Lateral flow assay for cattle age determination: Implications for diagnosing bovine spongiform encephalopathy

Quarantine authorities in East Asia prohibit the import of cattle over 30 months old due to concerns about bovine spongiform encephalopathy (BSE). Accurately determining the age of cattle is critical for complying with these regulations. However, current methods for determining cattle age are time-consuming and require laboratory analysis, making them impractical for real-time use during import inspections. This creates a pressing need for a point-of-care test (POCT) method that can rapidly determine cattle age from beef samples in the field. 4To develop a portable POCT method capable of quickly and accurately determining the age of cattle from beef samples in the field. We investigated the expression of cell cycle proteins p53 and p21, and based on these findings, designed a lateral flow assay (LFA) to measure cattle age using the expression patterns of these proteins. We investigated the expression of cell cycle proteins p53 and p21 using immunoblot analysis to determine the age of beef and applied these findings to lateral flow assay (LFA). The expression pattern of p53 remains at baseline levels up to 28 months and then increases sharply after 35 months. In contrast, p21 expression gradually decreases, reaching baseline levels after 30 months. The SPR sensogram of p21 using surface plasmon resonance (SPR) clearly distinguished beef samples between 27-month-old and 31-month-old cattle. We constructed a calibration curve using recombinant p21 protein to determine the age of beef samples from cattle aged 9 to 33 months. The LFA sensor strip, designed with bovine p21 epitope-based peptide antibody, improved sensitivity and limit of detection (LOD) by more than 10 times. • The p21 protein sharply decreases at 30 months of age, making it highly effective for identifying BSE-infected beef. • Developed customized p21 antibody using peptides to enhance the sensor's sensitivity. • The LFA sensor utilizing the custom p21 peptide antibody demonstrated a tenfold improvement compared to the full-length antibody.