2.6. Immunological Analysis of Milk Samples

The concentrations of 23 immune factors, including innate immune factors (IL1β, IL6, IL12, IFNγ, TNFα), acquired immunity factors (IL2, IL4, IL10, IL13, IL17), immunoglobulins (Igs) (IgA, total IgG [IgGt], IgM), chemokines (IL8, Groα, MCP1, MIP1β), and growth factors (IL5, IL7, granulocyte colony-stimulating factor (GCSF), granulocyte–macrophage colony-stimulating factor (GMCSF), TGFβ₂, and epidermal growth factor [EGF]) were determined by magnetic bead-based multiplex immunoassays using a Bioplex 200 instrument (Bio-Rad, Hercules, California, USA) and the Bio-Plex Pro Human Cytokine, Chemokine, and Growth Factor Assays (Bio-Rad), according to manufacturer’s instructions. EGF was determined by ELISA using the RayBio Human EGF ELISA kit (RayBiotech, Norcross, GA, USA). Concentrations of Igs (IgA, IgGt, and IgM) were determined using the Bio-Plex Pro Human Isotyping Assay kit (Bio-Rad) in the Bioplex system instrument.

Prior to their analysis, milk samples (1 mL) were centrifuged (800× g, 15 min, 4 °C), and after removing the upper fat layer, the supernatant was transferred and aliquoted into different tubes for subsequent immunological analysis. A fresh aliquot was used for each assay, avoiding defrosting cycles. Every assay was run in duplicate according to the manufacturer’s instructions, and standard curves were performed for each analyte in every assay. The inter-assay coefficients of variation were below manufacturers’ instructions for all the immune markers. Concentrations of immunological compounds were expressed as the weight per volume of whole milk.