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Method Article
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July 8th, 2025
Source: Fhied, C. L. et al., Dynamic Monitoring of Seroconversion using a Multianalyte Immunobead Assay for Covid-19. J. Vis. Exp. (2022).
This video describes an immunoassay technique using fluorescent magnetic beads or microspheres coupled with different antigens to simultaneously detect various antibody isotypes in a serum sample. The assay is helpful in understanding seroconversion during an infection or post-vaccination.
All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.
1. Preparation of antigen-conjugated microspheres
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| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| 384-well black side polystyrene microtiter plates | Thermo Fisher | 12-565-346 | |
| Activation buffer (0.1 M NaH2PO4, pH 6.2) | Prepared in house | ||
| Assay buffer (PBS, 1% Bovine Serum Albumin, 0.01%Polysorbate-20) | Prepared in house | ||
| Bovine Serum Albumin, heat shock fraction | MilliporeSigma | A-7888-50G | |
| Coupling buffer (50 mM MES, pH 5.0) | Prepared in house | ||
| COVID 19 M Coronavirus Recombinant Matrix Protein (6xHistag) | MyBioSource | MBS8574735 | |
| Disposable pipette tips | |||
| EDC (1-Ethyl-3-[3- dimethylaminopropyl]carbodiimide hydrochloride) | Thermo Fisher | PIA35391 | |
| Goat Albumin, Fraction V Powder | Millipore Sigma | A2514-1G | |
| IgA Goat anti-Human, R-PE, Polyclona | Thermo Fisher | OB205009 | |
| IgG Goat anti-Human, R-PE, Polyclonal | Thermo Fisher | OB204009 | |
| IgG Goat anti-Rabbit, R-PE, Polyclonal | Thermo Fisher | OB403009 | |
| gM Goat anti-Human, R-PE, Polyclonal | Thermo Fisher | OB202009 | |
| IgM Mouse anti-Human, SB 436, Clone SA-DA4 | Thermo Fisher | 62999842 | |
| Instrument Analyzer | Luminex Corp. | INTELLIFLEX-RUO | |
| Low-Bind microcentrifuge tubes (1.5 mL) | Thermo Fisher | 13-698-794 | |
| MagPlex-C Microspheres, Region XXX | Luminex Corp. | MC10XXX-01 | |
| MES hydrate (2-(N-Morpholino) ethane sulfonic acid hydrate) | MilliporeSigma | M2933 | |
| Microplate aluminum sealing tape | Thermo Fisher | 07-200-683 | |
| Polysorbate-20 | Thermo Fisher | BP337-500 | |
| Quality Biological Inc. PBS, pH 7.2 | Thermo Fisher | 50-751-7328 | |
| Quench buffer (PBS, 1% Goat Serum Albumin, 0.01% Polysorbate-20) | Prepared in house | ||
| SARS-CoV-2 (2019-nCoV) Nucleocapsid Protein (His tag) | Sino Biologicals | 40588-V08B | |
| SARS-CoV-2 (2019-nCoV) Spike Protein (S1 Subunit, His tag) | Sino Biologicals | 40591-V08H | |
| SARS-CoV-2 (2019-nCoV) Spike RBD Antibody, Rabbit pAb | Sino Biologicals | 40592-T62 | |
| SARS-CoV-2 (2019-nCoV) Nucleocapsid Antibody, Rabbit mAb | Sino Biologicals | 40588-R0004 | |
| SARS-CoV-2 (COVID-19) Membrane Antibody (IN), Rabbit pAb | ProSci | 10-516 | |
| Sulfo-NHS (N-hydroxysulfosuccinimide) | Thermo Fisher | PIA39269 | |
| Wash Buffer (PBS, 0.01% Polysorbate-20) | Prepared in house | ||
| Water, LC/MS grade | Thermo Fisher | W-64 |
