Harbor seal (Phoca vitulina) C-reactive protein (C-RP): purification, characterization of specific monoclonal antibodies and development of an immuno-assay to measure serum C-RP concentrations.
C-reactive protein (C-RP) was purified from harbor seal (Phoca vitulina) serum by calcium dependant phosphoryl-choline and protein A affinity chromatography. Polyacrylamide gel electrophoresis under reducing conditions revealed a single protein moiety with a molecular weight of approximately 25 kDa. An internal peptide derived from this purified protein was subjected to N-terminal amino acid sequencing. A high amino acid sequence similarity was obtained with other published mammalian C-RP molecules confirming that the purified protein was a C-RP homologue. Eight specific monoclonal antibodies (P13, P51, P87, P101, P106, P130, P157 and P219) were raised against this purified protein. All 8 monoclonal antibodies immunoblotted with the 25 kDa C-RP subunit under reducing conditions. A competitive immunoassay was developed identifying elevated C-RP concentrations in harbor seal serum samples with clinical evidence of inflammatory disease. Application of this immunoassay for the measurement C-RP may provide valuable information for the clinical assessment of harbor seal health.
Funke
C
C
Marine Mammal Center, Marin Headlands, GGNRA, Sausalito, CA 94965, USA.
King
D P
DP
Brotheridge
R M
RM
Adelung
D
D
Stott
J L
JL
D000210:Acute-Phase Reaction / Q000097:blood; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D002097:C-Reactive Protein / Q000032:analysis / Q000276:immunology* / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000662:veterinary; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D007118:Immunoassay / Q000662:veterinary*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D010446:Peptide Fragments / Q000276:immunology; D046048:Seals, Earless; D017386:Sequence Homology, Amino Acid
D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D002097:C-Reactive Protein