Multianalyte Immunobead Assay: A Flow-Based Immunoassay Technique for Concurrent Detection of Multiple Antibody Subtypes in Human Serum Sample

Published: April 30, 2023

Abstract

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.

Protocol

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 Select three different vials of magnetic microspheres with unique bead regions, recording bead ID and lot information for each vial used. NOTE: The following steps will be followed for each…

Divulgaciones

The authors have nothing to disclose.

Materials

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

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Multianalyte Immunobead Assay: A Flow-Based Immunoassay Technique for Concurrent Detection of Multiple Antibody Subtypes in Human Serum Sample. J. Vis. Exp. (Pending Publication), e21151, doi: (2023).

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