ELISpot Assay for Detecting Autoantibodies

ELISpot assays were performed as previously described (25 (link), 43 (link)). Briefly, splenocytes in RPMI containing 10% fetal bovine serum were plated at a concentration of 1 × 10⁶ cells/well onto salmon sperm dsDNA- (Invitrogen), nucleosome- (histone from Sigma Aldrich plated on a layer of dsDNA coating)- or SmRNP- (Arotec Diagnostics) coated multiscreen 96-well filtration plates (Millipore, Bedford, MA). Serially diluted (1:2) cells were incubated for 12 h at 37°C. dsDNA-, nucleosome-, and SmRNP-specific AFCs were detected by biotinylated anti-kappa Ab (Invitrogen) and streptavidin (SA)-alkaline phosphatase (Vector Laboratories) or alkaline phosphatase-conjugated anti-mouse IgG (Molecular Probes). Plates were developed using the Vector Blue Alkaline phosphatase Substrate Kit III (Vector Laboratories). ELISpots were enumerated and analyzed using a computerized ELISpot plate imaging/ analysis system (Cellular Technology).

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Chodisetti S.B., Fike A.J., Domeier P.P., Singh H., Choi N.M., Corradetti C., Kawasawa Y.I., Cooper T.K., Caricchio R, & Rahman Z.S. (2020). Type II but not type I interferon signaling is indispensable for TLR7-promoted development of autoreactive B cells and systemic autoimmunity. Journal of immunology (Baltimore, Md. : 1950), 204(4), 796-809.

Publication 2019

salmon sperm dsDNA nucleosome SmRNP dsDNA biotinylated anti-kappa Ab streptavidin (SA)-alkaline phosphatase alkaline phosphatase-conjugated anti-mouse IgG Vector Blue Alkaline phosphatase Substrate Kit III ELISpot plate imaging/ analysis system

Alkaline phosphatase Anti igg Assays Cellular Diagnostics Dsdna Elispot Fetal bovine serum Filtration Histone Molecular probes Mouse Nucleosome Salmon Sperm Streptavidin Vector

Corresponding Organization :

Other organizations : Pennsylvania State University, J. Craig Venter Institute, Temple University

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Variable analysis

independent variables

Stimuli used to coat the multiscreen 96-well filtration plates: salmon sperm dsDNA, nucleosome (histone from Sigma Aldrich plated on a layer of dsDNA coating), or SmRNP (Arotec Diagnostics)

dependent variables

Frequencies of dsDNA-, nucleosome-, and smRNP-specific antibody-forming cells (AFCs)

control variables

Splenocytes plated at a concentration of 1 × 10^6 cells/well
Cells incubated for 12 h at 37°C
Detection of AFCs using biotinylated anti-kappa Ab and streptavidin (SA)-alkaline phosphatase or alkaline phosphatase-conjugated anti-mouse IgG
Plate development using the Vector Blue Alkaline phosphatase Substrate Kit III

controls

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

Annotations

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