Production and Characterization of Monoclonal Antibodies against Human Ceruloplasmin

Abstract

Ceruloplasmin (CP) is the major plasma antioxidant and copper transport protein. Monoclonal antibodies (mAbs) against human CP were produced and characterized. A total of five hybridoma cell lines were established (CP2, CP10, CP20, CP25, CP30). From the epitope mapping analysis, two subgroups of mAbs recognize different peptide fragments were identified. When the purified CP was incubated with the mAbs, the ferroxidase activity of CP was inhibited up to a maximum 57 %. Immunoblotting with various tissue homogenates indicated that all the mAbs specifically recognize a single protein band of 130 kDa. They also appear to be extensively cross-reactive among different mammalian including human and avian sources. These results demonstrated that only one type of immunologically similar CP is present in all of the mammalian tissues including human. The CP mAbs could be of great benefit to design the diagnostic kit for CP-related diseases such as Wilson's disease.

Structural and Functional Analysis of Hypothetical Proteins in Mouse Hippocampus from Two-Dimensional Gel Electrophoresis

“…Production of Monoclonal Antibody for HP Q8CHP8. The production of a monoclonal antibody against HP Q8CHP8 was performed as previously described. , Briefly, the purified enzyme was mixed with an equal volume of complete Freund's adjuvant and was injected intraperitoneally into a BALB/c mouse (6−8 weeks old). Two booster injections with incomplete Freund's adjuvant at 3-week intervals were followed by a final injection without adjuvant at 3 days before the cell fusion experiment.…”

“…The HAD superfamily was discovered by computer analysis of bacterial HADs, 32 catalyzing the cleavage of substrate C-Cl, P-C, and P-OP bonds via nucleophilic substitution pathways and typically showing low sequence identity <15% to family members. 33 All sequence alignment searches showed low sequence identity (22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40).8%) of HP Q9CXN1 to HAD superfamily members ( Table 2). HAD superfamily catalytic sites forming the so-called "core" domain 32 in HP Q9CXN1 with typical conserved amino acids is shown in Figure 5: motif I (sequence position 8-17, AVLVDLNGTL) with conserved aspartate, motif II (sequence position 41-45, VRFVT) with a conserved arginine for phosphate binding, and motif III (sequence position 182-208, KTFFLEALRDADCAPEEAVMIGDDCRD) with conserved carboxylate residues for metal binding ( Figure 5).…”