Hydatid Cyst Fluid Antigen Research Articles

Evaluation of Hydatid Cyst Antigen for Serological Diagnosis.

Hydatidosis, a chronic zoonotic disease, has a distribution worldwide and is caused by the larval stage of the Echinococcus helminth. The Dot-ELISA test can diagnose hydatidosis quickly and accurately. Additionally, unlike other hydatid disease tests now used, this quick and affordable enzyme immunoassay is very serum-conservative and antigen-conservative, needing just nanogram levels of parasite antigen. In the present cross-sectional study, crude and B antigens of hydatid cyst fluid were obtained to diagnose human hydatidosis using CIEP (Counter Immunoelectrophoresis), ELISA (Enzyme-linked Immuno Sorbent assay), and Dot- ELISA (Dot Enzyme linked Immuno Sorbent Assay) methods. Infected liver with a hydatid cyst was collected from Tehran's slaughterhouses to prepare cyst fluid in different stages. After extracting and purifying the Cyst fluid, it is centrifuged at 4ºc, then prepared to concentrate. The study also included sera from hydatidosis (n=60), samples of helminth parasites (n=55), fascioliasis (n=35), toxocariasis (n=20) and negative control (n=35) were tested by CIEP (Counter Immunoelectrophoresis), ELISA (Enzyme-linked Immune Sorbent assay), and Dot- ELISA (Dot Enzyme linked Immuno Sorbent Assay) methods. All statistical analyses were performed using Statistical Package for the Social Sciences (SPSS) for Windows release 25.0 (SPSS Inc., Chicago, IL, USA). Crude antigen of hydatid cyst showed a specificity of 76.7%, a sensitivity of 93.3% using the ELISA method, and B antigen showed a specificity of 96.7% and sensitivity of 88.3% using the same method. The crude antigen of the hydatid cyst exhibited a specificity of 68.9% and a sensitivity of 86.7% using CIEP. The B antigen showed a specificity of 87.8% and sensitivity of 83.3% using the same method.The crude antigen of hydatid cyst having serum dilution at 1:800 exhibited a specificity of 83.3% and sensitivity of 100% using the Dot-ELISA method and B antigen having serum dilution at 1:800 serum showed a specificity of 100% and sensitivity of 98.3% using the same method. The results of this finding showed that B antigen has the maximum specificity to diagnose hydatid test using the Dot- ELISA method. Hydatid cysts present with varied symptomatology. History of exposure to infected animals may not be present. A high degree of clinical suspicion combined with meticulous history and clinical examination supported by laboratory investigations are required for its diagnosis. The Dot-ELISA system with native antigen B is a viable approach for the immunodiagnosis of human hydatidosis that is preferred to infection.

IEg67 kDa Bovine Hydatid Cyst Antigen: A Candidate for Developing Sero-Diagnostic Assays for Cystic Echinococcosis, a Disease of One Health Importance.

Cystic echinococcosis (hydatidosis) is a world-wide zoonotic disease of mainly humans, livestock and dogs, caused by Echinococcus granulosus. The disease can negatively impact food production and animal welfare and causes socio-economic hardship. Here, we aimed to identify the local bovine hydatid cyst fluid (BHCF) antigen for developing a sero-diagnostic assay to be used for the pre-slaughter screening of food animals. In total, 264 bovines approved for slaughter in Pakistan were subjected to serum collection and post-mortem screening for hydatid cysts. These cysts were assessed microscopically to assess fertility and viability, and by PCR for molecular confirmation of species. A BHCF antigen was identified from positive sera via SDS-PAGE, confirmed by Western blot, and quantified via a bicinchoninic acid (BCA) assay. The quantified crude BHCF antigen (iEg67 kDa) was then used in ELISA screening to test all sera collected from known positive and negative animals based on hydatid cyst presence/absence. Of the 264 bovines examined, 38 (14.4%) showed hydatid cysts during post-mortem examination. All of these individuals, plus an additional 14 (total: 52; 19.6%) tested positive based on less time-consuming ELISA examination. Based on ELISA, occurrence in females (18.8%) was significantly higher than in males (9.2%) and was higher in cattle (19.5%) compared to buffalo (9.5%). The infection rate increased with age in both host species: cumulatively, 3.6% in animals aged 2-3 years, 14.6% in 4-5-year-olds and 25.6% in 6-7-year-olds. The occurrence of cysts in cattle was significantly higher in the lungs (14.1%) compared to their livers (5.5%), whereas the opposite was true in buffalo (6.6% livers, 2.9% lungs). For both host species, most cysts in the lungs were fertile (65%), while the majority in the liver were sterile (71.4%). We conclude that the identified iEg67 kDa antigen is a strong candidate for the development of a sero-diagnostic screening assay for the pre-slaughter diagnosis of hydatidosis.

