Development of enzyme-linked immunosorbent assay for early pregnancy diagnosis in cattle

The prerequisite for enhancing reproductive efficacy and successful calving is an early and accurate diagnosis of pregnancy. A highly sensitive enzyme-linked immunosorbent assay (ELISA) utilizing the second antibody coating technique and the horseradish peroxidase conjugate as a label for the determination of Interferon-stimulated protein, 15 kDa (ISG15) in dairy cows was developed. To validate a neutrophil lysate based ELISA for early pregnancy diagnosis, blood samples from healthy multiparous Karan Fries (KF) cows were collected on day 0 (day of AI), 10, 14, 16 and 21 post artificial insemination. The pregnancy and non-pregnancy in cows were confirmed by plasma progesterone assay, ultrasonography and per rectal palpation. The detection range of the assay was 0.312 to 25 ng/ml with a sensitivity of 0.13 ng/ml. The inter-assay and intra-assay coefficient of variation was 12.1% and 10.1%, respectively. The parallelism for measured and expected concentrations had an r² value of 0.90 and 0.93, respectively. It was observed that ISG15 concentration was greater (P < 0.01) in pregnant as compared to non-pregnant cows, the greatest being 9.36 ± 0.50 ng/ml on day 16 in pregnant cows. With the use of immunocytochemistry, there was a positive staining for ISG15 with greater staining in the neutrophils of day 16 pregnant cows. The study reveals that ISG15 protein in a neutrophil lysate can be utilized as a biomarker for early pregnancy diagnosis in cows.