The establishment of an ELISA for the detection of pregnancy-associated glycoproteins (PAGs) in the serum of pregnant cows and heifers
Introduction
Pregnancy diagnosis is an essential component of sound reproductive management, particularly in the dairy industry [1] where a high proportion of AI fails to produce a calf [2], [3]. A reliable yet simple pregnancy test for cattle has long been sought. Several procedures are available, including a milk progesterone assay [1], [4], [5], estrone sulfate analysis [5], [6], palpation per rectum [7], ultrasound [8], [9], and blood tests for pregnancy-specific antigens [10], [11], [12], [13], [14], [15], which is the focus of this paper.
In cattle, binucleate cells are the invasive components of the trophoblast. By day 25, they constitute up to 20% of cells in the trophectoderm, and some of them can be observed fusing with maternal uterine epithelial cells [16], [17], [18]. After fusion, the fused cells release the contents of their secretory granules towards the maternal stroma and its network of capillaries [18], [19], [20], [21]. Such secretory products are attractive candidates for development of a pregnancy test.
Pregnancy-specific protein B (PSP-B) [22], a placental antigen, can be detected in the blood of pregnant cows in the fourth week of pregnancy [13], [23]. Two other groups developed immunoassays that are likely based on either similar or identical antigens [24], [25], [26], [12]. In one case the antigen (Mr ∼ 67 kDa) was called pregnancy-associated glycoprotein (PAG) [12], [26]; in the second it was named pregnancy serum protein60 (PSP60) to denote its molecular weight [24], [25]. To avoid confusion, PSP-B, PSP60 and PAG will be referred to as bovine PAG-1 (boPAG-1) since they all have been reported to have similar, if not identical, amino-terminal amino acid sequences [27], [28]. Importantly, boPAG-1 is a product of binucleate trophoblast cells [29], [30], [31], which is believed to be the explanation for how it can reach the maternal bloodstream at the time of binucleate cell migration. On the other hand, the PAG family of trophoblast-expressed genes is large and complex [31], [32], [33], [34], [35], [36], [37] and the various PAGs are not expressed coordinately throughout pregnancy. Some, for example, are expressed early, others only as pregnancy progresses [31]. Indeed, it has become clear that boPAG-1 may not be expressed at all in early pregnancy (e.g. day 25); presumably the boPAG-1 antiserum instead recognizes another PAG or PAGs [31].
The current immunoassay based on antiserum raised against purified boPAG-1 has two strong points. First, it is a relatively early test and is reliable by the end of the first month of pregnancy. Second, interpretation of the assay does not require knowledge of the exact date of service, since boPAG-1-like immunoreactivity is always present in the maternal serum of pregnant cows by day 30, and PAG amounts tend to increase as pregnancy advances. A crucial disadvantage of the test, however, is that boPAG-1 immunoreactivity rises dramatically at term [12], [13], [38], [39], often to well above 2 μg/mL. Because of the long circulating half-life of these antigens, PAG-1 immunoreactivity can still be detected 80 to 100-d post-partum, thus compromising accurate pregnancy diagnosis in cows bred within this early post-partum period due to false positives [12], [39], [40]. The goals of the work presented in this paper were to produce antibodies against PAG molecules predominantly expressed in early pregnancy and to establish an assay for PAGs in cattle that was not compromised by the persistence of circulating antigen in the post-partum period.
Section snippets
Preparation of proteins for immunizations
Placental tissue from three stages of pregnancy was used to produce antibodies. The earliest stage consisted of secretory proteins from the explant culture of pools of days 24 to 34 trophoblast. These samples were collected after slaughter in a local abattoir 24-, 27-, 29- and 34-d after insemination. The other sources of protein were from explant cultures of cotyledons from day 80 and day 150 pregnant cows collected from a local slaughterhouse. Developmental stage was determined by measurement
PAG isolation and antibody production
The aspartic proteinase inhibitor, pepstatin A, was used to affinity purify PAGs from trophoblast-conditioned culture medium [53], [54], [55]. The PAGs isolated from the day 150 cotyledon trophoblast culture gave three major bands (relative Mr varied from 40,000 to 80,000) when analyzed by one-dimensional SDS–PAGE (Fig. 1A). Such variability in molecular weights of crude PAG populations is consistent with earlier data including the presence of a low molecular weight component [33], [52], [56].
Discussion
Since the initial molecular cloning of boPAG-1 [29], many proteins, somewhat similar to, but clearly not identical to boPAG-1 have been identified as products of the bovine placenta. Over 21 distinct boPAG members, all of which belong to the aspartic proteinase gene family, have so far been cloned. However, others clearly exist [31], [33], [34]. Those members that have been cloned fall into two main groups, one evolutionarily more ancient, whose members are expressed in all cotyledonary
Acknowledgements
This work was supported by USDA/NRI grant 96-35203-3257 and funding from Monsanto Co. and the Missouri Agricultural Experiment Station. The authors thank Dr. George Perry (South Dakota State University) for his helpful discussions and assistance in the analysis of the assay results. The authors also thank the staff at the UMC Dairy Farm for help with blood collections and Ryan Egen, Kelli E. Valdez and Gail L. Winkelman for their assistance with sample cataloging. The authors acknowledge The
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