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

Abstract

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 > 5 × 10(5) cells/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 20 days were good for the immunosensor assay. Measurement of Hp was linear over a range of 15-100 mg/L, with a limit of detection of 0.63 mg/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.