Multiplexed Quantitative Bacterial Detection using Dye-Labeled Magnetic Microspheres

Abstract

This video demonstrates multiplexed quantitative bacterial detection employing carboxylated magnetic polystyrene microspheres dyed into distinct sets. The procedure encompasses the formation of bead-bacteria complexes, magnetic separation, and flow-based analysis, enabling precise and simultaneous quantification of multiple bacteria in the same reaction well.

Protocol

1. Covalent Coupling of Capture Antibodies to MagPlex Microspheres Each capture antibody is conjugated to a specific carboxylated magnetic microsphere set using a two-step procedure as described below. NOTE: A coupling kit including all of the reagents, buffers, and materials required for microsphere coupling is also available (see TABLE OF MATERIALS).  Resuspend the stock uncoupled microsphere susp…

Representative Results

Figure 1: Multiplexed capture sandwich immunoassay workflow. A multiplexed mixture of antibody-coupled microspheres is dispensed into each well, and the sample is added. After incubation for 1 hour, the reactions are washed, and the biotinylated detection antibodies are added. Following a 1 hour incubation, the reactions are washed again, and the SAPE reporter is added. Reactions…

Materials

MagPlex® Microspheres	Luminex®	MC100xx-YY	See website for individual catalog numbers (https://www.luminexcorp.com/magplex-microspheres/#order)
1.5 mL microcentrifuge tubes	Eppendorf™	22431081	Protein LoBind®
0.1 M monobasic sodium phosphate (NaH2PO4), pH 6.2	MilliporeSigma	S3139	Activation buffer
1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC)	Thermo Scientific™	Pierce™ 77149
N-hydroxysulfosuccinimide (Sulfo-NHS)	Thermo Scientific ™	Pierce™ 24510
Phosphate-buffered saline (PBS), pH 7.4	MilliporeSigma	P3813	Coupling buffer
xMAP® Antibody Coupling Kit	Luminex®	40-50016	Optional kit available for microsphere coupling.
Anti-Escherichia coli O157:H7 antibody	SeraCare	5310-0326	Polyclonal, used for both capture and detection
Anti-Salmonella CSA-Plus antibody	SeraCare	5310-0321	Polyclonal, used for both capture and detection
Anti-Campylobacter spp. antibody	SeraCare	5310-0324	Polyclonal, used for both capture and detection
Anti-Listeria antibody	SeraCare	5310-0319	Polyclonal, genus-specific, used for both capture and detection
PBS, pH 7.4 containing 0.02% Tween-20, 0.1% BSA, 0.05% sodium azide (PBS–TBN)	MilliporeSigma	P3563 A7888 S8032	Up to 1% BSA may be used
Anti-Escherichia coli O157:H7 Antibody	SeraCare	5310-0326	Polyclonal
(EZ-Link Sulfo-NHS-LC-Biotin) (Pierce).	Thermo Scientific™	A39257
E. coli O157:H7	SeraCare	5370-0013	Heat-killed
Salmonella serotype Typhimurium	SeraCare	5370-0002	Heat-killed
Campylobacter jejuni	SeraCare	5370-0011	Heat-killed
Listeria monocytogenes	SeraCare	5370-0010	Heat-killed
Streptavidin, R-Phycoerythrin Conjugate (SAPE)	Thermo Scientific™	S866