The antioxidant haptoglobin (Hp) is an acute-phase protein responsive to infectious and inflammatory diseases. Hp and somatic cell counts (SCC) are sharply elevated in bovine milk following intramammary administration of endotoxin or bacteria. However, the sources of milk Hp responsible for such increases are not fully understood. The purpose of this study was to define the source of milk Hp from dairy cows with naturally occurring mastitis. Quarter milk samples selected from 50 dairy cows were separated into four groups according to SCC as group A: < 100 (n = 19); B: 100–200 (n = 10); C: 201–500 (n = 10); and D: > 500 × 103 (n = 11) cells/mL. Our results reveal that milk Hp concentrations were correlated with SCC (r = 0.742; P < 0.01), and concentrations in group D were ~10-fold higher than in group A. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis indicates that the milk somatic cells from group D were not only capable of synthesizing Hp but could also markedly increase Hp mRNA expression. Western blot, immunocytochemistry, double confocal immunofluorescence, and Hp releasing experiments demonstrate that neutrophils were associated with the biosynthesis and release of Hp in milk. It further shows that Hp was significantly elevated in the epithelium of mammary gland tissue with mastitis and was also expressed in the cultured mammary epithelial cells. We propose that neutrophils and epithelial cells may play an essential role in elevating milk Hp in addition to previous suggestions that Hp may be derived from mammary tissues and circulation.

Molten globules are thought to be general intermediates in protein folding and unfolding. beta-lactoglobulin (beta-LG) is one of the major bovine whey proteins, constituting approximately 10 to 15% of total milk proteins. We have recently identified beta-LG as a superior marker for evaluating thermally processed milk. Strand D of beta-LG participates in irreversible thermal unfolding as probed by a monoclonal antibody (mAb) specific to thermally denatured beta-LG. In the present study, we used native beta-LG as an immunogen to test the hypothesis that a specific mAb against the native beta-LG could be established. As result, a mAb (4H11E8) directed against the native structure of beta-LG was made. The antibody did not recognize the heat-denatured form of beta-LG, such as its dimer and aggregates. Immunoassay using this "native" mAb showed that the stability of beta-LG was at temperatures < or =70 degrees C. beta-Lactoglobulin began to deteriorate between 70 and 80 degrees C over time. The denaturation was correlated with the transition temperature of beta-LG. Further chemical modification of Cys (carboxymethylation) or positively charged residues (acetylation) of beta-LG totally abolished its immunoreactivity, confirming the conformation-dependent nature of this mAb. Using competitive ELISA, the 4H11E8 mAb could determine the native beta-LG content in commercially processed milks. Concentrations of native beta-LG varied significantly among the local brands tested. From a technological standpoint, the mAb prepared in this study is relevant to the design and operation of appropriate processes for thermal sanitation of milk and of other dairy products.