Early detection of pregnancy status in cattle using ISG17 polyclonal antibody recombinant

Abstract

Abstract Optimization of female cattle reproduction requires reliable and accurate systems for early pregnancy detection. Identifying pregnant status as early as possible is essential in managing reproductive cycles because it allows farmers to make early decisions about rebreeding strategies. Interferon stimulated gen (ISG) 17 is a gene expressed during maternal recognition pregnancy through Interferon tau (IFN-T) secretion signal, which begins on days 8 to 9 of pregnancy and peaks on days 16 to 17 (day 0 = day of estrus). This study aims to detect ISG17 expression using ISG17 polyclonal antibodies as a determinant of pregnancy in cattle that have received artificial insemination. The polyclonal antibody of ISG17 was produced in New Zealand white rabbits using ISG17 recombinant protein expressed by pET-32b expression vector in Escherichia coli systems of strain pLys BL21-Origami. ISG17 polyclonal antibody (ISG17-pAb) was used to detect ISG17 proteins expressed in blood serum obtained from 2 to 10 weeks-inseminated cattle (Frisian Holstein and Ongole) by NCM-ELISA method. ISG17-pAb was also labeled with gold nanoparticles and assembled into a lateral flow system for easier pregnancy detection in the field. NCM-ELISA test results showed a typical reaction between ISG17 antigen and ISG17-pAb in the form of brown dots after adding DAB substrate. Detection of pregnancy status showed that the antibody reaction to blood serum from dairy cows indicated 13.2% false negatives, 15.8% false positives, and 71.0% accuracy. In comparison, blood serum from beef cattle showed 7.5% false negatives, 0% false positives, and 92.5% accuracy. Interestingly, a lateral flow kit based on ISG17-pAb-gold nanoparticles showed good antibody reactions against blood serum from pregnant and non-pregnant cows. This indicates that the development of early pregnancy detection based on ISG17-pAb is prospective for optimizing productive cows by continuing to increase accuracy.