Publication Date:
2021-07-28
Description:
The interest of marine mammals grew enormously in the last 10 years. This general interest reflects the increased interest of scientists in evolution, behaviour, and medicine of marine mammals. Marine mammal medicine runs a little bit behind and has by any means not the possibilities, f.e. in diagnostic questions than in humans. Veterinarians of wild and of rehabilitation animals are more than once with the problem conf ronted to lack quick informations for the assessment of the health of marine mammals. This thesis describes the result of the purification and characterization of harbour seal (Phoca vitulina) C-reactive protein (CRP) and the additional production of specific reagents for the development of an assay to quantify CRP concentrations in harbour seal plasma. C-reactive protein (CRP) is an acute phase protein found in serum. Rapid increases in serum concentrations can be correlated with a variety of inflammatory conditions in multiple mammalian species. Human and canine CRP are compromised of pentameric structures with approximate molecular weights of 118 and 100 kDa for the intact molecule and about 22 and 20 kDa for individual subunits, respectively. Phosphoryl-choline Sepharose based affinity chromatography was used to purify CRP from harbour seal serum. In the presence of calcium, phosphoryl-choline has a high affinity for CRP of a number of different species. Upon loading, the affinity column was washed with Tris-salinecalcium buff er to remove unbound and weakly bound proteins and then the CRP was eluted with Tris-saline-EDTA buffer. Contaminating lmmunoglobulin was separated from the putative CRP by adsorption with lmmobilized Protein A beads. The purified protein was applied to 12% SOS-PAGE (under reducing conditions) for identification of a 25 kDa protein. The identification of the purified protein as a harbour seal CRP subunit was achieved by internal sequencing of a 20 residues long peptide. Line-up with correspondent human peptide showed an amino acid identity of 65% and an amino acid similiarity of 70%. A panel of monoclonal antibodies (McAbs) were produced, of which, five had the isotype lgG1 and two lgM. The antibodies were divided into two groups corresponding their colour reaction on the western blot. Group A represented clones with a binding reaction exclusively at 25 kDa and group B represented clones with additional binding reactions at about 55 kDa and 〉100 kDa. Purification of harbour seal C-RP, with subsequent production of CRP specific antibody, will facilitate development of a routine diagnostic ELISA for identification of an inflammatory response. Such an ELISA would represent a valuable tool for assessment of the health of wild and rehabilitated seals.
Type:
Thesis
,
NonPeerReviewed
Format:
text
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