Multiplex Luminex Assay for Antibody Profiling

Antigen-specific antibody isotype, subclass, and Fc receptor binding profiles were analyzed using a custom multiplex Luminex assay^{38 (link)}. The antigens were coupled directly onto the Luminex microspheres (Luminex Corp) via carboxy coupling. Coupled beads were incubated with diluted plasma samples overnight at 4 °C. Following overnight incubation, non-specific antibodies were washed off and the immune complexes were incubated with Ig isotypes or subclasses with a 1:100 diluted PE-conjugated secondary antibody for IgG1 (clone: HP6001), IgG2 (clone: 31-7-4), IgG3 (clone: HP6050), IgG4 (clone: HP6025), IgM (clone: SA-DA4), IgA1 (clone: B3506B4), or IgA2 (clone: A9604D2) (all Southern Biotech). For avidity analysis. immune-complexes were incubated for 10 min with 2.5 M urea or PBS (no urea control) prior to PBS wash and addition of the IgG1 secondary probe. For the FcγR binding, the secondary probe is a PE-streptavidin (Agilent Technologies) coupled recombinant and biotinylated human FcγR protein. Beads with secondary antibodies were incubated on a shaker at room temperature for 1 hour. Following incubation, excessive antibodies were washed off and relative antigen-specific antibody levels were determined on an iQue analyzer (Intellicyt).

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Bartsch Y.C., Chen J.W., Kang J., Burns M.D., St Denis K.J., Sheehan M.L., Davis J.P., Edlow A.G., Balazs A.B., Yonker L.M, & Alter G. (2022). BNT162b2 induces robust cross-variant SARS-CoV-2 immunity in children. NPJ Vaccines, 7, 158.

Publication 2022

PE-conjugated secondary antibody for IgG1 PE-streptavidin iQue analyzer

Antibodies Antibody Antigen Clone Fc receptor Human protein Ig isotypes Iga1 Iga2 Igg1 Igg2 Igg3 Igg4 Immune complexes Microspheres Plasma Streptavidin Urea

Corresponding Organization :

Other organizations : Ragon Institute of MGH, MIT and Harvard, Massachusetts General Hospital, Reproductive Science Center

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Variable analysis

independent variables

Antigen-specific antibody isotype
Antigen-specific antibody subclass
Antigen-specific antibody Fc receptor binding profiles

dependent variables

Relative antigen-specific antibody levels

control variables

Coupled beads incubated with diluted plasma samples overnight at 4 °C
Non-specific antibodies washed off
Immune complexes incubated with Ig isotypes or subclasses with a 1:100 diluted PE-conjugated secondary antibody
Immune-complexes incubated for 10 min with 2.5 M urea or PBS (no urea control) prior to PBS wash and addition of the IgG1 secondary probe
Beads with secondary antibodies incubated on a shaker at room temperature for 1 hour
Excessive antibodies washed off

positive control

Not explicitly mentioned

negative control

Not explicitly mentioned

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