Antigen B modulates anti-inflammatory cytokines in the EAE model of multiple sclerosis.

Multiple sclerosis (MS) is characterized by the destruction of the blood-brain barrier, loss of myelin sheath, and contribution of inflammatory interleukins such as TNF-alpha, interleukin-17, and interleukin-6. The current study investigated the effect of antigen B of hydatid cyst fluid on the reduction of anti-inflammatory cytokines and nerve conduction velocity in rats with experimental autoimmune encephalomyelitis (EAE)-induced MS. After isolation of antigen B from sterile cyst fluid, the rats were randomly divided into four groups: saline, EAE, EAE + teriflunomide (EAE + TF), and EAE + antigen B (EAE + AngB). The EAE model was induced using cow spinal cord homogenization, in combination with Freund's complete adjuvant. The serum concentration of cytokines including IL-1B and IL-17, IL-10, IL-6, and TNF-X was measured by the ELISA method, and real-time PCR was performed to study gene expression. Electrophysiological, behavioral, and neuropathological tests were also conducted. Nerve conduction velocity and IL-10 concentration were increased in the antigen B group. The results of this study showed that antigen B reduced the inflammatory component of the EAE MS animal model by modulating the immune system compared to teriflunomide, which eventually led to a reduction in symptoms at the behavioral and electrophysiological level. It seems that antigen B plays a critical role in regulating immunity and it can be used as a possible therapeutic agent to modulate the immune system in MS patients. It might be rational to consider hydatid cyst fluid antigen as a modifier in MS.

A Comparative Evaluation of Four Different Immunoassays in the Diagnosis of Cystic Echinococcosis Using a Crude and Purified Hydatid Cyst Fluid Antigen.

Cystic echinococcosis (CE) is one of the most neglected tropical diseases as per WHO which has an immense public health significance. Diagnosis of CE is difficult as specific clinical signs are manifested only after the hydatid cyst attains a considerable size. Immunodiagnosis is a reliable method of diagnosing CE. SDS-PAGE was performed for the hydatid cyst fluid antigens. The antigen purity was tested by Western blotting and four different immunoassays were evaluated using these two antigens in sheep and buffalo in diagnosis of CE. SDS-PAGE revealed four bands of 72, 64, 48 and 24kDa for crude antigen and a single 72kDa band for purified antigen. Among sheep sera, ELISA was most sensitive (70%) using crude antigen and also while using the purified antigen (80%). In case of buffaloes, ELISA, DID and CIEP were more sensitive (83.3%) using crude antigen, whereas DID and CIEP were more sensitive (83.3%) using purified antigen. In sheep, while using the crude antigen ELISA was the most sensitive assay and IHA was the least sensitive assay. While using the purified antigen also, ELISA was the most sensitive and others were absolutely specific except for IHA being less sensitive. In buffaloes, using crude antigen, all the immunoassays CIEP, DID and ELISA were highly sensitive in diagnosing CE infection except IHA, whereas using the purified antigen, both CIEP and DID were more sensitive than ELISA and IHA which were comparatively less sensitive in detecting CE in buffalo sera.

