Characterization of GM7 mAb against PfGARP. (A) PfGARP-M was expressed as a fusion protein. TRX tag contributes ∼12.9 kDa, and PfGARP-M consists of 75 AAs. PfGARP-M1 consists of 9 repeats (47 AAs, red), and PfGARP-M2 consists of 5 repeats (28 AAs, green). Blue segments signify the flanking nonrepeat AAs (TPE and VVK) derived from PfGARP-M. (B) Ponceau staining of TRX, PfGARP-M1, PfGARP-M2, and PfGARP-M fusion proteins (left panel). Western blotting results of proteins shown in left panel (right panel). GM7 monoclonal specifically recognized PfGARP-M1 and PfGARP-M but not PfGARP-M2. (C) Detection of PfGARP in infected RBC ghosts. Normal RBC ghosts (lanes 1,3,5) and iRBC ghosts (lanes 2,4,6) were tested by western blotting using preimmune (lanes 1,2) and immunized (lanes 3-4) mouse serum against PfGARP-M. Immune serum detected an ∼48 kDa band (arrow, lane 3) but this band was not detected by preimmune serum (lane 2) in iRBC ghosts. Preincubation of immune serum with excess PfGARP-M prior to western blotting specifically blocked the detection of the 48 kDa band (lane 6). Preimmune serum detected several nonspecific bands. (D) Detection of 48-kDa PfGARP in iRBC ghosts. Normal RBC ghosts (lane 1) and iRBC ghosts (lane 2) were evaluated by western blotting using purified GM7 monoclonal. A single ∼48-kDa band was detected (lane 2, arrow). In fact, all hybridoma clones recognized the same band. Hybridoma clone #7 was selected and designated as GM7 for all subsequent studies. (E) Detection of native PfGARP in the P falciparum 3D7 culture supernatant of magnetically purified schizonts. Western blotting using GM7 detected 2 bands at ∼48 kDa and ∼38 kDa (arrows). The 38-kDa band represents a secondary processed secretory form of PfGARP or a degradation product of 48-kDa polypeptide detected in iRBC ghosts.