Development of a blood calcium test for hypocalcemia diagnosis in dairy cows

Hypocalcemia, defined as total blood calcium concentrations below 2.1 mM, has detrimental impacts on welfare, production and reproduction in dairy cattle. Yet, no cow-side test exists for testing total bovine blood calcium. Here, we modified the split trehalase complementation assay to detect total calcium in serum by incorporating calmodulin and the M13 peptide as fusion partners to the trehalase fragments. In the presence of calcium, calmodulin undergoes a conformation change and gains strong affinity for M13 peptide. A high reactive assay for calcium was developed with detection threshold of 10 uM and dynamic range between 1 uM and 1 mM. The addition of a specific concentration of calcium chelator, EDTA, in mild acidic conditions, shifted the dynamic range to physiological calcium concentrations and transformed the sensor from ionized calcium sensor to total calcium sensor. The sensor was validated on a collection of 213 bovine serum samples by comparison with quantitative colorimetric calcium test. A correlation coefficient of 0.81 was achieved and the accuracy of detecting subclinical hypocalcemia was 0.86 and specificity of 100%. The area under the Receiver Operating Characteristic curve was 0.93. The concordance correlation coefficient (0.80), Bland-Altman plot and weighted Kappa coefficient (0.71) demonstrated a substantial agreement between both methods. In conclusion, a novel total calcium test was developed that can be used as a convenient high throughput laboratory test and with potential to be incorporated into a version compatible with on-farm testing.