Quantifying Antigen-Specific Antibody Responses

For the ADCD assays^{39 (link)}, biotinylated antigens were coupled to FluoSphere NeutrAvidin beads (ThermoFisher). Immune complexes were formed by incubating antigen coupled beads with 10 ul of 1:10 diluted plasma samples at 37 °C for 2 hours. Following incubation, non-specific antibodies were washed off followed by incubation of the immune complex with guinea pig complement in GVB + + buffer (Boston BioProducts) at 37 °C for 20 min. To stop the complement reaction, 15 mM EDTA (Corning) in PBS was added to the immune complex. C3 deposited on beads were stained with anti-guinea pig C3-FITC antibody (MP Biomedicals, 1:100, polyclonal) and analyzed 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

FluoSphere NeutrAvidin beads GVB + + buffer EDTA anti-guinea pig C3-FITC antibody iQue analyzer

Anti antibody Antibodies Antigen Buffer Edta Fitc Guinea pig Immune complex Neutravidin Plasma

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

Concentration of antigen-coupled beads
Dilution of plasma samples

dependent variables

Amount of C3 deposition on beads
Fluorescence intensity of anti-guinea pig C3-FITC antibody

control variables

Incubation temperature (37 °C)
Incubation time for immune complex formation (2 hours)
Incubation time for complement activation (20 minutes)
Buffer composition (GVB++, PBS with EDTA)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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