  Development of a new ELISA for measurement of pregnancy-associated glycoprotein.; Melo de Sousa, Noelita  ; et alin Proceedings of the 6th European Congress of Bovine Health and Management (ECBHM) (2011, September 07) In the present study we report the development of a new ELISA for PAG measurement (Ref. code E.G.7, CER, Maloie) in ruminant species. Purified boPAG67kDa protein was purified in our laboratory and used as ... [more ▼] In the present study we report the development of a new ELISA for PAG measurement (Ref. code E.G.7, CER, Maloie) in ruminant species. Purified boPAG67kDa protein was purified in our laboratory and used as standard. Antiserum raised against caprine PAG 55kDa+62kDa (AS707) was used as capture antibody (1:40,000). Antiserum raised against PAG purified from buffalo placentas (AS859) was used as detection antibody (1:32,000 dilution). Avidin-HRP and TMB completed the ELISA system. Reproducibility was determined by calculating the intra- and inter-assay CV. Accuracy was determined by adding increasing concentrations of purified boPAG67kDa to bovine sera containing low PAG concentrations. Specificity was tested regarding 25 different compounds: carbohydrates, aspartic proteinases, hormones and major plasma proteins. Parallelism was assessed by serially diluting pregnant cow serum containing high PAG concentrations. Sera was collected from pregnant cows (n=4) during the whole duration of gestation. Samples were tested by both RIA and ELISA. Samples giving high PAG concentrations were serially diluted in order to fit with optical density (OD) of the ELISA standard curve. Intra-and inter-assay CV ranged from 5.5 to 9.1%. Accuracy ranged from 92.9 to 105.5%. No cross reaction was observed with almost all tested compounds excepting lactic acid at supra-physiological levels (1 mg/ml). Dilutions of bovine maternal sera were parallel to standard curve. Profiles obtained by ELISA in pregnant cows were very similar to those obtained by RIA. In conclusion, a new PAG-ELISA is available for measurement of PAG concentrations in cattle. This new test was validated in vitro and can be used for pregnancy diagnosis and follow-up of gestation in cattle. [less ▲] Detailed reference viewed: 35 (3 ULg)Pregnancy diagnosis in cattle by using a new ELISA for pregnancy-associated glycoprotein: preliminary results.Beckers, Jean-François ; Melo de Sousa, Noelita ; et alin Proceedings of the 6th European Congress of Bovine Health and Management (ECBHM) (2011, September 07) In the present study we report the use of a new PAG-ELISA for routine pregnancy diagnosis in cattle. A total of 2777 samples were collected from pregnant or non-pregnant cows and assayed by both classical ... [more ▼] In the present study we report the use of a new PAG-ELISA for routine pregnancy diagnosis in cattle. A total of 2777 samples were collected from pregnant or non-pregnant cows and assayed by both classical PAG RIA-497 method and newly developed PAG-ELISA (Ref. code E.G.7, CER, Maloie). Pregnancy status was determined on the basis of RIA results. Samples were assumed to be issued from pregnant cows when PAG concentrations were higher than 0.8 ng/mL. PAG concentrations <0.8 ng/mL were considered negative (non-pregnant). PAG concentrations between 0.8 and 1.0 ng/mL were considered doubtful. With regard to ELISA, concentrations below 0.6 were considered negative. Concentrations between 0.6 and 0.8 ng/mL were considered doubtful and finally concentrations under 0.8 ng/mL were assumed as positive. Sensitivity(Se), specificity(Sp) and predictive values (PV) of pregnancy diagnosis were determined. A total of 143(5.2%), 137(4.9%) and 38(1.4%) samples gave doubtful results by RIA, ELISA or both systems, respectively. These samples (318/2777=11.5%) were not used for estimation of Se, Sp, PPV and NPV because clinical confirmation of pregnancy diagnosis could not be established by an additional method (rectal palpation, ultrasound or calving). Taking into account the remaining samples (2459), Se, Sp, PPV and NPV obtained by ELISA were respectively 98.9%, 89.9%, 98.7% and 96.4%. In conclusion, PAG-ELISA can be successfully used for pregnancy diagnosis in cattle. In the near future, further investigations are to be conducted in order to compare results obtained by ELISA test with those obtained by rectal palpation and ultrasound in field conditions. [less ▲] Detailed reference viewed: 41 (5 ULg)Diagnostico precoz de gestation en Ganado ovino mediante un kit enzimoimmunoanalisis (EIA) de la glicoproteina asociada a la gestation (PAG) plasmatica.; ; et al in Proceedings of the XXXV Congreso de la Sociedad Espanola de Ovinotechnia y Caprinotecnia (SEOC) (2010, September) Detailed reference viewed: 6 (3 ULg) |
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