Identification And Characterization Of Protein Markers And Development Of Immunoassay For The Detection Of Hydatid Cyst Disease
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Date
2015-01
Authors
ZOHREH KAZEMI, MOGHADAM KAKHKI
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Abstract
Hydatid cyst disease (HCD) or hydatidosis is caused by an infection with the larval stage of Echinococcus granulosus. It affects human and animals, including wildlife and commercial livestock. The parasite antigens induce host humoral and cellular immune responses which may serve as potential markers in early detection of HCD. Diagnosis of HCD is based on non-specific clinical signs and imaging of suspected organs such as liver and lungs. Imaging methods cannot differentiate between hydatid cysts and other lesions such as liver abscess and neoplasms, therefore, immunodiagnosis is an important additional tool to detect the infection. Currently, there is no standardized test with high sensitivity and specificity that is available for the immunodiagnosis of HCD. In this study parasite antigens were prepared from E. granulosus protoscolex and hydatid cyst fluid (HCF) of hydatid cysts from naturally infected sheep. Serum samples were obtained from 130 HCD patients, 38 individuals infected with other parasitic infections and 30 healthy people. Peroxidase (HRP)-conjugated anti-human IgG and IgG4 were used as secondary antibodies. Two-dimensional gel electrophoresis and western/dot blot were used to identify protein bands of protoscolex and HCF that exhibited good diagnostic sensitivity and specificity.
A protoscolex antigenic band of 60 kDa was found to be 82% (40/49) sensitive and 100% (68/68) specific for detection of HCD when probed with anti-
human IgG4-HRP. Meanwhile the diagnostic sensitivity and specificity were 33% (10/30) and 100% (68/68) respectively when probed with anti-human IgG-HRP. By mass spectrometry analysis using MALDI TOF-TOF, the band was identified as protoscolex tegument paramyosin. The full length recombinant form of the paramyosin protein was produced; and the diagnostic sensitivity and specificity in IgG4 blots were found to be 86% (42/49) and 98% (58/59) respectively.
Two antigenic bands from hydatid cyst fluid (HCF) were identified namely 8 kDa and 37 kDa; the diagnostic sensitivities were found to be 86% (42/49) and 67% (33/49) respectively; while the diagnostic specificities were determined to be 100% (67/67) and 98% (66/67) respectively when probed with anti-human IgG4-HRP. When analysed using anti-human IgG-HRP, the sensitivity and specificity of the 8kDa antigenic band was 88% (43/49) and 37% (10/27), respectively. By mass spectrometry using MALDI TOF-TOF, the 8kDa band was identified as AgB/1 and the 37 kDa band as Ag5. Based on the AgB sequence identified in this study, a peptide of 54 residues with molecular weight of 6415 Da was selected and custom-synthesized. Using dot blot, the synthetic peptide (pRZ) showed diagnostic sensitivity of 90% (18/20) and specificity of 40% (8/20) when probed with anti-human IgG-HRP; and sensitivity of 88% (43/49) and a specificity of 100% (42/42), when probed with anti-human IgG4-HRP. Nitrocellulose membrane cards were separately lined with the pRZ at 0.75, 1, 1.25, 1.5 and 2 mg/ml and cut into lateral flow dipstick strips. The dipsticks using the last two concentrations showed high diagnostic sensitivity of 95% (40/42) and 98% (41/42) respectively, and specificity of 100% (85/85).
In conclusion, IgG4 detection of two hydatid cyst protein markers i.e. paramyosin from protoscolex and AgB/1 from HCF, were found to be useful for
the diagnosis of HCD. The recombinant form of paramyosin and the synthetic peptide of AgB/1 (pRZ) showed good diagnostic value. This study has successfully produced a rapid dipstick test using pRZ based on IgG4 detection and lateral flow technology. The test shows high potential for rapid, accurate diagnosis of HCD and would be useful in low resource settings of HCD endemic areas.
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Identification And Characterization Of Protein Markers And Development Of Immunoassay , For The Detection Of Hydatid Cyst Disease