Quantification of Serum Acute Phase Proteins

Measurements of the level of serum amyloid A in blood serum and uterine flush was performed using a commercial ELISA kit (Tridelta Development Ltd., Maynooth, Kildare, Ireland). The inter- and intra-assay coefficients of variation for SAA analysis were <12.1 and <7.5 %, respectively. The determination of haptoglobin in blood serum and uterine flush was performed using a commercial colorimetric assay kit (Tridelta Development Ltd., Kildare, Ireland). The inter- and intra-assay CVs for Hp analysis were <5.7 and <6.3 %, respectively. Procedures were performed according to the manufacturers’ instructions and literature methods (Suojala et al. 2008 (link) and Tothova et al. 2012 (link)). Absorbance readings and subsequent calculations of final concentrations were performed on an automatic microplate reader (Asys Expert Plus, Biochrom Ltd., Cambridge, England) at 450 nm, 630 nm for Hp, and 630 nm as a reference for SAA. Lyophilized bovine acute phase serum was used as a standard; calibration was performed according to the European Union concerted action on standardization of animal APPs (No. QLK5-CT-1999-0153).