Department of Animal Science, Iowa State University, Ames Iowa, 50014, USA.
J Dairy Sci. 2013 Jul;96(7):4535-41. doi: 10.3168/jds.2012-5843. Epub 2013 May 9.
Objectives of this study were to develop a rapid calf-side test to determine serum IgG concentrations using caprylic acid (CA) fractionation, followed by refractometry of the IgG-rich supernatant and compare the accuracy of this method with results obtained using refractometry using raw serum. Serum samples (n=200) were obtained from 1-d-old calves, frozen (-20°C), and shipped to the laboratory. Samples were allowed to thaw for 1h at room temperature. Fractionation with CA was conducted by adding 1mL of serum to a tube containing 45, 60, or 75µL of CA and 0.5, 1.0, or 1.5mL of 0.06 M acetic acid. The tube contents were mixed well, allowed to react for 1 min, and then centrifuged at 3,300 × g for 0, 10, or 20 min at 25°C. The %Brix and refractive index of the fractionated supernatant were determined using a digital refractometer. Nonfractionated serum was analyzed for %Brix (BRn), refractive index (nDn), and IgG concentration by radial immunodiffusion. The mean serum IgG concentration was 19.0 mg/mL [standard deviation (SD)=9.7], with a range of 3.5 to 47.0 mg/mL. The mean serum BRn was 8.6 (SD=0.91), with a range of 6.8 to 11.0. The mean serum nDn was 1.34566 (SD=0.00140), with a range of 1.34300 to 1.34930. Serum nDn was positively correlated with IgG concentration (correlation coefficient=0.86; n=185). Fractionated samples treated with 1mL 0.6 M acetic acid and 60µL of CA and not centrifuged before analysis resulted in a strong relationship between the refractive index of the fractionated supernatant and IgG (correlation coefficient=0.80; n=45). Regression was used to determine cut points indicative of 10, 12, and 14 mg of IgG/mL to determine the sensitivity and specificity of refractometry to identify failure of passive transfer (serum IgG <10 mg/mL at 24 h old). The nDn were 1.34414, 1.34448, and 1.34480 to predict 10, 12, and 14 mg of IgG/mL of serum, respectively. The BRn cut points were 7.6, 7.8, and 8.0, respectively. The nDn cut points of 1.34448 and 1.34480 resulted in similar specificities (82.9%), whereas the 1.34414 cut point had a specificity of 60.0%. The BRn cut point of 7.6 and 7.8%Brix resulted in a similar percentage of correctly classified samples (89.7 and 90.8%, respectively); however, the 7.8% Brix cut point resulted in fewer false positives. These results suggest that Brix refractometry of nonfractionated calf serum provides a strong estimate of IgG concentration and 7.8% Brix may be used as the cut point to identify failure of passive transfer in 1-d-old calves.
本研究的目的是开发一种快速的小腿侧测试，使用辛酸（CA）分级来确定血清 IgG 浓度，然后对富含 IgG 的上清液进行折射度测定，并将该方法的准确性与使用原始血清进行折射度测定的结果进行比较。从 1 日龄的小牛中获得血清样本（n=200），冷冻（-20°C），并运送到实验室。样本在室温下解冻 1 小时。通过向含有 45、60 或 75µL CA 和 0.5、1.0 或 1.5mL 0.06 M 乙酸的管中加入 1mL 血清来进行 CA 分级。将管内容物充分混合，反应 1 分钟，然后在 25°C 下以 3300×g 离心 0、10 或 20 分钟。使用数字折射计测定分级上清液的 Brix 值（%Brix）和折射率（nDn）。非分级血清用于通过放射免疫扩散分析 %Brix（BRn）、折射率（nDn）和 IgG 浓度。血清 IgG 浓度的平均值为 19.0mg/mL[标准差（SD）=9.7]，范围为 3.5 至 47.0mg/mL。血清 BRn 的平均值为 8.6（SD=0.91），范围为 6.8 至 11.0。血清 nDn 的平均值为 1.34566（SD=0.00140），范围为 1.34300 至 1.34930。血清 nDn 与 IgG 浓度呈正相关（相关系数=0.86；n=185）。用 1mL 0.6 M 乙酸和 60µL CA 处理的分级样本，在分析前不离心，导致分级上清液的折射率与 IgG 之间存在很强的关系（相关系数=0.80；n=45）。使用回归确定指示 10、12 和 14mg/mL IgG 的切点，以确定折射度测定法识别被动转移失败（24 小时龄时血清 IgG<10mg/mL）的灵敏度和特异性。nDn 分别为 1.34414、1.34448 和 1.34480 以预测 10、12 和 14mg/mL 的血清 IgG。BRn 切点分别为 7.6、7.8 和 8.0。nDn 切点为 1.34448 和 1.34480 导致相似的特异性（82.9%），而 1.34414 切点的特异性为 60.0%。BRn 切点为 7.6 和 7.8%Brix 导致正确分类样本的百分比相似（分别为 89.7%和 90.8%）；然而，7.8%Brix 切点导致假阳性更少。这些结果表明，小牛非分级血清的 Brix 折射度可很好地估计 IgG 浓度，7.8% Brix 可作为识别 1 日龄小牛被动转移失败的切点。
