In vitro production of peroxynitrite by haemocytes from marine bivalves: C-ELISA determination of 3-nitrotyrosine level in plasma proteins from Mytilus galloprovincialis and Crassostrea gigas

The present article describes a competitive ELISA for the measurement of 3-nitrotyrosine contents of plasma proteins from marine bivalves by means of a monoclonal anti 3-nitrotyrosine antibody mouse IgG.This assay is sensitive enough to determine the amounts of 3-nitrotyrosine in plasma proteins from one animal only.Using the C-ELISA, we have shown that the phagocytosis of zymosan particles increased the 3-nitrotyrosine levels of plasma proteins from mussel M. galloprovincialis and oyster C. gigas 5.8 and 7.5 times respectively.Bivalves, unlike vertebrates, do not have humoral antigen specific active compounds such as antibodies and their self-defence systems are based on non-specific defensive compounds and phagocytosis by haemocytes [1, 2].During phagocytic burst or after in vitro stimulation with PMA or LPS, haemocytes produce superoxide anions, i.e. the initial species of reactive oxygen intermediates (ROI) and nitric oxide (NO).ROI generation has been reported in Patinopecten vessoensis [3] Crassostrea virginica [4], Crassostrea gigas [5], Ostrea edulis, Pecten maximus [6], Mytilus edulis [7] and Mytilus galloprovincialis [8]. NO-synthase activity was detected in haemocytes of M. edulis [9] and C. gigas [10] and peroxynitrite production by M. galloprovincialis haemocytes has been recently reported [8, 11, 12].In the presence of superoxide anions, nitric oxide generates peroxynitrite, a strong oxidant which kills bacteria [13] and parasitic protozoa [10, 14, 15]. Moreover, peroxynitrite is a nitrating agent, that converts tyrosine in 3-nitrotyrosine [16]. Such nitration has been observed in proteins from human polymorphonuclear cells [17] and 3-nitrotyrosine has been used as a marker to assess peroxynitrite involvement in pathological processes such as adult respiratory distress syndrome [18], rheumatoid arthritis [19] and celiac disease [15].To determine levels of protein-associated 3-nitrotyrosine in human plasma or serum, Khan et al. [20] developed a competit