142 DESIGN OF ANTIBODIES SPECIFIC FOR PEPTIDES PRESENT EXCLUSIVELY IN DIFFERENT MEMBERS OF THE PREGNANCY ASSOCIATED GLYCOPROTEINS FAMILY

Abstract

Accurate diagnosis of non-pregnancy and prompt re-enlistment of cattle into an appropriate breeding protocol are essential components of successful reproductive programs. The search for biochemical markers of early pregnancy has led to the characterization of the pregnancy-associated glycoproteins (PAG), a large family expressed exclusively in the placenta. In cattle, the PAG family is composed of at least 22 translated genes, with different spatiotemporal expression (Telugu B. et al. 2009 BMC Genomics 10, 185). The PAG may be detected in placenta and some members have been detected in serum by immunological methods (Garmo B et al. 2008 J. Dairy Sci. 91, 3025-3033). However, the lack of antibodies specific for different PAG has prevented the identification of any member of the family as being present in serum at early pregnancy and undetectable after parturition. The objective of this study was to develop a method that allows the preparation of antibodies specific for different PAG, as candidates of early pregnancy markers. The method employed was in silico analysis of the bovine PAG family members that have been reported as of early expression in placenta and in binucleated cells (which are fusion cells between maternal and embryonic tissues), searching for peptide regions that differ between them. Selected peptides were analyzed for localization in reported exposed regions, antigenicity in the protein context, lack of consensus sequences for post-translational modifications and of putative homologous peptides using the reported bovine genome, and feasibility of chemical synthesis with low rates of contamination. Chosen peptides were synthesized by a facility and coupled to rabbit albumin. Rabbits were immunized and the serum was analyzed for the presence of specific antibodies. After in silico analysis, 43 candidate peptides contained in 13 different PAG were considered suitable. Four of these were chosen for synthesis and coupled successfully to rabbit albumin. Rabbits were immunized with peptide-rabbit albumin, according to procedures used by Bioterio of Facultad de Ciencias Bioquímicas y Farmacéuticas, UNR. Analysis of the obtained serum by dot-blot showed the presence of anti-peptide antibodies only for one of the peptides. This result is in accordance with the efficiency of anti-peptide antibodies development in rabbits. In conclusion, we designed a method for selection of peptides specific for different members of the PAG family and with high chances of being exposed in the surface of the molecule, which would allow immunological detection of the native glycoprotein in cow serum. These antibodies are being used for analysis of serum from pregnant and non-pregnant cows.

Fundación Nuevo Banco de Santa Fe Programa de fortalecimiento de las capacidades de investigación y desarrollo de la provincia de Santa Fe, Secretaria de Estado de Ciencia, Tecnología e Innovacón, Provincia de Santa Fe.