Immunopurification of T-cells from sea bass Dicentrarchus labrax (L.)

G Scapigliati, N Romano, L Abelli, S Meloni, A G Ficca, F Buonocore, S Bird, C J Secombes

Research output: Contribution to journalArticle

52 Citations (Scopus)

Abstract

The monoclonal antibody DLT15, specific for thymocytes and peripheral T-cells of the teleost fish Dicentrarchus labrax: (sea bass), was used to purify immunoreactive cells from blood and gut-associated lymphoid tissue. The purification was performed by immune-magnetic sorting of leucocyte fractions enriched by Percoll density gradient centrifugation, and the purity of the isolated cells was estimated by cytofluorimetric analysis. Following a single step, the percentage of DLT15-purified cells was 88 +/- 10% for gut-associated lymphoid tissue and 79 +/- 18% for blood leucocytes. DLT15-purified cells from gut-associated lymphoid tissue were employed for RNA extraction and cDNA synthesis. In RT-PCR experiments using as primers degenerate oligonucleotides corresponding to the peptide sequence MYWY and VYFCA of the trout TcR beta chain, a 203 bp product was amplified. When sequenced, the cDNA was found to show 60% nucleotide identity to the tr out TcRV beta 3. By 3'-RACE the cDNA was elongated to obtain the TcR constant region, with high similarity to other fish TcR sequences. These results strongly suggest that cells recognised by DLT15 are putative T lymphocytes. (C) 2000 Academic Press.

Original languageEnglish
Pages (from-to)329-341
Number of pages13
JournalFish & Shellfish Immunology
Volume10
Publication statusPublished - 2000

Keywords

  • fish
  • sea bass
  • Dicentrarchus labrax
  • monoclonal antibody
  • T-cells
  • T-cell receptor
  • TROUT ONCORHYNCHUS-MYKISS
  • THYMOCYTE MONOCLONAL-ANTIBODIES
  • RECEPTOR-BETA-CHAIN
  • CYPRINUS-CARPIO L
  • RAINBOW-TROUT
  • LYMPHOCYTE HETEROGENEITY
  • ANTIGENIC DETERMINANTS
  • CHANNEL CATFISH
  • TELEOST FISH
  • IMMUNOCYTOCHEMICAL DETECTION

Cite this

Scapigliati, G., Romano, N., Abelli, L., Meloni, S., Ficca, A. G., Buonocore, F., ... Secombes, C. J. (2000). Immunopurification of T-cells from sea bass Dicentrarchus labrax (L.). Fish & Shellfish Immunology, 10, 329-341.

Immunopurification of T-cells from sea bass Dicentrarchus labrax (L.). / Scapigliati, G ; Romano, N ; Abelli, L ; Meloni, S ; Ficca, A G ; Buonocore, F ; Bird, S ; Secombes, C J .

In: Fish & Shellfish Immunology, Vol. 10, 2000, p. 329-341.

Research output: Contribution to journalArticle

Scapigliati, G, Romano, N, Abelli, L, Meloni, S, Ficca, AG, Buonocore, F, Bird, S & Secombes, CJ 2000, 'Immunopurification of T-cells from sea bass Dicentrarchus labrax (L.)', Fish & Shellfish Immunology, vol. 10, pp. 329-341.
Scapigliati G, Romano N, Abelli L, Meloni S, Ficca AG, Buonocore F et al. Immunopurification of T-cells from sea bass Dicentrarchus labrax (L.). Fish & Shellfish Immunology. 2000;10:329-341.
Scapigliati, G ; Romano, N ; Abelli, L ; Meloni, S ; Ficca, A G ; Buonocore, F ; Bird, S ; Secombes, C J . / Immunopurification of T-cells from sea bass Dicentrarchus labrax (L.). In: Fish & Shellfish Immunology. 2000 ; Vol. 10. pp. 329-341.
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abstract = "The monoclonal antibody DLT15, specific for thymocytes and peripheral T-cells of the teleost fish Dicentrarchus labrax: (sea bass), was used to purify immunoreactive cells from blood and gut-associated lymphoid tissue. The purification was performed by immune-magnetic sorting of leucocyte fractions enriched by Percoll density gradient centrifugation, and the purity of the isolated cells was estimated by cytofluorimetric analysis. Following a single step, the percentage of DLT15-purified cells was 88 +/- 10{\%} for gut-associated lymphoid tissue and 79 +/- 18{\%} for blood leucocytes. DLT15-purified cells from gut-associated lymphoid tissue were employed for RNA extraction and cDNA synthesis. In RT-PCR experiments using as primers degenerate oligonucleotides corresponding to the peptide sequence MYWY and VYFCA of the trout TcR beta chain, a 203 bp product was amplified. When sequenced, the cDNA was found to show 60{\%} nucleotide identity to the tr out TcRV beta 3. By 3'-RACE the cDNA was elongated to obtain the TcR constant region, with high similarity to other fish TcR sequences. These results strongly suggest that cells recognised by DLT15 are putative T lymphocytes. (C) 2000 Academic Press.",
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N2 - The monoclonal antibody DLT15, specific for thymocytes and peripheral T-cells of the teleost fish Dicentrarchus labrax: (sea bass), was used to purify immunoreactive cells from blood and gut-associated lymphoid tissue. The purification was performed by immune-magnetic sorting of leucocyte fractions enriched by Percoll density gradient centrifugation, and the purity of the isolated cells was estimated by cytofluorimetric analysis. Following a single step, the percentage of DLT15-purified cells was 88 +/- 10% for gut-associated lymphoid tissue and 79 +/- 18% for blood leucocytes. DLT15-purified cells from gut-associated lymphoid tissue were employed for RNA extraction and cDNA synthesis. In RT-PCR experiments using as primers degenerate oligonucleotides corresponding to the peptide sequence MYWY and VYFCA of the trout TcR beta chain, a 203 bp product was amplified. When sequenced, the cDNA was found to show 60% nucleotide identity to the tr out TcRV beta 3. By 3'-RACE the cDNA was elongated to obtain the TcR constant region, with high similarity to other fish TcR sequences. These results strongly suggest that cells recognised by DLT15 are putative T lymphocytes. (C) 2000 Academic Press.

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