ELISA for LCMV-specific IgG1 Antibodies

For the measurement of LCMV-specific IgG1 antibodies, ELISA was performed as previously described with minor adaptations (Baumjohann et al., 2013 (link)). Briefly, 96-well Nunc MaxiSorp flat-bottom plates (Thermo Fisher Scientific) were coated with lysates of LCMV Armstrong-infected baby hamster kidney cells overnight. Nonspecific binding was blocked with PBS + 0.5% Tween 20 (Promega) + 10% FBS for 2 h at room temperature. Subsequently, serially diluted serum was added and incubated for another 2 h at room temperature. Rat anti-mouse IgG1 (Clone A85-1; BD Biosciences) or Rat anti-mouse IgG2c horseradish peroxidase (HRP; pooled anti-sera; SouthernBiotech) and ECL anti-rat IgG HRP linked whole antibody from goat (GE Healthcare UK Limited) were used to detect IgG1 antibodies upon the addition of TMB substrate (TMB substrate set; BioLegend). The O.D. was measured with an ELISA Reader (Multiscan GO, SkanIt Software for Microplate Readers RE) at 450 nM.

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Sandner L., Alteneder M., Rica R., Woller B., Sala E., Frey T., Tosevska A., Zhu C., Madern M., Khan M., Hoffmann P., Schebesta A., Taniuchi I., Bonelli M., Schmetterer K., Iannacone M., Kuka M., Ellmeier W., Sakaguchi S., Herbst R, & Boucheron N. (2023). The guanine nucleotide exchange factor Rin-like controls Tfh cell differentiation via CD28 signaling. The Journal of Experimental Medicine, 220(11), e20221466.

Publication 2023

Nunc MaxiSorp Tween 20 Rat anti-mouse IgG1 (Clone A85-1;

Corresponding Organization : Medical University of Vienna

Other organizations : Vita-Salute San Raffaele University, Istituti di Ricovero e Cura a Carattere Scientifico, RIKEN Center for Integrative Medical Sciences

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Variable analysis

independent variables

Serum dilution

dependent variables

LCMV-specific IgG1 antibody levels

control variables

LCMV Armstrong-infected baby hamster kidney cells used for coating the 96-well plates
PBS + 0.5% Tween 20 + 10% FBS used for blocking nonspecific binding
Rat anti-mouse IgG1 (Clone A85-1; BD Biosciences) and Rat anti-mouse IgG2c horseradish peroxidase (HRP; pooled anti-sera; SouthernBiotech) used for detecting IgG1 antibodies
ECL anti-rat IgG HRP linked whole antibody from goat (GE Healthcare UK Limited) used for detecting IgG1 antibodies
TMB substrate (TMB substrate set; BioLegend) used for colorimetric detection
ELISA Reader (Multiscan GO, SkanIt Software for Microplate Readers RE) used for measuring the O.D. at 450 nM

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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