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

Abstract

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.

FIG. 1.

Percentage of the constitutive milk chemokine CXCL1 and CXCL3 concentrations (100%, concentrations in skim milk) as a function of milk treatment. Milk wheys were prepared by lowering the pH to 4.3 and bringing it back to 7.0 (acidic), by ultracentrifugation (100,000 × g for 30 min), by centrifugation (18,000 × g for 90 min), or with rennet. Chemokine concentrations were measured by ELISAs. Results are medians from 8 milk samples (bars are for the upper and lower deciles). Asterisks indicate statistical significance (P < 0.05) of difference from concentrations in skim milk (Friedman test followed by multiple comparisons).

FIG. 2.

Effect of skim milk or whey prepared by acidification on CXCL2 ELISA results. (a) Various concentrations of CXCL2 were prepared in either PBS-G, skim milk, or acidic whey, as they were for establishing reference curves. The ELISAs were performed as described in Materials and Methods. (b) The same concentrations of CXCL2 were prepared in parallel in PBS-G at pH 6.2, 6.8, and 7.4, and these samples were used in the ELISA to determine the effect of sample pH on the results. The chromogenic substrate was TMB. The three conditions were tested in parallel in the same ELISA plate to reduce factors of variation. Results are from 4 experiments made in duplicate and are expressed as median values. Variation bars indicate the first and third quartiles.

FIG. 3.

Effect of skim milk or whey prepared by acidification on C5a ELISA results. (a) Various concentrations of C5a were prepared in either PBS-G, skim milk, or acidic whey, as they were for establishing reference curves. The ELISAs were performed as described in Materials and Methods. (b) Conditions were the same as those described above for panel a, except that ABTS was used as the chromogenic substrate instead of TMB.

FIG. 4.

Effect of skim milk or whey prepared by acidification on CXCL8 ELISA results. (a) Various concentrations of CXCL8 were prepared in either PBS-G, skim milk, or acidic whey, as they were for establishing reference curves. The ELISAs were performed as described in Materials and Methods. (b) Conditions were the same as those described above for panel a, except that ABTS was used as the chromogenic substrate instead of TMB.