Production and characterization of two monoclonal antibodies to bovine tumour necrosis factor alpha (TNF-alpha) and their cross-reactivity with ovine TNF-alpha.
Tumour necrosis factor alpha (TNF-alpha) is an innate pro-inflammatory cytokine involved in protection against intracellular pathogens. Existing methods for measuring TNF-alpha production and function in ruminants are limited to ELISA and many rely on polyclonal antisera. With a view to developing improved detection methods for bovine (bov) TNF-alpha, monoclonal antibodies (mAb) were produced by immunising mice with a plasmid encoding bov TNF-alpha. Two of the resulting mAb, termed CC327 and CC328, were used to develop a sandwich ELISA capable of detecting both native and recombinant bov TNF-alpha. This ELISA did not detect recombinant ovine (ov) TNF-alpha. A luminometric method was applied to the ELISA to improve sensitivity for detection of native bov TNF-alpha in culture supernatants derived from bovine monocyte-derived dendritic cells (DC) infected with Mycobacterium bovis. Both CC327 and CC328 detected intracytoplasmic expression of TNF-alpha in mitogen-activated bovine T lymphocytes. However, only CC328 detected intracytoplasmic ovine TNF-alpha in transfected cells, explaining the failure of the sandwich ELISA to detect recombinant ov TNF-alpha. These mAbs have generated the capability to study the role of TNF-alpha in host immune protection and disease pathogenesis in ruminants.
Kwong
L S
LS
Institute for Animal Health, Compton, Near Newbury, RG20 7NN, UK.
Thom
M
M
Sopp
P
P
Rocchi
M
M
Wattegedera
S
S
Entrican
G
G
Hope
J C
JC
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D057134:Antibodies, Neutralizing / Q000096:biosynthesis / Q000276:immunology; D001483:Base Sequence; D002417:Cattle / Q000235:genetics / Q000276:immunology*; D003429:Cross Reactions; D017931:DNA Primers / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000662:veterinary; D051379:Mice; D010957:Plasmids / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000276:immunology; D012756:Sheep / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis / Q000235:genetics / Q000276:immunology*
D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D017931:DNA Primers; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha