Apoptosis is an active cell death process mediated by caspases activation, in which
different extrinsic or intrinsic signalling pathways result in direct activation of effector caspases.
Caspase-3 is considered to be the most important of the executioner caspases, which cause the
morphological and biochemical changes detected in apoptotic cells. Different bacterial and virus
pathogens have developed different strategies to survive inside the host and overcome natural
protections, one of them is inducing apoptotic death in infected cells. We have demonstrated previously
that Piscirickettsia salmonis
activates this process in monocytes/macrophages from salmonid RTS11
cell line both by morphological and caspase detection assays; nevertheless, recognition of caspase
activation by western blot was impossible since most of the commercially available antibodies for
mammalian caspases are not cross-reacting. Results:
We have generated a monospecific polyclonal
antibody directed to an epitope region of salmonid caspase-3; the selected epitope present high
homology with caspase-3 from others teleost species and includes the active site of the enzyme. The
peptide was designed using bioinformatics tools and was chemically synthesized using the Fmoc
strategy, analysed by RP-HPLC, its molecular weight confirmed by mass spectrometry and its structure
analyzed by circular dichroism. The synthetic peptide was immunized and antibodies from ascitic fluid
were enriched for immunoglobulins using caprylic acid and then purified by activated affinity columns.
The anti-peptide activity of purified antibodies was verified by ELISA, and the ability of the anti-peptide
to recognize salmonid caspase-3 activation was demonstrated with the molecule in P. salmonis
infected cells by western blotting, ELISA and immunocytochemistry. Conclusions:
This is the first
antibody available for a fish caspase, specifically for trout caspase-3, whose applications were
validated by different immunological assays.