The Intervention of the Pirfenidone with Pericyst Layer Building of the Hydatid Cyst

Hydatid disease is a zoonosis caused by Echinococcus granulosus larval stage, characterized by developing a fluid-filled hydatid cyst in any part in the body, with a particular liver and lung localization. The released hydatid cyst fluid (HCF) antigens actively induce epithelial to mesenchymal transition (EMT) in the epithelial cells surrounding the mass of the hydatid cyst, result in pericyst layer construction, which plays a vital role in preventing the antiparasitic medications from being reached to the parasite. Therefore, the current study attempted to employ the antifibrotic effect of the Pirfenidone to hinder the induced EMT by HCF antigens. For this reason, the adenocarcinomic human alveolar basal epithelial cells (A549 cell line) was used as a model system to human alveolar epithelial cells type II (AEC II) to assess the effect of the Pirfenidone on the induced EMT by HCF antigens. Different assays used to investigate the EMT induction, then Pirfenidone effect assessed using immunofluorescence assay to detect cytoskeletal markers expression prior to and after treatment. The results reached that the Pirfenidone interrupting the induced EMT in A549 cells, and thus, it may interrupt the EMT and pericyst fibrosis in the same way in the infected tissues, which would facilitate the antiparasitic drugs delivery across the cyst wall and help in parasite killing.

Immunological detection of human and camel cystic echinococcosis using different antigens of hydatid cyst fluid, protoscoleces, and germinal layers.

Background and Aim:Cystic echinococcosis (CE)/hydatidosis is one of the most prevalent neglected zoonotic diseases. It is initially asymptomatic and does not produce any clinical signs until the cyst becomes enlarged, causing localized pressure on internal organs and tissues. Therefore, the detection of Echinococcus granulosus antibodies is highly essential. This study evaluated the antigens of hydatid cyst fluid, protoscoleces, and germinal layers for efficient immunological diagnosis of CE in humans and camels.Materials and Methods:Hydatid cyst fluid (FLc), protoscoleces (Psc), and the germinal layer (GLc) antigens were prepared from camel-lung hydatid cysts. In the same way, hydatid cyst fluid (FLh) and protoscoleces (Psh) antigens from human-liver cyst aspirate were produced. The comparative immunodiagnostic efficacy of the prepared antigens was verified using indirect enzyme-linked immunosorbent assay (ELISA), SDS-PAGE, and immunoblotting.Results:ELISA proves that FLc and GLc antigens were higher than FLh and Psh antigens. This shows that binding reactivity in naturally infected human sera, camel sera, and Psc is the most potent, exhibiting 100% sensitivity with 78.26% and 76.47% specificity in camel and human sera, respectively. The CE prevalence using diagnostic Psc was 54.79% and 61.32% in tested human and camel sera, respectively. The electrophoretic profiles of all shared antigens showed similarities at 52, 41, and 22 kDa. Immunoblotting demonstrated common immune-reactive bands in all antigen types at 52 and 41 kDa against positive human and camel sera.Conclusion:This immunological study introduces camel hydatid cyst Psc as a potent diagnostic antigen and new immune-reactive fractions of 52 and 41 kDa for diagnosing hydatidosis in humans and camels.

A trial treatment of Hydatidosis in white mice by Immunization and Chemicals

The present study was conducted as atrial to treat secondary hydatidosis in white mice of species Mus musculus. (Balb/c strain), experimentally infected with secondary hydatid cysts of sheep origin. The immunization process was carried by using CF/PS antigen which consist hydatid cyst fluid (CF) antigen, and protoscolices (PS) antigen mixed together and by using two concentrations 7.5, 15 mg/ml. Then derivatives of Benzimidazole, Mebendazole (M) and drugs Albendazole (A) were used 10 and 40 μm/gm body weight respectively. The mixture of drugs was used one week after challenged dose. Criteria taken into consideration to define the activity of the immunization and chemotherapy in this study were reduction in the numeral of cysts, the coefficient of split cells in spleen and boon marrow, and measurement cellular and humoral immune response. The results of the present consider demonstrated those portray the viability about immunization also chemotherapy all the while over decay those numeral of growing cyst, toward that perspective stately both humoral and cell-mediated immunity. Those outcomes indicated raise those safe reactions that replicated diminish numeral Also breadth for hydatid growth. Those goal that viability of immunotherapeutic state over reversal those developing from growing hydatid cysts to mice.

