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

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.

Utilização das proteínas associadas à gestação (PAG’s - Bovine Pregnancy Test Idexx®) como método de diagnóstico precoce de gestação em vacas leiteiras da raça girolando

Objetivou-se com este estudo observar a eficácia da utilização das Proteínas Associadas à Gestação (PAG´s) como método de diagnóstico precoce de gestação em vacas leiteiras da raça Girolando. Por meio de um teste de reação colorimétrica, recomendado para uso em vacas a partir do 28º dia pós-serviço, o VISUAL-PAG do “IDEXX Laboratories”. Verificou-se a sensibilidade e especificidade do teste aos 25, 26, 27 e 28 dias pós-Inseminação Artificial em Tempo Fixo (IATF), utilizando-se 57 fêmeas multíparas, submetidas a quatro coletas sequenciais de amostras de sangue nos dias descritos. O material foi coletado através de venopunção dos vasos coccígenos em tubos de vácuo sem anticoagulante, processado e armazenado a -20ºC para posterior análise. Aos 32 dias da IATF todas as fêmeas foram submetidas ao diagnóstico de gestação por ultrassonografia (US), teste padrão do experimento. Os resultados foram obtidos através de um estudo de concordância, onde revelou sensibilidade de 100% e especificidade de 94,7% iguais para os dias 25, 26, 27 e 28 pós-IATF. Diante destes resultados pode-se considerar que o kit VISUAL-PAG pode ser utilizado como um método de diagnóstico precoce e seguro de gestação em vacas leiteiras da raça Girolando aos 25 dias pós-inseminação artificial.

Validation of Buffalo PAG Antisera for Early Pregnancy Diagnosis in Bovine

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 As#706 and As#859. On the other hand, the coefficients of correlations between concentrations obtained by the use of As#859 and As#497 were highly comparable to those between As#859 and As#706 or As#497 and As#706. In conclusion, the present study clearly shows that antisera against buffalo PAG can be used to develop new PAG-RIA system for bovine pregnancy diagnosis with high comparable results.