Department of Animal Science, Cornell University, Ithaca, NY 14853.
Sibley School of Mechanical and Aerospace Engineering, Cornell University, Ithaca, NY 14853.
J Dairy Sci. 2020 May;103(5):4743-4753. doi: 10.3168/jds.2019-17517. Epub 2020 Mar 18.
Our objective was to develop and validate a tool integrating a disposable fluorescence-based lateral flow immunoassay (LFIA) coupled with a portable imaging device for estimating circulating plasma concentrations of progesterone (P4). First, we developed and optimized a competitive LFIA test strip to measure P4 in bovine plasma. The LFIA design included a sample pad, a conjugate pad that stores R-phycoerythrin-anti-P4 conjugates, a glass-fiber spacer pad, a nitrocellulose membrane with printed test and control lines, and a cellulose-fiber absorbent pad. To perform a test, 20 µL of plasma and 50 µL of running buffer were added on the sample pad. After 3 min, 45 µL of running buffer was added to initiate sample flow. After allowing 15 min to stabilize the colorimetric signal, strips were introduced in an LFIA portable reader wirelessly linked to a laptop to determine P4 concentration based on test-to-control-line signal (T/C ratio). In a series of experiments (n = 6), the ability of the LFIA to differentiate plasma samples with ≥1 or <1 ng/mL of P4 was evaluated. For each experiment, a calibration curve was constructed using plasma with known concentrations of P4 (0.1 to 3.7 ng/mL; n = 5). The resulting linear equation was then used to determine a T/C ratio cutoff to differentiate samples with ≥1 or <1 ng/mL of P4. In addition, to evaluate the ability of the platform to assign samples to P4 concentration groups without a calibration curve for individual batches, we performed a receiver operating characteristic analysis to identify a single cutoff value for T/C ratio that could potentially be used for all batches. Overall, calibration curves showed a linear relationship between T/C ratio and P4 levels (mean coefficient of determination = 0.74; range 0.42 to 0.99). Next, plasma samples from lactating dairy cows (n = 58) were tested in triplicate to determine the ability of the LFIA system to differentiate plasma samples with ≥1 or <1 ng/mL of P4 using a RIA for P4 as reference test. Overall, the LFIA assay correctly classified 90% of the samples, with 97% sensitivity, 83% specificity, 85% positive predictive value, and 96% negative predictive value. Agreement between the tests was substantial (kappa = 0.79; 95% confidence interval 0.64 to 0.95). When using a single cutoff value for T/C ratio selected by receiver operating characteristic analysis, sensitivity and specificity to determine CL presence were 97 (95% confidence interval 82 to 99) and 79% (95% confidence interval 60 to 92), respectively. These data suggest that the developed portable LFIA system can accurately differentiate plasma samples with ≥1 or <1 ng/mL of P4.
我们的目标是开发和验证一种工具，该工具将基于一次性荧光侧向流动免疫测定 (LFIA) 与便携式成像设备集成在一起，用于估计孕酮 (P4) 的循环血浆浓度。首先，我们开发并优化了一种竞争性 LFIA 测试条，用于测量牛血浆中的 P4。LFIA 的设计包括样品垫、储存 R-藻红蛋白-抗 P4 结合物的结合垫、玻璃纤维间隔垫、带有打印测试线和控制线的硝酸纤维素膜以及纤维素纤维吸收垫。要进行测试，将 20µL 血浆和 50µL 运行缓冲液添加到样品垫上。3 分钟后，添加 45µL 运行缓冲液以启动样品流动。在稳定比色信号 15 分钟后，将条带引入 LFIA 便携式读取器中，该读取器通过无线方式与笔记本电脑连接，以根据测试线到控制线信号 (T/C 比) 确定 P4 浓度。在一系列实验 (n=6) 中，评估 LFIA 区分具有 ≥1 或 <1ng/mL P4 的血浆样本的能力。对于每个实验，使用具有已知 P4 浓度 (0.1 至 3.7ng/mL；n=5) 的血浆构建校准曲线。然后使用所得线性方程确定区分具有 ≥1 或 <1ng/mL P4 的样本的 T/C 比截止值。此外，为了评估该平台在没有针对每个批次的校准曲线的情况下将样本分配到 P4 浓度组的能力，我们进行了接收者操作特征分析，以确定 T/C 比的单个截止值，该值可能适用于所有批次。总体而言，校准曲线显示 T/C 比与 P4 水平之间存在线性关系 (平均确定系数 =0.74；范围 0.42 至 0.99)。接下来，使用放射免疫分析 (RIA) 作为参考测试，对来自哺乳期奶牛的 58 个血浆样本进行了三次重复测试，以确定 LFIA 系统区分具有 ≥1 或 <1ng/mL P4 的血浆样本的能力。总体而言，LFIA 检测法正确分类了 90%的样本，具有 97%的敏感性、83%的特异性、85%的阳性预测值和 96%的阴性预测值。两种检测方法之间的一致性很高 (kappa=0.79；95%置信区间 0.64 至 0.95)。当使用接收器操作特征分析选择的 T/C 比的单个截止值时，确定 CL 存在的灵敏度和特异性分别为 97%(95%置信区间 82 至 99)和 79%(95%置信区间 60 至 92)。这些数据表明，开发的便携式 LFIA 系统可以准确地区分具有 ≥1 或 <1ng/mL P4 的血浆样本。