Cytokine profiles in peripheral blood mononuclear cells from patients with cystic echinococcosis

IntroductionCystic echinococcosis (CE) is a chronic zoonotic disease caused by the larval stage of Echinococcus granulosus (E. granulosus), which affects domestic and wild carnivores as the definitive host and ungulates as intermediate hosts. In intermediate hosts, both Th1 and Th2 cells are involved in the immune responses to an echinoccocal infection. This study aimed to investigate production of IL-4, IL-10, and IFN-γ cytokines in peripheral blood mononuclear cells (PBMCs) of CE patients before and after surgical treatment. MethodsTo evaluate cytokine production in response to E. granulosus antigens, we investigated IL-4, IL-10, and IFN-γ production in PBMCs of 20 CE patients in response to hydatid cyst fluid antigen (HCF-Ag) before and after surgical treatment using ELISA. ResultsThe mean IL-4 production from HCF-Ag stimulated PBMCs was significantly decreased (p < 0.05), while IFN-γ was significantly increased in HCF-Ag stimulated PBMCs in patients after surgery (p = 0.005).Furthermore, our results showed that there is no significant difference between IL-10 production in patients before and after treatment (p = 0.562). ConclusionsOur data Indicated production of IL-4 in cultured PBMCs of CE patients stimulated with HCF-Ag was decreased significantly. While, production of IFN-γ was increased significantly in responses to HCF Ag after surgery. We concluded that the evaluation of IL-4 and IFN-γ in HCF-Ag stimulated PBMCs of CE patients should be considered as a useful marker in the follow up of patients with cystic echinococcosis.

A simple and efficient purification method of native immunoreactive antigen for diagnosis of camel hydatidosis.

Background:Cystic echinococcosis (CE), a zoonotic disease that affects animal and human health, is of increasing economic importance due to high morbidity rates and high economic losses in the livestock industry.Aim:The present study was conducted to purify the antigen from hydatid cyst fluid (HCF) with high diagnostic efficacy of camel hydatidosis using indirect enzyme-linked immunosorbent assay (ELISA).Materials and Methods:The HCF antigen was purified using Sephacryl S-300 column chromatography. Characterization of fractions was performed using reducing and non-reducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. Further, antibodies against Echinococcus granulosus cysts in camel serum were detected using indirect ELISA.Results:The purification process resulted in three fractions of antigens: FI, FII, and FIII. Indirect ELISA showed that higher diagnostic efficacy was observed in FI than in FII and FIII. Indirect ELISA, in which FI was utilized, showed 88% sensitivity and 91.7% specificity. Non-reducing SDS-PAGE showed that FI had two bands of molecular weights 120 and 60 kDa. Western blot analysis of FI demonstrated that 60, 38, and 22 kDa were antigenic bands when reacted with naturally infected camel sera with E. granulosus cysts. Using indirect ELISA, F1 recorded an infection percentage of 81.7% in randomly collected camel serum samples.Conclusion:FI is a promising antigen for accurate diagnosis of camel CE using indirect ELISA.

Bone marrow-derived mesenchymal stem cell (BM-MSC): A tool of cell therapy in hydatid experimentally infected rats.

This study aimed to clarify the potentiality of bone marrow mesenchymal stem cells (BM-MSC) transplantation with albendazole (ABZ) on the modulation of immune responses against hydatid cyst antigens and the regeneration of injured livers in experimentally infected rats. Three different antigens of hydatid cyst fluid (HCF), hydatid cyst protoscolex (HCP) and hydatid cyst germinal layer (HCG) were isolated and their antigenic potencies were determined. The ultrasound, immunological and pathological criteria were investigated. Counting of 80% confluence BM-MSC was 4.68 × 104 cells/cm2 with 92.24% viability. Final population doublings score was 65.31 that indicated proliferation and self-renewability. Phenotyping of BM-MSC showed expression of CD73 and CD29 without exhibition of CD34 and CD14. Ultrasound examination showed multiple hydatid cysts in liver with low blood flow and spleenomegaly 8 weeks’ post infection. No significant differences were noted in cystic diameter in uni-cyst liver at 2nd and 4th weeks following ABZ treatment while it was significantly decreased (P < 0.05) following transplantation of BM-MSC + ABZ treatment comparing to experimentally infected untreated group. Igs and IgG responses to the three antigens were significantly elevated while elevation in IgM response was only to HCG (P < 0.05). ABZ treatment accompanied with significant decrease in Igs and IgG titers against HCF and HCG only at 4th week post treatment (P < 0.05). However, Igs titer against HCF, HCP and HCG was significantly decreased at the 4th week following transplantation of BM-MSC + ABZ. Interestingly, the combination of BM-MSC + ABZ treatment resulted in reduction of Igs response to HCP to normal level as that of healthy control. Experimental infection resulted in elevation of TNF-α and IL-6 (P < 0.05) while, IL-4 and IL-10 decreased (P < 0.01). After transplantation of BM-MSC + ABZ treatment, serum TNF-α and IL-6 concentrations were reduced (P < 0.05) at both the 2nd and 4th weeks. However, IL-4 and IL-10 concentrations were significantly elevated (P < 0.05) only at 4th week following transplantation of BM-MSC + ABZ treatment. In conclusion, BM-MSC transplantation following ABZ administration can regenerate injured liver tissue without complete disappearance of hydatid cyst. In addition, it can modulate host protective humeral and cell mediated immune responses against hydatid cyst antigens. Therefore, the current study encourages to move to the step of performing clinical trials in humans.

