STANDARDIZATION OF ELISA FOR DETECTION OF LEPTOSPIRAL ANTIBODIES IN BOVINES
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Date
2010-08
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Sri Venkateswara Veterinary University, TIRUPATI – 517 502,A.P
Abstract
ABSTRACT:
Leptospirosis is an emerging zoonosis caused by pathogenic spirochetes belonging to the genus Leptospira. In the present study, we used the humoral immune response to detect leptospiral protein antigens expressed during infection.
The hyperimmune serum raised against whole cell antigen and bovine serum positive for Leptospira were used for western blotting. Whole cell, sonicate antigen and outer membrane proteins extracted by Triton X-114 method were characterized by SDS-PAGE and western blotting immunoanalysis. SDS-PAGE analysis of the whole cell and sonicated antigens revealed ten protein bands with molecular weights ranging from 18 to 67 KDa i.e. 18, 22, 32, 36, 41, 43, 45, 63, 65 and 67 KDa where as outer membrane proteins extracted by Triton X-114 revealed protein bands of 22, 32, 36, 43, 45 and 63 KDa. Westernblot analysis of outer membrane proteins using hyperimmune rabbit serum revealed protein bands of 22, 32, 41, 43 and 63 KDa as major immunogens, where as analysis using positive bovine serum of L.grippotyphosa revealed protein bands of 25, 32 and 45 KDa as major immunogens. A total of 1100 serum samples available at Leptospira research centre C.V.Sc., Tirupati, were screened against different leptospiral serovars using MAT. The MAT titre value ≥ 1:80 dilution is considered as positive as standardized in the research centre. A total of 157(14.27%) samples were found positive for Leptospira. The MAT titres varied between 1:80 to 1:640. Indirect ELISA was standardized using heat extracted antigen and evaluated for measuring the antibody titres in bovine sera. The specificity, sensitivity and efficacy of the ELISA relative to the MAT were found to be 89.8%, 92.9%, 92.1% respectively. 19
Once ELISA was standardized, a random of 200 field serum samples available at research centre were screened by ELISA. Out of 200 serum samples, 32 (16%) were found positive by ELISA. The ELISA used in the present study was easily standardized and was found to be sensitive, rapid, specific, easy to perform, semi- automated and used a non-hazardous antigen which can be routinely prepared in large amounts.
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Keywords
BOVINES; Leptospirosis; zoonosis; ELISA