title: The establishment of an ELISA for the detection of pregnancy-associated glycoproteins (PAGs) in the serum of pregnant cows and heifers.
abstract: The pregnancy-associated glycoproteins (PAGs) are a large gene family expressed in trophoblast cells of ruminant ungulates. The detection of PAGs (more specifically, PAG-1) in maternal serum has served as the basis for pregnancy detection in cattle. Unfortunately, PAG-1 and/or antigenically-related PAGs exhibit a long half-life in maternal serum (>8 d) and can be detected 80-100 d post-partum, thereby producing false positives in animals bred within 60-d of calving. The goal of the present studies was to develop a monoclonal-based assay that targeted early-pregnancy PAGs whose persistence in maternal serum post-partum might be relatively short-lived. Three anti-PAG monoclonal antibodies that recognized distinct subsets of PAGs were selected and used as trapping reagents in a 'sandwich' type of enzyme-linked immunosorbant assay (ELISA). A polyclonal antiserum with broad specificity was used for detecting bound PAGs. A total of 42 cows and heifers were bled daily on day 15, days 22 to 28, and then weekly throughout pregnancy and for 10 weeks (approximately 70 d) into the post-partum period. The ELISA was able to detect PAG in maternal serum of all animals unambiguously by day 28 post-insemination (PAG concentration: 8.75 +/- 3.04 ng/mL). In maternal serum, PAG concentrations peaked during the week of parturition at 588.9 +/- 249.9 ng/mL, and after calving, PAG was completely cleared (half-life: 4.3 d) by eight-week post-partum in 38 of 40 of the animals tested and was at very low concentrations in the remaining two (1.4 and 4.9 ng/mL, respectively). In summary, a monoclonal-based assay has been established that is sensitive enough to detect PAG in maternal serum by the forth week of pregnancy, but does not suffer from carry-over of antigen from a previous pregnancy.
authors.affiliation: Department of Animal Sciences, University of Missouri-Columbia, 163 ASRC, 920 East Campus Drive, Columbia, MS 65211, USA. GreenJo@missouri.edu
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Green
authors.forename: Tina E
authors.initials: TE
authors.lastname: Parks
authors.forename: Mary Pavlo
authors.initials: MP
authors.lastname: Avalle
authors.forename: Bhanu Prakash
authors.initials: BP
authors.lastname: Telugu
authors.forename: April L
authors.initials: AL
authors.lastname: McLain
authors.forename: A James
authors.initials: AJ
authors.lastname: Peterson
authors.forename: William
authors.initials: W
authors.lastname: McMillan
authors.forename: Nagappan
authors.initials: N
authors.lastname: Mathialagan
authors.forename: Reuel R
authors.initials: RR
authors.lastname: Hook
authors.forename: Sancai
authors.initials: S
authors.lastname: Xie
authors.forename: R Michael
authors.initials: RM
authors.lastname: Roberts
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D002417:Cattle / Q000097:blood*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000662:veterinary*; D005189:False Positive Reactions; D005260:Female; D005865:Gestational Age; D006023:Glycoproteins / Q000097:blood* / Q000737:chemistry; D006207:Half-Life; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010802:Phylogeny; D049590:Postpartum Period / Q000097:blood; D011247:Pregnancy; D011257:Pregnancy Proteins / Q000097:blood* / Q000737:chemistry; D011258:Pregnancy Tests / Q000662:veterinary; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014357:Trypsin
chemical_list: D000911:Antibodies, Monoclonal; D006023:Glycoproteins; D010446:Peptide Fragments; D011257:Pregnancy Proteins; D014357:Trypsin