Advances in Bioscience and Biotechnology, 2011, 2, 85-88 ABB doi:10.4236/abb.2011.22013 Published Online April 2011 (http://www.SciRP.org/journal/abb/ ). Published Online April 2011 in SciRes. http://www.scirp.org/journal/ABB Preparation of monospecific anti-PAG antibodies for cattle pregnancy detection: use of synthetic peptides to improve specificity Jimena Inés Ruiz Álvarez, Juan Manuel Teijeiro, Patricia Estela Marini * Instituto de Biología Molecular y Celular de Rosario, IBR-CONICET and Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina. Email: marini@ibr.gov.ar ; pmarini@fbioyf.unr.edu.ar Received 16 February 2011; revised 7 April 2011; accepted 10 April 2011. ABSTRACT Immunological methods involving pregnancy associ- ated glycoproteins (PAG) are used for cattle preg- nancy detection. The faults of these methods could be overcome by using antibodies specific for each member of the PAG family. In order to differentiate between very similar proteins, preparation of anti- bodies specific for peptides is a method of choice. In this work, we summarize a series of considerations regarding peptide design and choose free access NCBI, Antigenicity Plot, EMBOSS Antigenic and Expasy tools to apply them. We design peptides spe- cific for different reported PAG members and obtain the corresponding polyclonal antibodies for five of them. Keywords: Anti-peptide; Antibodies; Pregnancy; Cattle 1. INTRODUCTION Diagnosis of pregnancy and prompt re-enlistment of ‘‘non-pregnant’’ cattle into an appropriate reproduction protocol are essential components of successful breeding programs. Thus, early and accurate pregnancy detection is prompted and several systems that use proteins from the pregnancy associated glycoprotein (PAG) family have been developed [1]. This family can be separated in two groups, one of which is expressed only in tropho- blastic binucleated cells, being its members detectable as a group in maternal blood from the time of implantation. The concentration of this group of proteins increases steadily in plasma, peaks before parturition, and remains detectable for 2 - 3 months after calving [2]. Immu- nological reactions involving PAGs have been used to design commercial kits for pregnancy detection [3], for review see [4]. Variations on detection between methods are significant and may be attributed to the quality of the anti-PAG antibodies used for each assay [4]. Anti-PAG antibodies have been developed using PAG preparations purified from placenta, which contain various PAG members, or whole PAG proteins as immunogenic mate- rial. Even when monoclonal antibodies are used, several members of the PAG family may be recognized by these antibodies, as PAGs may share the recognized epitope. Sensitivity and specificity of methods using radioim- munoassay were improved by the use of heterologous anti-PAG antibodies [3] and of monoclonal antibodies that recognize only a few members of the PAG family [5]. Persistence of some members of the PAG group pos-partum also limits the use of the currently existing methods. As different members of PAG have distinct temporal expression [2], the development of antibodies specific for them could help improve the currently ex- isting techniques. Also, as different PAGs have distinct half lives not all the members of the family persist in pos-partum serum, thus the use of antibodies that distin- guish between PAGs could permit accurate pos-partum pregnancy detection. The same would apply to the de- tection of wastage. The objective of this work is to de- velop antibodies against peptides unique to different members of the PAG family, which would presumably be specific for one reported PAG. Anti-peptide antibodies may be obtained using recom- binant proteins [6] or synthetic peptides coupled to car- rier proteins [7] as antigens. The production of antibodies using synthetic peptides allows the recognition of spe- cific regions of proteins and is often found advantageous for diagnostic purposes. The selection of the peptide se- quence is essential for the immunization of the recipient organism and for the future detection of the protein or domain in its native form. In this work, free access NCBI, Antigenicity Plot, EMBOSS Antigenic and Ex- pasy tools are used for antigenic peptide sequences se- lection.