A lateral flow-based portable platform for determination of reproductive status of cattle

摘要

Our objective was to develop and validate a toolintegrating a disposable fluorescence-based lateral flowimmunoassay (LFIA) coupled with a portable imagingdevice for estimating circulating plasma concentrationsof progesterone (P4). First, we developed andoptimized a competitive LFIA test strip to measure P4in bovine plasma. The LFIA design included a samplepad, a conjugate pad that stores R-phycoerythrin-anti-P4 conjugates, a glass-fiber spacer pad, a nitrocellulosemembrane with printed test and control lines, and acellulose-fiber absorbent pad. To perform a test, 20 μLof plasma and 50 μL of running buffer were added on thesample pad. After 3 min, 45 μL of running buffer wasadded to initiate sample flow. After allowing 15 min tostabilize the colorimetric signal, strips were introducedin an LFIA portable reader wirelessly linked to a laptopto determine P4 concentration based on test-to-controllinesignal (T/C ratio). In a series of experiments (n= 6), the ability of the LFIA to differentiate plasmasamples with ≥1 or <1 ng/mL of P4 was evaluated. Foreach experiment, a calibration curve was constructedusing plasma with known concentrations of P4 (0.1 to3.7 ng/mL; n = 5). The resulting linear equation wasthen used to determine a T/C ratio cutoff to differentiatesamples with ≥1 or <1 ng/mL of P4. In addition,to evaluate the ability of the platform to assign samplesto P4 concentration groups without a calibration curvefor individual batches, we performed a receiver operatingcharacteristic analysis to identify a single cutoffvalue for T/C ratio that could potentially be used forall batches. Overall, calibration curves showed a linearrelationship between T/C ratio and P4 levels (meancoefficient 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 ofthe LFIA system to differentiate plasma samples with≥1 or <1 ng/mL of P4 using a RIA for P4 as referencetest. Overall, the LFIA assay correctly classified 90% ofthe samples, with 97% sensitivity, 83% specificity, 85%positive predictive value, and 96% negative predictivevalue. 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 selectedby receiver operating characteristic analysis, sensitivityand specificity to determine CL presence were 97 (95%confidence interval 82 to 99) and 79% (95% confidenceinterval 60 to 92), respectively. These data suggest thatthe developed portable LFIA system can accurately differentiateplasma samples with ≥1 or <1 ng/mL of P4.