Anal. Chem. 2009, 81, 5783–5793
Rapid Multiplexed Flow Cytometric Assay for Botulinum Neurotoxin Detection Using an Automated Fluidic Microbead-Trapping Flow Cell for Enhanced Sensitivity Richard M. Ozanich, Jr.,*,† Cynthia J. Bruckner-Lea,† Marvin G. Warner,† Keith Miller,† Kathryn C. Antolick,† James D. Marks,‡ Jianlong Lou,‡ and Jay W. Grate† Pacific Northwest National Laboratory, P.O. Box 999, Richland, Washington 99352, and Department of Anesthesia, University of California, San Francisco, California 94110 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 antiBoNT/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 microbeadtrapping flow cell to capture antibody-coupled beads with subsequent sequential perfusion of sample, wash, dyelabeled 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 (∼50 pg/mL for BoNT/A-HC-fragment) for the 15 min fluidic assay in buffer. Botulinum neurotoxin (BoNT) is produced by the Grampositive bacterium Clostridium botulinum and two other Clostridium species and is the most toxic substance known to exist.1-4 By weight, it is approximately 1000 times more toxic than ricin, 15,000 times more toxic than VX nerve gas, and 100,000 times more toxic than sarin. There are seven known serotypes of BoNT (A-G), of which types A, B, E, and F cause virtually all human cases. Type A BoNT (BoNT/A) exposure causes the majority of food borne outbreaks and has been observed to cause more severe * Corresponding author e-mail:
[email protected]. † Pacific Northwest National Laboratory. ‡ University of California. (1) Sugiyama, H. Microbiol. Rev. 1980, 44, 419–448. (2) Lacy, D. B.; Stevens, R. C. J. Mol. Biol. 1999, 291, 1091–1104. (3) Scarlatos, A.; Welt, B. A.; Cooper, B. Y.; Archer, D.; DeMarse, T.; Chau, K. V. J. Food Sci. 2005, 70, R121–R130. (4) Gill, M. D. Microbiol. Rev. 1982, 46, 86–94. 10.1021/ac9006914 CCC: $40.75 2009 American Chemical Society Published on Web 06/16/2009
symptoms with a higher mortality.5,6 All of the BoNTs are structurally similar and consist of a 100 kDa heavy chain (BoNT/ A-HC) comprised of a 50 kDa translocation domain and a 50 kDa binding domain and a 50 kDa light chain comprised of the active proteolytic domain; the heavy and light chains are linked through a disulfide bond and noncovalent forces.7 The “gold-standard” measurement approach for BoNT is the mouse bioassay.3,8-10 While the mouse bioassay method can detect BoNT concentrations
