Isolation and characterization of a human alternative complement pathway-inhibiting protein from larval hemolymph of the silkworm, Bombyx mori

Presence of alternative complement pathway-inhibiting protein (ACPIP) from silkworm (Bombyx mori): changes in activity at various stages of fifth larval stadium

“…These results confirmed that the inhibition of ACPactivation by the given samples was due to ACPIP present in the silkworm hemolymph. In our previous paper (Sekijima et al, 2001), we showed that the size of the hemolysis-inhibition circle decreased linearly in 1-mm increments from the maximum size (13.0 mm) at each two-fold dilution. As seen in Table 1, the maximal ACPIP-activities were observed in fifth stadium larvae on the sixth to ninth days.…”

“…Using this hemolysis-inhibition plate assay and column chromatographies, we could isolate a nearly homogeneous preparation of the human alternative complement pathway-inhibiting protein (ACPIP) from hemolymph of the silkworm. This protein showed a nearly single band of a molecular weight of about 90 kDa on native polyacrylamide-gel-electrophoresis (PAGE), which consisted of two polypeptides (34 and 32 kDa) on SDS-PAGE (Sekijima et al, 2001). The amino acid compositions of these two polypeptides were nearly identical, and showed some similarities with that of a previously reported inhibitor from Spodoptera frugiperda (D'Cruz and Dayet, 1984).…”

“…Antibody (IgG) used for immunoblotting was prepared from rabbit serum immunized with purified ACPIP as described in a previous paper (Sekijima et al, 2001).…”

“…The inhibitory activities of ACPIP against ACP-activation of human serum were determined by using the hemolysis-inhibition plate assay as described previously (Fujikura and Sekijima, 1997;Sekijima et al, 2001). An outline of the method was as follows: Three milliliters of 0.75% (w/v) agarose containing 10 mM EGTA, 5 mM MgCl 2 , 150 ml of fresh normal human serum and 30 ml of 50% (v/v) guinea pig erythrocytes was poured on a slide glass and allowed to set at room temperature.…”

“…After the incubation, the diameter (in mm) of the opaque circle was measured. The details of the method including the preparation of the plate have been described previously (Sekijima et al, 2001). ACPIP-like activities in the hemolymph from larvae of other insect species were also determined as described above.…”