Multiplexed Quantitative Bacterial Detection using Dye-Labeled Magnetic Microspheres

Published: February 29, 2024

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…

Disclosures

The authors have nothing to disclose.

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  Pierce77149
N-hydroxysulfosuccinimide (Sulfo-NHS) Thermo Scientific ™ Pierce24510
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

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Cite This Article
Multiplexed Quantitative Bacterial Detection using Dye-Labeled Magnetic Microspheres. J. Vis. Exp. (Pending Publication), e22208, doi: (2024).

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