Ovine Pregnancy-Associated Glycoprotein (ovPAG) as a marker of pregnancy on Garut Sheep

Abstract

Pregnancy-Associated Glycoprotein (PAG) structurally related to aspartic proteinase, expressed in the outer epithelial cell layer (trophectoderm) of ungulate placenta. Ovine PAG synthesized by mono- and binucleic trophoblast before complete implantation at day 14-15. Of this, ovPAG could be used as a marker for early pregnancy and foetal wellbeing. The research was started with extraction and isolation of PAG from placenta of garut sheep collected at parturation and to characterize their molecular weight. The procedures included extraction of protein at neutral pH (cotyledon was thawed, minced, added PBS, blended and centrifuged), acidic (H3PO41M, pH 4,5; centrifuged) and ammonium sulfate (40% and 80% (NH4)SO4, centrifuged) precipitation ; gel filtration (Sephadex- G75), anion exchanged chromatography (DEAE- cellulose). Cotyledone’s extract was subjected to Sephadex-G75 and DEAE cellulose, and their fractions were measured their absorbances. Sephadex-G75 and DEAE fractions at peak absorbances were assayed for protein concentration (Bichinconinic protein assay). Continuously, these fractions were subjected to monogel SDS-PAGE and stained by Commassie Brilliant Blue. Then, isolate was used to produce polyclonal antibody ( rabbit anti-ovPAG). Polyclonal antibody production was carried out in accordance with Animal Care and Use Committee of PT Indoanilab No: 03-IAACUC- 08, 15th Nopember 2008. Rabbit anti-ovPAG DN32 gave the best immune response using Modified ELISA technique also could differenciate ovPAG in the urine of pregnant and non pregnant ewes using Western Blot Technique. Simultaneously, modified ELISA was developed started by producing ovine PAG standard, carried out by developing serial dilution at concentration 0,344; 0,172; 0,0688; 0,0344 and 0,0172 ng/ml, continued with intra- and interassay validation. Validation of standard showed coefficient variation intra-assay on QC-H and QC-L were 9,97 % and 5,87 %, respectively. Moreover, coefficient variation inter-assay on QC-H and QC-L were 16,95 % and 13,9 %, respectively. Using Western blot technique, protein at 31 kDa molecular weight appeared in pregnant’s urine whereas protein around 71 kDa became a common protein. Modified ELISA technique could differentiate the concentration of ovPAG in between pregnant and non pregnant urine at 55,56 % of nine ewes.

Pregnancy-associated glycoprotein (PAG) merupakan famili aspartik protease, terdapat di dalam sel trofektoderm plasenta ungulata. OvinePAG disintesa oleh sel mono- dan binukleat trofoblas sesaat menjelang implantasi pada hari ke 14-15. Oleh karena itu, PAG digunakan sebagai indikator penanda kebuntingan dini dan juga kesehatan fetus. Tujuan penelitia ini adalah ekstrasi dan isolasi ovPAG dalam kotiledon plasenta; memproduksi antibodi poliklonal (Rabbit anti-ovPAG); mendeterminasi ovPAG dalam urine domba bunting, dan pembuatan standar ovPAG. Kegiatan penelitian diawali dengan melakukan ekstraksi dan isolasi PAG dari plasenta domba garut yang dikoleksi pada saat melahirkan dan mengkarakterisasi PAG berdasarkan berat molekulnya. Pada saat melahirkan, kotiledon dikoleksi dari plasenta, disimpan pada -200C sampai seluruh induk domba (n=9 ekor) melahirkan. Selanjutnya kotiledon diekstrak dengan perlakuan presipitasi asam (H3PO41M, pH 4,5 ; sentrifus) dan garam (40% (NH4)SO4 dilanjutkan 80% (NH4)SO4, dan disentrifus). Berikutnya dilakukan isolasi protein melalui gel filtrasi (Sephadex G-75) dan kromatografi pertukaran anion (DEAE-cellulose). Fraksi yang ditampung diukur absorbansinya, fraksi yang memiliki absorbansi tinggi, diukur total proteinnya.