Consequences of interference of milk with chemoattractants for enzyme-linked immunosorbent assay quantifications.

Concentrations of the chemoattractants CXCL1, CXCL2, CXCL3, CXCL8, and C5a in milk were reduced by the preparation of milk whey by high-speed centrifugation or with rennet. About half of the chemoattractants (35 to 65%) were associated with the casein micelle sediment, except when whey was prepared by acidification. Consequently, quantification of chemoattractants should be carried out preferentially with skimmed milk samples or, whenever whey is needed, with acidic whey samples. The interference of milk or milk whey with the enzyme-linked immunosorbent assays (ELISAs) used to quantify the chemoattractants was moderate, as long as tetramethylbenzidine (TMB), not ABTS [2,2'-azino-bis-(3-ethylbenzthiazoline-sulfonate)], was used as the substrate of peroxidase. These considerations will help to assess more precisely a component of the immune response of the mammary gland to infection.