Development of an immunosensor assay for detection of haptoglobin in mastitic milk

Background Detection of subclinical mastitis in dairy cows is important, as it represents a major economic challenge for the dairy industry worldwide owing to propagation of mastitis-causing pathogens and to long-term reduction in milk yield and quality. Haptoglobin (Hp) is one of the most sensitive acute phase proteins in milk during udder inflammation and as an indicator of mastitis. Objectives The aim of this study was to develop a rapid and sensitive immunosensor assay for measuring Hp concentration in mastitic milk. Methods The immunosensor was constructed by immobilizing anti-bovine Hp antibody on a gold electrode through gold nanoparticles fabricated on self-assembled L-cysteine layers. The immunosensor assay was used to measure Hp concentration in 20 milk samples positive for bacteria with a somatic cell count>5x105cells/mL from cows without clinical signs of mastitis. Results were compared with those obtained using a commercial ELISA kit. Results Reproducibility of Hp measurement and stability after storage for 20days were good for the immunosensor assay. Measurement of Hp was linear over a range of 15100mg/L, with a limit of detection of 0.63mg/L. Agreement between results obtained with the immunosensor and ELISA methods was satisfactory as analyzed using the Wilcoxon signed-rank test (Z=1.739, P=.073). Conclusion An immunosensor assay for measuring Hp in milk provided rapid results and was easy to perform, facilitating its potential use in the field for the diagnosis of subclinical mastitis once a cutoff value for Hp concentration is established.