Identification of antigen B (AgB) Gene polymorphism in cattle and sheep isolates of Echinococcus granulosus and investigation of effects on serological diagnosis

Cystic Echinococcosis (CE) is a worldwide common helminth disease caused by the larval form of Echinococcus granulosus. The aim of this study is to determine the genetic differences between distinct isolates of E. granulosus obtained from cattle and sheep and determine the polymorphism of the AgB1 gene by DNA sequence analysis, as well as investigating its relationship with serological response using ELISA and Western Blot tests. For this aim, germinal membranes of hydatid cysts of 30 cattle and 30 sheep from the provinces of Elazig and Erzincan in Turkey and serum samples of these animals were collected. Following isolation of the total genomic DNA, the 12S rRNA gene of all isolates was amplified by PCR for genetic characterization, and the mt−CO1 gene region was examined by DNA sequence analysis. The gDNAs were then amplified by PCR using AgB1-specific primers, and genetic variation was investigated by DNA sequence analysis. At the final stage, all serum samples were analyzed by ELISA and Western Blot tests using a partially purified hydatid cyst fluid antigen. As a result, 114 (95%) of the 120 isolates were determined to be E. granulosus sensu stricto by using 12S rRNA-PCR. Subsequently, the DNA sequence analysis of the remaining 6 samples of the mt−CO1 gene revealed that all samples were E. granulosus sensu stricto. According to the DNA sequence analysis that followed, nucleotide changes in the AgB1 gene were observed in 13 (10.8%) of 120 samples. With this study, 9 (69.2%) out of 13 hydatid cysts in which polymorphism was detected by DNA sequence analysis in their AgB1 gene were found to be positive with ELISA, and 6 (46.1%) were found positive by WB. While 80 (74.7%) of 107 non-polymorphic samples in the AgB1 gene were found to be positive with ELISA, and 75 (70.9%) were positive with WB. As a result, variation in different ratios was determined in the AgB1 gene of E. granulosus sensu stricto, and it was determined that this had a partial effect on serological response.

Effect of Hydatid Cyst Fluid Antigens on Induction of Apoptosis on Breast Cancer Cells.

Background:Hydatid cyst is a zoonotic and parasitic disease with worldwide distribution. Anticancer effects of hydatid cyst have been shown in cell culture experiments and animal model investigations. The mechanism of anti-cancer effects of hydatid cyst fluid has not been clearly elucidated, and the induction of apoptosis may have a role in this regard. Hence, in this study, the effect of hydatid cyst fluid (HCF) on the induction of apoptosis on mouse breast cancer (4T1) cell line in cell culture medium has been investigated.Materials and Methods:Echinococcus granulosus HCF antigens including Antigen B (AgB), glycolipid, glycoprotein, and 78 KDa fractions were prepared. Breast cancer cell line (4T1) was cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and appropriate antibiotics. Apoptosis was determined by flow cytometry using the annexin V-fluorescein isothiocyanate apoptosis kit.Results:The 78 KDa and glycoprotein fractions induced more than 40% apoptosis. HCF and glycolipid antigens induced 39% and 34% apoptosis, respectively. However, less apoptosis observed after treatment with AgB fraction.Conclusion:Hydatid cyst antigens especially the 78 KDa and glycoprotein fractions induced apoptosis on mouse breast cancer cells.

