Abstract
<title>Abstract</title> <p>Early and accurate pregnancy diagnosis remains a key challenge in modern dairy farming. We developed monoclonal Fab antibodies against Preimplantation Factor (PIF), an embryo-secreted peptide absent in non-viable pregnancies, using high-diversity phage display libraries (approximately 2.2×10⁷ and 1.6×10¹⁰) derived from immunized rabbits. Biopanning yielded 22 PIF-specific clones with high affinity and strict specificity. Structural modeling revealed that CDR3 loops of both chains mediate recognition of the conserved RIKP motif in PIF. A competitive ELISA based on these Fab antibodies detected pregnancy in dairy cows as early as 10–20 days post-insemination with >98% accuracy, validated by ultrasonography. This study establishes PIF specific Fab antibodies as a reliable tool for early pregnancy diagnosis in dairy cattle, offering significant advantages over conventional methods.</p>