Serodiagnosis of fasciolosis by fast protein liquid chromatography-fractionated excretory/secretory antigens

Abstract

In several studies, different antigenic preparations and diverse immunological tests were applied for serodiagnosis of Fasciola hepatica infections. Most of these preparations showed cross-reactivity with proteins of other parasites. Application of purified antigens might reduce these cross-reactivities. Here, we used fast protein liquid chromatography (FPLC)-fractionated extracts of F. hepatica excretory/secretory antigens (E/S Ags) for serodiagnosis of human and sheep fasciolosis. To develop an improved diagnostic method, we fractionated F. hepatica E/S Ags by anion exchange chromatography on a Sepharose CL-6B column and then tested the serodiagnostic values of the fractions. We used sera from F. hepatica-infected human and sheep as positive controls. Sera from patients with hydatidosis and strongyloidiasis were used for cross-reactivity studies. Enzyme-linked immunosorbent assays (ELISA) of the second FPLC peak, containing 20, 25, and 70 kDa proteins, discriminated between F. hepatica-infected and uninfected human and sheep samples. Fractionation of F. hepatica E/S Ags by FPLC is a fast and reproducible way of obtaining antigens useful for serodiagnosis of human and sheep fasciolosis with acceptable sensitivity and specificity. Graphical abstract ᅟ.

Application of Dirofilaria immitis immunoreactive proteins in serodiagnosis

“…Dit33 is expressed by several parasites including D. immitis , Brugia malayi and Onchocerca volvulus ; immunogenic galectin is found in D. immitis , B. malayi and Haemonchus contortus ; and finally GAPDH reacted with sera of hosts infected with B. malayi and Wuchereria bancrofti . The P27 protein is an immunogenic component in D. immitis , but in this study, we did not detect it . We identified two main reactive enzymes, superoxide dismutase and peroxiredoxin (thioredoxin peroxidase), in the E/S extract.…”

“…Immunoblotting of chromatography‐resolved E/S proteins demonstrated several immunogenic bands at approximately 11, 20, 22 and 25 kDa, similar to our findings on immunoblots of unfractionated E/S extracts. Therefore, we regard anion exchange‐based FPLC as a fast, accurate and cost‐effective technique . One main advantage of FPLC over other routine protein immunological identification methods is its ability to rapidly and reliably identify adequate amounts of antigens, applicable for serodiagnosis of canine dirofilariasis.…”

“…Following Coomassie Blue staining of SDS‐PAGE‐resolved proteins, the gels were rinsed with distilled water and immunoreactive protein bands, based on the Western blots, were excised and analysed by mass spectrometry at the Department of Biological Science of York University, UK, as previously described . In brief, carbamidomethylation was considered as fixed modification, and oxidation/deamidation was considered as variable changes.…”

“…The lyophilized fractions were solubilized in 0.1 mL of distilled water and electrophoresed on 15% polyacrylamide gels. The resolved proteins were stained with silver nitrate, and their apparent molecular weights were compared with the pattern of total E/S content …”

Fractionation and identification of the allergic proteins in Aspergillus species

“…The final extract was agitated for 3-4 h on a shaker at 2000 rpm, and then the contents were centrifuged at a speed of 12,000 g. The supernatant was removed and transferred into a dialysis bag(4 kDa, in 10 mM potassium phosphate buffer, pH 8.0) in order to deplete the salts and impurities, while simultaneously being mixed on a magnetic shaker for 16 h at 4°C. The protein concentration of the crude extracts was measured using the Bradford method [ 17 ].…”