Identifying the Most Effective Hydatid Cyst Fluid Fraction for Anticancer Vaccination of 4T1 Breast Tumor-Bearing Mice.

Background:The hydatid cyst fluid antigens have high homology with cancer cell antigens and also exhibit considerable immunogenicity. Therefore, their utilization for cancer immunization can cause an effective antitumor immune response. However, the main challenge is identifying the most effective antigens for this purpose.Methods:Hydatid cyst fluid fractions including the glycolipid fraction, glycoprotein fraction, 78 kDa fraction, and antigen B fraction were prepared. Then, the BALB/c mice were immunized against different antigens and, subsequently, 4T1 cells were subcutaneously implanted. The tumors' growth, metastasis, and tumor-bearing mice survival were assessed in different immunized groups. In addition, IL-2, IL-4, IFN-γ, and TNF-α serum levels were estimated to evaluate the immune system response.Results:BALB/c mice immunization against the complete hydatid cyst fluid antigens exhibited more significant inhibition of the tumors' growth and metastasis and increase of tumor-bearing mice survival in comparison with its derived fractions. However, the 78 kDa fraction exhibited the best results according to the same factors in comparison with all the prepared fractions.Conclusions:The 78 kDa fraction of the hydatid cyst fluid was the most effective fraction of hydatid cyst fluid for immunization against 4T1 breast tumors.

Antigenic and genotypic characterization of Echinococcus granulosus larval isolates from Egypt

Background: Cystic echinococcosis (CE), caused by the larval stage of Echinococcus granulosus, is a global zoonotic infection of economic importance constituting a threat to public health in many countries. E. granulosus exists as a complex of different strains that have an impact on the epidemiology and control of CE, the most important of which are G1 and G6 strains. In Egypt, some studies confirm the predominance of G1 strain while others demonstrated the involvement of camel G6 strain in causing human infection.Objective: To study the diagnostic potential of purified antigenic yields of hydatid cyst fluid (HCF) from Egyptian CE patients and DNA corresponding to different recorded genotypes, in addition to the characterization of E. granulosus genotype in human and animal isolates in Egypt.Subjects and Methods: Crude HCF antigens from 30 patients were extracted and fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and probed by enzyme immunoelectrotransfer blot (EITB) against sera from CE patients and 50 controls. HCF from human and animal isolates was obtained and prepared for DNA extraction and the amplification of the predominant genotype bands at 254 bp.Results: PCR applied to HCF protoscolices of human and camels showed a typical 254 bp band of 12S fragment of mitochondrial gene belonging to G6 genotype (camel strain). SDS-PAGE fractionation of crude HCF antigen gave a protein profile composed of 11 bands. Immunoblotting assay showed that anti-E. granulosus IgG of patients' sera recognized 9 antigenic bands, varying in molecular weight from 12-110 kDa. The 48 and 12 kDa bands detected in all patients' sera disappeared after treatment.Conclusion: This study confirmed the predominance of G6 genotype (camel strain) of cystic echinococcosis in Egypt. PCR using the amplification primers of G6 genotype is a promising tool in the diagnosis of CE using either patients’ HCF or sera. The use of EITB in the diagnosis and post-treatment follow up of G6 genotype CE patients proved of high sensitivity and specificity. The recognition of 48 and 12 kDa antigenic proteins in 100% of CE cases’ sera and their disappearance after treatment marks their usefulness in diagnosis and follow up of CE cases.

Design and construction of a new recombinant fusion protein (2b2t+EPC1) and its assessment for serodiagnosis of cystic echinococcosis.

