ELISA-Based Serum Antibody Detection

Anti-protein and anti-polysaccharide serum antibodies were detected by indirect ELISA as described previously (12 (link),13 (link)). Briefly, Microlon high binding plates (Greiner Bio-One) were coated overnight at 4°C with either 10 ug/ml ovalbumin (Sigma) or S. pneumoniae serotype 14 polysaccharide (kindly supplied by John Schreiber, Tufts University, Boston, MA) diluted in PBS. Serial dilutions of serum were used to probe antigen bound plates. Detection was performed using biotinylated anti-mouse IgG polyclonal antibody (Jackson ImmunoLabs) and europium-conjugated streptavidin (PerkinElmer) followed by quantification by time-resolved fluorescence on a Victor3V Multilabel Counter using DELFIA Enhancement Solution according to the manufacturer’s protocol (PerkinElmer).

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Ryan S.O., Abbott D.W, & Cobb B.A. (2014). Myeloid Glycosylation Defects Lead to Spontaneous a CVID-Like Condition with Associated Hemolytic Anemia and Anti-Lymphocyte Autoimmunity. Journal of immunology (Baltimore, Md. : 1950), 192(12), 5561-5570.

Publication 2014

ovalbumin europium-conjugated streptavidin Victor3V Multilabel Counter DELFIA Enhancement Solution

Anti antibodies Anti antibody Antigen Dilutions Elisa Europium Fluorescence Microlon Mouse Ovalbumin Polysaccharide Protein S pneumoniae Serum Streptavidin

Corresponding Organization :

Other organizations : Case Western Reserve University, University School

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Variable analysis

independent variables

Coating of microlon high binding plates with either 10 ug/ml ovalbumin or S. pneumoniae serotype 14 polysaccharide diluted in PBS

dependent variables

Detection of anti-protein and anti-polysaccharide serum antibodies by indirect ELISA

control variables

Coating of microlon high binding plates overnight at 4°C
Use of serial dilutions of serum to probe antigen bound plates
Detection using biotinylated anti-mouse IgG polyclonal antibody and europium-conjugated streptavidin, followed by quantification by time-resolved fluorescence

controls

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

Annotations

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