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