Purification of Two Different Immunoglobulins (Igs) from Olive Flounder Paralichthys olivaceus and Analysis of Lactococcus garvieae Antigens by the Igs

Abstract

ABSTRACT-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.

Development of an indirect ELISA assay to evaluation of the adaptive immune response of pacu (Piaractus mesopotamicus)

“…Moreover, the production of pacu anti-bovine IgG hyperimmune serum, which was added to the MBP column, contributed to the yield of purified pacu IgM. This is supported by our observations that bovine IgG have high immunogenicity for pacu and was able to induce a high production of IgM antibodies in the immunized fish, and similar observations from others who studied purification of immunoglobulins from other fish species (Palenzuela et al 1996, Shin et al 2007, Watts et al 2001a.…”

Evaluation of non-specific immune components from the skin mucus of olive flounder (Paralichthys olivaceus)

“…ELISA plates (Costar â , Corning Inc., USA) were coated with 100 ml (10 mg well À1 ) of CME, 2-fold serially diluted in the same buffer and incubated overnight at 4 C. Non-specific binding sites were blocked with 200 ml well À1 of 5% skim milk in low salt wash buffer (LSWB, 0.02 M Tris, 0.38 M NaCl, 0.05% Tween-20, pH 7.3) for 1 h at room temperature (RT) and washed three times with LSWB for 15 min each. The plates were then incubated with 100 ml of anti-flounder IgM monoclonal antibody raised against the mannan-binding protein (MBP) affinity purified olive flounder serum IgM [18] for 1 h at RT. The wells were washed five times with high salt wash buffer (HSWB, 0.02 M Tris, 0.5 M NaCl, 0.05% Tween-20, pH 7.8) and incubated for 5 min at RT with the final wash.…”

“…The membrane was blocked with 5% skim milk in PBS-T (3 mM KCl, 137 mM NaCl, 1.5 mM KH 2 PO 4 , 8 mM Na 2 HPO 4 , pH 7.5 and 0.05% Tween-20) for 60 min at RT. Membrane was washed with PBS-T then incubated with anti-flounder IgM monoclonal antibody raised against the MBP purified olive flounder serum IgM [18] overnight at 4 C. The membrane was then washed 3 times with PBS-T and incubated with goat anti-mouse IgG-HRP (Jackson. USA; 1:4000) for 1 h at RT.…”

Mucosal immunoglobulins and B cells of teleost fish

“…The ratio of LMW to HMW Ig was 3 times higher in the skin mucus than serum in sheepshead (Lobb and Clem, 1981a). In olive flounder ( Paralichthys olivaceus ), a monomeric Ig was detected in the cutaneous mucus with a monoclonal antibody (mAb) against a L chain of serum Ig purified with mannan-binding protein (MBP) affinity column (Palaksha et al, 2008; Shin et al, 2007). In carp, the Ig from cutaneous mucus had different protein/carbohydrate composition and antigenicity from that of serum IgM, and the electophoretic analysis revealed that the majority of both Igs were tetrameric (Rombout et al, 1993b).…”