FIG. 5.

Detection of endogenous chlamydial proteins in chlamydia-infected cells by human antibodies purified with chlamydial GST fusion proteins. The chlamydial GST fusion proteins (including GST-CPAF, GST-HSP60, and GST-MOMP, as indicated on the left of the figure) were immobilized onto glutathione-agarose beads, and each fusion protein bead complex was used to purify the corresponding antibodies from the pooled human sera. The fusion protein-purified human antibodies were used to stain HeLa cells infected with C. trachomatis serovar D organisms for 72 h for CPAF (a and d), HSP60 (b and e), and MOMP (c and f). Parallel cultures were stained with specific antibodies to CPAF (clone 100a) (g and j), HSP60 (BC7.1) (h and k), or MOMP (mouse antiserum) (i and l) as staining controls. The first antibody binding was revealed by either fluorescein isothiocyanate-conjugated goat anti-human IgGs (a to f) or mouse IgGs (g to l) and was displayed as either single-color (gray) (a to c and g to i) or dual-color (green) (d to f and j to l) images. The DNA was costained with the Hoechst dye (blue). Note that GST-CPAF-purified human antibodies (Ab) stained cytosolic signals similarly to the CPAF-specific MAb 100a.