Journal: The Journal of Experimental Medicine

Article Title: Antiidiotype Antibody against Platelet Anti-Gpiiia Contributes to the Regulation of Thrombocytopenia in HIV-1–Itp Patients

doi:

Figure Lengend Snippet: Presence of IgG and IgM antiidiotype reactivity against anti–GPIIIa 49–66. (A) IgG antiidiotype. Various molar ratios of peptide/F(ab′) 2 anti–GPIIIa 49–66 fragments were preincubated as described in Materials and Methods before addition of antiidiotype Ab. The mean is given for five experiments. (B) IgM antiidiotype. Various molar ratios of peptide/F(ab′) 2 fragments were preincubated before addition of the IgM antiidiotype. The mean is given for five experiments (▪). The mean of five additional experiments with irrelevant peptide is also given (•). (C) Inhibition of anti–GPIIIa 49–66 binding to platelets with IgM antiidiotype. Binding of anti–GPIIIa 49–66 was measured by solid-phase ELISA, using goat F(ab′) 2 anti–human IgG coupled to alkaline phosphatase . Patient IC-IgM (•) or control IgM (○) or patient IC-IgG (▴) was preincubated with affinity-purified anti–GPIIIa 49–66 at various molar ratios (IgM/IgG or IgG/IgG) for 2 h at room temperature, then was applied to platelets bound to the microtitre plate, blocked, and washed with 5% BSA-PBS. Symbols in the lower corner of the right vertical line refer to various control measurements: platelets plus IC-IgM plus detection Ab (▵), blocked plates plus anti–GPIIIa 49-66 plus IC-IgM plus detection Ab (+), and platelets plus detection Ab (□). Representative of four different IgM antiidiotype preparations.

Article Snippet: The second Ab, used to detect IgM binding, was a 1:1,000 dilution of goat F(ab′) 2 anti–human IgM (μ chain specific) coupled to alkaline phosphatase (ICN Pharmaceuticals, Inc.).

Techniques: Inhibition, Binding Assay, Enzyme-linked Immunosorbent Assay, Control, Affinity Purification