Skip to main content
Download PDF
  • Published:

Purification and Characterization of IgM-like Immunoglobulin from Turbot (Scophthalmus maximus L.)

Acta Veterinaria Scandinavica volume 35, pages 1–10 (1994)Cite this article

Abstract

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.

References

  • Al-Harbi AH, Austin B: Purification of macroglobulins from the serum, and skin and gut mucus of turbot (Scophthalmus maximus L.) immunized with lipopolysaccharide (LPS) from a fish-pathogenic cytophaga-like bacterium (CLB). Bull. Eur. Ass. Fish Pathol., 1993, 13, (40–44).

    Google Scholar 

  • Alsenz J, Avila D, Huemer HP, Esparza I, Becherer JD, Kinoshita T, Wang Y, Oppermann S, Lambris JD: Phylogeny of the third component of complement, C3: Analysis of the conservation of human CRI, CR2, H, and B binding sites, concanavalin A binding sites, and thiolester bond in the C3 from different species. Dev. comp. Immunol. 1992, 16, (63–76).

    Article  CAS  Google Scholar 

  • Axelsen NH: (ed.). Handbook of immunoprecipitation-in-gel techniques. Scand. J. Immunol. 1983, 17, suppl. no. 10.

    Google Scholar 

  • Buchmann K, Ostergaard L, Glamann J: Affinity purification of antigen-specific serum immunoglobulin from the European eel (Anguilla anguilla). Scand. J. Immunol. 1992, 36, (89–97).

    Article  CAS  Google Scholar 

  • Clem LW, McLean WE: Phylogeny of immunoglobulin structure and function. VIL Monomeric and tetrameric immunoglobulin of the margate, a marine teleostfish. Immunology 1975, 29, (791–799).

    CAS  Google Scholar 

  • Cottrell B: The immune response of plaice (Pleuronectes platessa L.) to the metacercariae of Cryp- tocotyle lingua and Rhipidocotyle johnstonei. Parasitol. 1977, 74, (93–107).

    Article  Google Scholar 

  • Elcombe BM, Chang RJ, Taves CJ, Winkelhake JL: Evolution of antibody structure and effector functions: comparative hemolytic activities of monomeric and tetrameric IgM from rainbow trout, Salmo gairdnerii. Comp. Biochem. Physiol. 1985, 80B, 697–706.

    CAS  Google Scholar 

  • Hävarstein LS, Aasjord PM, Ness S, Andresen C: Purification and partial characterization of an IgM-like serum immunoglobulin from salmon (Salmo salar). Dev. comp. Immunol. 1988, 12, (773–785).

    Article  Google Scholar 

  • Israelsson O, Petersson A, Bengtén E, Wiersma EJ, Andersson J, Gezelius G, Pilström L: Immunoglobulin concentration in Atlantic cod, Gadus morhua L., serum and cross-reactivity between anti-cod-antibodies and immunoglobulins from other species. J. Fish Biol. 1991, 39, (265–278).

    Article  CAS  Google Scholar 

  • Kyhse-Andersen J: Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J. biochem. biophys. Meth. 1984, 10, (203–209).

    Article  CAS  Google Scholar 

  • Laemmli UK: Clevage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227, (680–685).

    Article  CAS  Google Scholar 

  • Lobb CJ, Clem LW: Phylogeny of immunoglobulin structure and function. XI Secretory immunoglobulins in the cutaneus mucus of the sheepshead, Archosargus probatocephalus. Dev. comp. Immun. 1981, 5, (587–596).

    CAS  Google Scholar 

  • Lobb CJ, Olson MOJ: Immunoglobulin heavy H chain isotypes in a teleost fish. J. Immunol. 1988, 141, (1236–1245).

    CAS  PubMed  Google Scholar 

  • Lobb CJ, Olson MOJ, Clem LW: Immunoglobulin light chain classes in teleost fish. J. Immunol. 1984, 132, (1917–1922).

    CAS  PubMed  Google Scholar 

  • Marchalonis JJ: Emergence of IgM immunoglobulins. In: Immunity in evolution, p.74, Harward University Press, Cambridge, Massachusetts, 1977.

    Google Scholar 

  • Marchalonis JJ, Warr GW: Phylogenetic origins of immune recognition: naturally occurring DNP-binding molecules in chordate sera and hemolymph. Dev. comp. Immunol. 1978, 2, (443–460).

    Article  CAS  Google Scholar 

  • Matsudaira PT, Burgess DR: SDS microslab linear gradient polyacrylamide gel electrophoresis. Anal. Biochem. 1978, 386, (386–396).

    Article  Google Scholar 

  • Ouchterlony O: Diffusion-in-gel methods for immunological analysis. In: P. Callos (ed.): Progress in Allergy, 1958, vol. V, p 1. Karger, Basel.

    Google Scholar 

  • Pilström L, Petersson A: Isolation and partial characterization of immunoglobulin from cod (Gadus morhua L.). Dev. comp. Immunol. 1991, 15, (143–152).

    Article  Google Scholar 

  • Secombes CJ, White A, Fletcher TC, Houlihan DF: The development of an ELISPOT assay to quantify total and specific antibody-secreting cells in dab (Limanda limanda (L)). Fish and Shellfish Immunol. 1991, 1, (87–97).

    Article  Google Scholar 

  • Shelton E, Smith M: The ultrastructure of carp (Cyprinus carpio) immunoglobulin: a tetrameric macroglobulin. J. molec. Biol. 1970, 54, (615–617).

    Article  CAS  Google Scholar 

  • Vilain C, Wetzel M-C, Pasquier LD, Charlemagne J: Structural and functional analysis of spontaneous anti-nitrophenyl antibodies in three cyprinid fish species: carp (Cyprinus carpio), goldfish (Carassius auratus) and tench (Tinea tinea). Dev. comp. Immunol. 1984, 8, (611–622).

    Article  CAS  Google Scholar 

  • Weber K, Osborn M: The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. biol. Chem. 1969, 244, (4406–4412).

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by grants from the Carlsberg Foundation and The Danish Agricultural and Veterinary Research Council grant no. 13-4710-1 and 13-4508-1 NMR

Author information

Authors and Affiliations

  1. Department of Veterinary Microbiology, Section of Fish Diseases, Royal Veterinary and Agricultural University, Frederiksberg, Denmark

    H. Kofod, K. Pedersen, J. L. Larsen & K. Buchmann

Authors
  1. H. Kofod
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. K. Pedersen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. J. L. Larsen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. K. Buchmann
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kofod, H., Pedersen, K., Larsen, J.L. et al. Purification and Characterization of IgM-like Immunoglobulin from Turbot (Scophthalmus maximus L.). Acta Vet Scand 35, 1–10 (1994). https://doi.org/10.1186/BF03548350

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1186/BF03548350

Keywords

Acta Veterinaria Scandinavica

ISSN: 1751-0147