Detection of cellular prion protein in animals of economic importance of Thailand

Abstract

Prion protein is the infectious causative agent of a fatal neurodegenerative disease belonging to a group of transmissible spongiform encephalopathies (TSEs) in various mammalian species including human. In this research polyclonal antisera were raised against three synthetic peptides corresponding to amino acids sequences in three different regions of sheep PrP; 40-56 (PI), 145-163 (PII) and 209-228(PIII). These peptides were custom-synthesized using an automated peptide synthesizer and multiple antigenic peptides (MAPs). The polclonal antisera were used to detect the cellular prion protein in animals of economic importance of Thailand: catfish, pigs, and cows. In this study nine BALB/cA mice were divided into 3 groups, each group was separately immunized with synthetic peptides PI, PII, and PIII. The antiserum elicited by each peptide displayed antibody binding to the homologous peptide. Antibody titers (OD[subscript 50] values) were determined by testing in an enzyme-linked immunosorbent assay (ELISA). Antisera against sequence PI (Ab PI), PII (Ab PII), and PIII (Ab PIII) showed the antibody titer at dilutions of 1:1,600, 1: 51,200 and 1:1,638,400, respectively. PrPC was purified from microsomal fraction by detergent extraction with Zwittergent 3-12 (ZW 3-12), separated by differential centrifugation and partially purified by immobilized Cu[superscript 2+] ion affinity chromatography (IMAC) owing to the binding capacity of PrP to Cu[superscript 2+] ions. Determination by Coomassie blue staining and immunostaining with mouse polyclonal antisera and monoclonal antibody (mAb L42) showed protein bands at 30 and 40 kDa in crude brain homogenates of the pig and the cow, while crude brain homogenate of fish yielded protein bands at 40 and 42 kDa. During the partial purification by IMAC, PrPC was eluted by either imidazole or EDTA. When subjected to immunostaining by Ab PII, this imidazole eluent showed the same protein bands as found in crude brain homogenates of fish, the pig, and the cow. The EDTA eluent, however, showed an immunostained protein band at 21 kDa in cases of the pig and the cow but not in the case of fish.