Identification of immunodominant fraction of Paramphistomum epiclitum and its evaluation for use in the serodiagnosis of paramphistomosis by ELISA

Abstract

The present study was undertaken to identify and purify the immunodominant fractions from the excretory secretory (ES) antigen of Paramphistomum epiclitum, a predominant amphistome species infecting ruminants in India. ES antigen was prepared and characterized using SDS-PAGE and Western blot analysis. Major polypeptides of molecular weight 11, 22, 28, 31, 33, 39, 52, 59, 63 and 72 kDa were visualized in SDS-PAGE. Polypeptides (9) of 11, 14, 16, 22, 31, 33, 39, 63 and 72 kDa showed immunoreactivity in Western blot analysis. The whole ES antigen of P. epiclitum was initially concentrated using PEG-8000 followed by spin-X UF concentrator with 10 kDa cutoff range and subsequently fractionated by size exclusion chromatography using Sephadex G-25. Cross reactivity of the P. epiclitum ES antigen was studied with positive sera of F. gigantica and H. contortus. Based on the cross reactivity profile, the low molecular weight antigenic fraction with 11 kDa polypeptide was selected for further use in indirect-ELISA. Bovine serum samples (258) were tested with optimized ELISA. Sensitivity of the ELISA was calculated as 75.0%, while the specificity was 85.0%. The percent positive and negative predictive values for the test were 70.78 and 87.57%, respectively.

Keywords

• ES antigen
• Immunodiagnosis
• Low molecular weight protein
• Paramphistomosis