Immunopurification of B-lymphocytes from seabass Dicentrarchus labrax (L.)

Giuseppe Scapigliati, S. Meloni, F. Buonocore, P. Bossu, D. Prugnoli, Christopher John Secombes

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

The monoclonal antibody DLlg3, specific for immunoglobulins and B cells of the teleost fish Dicentrarchus labrax (Mediterranean sea bass), was used to enrich immunoreactive cells from peripheral blood, spleen, and head kidney leukocytes. The purification was performed by immunomagnetic sorting of leukocyte fractions enriched by Percoll density gradient centrifugation, and the purity of the isolated cells was estimated by immunofluorescence and cytofluorimetric analysis. Following a single immunopurification step, the percentages of DLIg3-purified cells were 61% +/- 6% from peripheral blood leukocytes, 66% +/- 5% from splenocytes, and 77% +/- 9% from head kidney cells. DLIg3-immunopurified cells, from the head kidney of antigen-primed fish displayed an enhanced proliferation index when incubated with the immunization antigen. DLIg3-purified cells from head kidney lymphoid tissue were employed for RNA extraction and complementary DNA synthesis, and in reverse transcriptase polymerase chain reaction experiments using specific primers corresponding to the sequences of the sea bass immunoglobulin light chain, and of T-cell receptor. DLIg3-purified cells displayed enhanced expression of the immunoglobulin gene, and lower expression of T-cell receptor.

Original languageEnglish
Pages (from-to)214-221
Number of pages7
JournalMarine Biotechnology
Volume5
DOIs
Publication statusPublished - 2003

Keywords

  • fish
  • sea bass
  • Dicentrarchus labrax
  • monoclonal antibody
  • B cells
  • aquaculture
  • TROUT ONCORHYNCHUS-MYKISS
  • CARP CYPRINUS-CARPIO
  • PERIPHERAL-BLOOD LEUKOCYTES
  • ANTIBODY-PRODUCING CELLS
  • HEAD-KIDNEY LEUKOCYTES
  • RAINBOW-TROUT
  • VIBRIO-ANGUILLARUM
  • TELEOST FISH
  • IN-VITRO
  • IMMUNOCYTOCHEMICAL DETECTION

Cite this

Immunopurification of B-lymphocytes from seabass Dicentrarchus labrax (L.). / Scapigliati, Giuseppe; Meloni, S.; Buonocore, F.; Bossu, P.; Prugnoli, D.; Secombes, Christopher John.

In: Marine Biotechnology, Vol. 5, 2003, p. 214-221.

Research output: Contribution to journalArticle

Scapigliati, Giuseppe ; Meloni, S. ; Buonocore, F. ; Bossu, P. ; Prugnoli, D. ; Secombes, Christopher John. / Immunopurification of B-lymphocytes from seabass Dicentrarchus labrax (L.). In: Marine Biotechnology. 2003 ; Vol. 5. pp. 214-221.
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abstract = "The monoclonal antibody DLlg3, specific for immunoglobulins and B cells of the teleost fish Dicentrarchus labrax (Mediterranean sea bass), was used to enrich immunoreactive cells from peripheral blood, spleen, and head kidney leukocytes. The purification was performed by immunomagnetic sorting of leukocyte fractions enriched by Percoll density gradient centrifugation, and the purity of the isolated cells was estimated by immunofluorescence and cytofluorimetric analysis. Following a single immunopurification step, the percentages of DLIg3-purified cells were 61{\%} +/- 6{\%} from peripheral blood leukocytes, 66{\%} +/- 5{\%} from splenocytes, and 77{\%} +/- 9{\%} from head kidney cells. DLIg3-immunopurified cells, from the head kidney of antigen-primed fish displayed an enhanced proliferation index when incubated with the immunization antigen. DLIg3-purified cells from head kidney lymphoid tissue were employed for RNA extraction and complementary DNA synthesis, and in reverse transcriptase polymerase chain reaction experiments using specific primers corresponding to the sequences of the sea bass immunoglobulin light chain, and of T-cell receptor. DLIg3-purified cells displayed enhanced expression of the immunoglobulin gene, and lower expression of T-cell receptor.",
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AU - Bossu, P.

AU - Prugnoli, D.

AU - Secombes, Christopher John

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N2 - The monoclonal antibody DLlg3, specific for immunoglobulins and B cells of the teleost fish Dicentrarchus labrax (Mediterranean sea bass), was used to enrich immunoreactive cells from peripheral blood, spleen, and head kidney leukocytes. The purification was performed by immunomagnetic sorting of leukocyte fractions enriched by Percoll density gradient centrifugation, and the purity of the isolated cells was estimated by immunofluorescence and cytofluorimetric analysis. Following a single immunopurification step, the percentages of DLIg3-purified cells were 61% +/- 6% from peripheral blood leukocytes, 66% +/- 5% from splenocytes, and 77% +/- 9% from head kidney cells. DLIg3-immunopurified cells, from the head kidney of antigen-primed fish displayed an enhanced proliferation index when incubated with the immunization antigen. DLIg3-purified cells from head kidney lymphoid tissue were employed for RNA extraction and complementary DNA synthesis, and in reverse transcriptase polymerase chain reaction experiments using specific primers corresponding to the sequences of the sea bass immunoglobulin light chain, and of T-cell receptor. DLIg3-purified cells displayed enhanced expression of the immunoglobulin gene, and lower expression of T-cell receptor.

AB - The monoclonal antibody DLlg3, specific for immunoglobulins and B cells of the teleost fish Dicentrarchus labrax (Mediterranean sea bass), was used to enrich immunoreactive cells from peripheral blood, spleen, and head kidney leukocytes. The purification was performed by immunomagnetic sorting of leukocyte fractions enriched by Percoll density gradient centrifugation, and the purity of the isolated cells was estimated by immunofluorescence and cytofluorimetric analysis. Following a single immunopurification step, the percentages of DLIg3-purified cells were 61% +/- 6% from peripheral blood leukocytes, 66% +/- 5% from splenocytes, and 77% +/- 9% from head kidney cells. DLIg3-immunopurified cells, from the head kidney of antigen-primed fish displayed an enhanced proliferation index when incubated with the immunization antigen. DLIg3-purified cells from head kidney lymphoid tissue were employed for RNA extraction and complementary DNA synthesis, and in reverse transcriptase polymerase chain reaction experiments using specific primers corresponding to the sequences of the sea bass immunoglobulin light chain, and of T-cell receptor. DLIg3-purified cells displayed enhanced expression of the immunoglobulin gene, and lower expression of T-cell receptor.

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KW - ANTIBODY-PRODUCING CELLS

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KW - IMMUNOCYTOCHEMICAL DETECTION

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