DEVELOPMENT OF IN-HOUSE ELISA FOR THE DETECTION OF Mycobacterium avium subspecies paratuberculosis INFECTION IN ANIMALS
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Date
2017-10
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KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCIES UNIVERSITY, BIDAR
Abstract
The present study was under taken to develop an in-house ELISA using a local
Mycobacterium avium subspecies paratuberculosis (MAP) isolate and to investigate the
seroprevalance of Paratuberculosis in animals. The MAP protoplasmic antigen was
extracted by sonication and quantified by ‘Nanodrop’ spectrophotometer and 4.193
mg/ml antigen was obtained. Extracted MAP antigen was characterized by SDS - PAGE
and immune-blot analysis. The SDS-PAGE profile of MAP revealed 25, 35, 57, 72, 100
and 193 kDa proteins and immunoblotting revealed that 20, 29, 35, 45, 70 and 193 kDa as
immunogenic proteins. ELISA was standardized with MAP antigen of 0.125 μg / well,
serum dilution of 1:50 and Protein A-HRP conjugate dilution of 1:2500. 1032 serum
samples from sheep, goat and cattle were screened for MAP infection with in-house
ELISA and IDEXX ELISA kit. 113 (10.94 %) serum samples were positive by in-house
ELISA and 111 (10.75 %) serum samples were positive by IDEXX ELISA kit. Cut off
values were determined as SP ratio ≥ 80 per cent as Positive, ≤ 60 per cent as Negative
and 60 - 80 per cent as suspected. Seroprevalence of MAP in sheep, goat and cattle was
8.52, 5.95 and 6.6 per cent respectively. The relative sensitivity and specificity of in
house ELISA was 76.58 per cent and 96.96 per cent respectively with Kappa value of
0.7296 showing good agreement. Comparative evaluation of in-house ELISA with
IDEXX ELISA kit was statistically analysed by Pearson’s correlation with significant
correlation of 0.7725 (P=0.0001) between in-house ELISA and IDEXX ELISA kit.
Receiver operating characteristics (ROC) curve was drawn with excellent area under
curve of 0.992 was obtained. The study indicated the superiority of in-house indirect
ELISA using native MAP antigen as against purified protoplasmic MAP antigens used in
commercial ELISA kit in terms of cost effectiveness.