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.

中文翻译：

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基于侧向流动的便携式平台，用于确定牛的繁殖状况。

我们的目标是开发和验证一种工具，该工具将一次性的基于荧光的侧向流免疫测定（LFIA）与便携式成像设备结合在一起，用于估计孕酮（P4）的循环血浆浓度。首先，我们开发并优化了竞争性LFIA测试条，以测量牛血浆中的P4。LFIA设计包括样品垫，存储R-藻红蛋白-抗P4结合物的结合物垫，玻璃纤维间隔垫，带有印刷测试线和对照线的硝酸纤维素膜以及纤维素纤维吸收垫。为了进行测试，在样品垫上添加了20 µL血浆和50 µL运行缓冲液。3分钟后，添加45 µL运行缓冲液以启动样品流。静置15分钟以稳定比色信号后，将试纸条引入与笔记本电脑无线链接的LFIA便携式阅读器中，以根据测试对控制线信号（T / C比）确定P4浓度。在一系列实验（n = 6）中，评估了LFIA区分P4≥1或<1 ng / mL的血浆样品的能力。对于每个实验，使用血浆中已知P4浓度（0.1至3.7 ng / mL； n = 5）构建校正曲线。然后将所得的线性方程式用于确定T / C比截止值，以区分P4≥1或<1 ng / mL的样品。此外，为评估平台将样品分配给P4浓度组而无需单独批次的校准曲线的能力，我们执行了接收器操作特性分析，以识别可能适用于所有样品的T / C比的单个临界值。批次。总体而言，校准曲线显示T / C比与P4水平之间呈线性关系（平均测定系数= 0.74；范围为0.42至0.99）。接下来，一式三份测试来自泌乳奶牛（n = 58）的血浆样品，以确定LFIA系统使用PIA的RIA作为参考测试，区分P4≥1或<1 ng / mL的血浆样品的能力。总体而言，LFIA分析正确分类了90％的样品，灵敏度为97％，特异性为83％，阳性预测值为85％，阴性预测值为96％。测试之间的一致性很高（kappa = 0.79； 95％置信区间0.64至0.95）。当使用接收器工作特性分析选择的T / C比的单个截止值时，确定CL存在的敏感性和特异性分别为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系统可以准确区分血浆P4≥1或<1 ng / mL。