Figure 3

Confirmation of IgG accumulation by additional methods. Blood smears from two human donors, both with laboratory-confirmed chlamydial infections, were immunostained for Chlamydia (TRITC, red) and human IgG (FITC, green). Microscopy fields A and B contain three unidentified leukocytes each, two of which are infected and representative of all infected white cells observed in vivo. Probing with a monoclonal mouse anti-human IgG antibody (detected using FITC-conjugated goat anti-mouse IgG) demonstrates that detectable levels of human IgG are present in infected leukocytes. Yellow coloration represents instances of co-localization between IgG and chlamydial inclusions. Inclusions were labeled with a rabbit anti-Chlamydia polyclonal antibody followed by TRITC-conjugated goat anti-rabbit IgG. Nomarski DIC imaging on a portion of each field demonstrates the membrane integrity of both erythrocytes and infected leukocytes (insets) (bar = 20 μm). (C) Western blot hybridization analysis probing for bovine IgG was performed on renografin-purified C. trachomatis EBs (EBs) and lysates from uninfected (Uninf.) and infected cultures (Ser K). Cell lysates were matched to ensure equal numbers of cells per lane (~2.0 × 10⁷ cells/mL) and purified EBs were diluted to the same IFU/mL as the infected the cell lysate (~1 × 10⁶IFU/mL).