Establishment of a Sandwich-ELISA for simultaneous quantification of bovine pregnancy-associated glycoprotein in serum and milk

Abstract

Bovine pregnancy-associated glycoproteins (boPAG) are expressed by trophoblast cells in the bovine placenta. The multigene family of boPAG belongs to the group of aspartic proteases. The accumulation and circulation in maternal blood and milk has made boPAG very useful and important for pregnancy diagnosis in cattle. The goal of the present study was to develop and validate a new Sandwich-ELISA which allows the detection of boPAG in maternal serum and whole milk. Therefore, 984 serum and 928 milk samples were collected monthly from 231 Holstein Friesian cows (Bos Taurus) from one week after insemination (p.i.) until six weeks postpartum. The ELISA is able to identify a cow as being pregnant at day 30 p.i. in serum and at day 40 p.i in milk with threshold values of 1.0 ng/ml in serum and 0.0165 ng/ml in milk. The postpartum half-life of boPAG was estimated to be 6.4 days in serum and 7.1 days in milk. The boPAG profile established during pregnancy in serum and milk showed a typical pattern. The amount of boPAG found in milk was 1.5 % of the amount of boPAG present in serum. In conclusion, a Sandwich-ELISA has been developed to quantify boPAG in serum and in whole milk simultaneously with the same test procedure. This is time saving for farmers and more efficient for laboratories.

Using Pregnancy-Associated Glycoproteins (PAGs) to Improve Reproductive Management: From Dairy Cows to Other Dairy Livestock

“…Recently, Krebs et al [ 59 ], using ELISA newly developed in their lab, reported that the amount of PAG found in milk was 1.5% of the amount present in serum. The milk test identifies pregnancy at day 40 post-insemination, with a threshold value of 0.0165 ng/mL.…”

A multiplexed parallel reaction monitoring assay to monitor bovine pregnancy-associated glycoproteins throughout pregnancy and after gestation

“…BoPAGs were purified from cotyledons from different gestation stages by FPLC. A detailed description of the purification process can be found in Krebs et al (2021) [29]. A scheme of the different FPLC steps is shown in S1 Fig in S2 File.…”

“…A scheme of the different FPLC steps can be found in S1 Fig in S2 File. The resulting boPAG-containing fractions were stored at -20˚C until further analysis [29].…”

“…Polyclonal boPAG antisera were produced using seven boPAGfractions of different pregnancy stages from chromatography. A detailed description of the polyclonal antibody production is given in Krebs et al (2021) [29]. Afterwards, membranes were incubated overnight at 4˚C while shaking.…”

Development of a Double-Antibody Sandwich ELISA for Rapid Detection of the MCP Antigen Concentration in Inactivated ISKNV Vaccines

“…It is more sensitive when different class antibodies were used as the detection and capture antibodies with a broad spectrum of epitopes. Furthermore, the detection of multiple epitopes offers better sensitivity for detecting proteins that are present in low concentrations in a sample [11,12]. The DAS ELISA was established to determine the effective antigen yields in inactivated vaccines and thus represents an alternative for assessing the potency of FMD vaccines in vitro [13,14].…”