The bovine pregnancy-associated glycoproteins (bPAGs) have been widely used as robust markers for early diagnosis of pregnancy in the cattle. The current immune recognition methods for detecting bPAGs are limited and, to a certain extent, are associated with high costs and poor stability of the antibody. Aptamers that are more stable and easily synthesized than antibodies might serve as suitable candidates for the development of rapid detection methods. This paper describes selection and characterization of bPAG4 aptamers and theirs applicability to detect bPAG4 in the serum. In this work, the recombinant bovine pregnancy-associated glycoproteins 4 (bPAG4) with a relative molecular mass of about 48 kDa was successfully expressed in human embryonic kidney 293 (HEK 293) cells. Subsequently, the ssDNA aptamers were selected by systematic evolution of ligands by exponential enrichment (SELEX) using magnetic beads (MB) coated with bPAG4 as target. After 9 rounds of selection, three aptamers with high affinity to bPAG4 (K_d = 11.7~40.2 nM) were identified. The selected aptamers were successfully used in enzyme-linked aptamer assay (ELAA) to detect bPAG4 at a detection limit of 0.09 ng/mL. Meanwhile, it has been successfully applied for the detection of bPAG4 in serum samples. This work demonstrated that the selected aptamers could be used as promising affinity probes in the development of inexpensive, simple, and sensitive analysis methods for detecting bPAGs.

Graphical abstract

牛妊娠相关糖蛋白（bPAG）已被广泛用作牛早期妊娠诊断的可靠标志物。当前用于检测bPAG的免疫识别方法是有限的，并且在一定程度上与抗体的高成本和差的稳定性有关。比抗体更稳定和更容易合成的适体可以作为开发快速检测方法的合适候选者。本文介绍了bPAG4适体的选择和表征及其在血清中检测bPAG4的适用性。在这项工作中，重组分子量与约48 kDa的牛妊娠相关糖蛋白4（bPAG4）在人胚胎肾293（HEK 293）细胞中成功表达。后来，通过使用涂有bPAG4的磁珠（MB）通过指数富集（SELEX）进行配体的系统进化来选择ssDNA适体。经过9轮筛选，获得了三个对bPAG4具有高度亲和力的适体（ 鉴定出K _d＝ 11.7〜40.2nM。所选的适体已成功用于酶联适体测定（ELAA）中，以0.09 ng / mL的检出限检测bPAG4。同时，它已成功地用于血清样品中bPAG4的检测。这项工作表明所选的适体可以用作开发廉价，简单，灵敏的分析方法检测bPAGs的有希望的亲和探针。

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