Determination of IgM Concentration

The concentrations of IgM in skim milk samples were determined by conventional ELISA. In detail, AffiniPure rabbit anti-human IgM antibody as a capture antibody (Jackson ImmunoResearch, Europe Ltd., Ely, UK) diluted 1:2,000 was taken, and the plates were incubated for 2 h at 37°C. Then, the plates were incubated for 2 h at 37°C and overnight at 4°C with TBS (pH 7.5) containing 0.5% Tween-20 as a blocking agent. For testing, 100 μL of 50-, 100-, and 200-fold diluted skim milk and human IgM standard preparation from 0.39 to 6.25 ng/100 μL (Jackson ImmunoResearch, Europe Ltd., Ely, UK) were taken. In the next step, horseradish peroxidase-conjugated goat anti-human IgM antibodies (Jackson ImmunoResearch, Europe Ltd., Ely, UK) diluted 1:20,000 in TBS containing 0.05% Tween-20 were added to each well, and the plates were incubated for 1 h at 37°C. The reaction was developed by adding orthophenylenediamine (Calbiochem, Denmark) in 0.1 M citrate buffer, pH 5.0 with H₂O₂, and the plates were incubated for 10 min at room temperature in the dark. The reaction was stopped with 12.5% H₂SO₄, and the absorbance was measured in a Stat Fax 2100 Microplate Reader (Awareness Technology Inc., Palm City, FL, USA) at 492 nm, with 630 nm as the reference filter. For dilution of skim milk samples and for all washing steps, TBS (pH 7.5) containing 0.1 or 0.05% Tween-20 was used.

The coefficients of variation were calculated for IgM-ELISA, namely, 5.4 and 2.5% for intra- and inter-assay, respectively.