Purification of two different immunoglobulins (Igs) from olive flounder Paralichthys olivaceus and analysis of Lactococcus garvieae antigens by the Igs

Immunoglobulins (Igs) were purified from sera obtained from olive flounder Paralichthys olivaceus immunized with goat IgG using immunoaffinity and mannan-binding protein (MBP) affinity columns and designated IMMIg and MBPIg respectively. SDS-PAGE and two-dimensional gel electrophoresis (2-DE) analyses demonstrated that the olive flounder serum contained at least two different types of Ig in terms of molecular weight and pI of heavy chain. 2-DE separation of the Igs followed by peptide mass fingerprinting using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) revealed that these two Igs were IgM and IgM precursor of olive flounder. Monoclonal antibodies (MAbs) were raised against the IMMIg and MBPIg and proteome analysis of Lactococcus garvieae using these MAbs and olive flounder immune sera demonstrated that IMMIg and MBPIg recognized different antigens of L. garvieae. This suggested that different Igs are possibly involved in protecting olive flounder against L. garvieae infection.