Comparison of Five Radioimmunoassay Systems for PAG Measurement: Ability to Detect Early Pregnancy in Cows (original) (raw)
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Validation of Buffalo PAG Antisera for Early Pregnancy Diagnosis in Bovine
Indian Journal of Animal Research
This study was designed ability using antibody against buffalo PAG to detect concentration of pregnancy-associated glycoprotein (PAG) in bovine plasma samples. Successful purification of buffalo PAG molecules with high enrichment degree by using Vicia villosa aûnity chromatography was recently reported. In this research, the RIA system using novel anti buffalo-PAG was used for bovine pregnancy diagnosis. In a total 437 blood sampling of dairy cow in Belgium were used. Three PAG-RIA systems were undertaken to measure PAG concentration in bovine routine blood samples: AS#497 (or RIA-1) using rabbit antisera against purified bovine PAG67kDa, AS#706 (or RIA-2) using rabbit antisera against caprine PAG55+62kDa and new AS#859 (or RIA-3) using rabbit antisera against buffalo PAG59.5-75.8kDa). The measured PAG concentrations were similar for the systems As#497 and As#859; although the dilution was shown parallel, the measured PAG concentrations were significantly different between systems A...
Small Ruminant Research, 2013
Pregnancy-associated glycoproteins (PAGs) are macromolecules produced by placental tissue and released into the maternal circulation where they allow pregnancy diagnosis and follow-up. The present study addresses the question to what extent plasma PAG determination may serve as a means of early pregnancy detection in goats in a similar way it is practiced in cows, and whether an ovine or bovine PAG-ELISA may be utilized to this end. Blood samples were collected from eight pregnant pluriparous Boer goat does twice weekly during the first seven weeks and the last four weeks of pregnancy and weekly in-between and during four weeks following parturition. Plasma PAG concentrations (mean ± SEM) were determined using a competitive enzyme-linked immunosorbent assay. Assays were conducted with polyclonal antisera raised in rabbits against purified preparations of caprine (AS#706), ovine (AS#780) and bovine PAG (AS#726). In the assay systems purified bovine PAG served as standard and tracer and goat anti-rabbit IgG served as coating antibody. With the antibody raised against caprine PAG (AS#706) a steep increase to a climax of 69 ± 9 ng/ml on day 56 of pregnancy was followed by a gradual decline to 16 ± 3 ng/ml at parturition and 0.3 ± 0.07 ng/ml four weeks postpartum. The results achieved with the antiovine PAG (AS#780) showed close similarity, a maximum of 92 ± 14 ng/ml being reached at 56 days of pregnancy. With anti-bovine PAG (AS#726), the PAG level increased to a maximum of 3.1 ± 0.2 ng/ml on day 105 of pregnancy and fluctuated around 3 ng/ml until the end of pregnancy. The difference between pregnant and non-pregnant does reached a significant level 21 days after conception, one week earlier than with caprine and ovine antisera.
Pre-Analytic Sources of Error with the Serum PAG-Pregnancy Test for Cows
Journal of Agricultural Science, 2015
The pregnancy associated glycoprotein (PAG) test for pregnancy detection in cows necessitates transportation of blood samples to the laboratory. This investigation addresses preanalytic sources of error that might compromise its reliability. During shipping blood samples undergo substantial temperature fluctuations (Experiment 1). Temperatures of whole blood beyond 0°C had no effect, whereas freezing reduced measurements by 22% at-10 °C and by 25% at-20 °C (Experiment 2). Freezing of blood with low PAG content (Experiment 3) caused an increase from 2.4 to 3.7 ng/ml (P < 0.01). Cryopreservation of serum with various PAG concentrations (Experiment 4) brought about increases to varying degrees. The presence of heparin and EDTA in collecting tubes had no effect on PAG measurements, whereas citrate caused an initial reduction, but remained stable thereafter (Experiment 5). In blood stored six months at chilling temperature no change in PAG values occurred as long as samples contained heparin or EDTA (Experiment 6). In Experiment 7 vortexing of whole blood showed no effect, whereas freezing and dilution with water seriously compromised results. In summary, to obtain reliable PAG measurements, contamination with water must be avoided; freezing of whole blood or serum and the use of collecting tubes containing citrate will result in inaccuracies without altogether distorting results. High ambient temperature, physical agitation and long term storage at chilling temperature in the presence of heparin or EDTA will have no impact. PAG determination in blood may thus be considered a reliable pregnancy test for cows in most situations.
Research in Veterinary Science, 2009
The measurement of serum or plasma PAG concentrations is currently used as a specific method for pregnancy diagnosis in cattle. In this study, the correlation between five radioimmunoassay systems (RIA-497, RIA-706, RIA-780, RIA-809 and RIA-Pool) developed for measurement of PAG concentrations in ruminant species was investigated in plasma from pregnant Friesian Holstein females. Plasma PAG concentrations (ng/mL) measured by different RIA systems were significantly correlated between them (P 0.81; P < 0.001). PAG concentrations increased significantly from Day 21 (n = 27) to 30 (n = 37) after AI by use of all PAG-RIA systems. From Day 30 to 80 after AI, lower PAG concentrations were observed when using the homologous system RIA-497. The addition of several proteinase inhibitors changed neither the non specific binding nor the B 0 binding to the tracer. Our results suggest that all tested PAG-RIA (RIA-497, RIA-706, RIA-780, RIA-809 and RIA-Pool) are highly correlated and can be useful to follow PAG concentrations in samples collected during the first trimester of gestation.
