Neonatal diarrhea: development and evaluation of a method of ELISA for rotavírus detection from fecal material.

Author(s)

Fábio Gregori

• Document type: Master's Dissertation
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina Veterinária e Zootecnia (FMVZ/SBD)
• Defense date: 1999-06-25

Examining board members

Jose Antonio Jerez; Rosa Maria Piatti; Leonardo Jose Richtzenhain

• Advisor: Jose Antonio Jerez

Abstract

Rotaviruses have been identified worldwide as a major etiologic agent of acute nonbacterial diarrhea in the young of many species, including humans. In this investigation was developed and evaluated a “double-sandwich" antibody ELISA method for detection of rotavirus from stool specimens. For that, the NCDV strain of rotavirus group A was serially cultivated in MA-104 cell culture. The virus was concentrated by ultra-centrifugation and inoculated in rabbits and sheeps. After that, the IgG of serum samples of the animals was purified by ion-exchange chromatography and absorbed with whole serum of both animal species using a glutaraldehyde polymer, in order to eliminate inespecific reactions. The presence of rotavirus was detected by the sheep’s IgG and revelated by the rabbit’s IgG, using a anti-rabbit IgG peroxidase conjugate developed in goat. The values of diluition of the components of the ELISA and the cut-off value were defined using 26 fecal samples (13 positive and 13 negative) of piglets. Following this procedure, the test was employed in a panel of 86 fecal samples from piglets with diarrhea, using as standard the polyacrilamide gel electrophoresis (PAGE) test. The results of the ELISA were: 100% of sensivity; 98.79% of specificity, with an agreement of 98.83%. The variance between 86 repetitions of the same sample were 0.001 (for one positive sample) and 0.0002 (for one negative sample). These results showed that this ELISA is an sensitive and specific screening test for rotavirus diagnosis from fecal material. (AU)