant. Three different anti-BoNT/A antibodies were attached to three different fluorescent dye encoded flow cytometry beads for multiplexing. The assay was conducted in two formats: a manual microcentrifuge tube format and an automated fluidic system format. Flow cytometry detection was used for both formats. The fluidic system used a novel microbead-trapping flow cell to capture antibody-coupled beads with subsequent sequential perfusion of sample, wash, dye-labeled reporter antibody, and final wash solutions. After the reaction period, the beads were collected for analysis by flow cytometry. Sandwich assays performed on the fluidic system gave median fluorescence intensity signals on the flow cytometer that were 2-4 times higher than assays performed manually in the same amount of time. Limits of detection were estimated at 1 pM (approximately 50 pg/mL for BoNT/A-HC-fragment) for the 15 min fluidic assay in buffer.
authors.affiliation: Pacific Northwest National Laboratory, P.O. Box 999, Richland, Washington 99352, USA. richard.ozanich@pnl.gov
authors.forename: Richard M
authors.initials: RM
authors.lastname: Ozanich
authors.forename: Cynthia J
authors.initials: CJ
authors.lastname: Bruckner-Lea
authors.forename: Marvin G
authors.initials: MG
authors.lastname: Warner
authors.forename: Keith
authors.initials: K
authors.lastname: Miller
authors.forename: Kathryn C
authors.initials: KC
authors.lastname: Antolick
authors.forename: James D
authors.initials: JD
authors.lastname: Marks
authors.forename: Jianlong
authors.initials: J
authors.lastname: Lou
authors.forename: Jay W
authors.initials: JW
authors.lastname: Grate
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology; D001331:Automation; D015374:Biosensing Techniques / Q000379:methods*; D019274:Botulinum Toxins, Type A / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D002021:Buffers; D001330:Electronic Data Processing; D000939:Epitopes / Q000276:immunology; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D055767:Immobilized Proteins / Q000032:analysis / Q000737:chemistry / Q000276:immunology; D051379:Mice; D008863:Microspheres; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis / Q000276:immunology; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry / Q000276:immunology; D013997:Time Factors
chemical_list: D000906:Antibodies; D002021:Buffers; D000939:Epitopes; D055767:Immobilized Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D019274:Botulinum Toxins, Type A