The immunodiagnostic tests for cystic echinococcosis (CE) are mostly serological tests based on ELISA that use hydatid cyst antigens for primary screening because of its simple preparation and availability. The challenge to develop new serological methods (as compared to those based on the hydatid cyst fluid antigens) to meet the gold standard remains. Appropriate sources of antigenic material are necessary for application to improve the efficacy of immunodiagnostic tests at a population level. In the current study, a fusion protein containing the coding sequence of antigen B2t and two sequences of EPC1 antigen with some modifications was reconstructed. Using bioinformatics tools, these sequences were joined together by applying the sequence of a rigid α-helix-forming linker to obtain an appropriate structure of a fusion protein. Synthetic recombinant fusion protein was expressed using pET28a as a vector and evaluated by indirect ELISA test for sera from patients with hepatic CE and other parasitic infections. The sensitivity of the fusion protein was lower (88.46%) than the available ELISA kit (96.15%). However, the differences in sensitivity were not statistically significant as compared to the recombinant fusion peptide with the commercial kit (p=0.269). The specificity of the recombinant fusion protein (95.45%) was not significantly lower than the commercial kit (96.59%; p=1.000). Moreover, surprisingly there was no difference in the cross-reactivity values of performance between the recombinant-ELISA and commercial kit. The positive and negative predictive values of the recombinant antigen were achieved as 92% and 93.33%, respectively, while for the commercial kit, they were obtained as 94.33% and 97.70%, respectively. In conclusion, as an early evaluation of these antigens the performance of our recombinant fusion protein in ELISA is relatively promising. Although, it seemed that this peptide with specific antigenic epitopes might be more appropriate for the serological evaluation of CE by use of bioinformatics tools, our findings showed that cross-reactions and a negative reaction could occur in clinical performance. This fusion protein may have utility for diagnosis in humans, but further evaluation is needed using the WHO ultrasound classification for CE.

Monoclonal Antibodies Production Against a 40KDa Band of Hydatid Cyst Fluid.

Hydatid cyst is the larval stage of the tapeworm Echinococcus granulosus. Hydatid cyst fluid, cyst membrane and Protoscolices, contain a complex mixture of antigens that can induce immune responses in the host. Anti-cancer properties of Protoscolices and hydatid cyst fluid has been shown. In order to identify antigens of hydatid cyst fluid that have anti-cancer effect, in this study production of monoclonal antibodies against one of the hydatid cyst fluid band (40KDa) has been investigated. There are many published patents about applications of monoclonal antibodies. In this experimental study, 40KDa band of hydatid cyst fluid that has cross reaction with sera of patients with breast cancer was used as antigen. A group of mice were immunized with this antigen, and then their spleen cells were extracted and fused with SP2 cells. Monoclonal antibodies production was checked in wells with signs of cell growth using ELISA and western blotting. The reaction of the produced monoclonal antibodies with breast cancer cells was tested using flow cytometry method. Finally, effect of the monoclonal antibodies on growth of breast cancer cells was investigated in vitro. The results of this study showed that in the first plate antibody against 40KDa was detected in several wells. In the second plate monoclonal antibodies with high titer was detected in one well. The produced monoclonal antibodies reacted with the surface of breast cancer cells. However, they had no significant effect on growth of breast cancer cells in culture medium. Monoclonal antibodies against hydatid cyst fluid 40KDa band were produced. These antibodies reacted with the surface of breast cancer cells but had no significant effect on growth of these cells.

Antibody response to glycan antigens of hydatid cyst fluid, laminated layer and protoscolex of Echinococcus granulosus.

Background: Hydatid disease is characterized by long-term growth of hydatid cysts in the human. The glycan antigens have an important role in the immunology of hydatid cyst. In this study immunological reaction of host sera to different glycan antigens of the cyst, has been investigated. Methods: The antibody responses were tested to glycoprotein and glycolipid of the laminated layer (LL), cyst fluid (CF) and protoscolex (PS) antigens of E. Granulosus using ELISA and western immunoblotting tests. Thin-layer chromatography and ß-elimination were used for glycan purification. Results: Both hydatid cyst and normal human sera reacted with hydatid cyst fluid, protoscolices, laminated layer, glycoprotein and glycolipid antigens. The most antigen-antibody reaction was related to CF and PS antigens, and LL antigens had the minimal reaction with the sera. Thin layer chromatography (TLC) of the antigens showed presence of many glycan bands in the laminated layer. Conclusion: The parasite may elaborate different glycan antigens in LL to evade host immune response.

