Diagnosis ofFasciolainfection by SDS–PAGE eluted excretory secretory (ES) protein fractions using dot-ELISA

Abstract

Fascioliasis is now recognized as an emerging zoonotic disease in Egypt. Diagnosis in suspected patients still needs some degree of accuracy. In the present study, three Fasciola gigantica execratory secretory (ES) protein bands of molecular weight (MW) ranging from 14 to 20 kDa, 25 to 32 kDa and 45 to 65 kDa were eluted after fractionation of the parasite antigen using SDS-PAGE. The extracted kDa protein bands were concentrated and evaluated in diagnosis of Fasciola infection. Moreover the level of their cross reaction with other parasitic infections in infected and suspected patients of known parasite eggs/gram stool was evaluated using the dot-ELISA technique. Protein bands in the range of 14-20 kDa and that of 25-32 kDa were markedly specific and sensitive in diagnosis of different levels of anti-Fasciola antibodies (Ab) in sera of infected cases. These two groups of bands were able to exclude cross-reaction between anti-Fasciola Ab and other parasites recorded in stool of selected patients suffering from Schistosoma mansoni, Ascaris, and Giardia, either in single or mixed conditions with Fasciola eggs. While that of 45-65 kDa appeared less specific than the other previously mentioned bands. Protein bands in the range of 25-32 kDa appeared more sensitive than the other protein bands in detection of anti-Fasciola Ab at higher serum dilutions. The Dot-ELISA technique was proved to be more economic and easy in application. The dotted very small amount of antigens can be stored in a freezer and used at request in diagnosis of large numbers of samples. ª 2014 Production and hosting by Elsevier B.V. on behalf of Faculty of Veterinary Medicine, Cairo University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/3.0/).

Design and expression of polytopic construct of cathepsin-L1, SAP-2 and FhTP16.5 proteins of Fasciola hepatica

“…The antigens commonly used in commercial diagnostic ELISA kits are the crude extracts or excretory–secretory antigens. However, the use of such complex antigens reduces the specificity of the serological tests due to cross-reactivity with parasites sharing common epitopes (Losada et al , 2005; Salimi-Bejestani et al , 2005; Sabry et al , 2014).…”

Antibody immunoglobulin G1 and immunoglobulin G2a responses against some cystic fluid proteins of Cysticercus bovis in Balb/c mice

“…The use of C. bovis proteins eluted from SDS-PAGE gel as antigens for ELISA and Western blotting assays has never been reported. However, a similar procedure has been used in the preparation antigen for ELISA and Western blotting assays of Fasciola gigantica excretory/secretory fluid [21]. The removal of SDS using acetone precipitation [18] appeared to be an important step in regaining the antigenicity of proteins eluted from SDS-PAGE gel.…”

Serological evaluation of antigen Tp0693 for diagnosis of syphilis

“…A bioinformatics analysis, including transmembrane, signaling peptide, subcellular localization and epitope analyses, indicated that it is a secretion protein with strong immunogenicity in the present study. Secretion proteins of other pathogens, such as leishmaniasis ( 19 ), tuberculosis ( 20 ), Fasciola ( 21 ) and gasterophilosis ( 22 ) have been found to serve as diagnostic antigens. However, whether Tp0693 is a diagnostic antigen for syphilis has remained elusive.…”