Purification and characterization of IgM-like immunoglobulin from turbot (Scophthalmus maximus L.).
A total of 40 turbot (Scophthalmus maximus) were immunized 3 times during a 3 months period using DNP-HSA whereafter serum samples were collected and pooled. Specific immunoglobulins (Ig) were affinity purified on an agarose column with immobilized DNP-BSA and further purified by gel filtration whereafter monospecific rabbit anti Ig serum was generated. Size exclusion chromatography and non-reduced SDS-PAGE indicated a MW of 8-900 kDa of the dominant antigen binding proteins from turbot serum. Reduced SDS-PAGE showed this fraction to be composed of disulphide linked heavy and light chains with MWs of 79 and 27-29 kDa, respectively, indicating a tetrameric structure. Isoelectric focusing of the 800-900 kDa Ig showed several bands between pH 5.5 and pH 5.8. Mean Ig concentration in serum of 10 turbot was measured to 6.48 mg/ml (SD 5.4) using rocket immunoelectrophoresis. Low molecular weight antigen binding molecules were copurified with the dominating immunoglobulins with an estimated MW of 500 kDa. Reducing SDS-PAGE of this fraction revealed molecules with MWs of 97, 79, 57, 29, and 27 kDa.
Kofod
H
H
Department of Veterinary Microbiology, Royal Veterinary and Agricultural University, Frederiksberg, Denmark.
Pedersen
K
K
Larsen
J L
JL
Buchmann
K
K
D000818:Animals; D004140:Dinitrophenols / Q000276:immunology; D005412:Flatfishes / Q000276:immunology*; D006241:Haptens / Q000276:immunology; D007075:Immunoglobulin M / Q000276:immunology* / Q000302:isolation & purification*; D007526:Isoelectric Point; D011817:Rabbits; D012709:Serum Albumin / Q000276:immunology
D004140:Dinitrophenols; D006241:Haptens; D007075:Immunoglobulin M; D012709:Serum Albumin; C058064:dinitrophenyl-human serum albumin conjugate