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Journal of Dairy Science Vol. 85 No. 4 765-773
© 2002 by American Dairy Science Association ®
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Development of Anti-Bovine TNF-alpha mAb and ELISA for Quantitating TNF-alpha in Milk After Intramammary Injection of Endotoxin

M. J. Paape 1, P. M. Rautiainen 2, E. M. Lilius 2, C. E. Malstrom 1, and T. H. Elsasser 3

1 Immunology and Disease Resistance Laboratory, ARS, USDA, Beltsville, Maryland 20705
2 Department of Biochemistry, University of Turku, FIN-20500 Turku, Finland
3 Growth Biology Laboratory, ARS, USDA, Beltsville, Maryland 20705

Murine mAb reactive with recombinant bovine tumor necrosis factor-alpha (r-boTNF-alpha) were produced. An ELISA using murine mAb and rabbit polyclonal antibodies, each reactive with r-boTNF-alpha to sandwich bovine TNF-alpha was developed.

Secretion of TNF-alpha in quarter milk increased 1 h after injection of 0.1 mg (four cows) or 0.5 mg (four cows) Escherichia coli lipopolysaccharide (LPS) into a mammary quarter, peaked 1 to 5 h later, and returned to control levels in 24 h. There were no differences in body temperature, SCC, TNF-alpha, and blood leukocyte responses between 0.1 and 0.5 mg of LPS. To determine effects of repeated injections of LPS into the same udder, a second injection of 0.1 mg of LPS into the same quarter (two cows) 24 h after the first injection produced a strongly attenuated TNF-alpha response. However, a normal TNF-alpha response was observed when LPS was injected into a contralateral quarter (two cows) 24 h after the first LPS injection. Leukocyte counts in blood decreased and body temperature increased substantially after each injection of LPS. Quarter milk SCC increased 200-fold 8 to 12 h after the LPS injections. It would appear that these changes were not regulated by TNF-alpha secretion because the changes were also similar after the second injection of LPS into the same mammary quarter.

Key Words: tumor necrosis factor-alpha • endotoxin • lipopolysaccharide • mastitis

Submitted on December 26, 2000
Accepted on August 15, 2001




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