Abstract
Detection of subclinical mastitis (SCM) in its initial stage can save great economic losses, improve milk quality and animal welfare. We have developed a semiquantitative lateral flow assay for the detection of SCM in dairy cows targeting myeloperoxidase (MPO) enzyme of milk neutrophils. A competitive immunoassay format was used, and colloidal gold nanoparticles (GNP) were prepared and used as a labelling agent. Monoclonal anti-MPO antibodies were used and assessed for its quality by enzyme-linked immunosorbent assay and dot blot. Conjugation method for GNP and anti-MPO antibodies was standardised, and the conjugate was placed over the conjugate pad. MPO coupled with a carrier protein (OVA) and the species-specific secondary antibodies were placed on test and control lines, respectively. The developed assay was verified with 75 milk samples collected from healthy, SCM and clinical mastitis cows. It displayed a high sensitivity as it could detect MPO as low as 1.5 ng/ml, an accuracy greater than 97% and showed no crossreactivity when crosschecked with other milk proteins. The developed assay can be used as an alternative for SCM diagnostic tests where lab structure are available for obtaining the lysate of milk SCC.
Highlights
Detection of subclinical mastitis (SCM) in its initial stage can save great economic losses, improve milk quality and animal welfare
If SCM is not diagnosed during early stage and proper management actions are not taken, it may develop into clinical mastitis (CM) and lead to substantial economic losses to the dairy sector[1]
There are many diagnostic approaches for SCM, the somatic cell counts (SCC) and California mastitis test are still the most common and preferable methods[2,7]. These tests are mainly dependent on the total number of milk SCC, which primarily increase during SCM due to an increase in different leucocytes number[8]
Summary
Detection of subclinical mastitis (SCM) in its initial stage can save great economic losses, improve milk quality and animal welfare. We have developed a semiquantitative lateral flow assay for the detection of SCM in dairy cows targeting myeloperoxidase (MPO) enzyme of milk neutrophils. The developed assay was verified with 75 milk samples collected from healthy, SCM and clinical mastitis cows. It displayed a high sensitivity as it could detect MPO as low as 1.5 ng/ml, an accuracy greater than 97% and showed no crossreactivity when crosschecked with other milk proteins. There are many diagnostic approaches for SCM, the somatic cell counts (SCC) and California mastitis test are still the most common and preferable methods[2,7]. Depreester et al.[15] developed a flow cytometric method for the determination of MPO in bovine blood neutrophils
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