Poster W7 - ReproPhone: A lateral flow based portable platform for the determination of cattle reproductive status

Introduction: Reproductive management is critical to dairy and beef farm profitability and sustainability, necessitating accurate timely ovarian status and pregnancy detection in cattle. Our objective was to develop and validate a tool integrating disposable fluorescence-based lateral flow immunoassays (LFIA) coupled with a portable fluorescence imaging device for estimating circulating blood concentrations of progesterone (P4) and pregnancy specific protein-b (PSPB). To perform a test, a few drops of blood and running buffer are added to the sample well. After a few minutes, additional running buffer was added to initiate sample flow. After allowing 25 min to stabilize the fluorescent signal, strips were introduced in an LFIA portable fluorescence reader complete with a custom application enabling image capture, image processing, and analyte concentration calculation. The tool offers farmers an accurate, rapid, user-friendly, and on-farm way to determine cattle pregnancy and ovarian status. By enabling timely and reliable reproductive management practices, this tool could significantly improve dairy farm productivity.

Materials and

Methods: First, a competitive LFIA was developed to measure P4 and an immune-sandwich LFIA developed to measure PSPB. For P4 detection, the design included plasma separation pads, a conjugate pad that stores R-phycoerythrin-anti-P4 conjugates, a glass-fiber spacer pad, a nitrocellulose membrane with printed test (P4-BSA; 1.5mg/ml) and control (anti-mouse IgG; 0.3mg/ml) lines, and a cellulose-fiber absorbent pad. Similarly, the PSPB detection LFIA includes Europium-anti-PSPB and biotin-anti-PSPB conjugates, a nitrocellulose membrane with printed test (Polystreptavidin; 1.5mg/ml) and control (anti-mouse IgG; 0.3mg/ml) lines, and a cellulose-fiber absorbent pad. The final system is complete with the developed imaging system, which is comprised of a raspberry pi model 4B, raspberry pi camera HD, a long-pass optical filter consistent with the emission wavelengths of R-phycoerythrin and Europium, a PCB holding LEDs with emitting light at appropriate wavelengths for the excitation of the two fluorophores, an LCD screen, and 3D printed parts to hold the system. System optimization and validation was achieved using standard concentrations of P4 and bPSPB antigen suspended in 1% BSA, and bovine blood samples respectively.

Results, Conclusions, and Discussions: The developed system, comprised of the two LFIA strips and fluorescence imaging device, is a novel method for the determination of circulating P4 and PSPB concentrations from bovine blood samples. The design of the P4 detection LFIA is a competitive-type assay where P4 in the sample and P4 immobilized on the test line compete for a limited number of fluorophore conjugated anti-P4 antibody binding sites. The competitive-type assay results in greater test to control line (T/C) ratios for lower concentrations of P4 in the sample or an inversely proportional relationship between P4 concentration and T/C ratio. For PSPB detection, the LFIA follows an immune-sandwich format where PSPB in the sample encounters and binds to fluorophore conjugated anti-PSPB antibodies and biotinylated anti-PSPB antibodies on the conjugate pad forming the immune-sandwich complex. Polystreptavidin immobilized on the test line then binds to the biotinylated antibody in the antigen-antibody complex. The immune-sandwich format of the assay results in a directly proportional relationship between PSPB concentration and T/C ratio. Upon applying blood sample and running buffer to the sample wells of the cassettes holding the test strips and incubation for 25-30 min, they are placed in the imaging device where a user-interface allows users to capture an image and provides a preview of the fluorescent signal on the test strip, process the captured image, and provide a calculated T/C ratio and estimated concentration of the target biomarker in the sample. Estimation of P4 or PSPB concentration relies on a previously created calibration curve, however, an option to recalibrate the image processing is available to allow users to re-calibrate the system in case of high-performance variation between different batches of LFIA strips or other environmental conditions affecting the test.

Acknowledgements (Optional):