Schweizer Archiv für Tierheilkunde, 2014
The aim of this study was to compare two methods of early pregnancy diagnosis by determining pregnancy-associated glycoprotein (PAG) concentration in blood and PAG concentration in milk. Blood and milk samples were obtained on days 0 (AI day), 14, 21, 28, 35, 49, 63, 77, 91 and 105 of gestation from 60 lactating Holstein Frisian cows from one herd, carrying live fetuses. To determine PAG concentration a specific RIA system (RIA-706) was used. PAG concentration in blood and milk increased after 28 days of pregnancy, with blood concentrations being significantly higher than in milk. However, the accuracy of both tests at this time point was similar (sensitivity: 92 % in blood, 93 % in milk; specificity 53 % and 60 % respectively). None of the tests were able to detect open cows properly at this stage. On day 35 of gestation sensitivity (100 % for blood, 97 % for milk) and specificity (100 % for blood, 100 % for milk) were high enough to be used for reliable pregnancy diagnosis. The ac...
Visual ELISA for detection of pregnancy-associated glycoproteins (PAGs) in ewe serum
Theriogenology, 2017
The aim of the present study was to evaluate the efficiency of visual ELISA-PAG for early pregnancy diagnosis based on the presence of PAG (pregnancy-associated glycoprotein) using sheep blood serum. Experiment 1: 140 ewes were divided into three groups with different stages of pregnancy. In the first group, 41 pregnant ewes were sampled at 26, 28, 30, and 32 days of pregnancy; in the second group, 65 ewes (49 pregnant with 30 days and 16 non-pregnant) were sampled; in the third group, 34 nonpregnant ewes were sampled. Experiment 2: 10 pregnant ewes were sampled weekly from day 35 of gestation until day 70 post-partum to verify the total period in which PAG can be detected in the blood serum by the test. Transrectal ultrasound was used as a gold standard. The detection or non-detection of PAG was analyzed by the logistic model PROC GENMOD of SAS; differences were detected by the chisquared test. In group 1, there were no differences between the results from 28, 30, and 32 days of gestation, but samples from 30 days were easier to interpret in comparison to 28 days, with a sensitivity of 97.56%. In the second group, using 65 sheep, visual ELISA-PAG showed 100% sensitivity and 93.75% specificity, which indicates the diagnosis of an animal as a false positive. In the third group, 97.06% of the sheep were confirmed as negative and 2.94% as positive, again indicating the presence of a false positive. In 100% of the sheep, the PAG remained in the blood circulation throughout the antepartum period until birth and seven days post-partum, declining thereafter. Based on our results, the visual ELISA-PAG is an effective method for the early diagnosis of pregnancy in sheep and can be performed from day 30 of gestation.
European Journal of Obstetrics & Gynecology and Reproductive Biology, 2002
Background: Circulating human chorionic gonadotropin (hCG) and progesterone are commonly used as markers of abnormal pregnancies. Previous studies have shown that pregnancy-associated plasma protein-A (PAPP-A) was also depressed in extrauterine pregnancies (EUP). Previously, PAPP-A was measured with polyclonal antibodies which were later shown to recognise also the pro-form of major basic protein (pro-MBP). Objective: To evaluate the clinical usefulness of PAPP-A measurements in early pregnancy. Study Design: Circulating PAPP-A, hCG and progesterone were measured in patients with EUP (n ¼ 68), abnormal intrauterine pregnancies (abIUP, n ¼ 31) and normal intrauterine pregnancies (nIUP, n ¼ 72). Gestational age was 30-70 days from the last menstruation. Results: For PAPP-A and hCG, a steep increase was observed from day 30 after last menstrual period onwards, this increase being much less important for abIUP and EUP. The values of PAPP-A and hCG were significantly decreased in abIUP and EUP, from 42 days after LMP onwards. There were no significant differences between abIUP and EUP. Progesterone concentration does not vary with amenorrhoea and was significantly lower in abIUP and EUP. Values in abIUP were significantly (P ¼ 0:02) lower compared with EUP for amenorrhoea above 42 days. ROC curves were constructed for amenorrhoea above 42 days. For a specificity of 99%, the sensitivity of PAPP-A, hCG and progesterone were 64.5, 93.3 and 76%, respectively. The threshold values were 14.3 mIU/l, 10,400 IU/l and 10.1 ng/ml for PAPP-A, hCG and progesterone. Conclusion: We confirm the decrease of PAPP-A concentrations in pregnancy failure, but hCG and progesterone remain the best clinical tools.
Animal Reproduction Science, 2018
The aim of the present study was to compare a Bovine Pregnancy Rapid Test (Ubio quickVET; BPRT) with a commercial ELISA-PAG test (Bovine Pregnancy Test DG29®) for early pregnancy diagnosis based on the presence of the PAG (pregnancy-associated glycoprotein) in dairy cattle between 30 and 40 days after artificial insemination (AI). Blood samples were collected from 212 cows between 30 and 40 days after artificial insemination (AI) to quantify the concentrations of PAG in each sample. Transrectal ultrasonography (TRUS) diagnosis of pregnancy was conducted at day 45 ± 3 after AI as the reference standard for the two PAG tests. The results indicated the sensitivity (Se) of the BPRT and DG29 for diagnosing pregnant cattle were 89.4% and 100%, respectively while the specificity (Sp) of the two tests for diagnosis of non-pregnant animals was 89.8% and 81.3%, respectively. Based on these results, the BPRT has a slightly lesser sensitivity and greater specificity than the DG29 test. Moreover, the accuracy of both tests was 94% and 90%, respectively, for DG29 and BPRT. This implies that the BPRT test could be considered an accurate test when compared to PAG-ELISA test and TRUS at days 30 to 40 after AI. The BPRT test, therefore, can be used as an alternative to the PAG-ELISA test with some constraints that need to be considered with its use.