Development of Anti-Bovine TNF-α mAb and ELISA for Quantitating TNF-α in Milk After Intramammary Injection of Endotoxin

Abstract

Murine mAb reactive with recombinant bovine tumor necrosis factor-α (r-boTNF-α) were produced. An ELISA using murine mAb and rabbit polyclonal antibodies, each reactive with r-boTNF-α to sandwich bovine TNF-α was developed.Secretion of TNF-α in quarter milk increased 1h after injection of 0.1mg (four cows) or 0.5mg (four cows) Escherichia coli lipopolysaccharide (LPS) into a mammary quarter, peaked 1 to 5h later, and returned to control levels in 24h. There were no differences in body temperature, SCC, TNF-α, and blood leukocyte responses between 0.1 and 0.5mg of LPS. To determine effects of repeated injections of LPS into the same udder, a second injection of 0.1mg of LPS into the same quarter (two cows) 24h after the first injection produced a strongly attenuated TNF-α response. However, a normal TNF-α response was observed when LPS was injected into a contralateral quarter (two cows) 24h 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 12h after the LPS injections. It would appear that these changes were not regulated by TNF-α secretion because the changes were also similar after the second injection of LPS into the same mammary quarter.