Dept. Production Animal Health, Faculty of Veterinary Medicine, University of Calgary, Calgary, Alberta, Canada.
Dept. of Agricultural Food and Nutritional Science, University of Alberta, Edmonton, AB, Canada.
Res Vet Sci. 2022 Oct;147:60-67. doi: 10.1016/j.rvsc.2022.04.003. Epub 2022 Apr 12.
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.
低钙血症定义为全血钙浓度低于 2.1 mM，对奶牛的福利、生产和繁殖有不利影响。然而，目前还没有用于检测牛全血钙的牛场检测方法。在这里，我们通过将钙调蛋白和 M13 肽作为融合伴侣与海藻糖酶片段结合，对分离的海藻糖酶互补测定法进行了修改，以检测血清中的总钙。在钙存在的情况下，钙调蛋白构象发生变化，与 M13 肽的亲和力增强。通过加入特定浓度的钙螯合剂 EDTA 在温和酸性条件下，将动态范围从 10 μM 扩展到 1 μM 至 1 mM 之间，开发了一种高反应性的钙检测方法。该传感器在 213 份牛血清样本的集合上进行了验证，并与定量比色钙测试进行了比较。实现了 0.81 的相关系数，亚临床低钙血症的检测准确性为 0.86，特异性为 100%。接受者操作特性曲线下的面积为 0.93。一致性相关系数（0.80）、Bland-Altman 图和加权 Kappa 系数（0.71）表明两种方法之间存在实质性一致性。总之，开发了一种新的总钙测试方法，可作为一种方便的高通量实验室测试方法，并有可能与适合于农场测试的版本相结合。