Detection of specific IgG, IgM, IgE and IgG subclass antibodies for serological diagnosis of human cystic echinococcosis

Summary An enzyme linked immunosorbent assay (ELISA), based on sheep hydatid cyst fluid antigen was used for the detection of specific antibodies of IgG, IgM, IgE and IgG subclass in the serum samples of 62 clinically and radiologically diagnosed cystic echinococcosis (CE) patients, 8 clinically suspected cases of CE, 25 other parasitic disease controls and 25 healthy controls. The diagnostic sensitivity in the clinically and radiologically suggestive cases (n = 62) for IgG antibody detection was highest (93 %), followed by IgE, IgG4, IgG1, IgG2, IgM and IgG3 with 89 %, 87 %, 85 %, 76 %, 70 % and 55 % respectively. The detection of specific IgE, IgG1 and IgG4 antibody showed the higher diagnostic sensitivity and specificity to the extent that they can be safely used as better substitute to IgG. Even though, the diagnostic sensitivity of IgG was highest (93%) but was less specific (88 %) due to the frequent non-specific reactions in the sera of patients with other parasitic infections and healthy controls. None of the sera samples from healthy controls gave non-specific reaction with IgE, IgG1 and IgG4 and there was a considerably reduced cross-reaction with these antibodies. The most discriminatory and specific antibodies found in this study belonged to IgE, IgG1 and IgG4; therefore, these antibodies may serve as useful diagnostic markers for CE.

Immunodiagnosis of sheep infections with Echinococcus granulosus: in 35 years where have we come?

W. K. Yong and D. D. Heath published in 1979 a seminal paper in the first issue of Parasite immunology describing their efforts to determine whether the arc 5 precipitin band, formed when test human serum is reacted against electrophoresed hydatid cyst fluid antigen, would be a suitable immunodiagnostic test for the identification of sheep infected with Echinococcus granulosus. Although they found antibodies to arc 5 in the sera of hydatid-infected sheep, the sera of some sheep harbouring Taenia ovis and T. hydatigena also precipitated the hydatid cyst fluid arc 5 antigen, so they concluded arc 5 antibodies were not suitable for the specific immunodiagnosis of E. granulosus infection in sheep in New Zealand. Subsequent work has shown that the existence of multiple infections with different taeniid species, antigenic cross-reactivity between these related parasites and the low level of specific antibody response to infection continue to hinder efforts to improve the diagnosis of hydatid infection in sheep and other natural intermediate hosts, thereby preventing the development of any practical test. In particular, the poor antibody response of ruminants to naturally acquired hydatid infection may prove an insurmountable barrier in future efforts to develop a reliable and accurate immunological test.

Determination of Immunodominant Antigens of Dicrocoelium Dendriticum by Hyperimmune Sera

Dicrocoeliasis caused by the small liver fluke Dicrocoelium dendriticum (syn. D.lanceolatum), which live in the bile ducts and gall bladder of wild and domesticated mammals, particularly livestock farming. Parasitological methods are not reliable enough for diagnosis of this trematode infection. In the present study, the sera from rabbits immunized with somatic and excretory-secretory antigens of D.dendriticum were assayed with immunoblot technique. Adult worms were collected from infected livers of sheep at a local abattoir. The excretory-secretory product and somatic antigens were prepared through homogenization and incubation of trematodes, respectively. Polyclonal antibodies were raised in rabbits after inoculation of the two antigens with emulsified Freunds adjuvant followed by booster injection. Comparative study between D.dendriticum antigens and those of Fasciola, cysticercus tenuicollis and hydatid cyst fluids antigens was carried out. SDS-PAGE of antigens was performed and for immunoblotting analysis, hyperimmune sera were obtained from immunized rabbits of case and control groups. Immunoblot analysis of rabbits antisera against somatic and excretory-secretory antigens of D.dendriticum were showed six and four protein bands ranging from 25 to more than 170 and 25 to 70 kDa molecular weights, respectively. However cross-reaction with other specific proteins was observed in Fasciola excretory-secretory, hydatid cyst and cysticercus tenuicollis fluid antigens. The results demonstrated that the 25-27 kDa proteins were strongly recognized by both hyperimmune sera and was common in both somatic and excretory-secretory antigens. According to the results of this study performed with two antigens of D.dendriticum shows that the 25-27 kDa polypeptide as immunodominant protein, could be considered for the immunodiagnosis of dicrocoeliasis and it probably induces protective immunity against Dicrocoelium infection.