detection of doth fasciolu gigantica circulating antigen and coproantigdn by monoctonal-based sandwich ELISA in diagnosis of human fascioliasis

Human fascioliasis is another important health challenge in Egypt. lip till now and due to many factors. its diagnosis is a problematic issue. In many eases, a considerable damage of the hepatic tissue often occurs before a proper diagnosis could he done. This in turn necessitates finding a reliable. easily applicable and locally affordable diagnostic test to overcome the diagnostic difficulties that handicap prevention and control efforts. The detection of E/S antigens in stool specimens [coproantigens] and in sera of infected humans using a MAb-based sandwich ELISA system. Stool and serum samples were collected from 35 fascioliasis patients having clinical and parasitological evidences of infection, 20 patients harboring other parasites [Schistosoma mansoni and haematobium Wuchereria bancuofti and hydatid cysts] and 25 healthy subjects. A pair of monoelonal antibodies [MAbs: 9F/10B and 5F/6H], raised against both FascioIa gigantica excretory-Secretor [E/S] products and crude antigens respectively, were employed in sandwich ELISA. The lower detection limit of E/S coproantigen assay corresponded to 15 ng/ml, while that of E/S antigen assay in serum corresponded to 50ng/ml. The anti-F gigantica MAb-based sandwich ELISA for antigen detection in collected sera showed 77% sensitivity and 100% specificity with 87% diagnostic accuracy. Coproantigen detection in stool samples showed 94% sensitivity and 100% specificity with 97% diagnostic accuracy. A positive correlation was detected between antigen level in stool samples and its level in corresponding serum samples. This study showed that the use of anti-F gigantica MAb-based sandwich ELISA was more ssensitive for antigen detection in stool samples of fascioliasms patients. Than its detection in their corresponding serum samples, providing a simple, reliable, non-invasive diagnostic method for active human infection

detection of Giardia lamblia coproantigen as a sensitive immunodiagnostic method for human giardiasis

This study was designed to develop a sandwich ELISA for detection of G. Iambiia antigens in stool and sera of giardiasis patients as a better diagnostic alternative to routine parasitological methods. Anti-G. Iamblia antibodies were produced by immunization of rabbit with G. lamblia antigen obtained from cultured trophozoites. Raised antibodies were then employed in sandwich ELISA for detection of G. lamblia antigen in collected sera and stool samples. In this study sera and stool samples from 80 G. lamblia infected patients, 71 patients infected with other parasites [Entamoeba histolytica, Schistosoma mansoni and Fasciola hepatica] and 30 uninfected individuals were tested by sandwich-ELISA for detection of G. lamblia antigen. The sensitivity of coproantigen assay reached 98.8% for detection of Giardia antigens in stool and 87.5% for detection of Giardia antigen in sera of giardiasis patients. The specificity of the assay was 94.1% for stool samples and 91% for sera of negative controls and patients harboring other parasites collectively. A positive correlation between age of patients and the antigen levels in both sera and stool samples of G. lamblia infected patients was observed. The sensitivity of antigen detection assay was directly related to the intensity of infection. The positivity rate for detection of coproantigen in stool was compared to the number of cysts in stool. Patients passing < 8 cysts showed false negativity in stool samples [one patient] compared to 100% positivity in patients passing > 50 cysts of stool [79 patients]. Moreover, a positive correlation was found between coproantigen level in stool and number of cysts in stool of G. lamblia infected patients [r=0.887, p< 0.001]. In conclusion, our data demonstrated that the employment of rabbit anti-G. lamblia IgG antibodies in sandwich ELISA for the detection of G. lamblia coproantigen in stool provided a sensitive and specific tool for immunodiagnosis of G. lamblia infection

Study of kinetics of circulating schistosome antigens in tissues and sera of infected mice using anti-schistosoma mansoni soluble egg antigen monoclonal antibody

In the present study, the kinetics of uptake and deposition of Schistosoma mansoni antigens in liver, spleen, kidney and intestine of C57BL/6 mice infected with 100 Schistosoma mansoni cercariae as well as their levels in sera have been investigated during the course of infection [12 weeks]. The presence of antigen was demonstrated by indirect immunofluorescence using IgM anti-soluble egg antigen monoclonal antibody [anti-SEA MAb]. Immunofluorescence reactivity was evident in both renal and spleen tissues 3 weeks post-infection [p.i.], in Kupffer cells of liver 4 weeks p.i. and in intestinal mucosa [5 weeks p.i.]. Maximal immunofluorescence staining was reached during the patent phase [5-9 weeks p.i.]. During the chronic stage of infection [9-12 weeks pi.], diminution of immunofluorescent intensity was evident in liver tissue, while it remained constant in other studied organs. Circulating schistosome antigen [CSA] level in mice sera was determined using a sandwich ELISA with a MAb for both antigen capture and detecting antibody. CSA was demonstrated in mice sera [one week p.i.] reaching its peak at 6 weeks p.i. and remained at a detectable level until the end of the study [12th week p.i.]

Expression of intercellular adhesion molecule ICAM-1 and cytokeratin as Potential markcrs of disease activity in the Egyptian pattern of chronic hepatitis C

Chronic hepatitis C [ch.HCV] and schistosomal hepatic fibrosis or both as a mixed hepatic lesion [MHL] are among the most common causes of endemic chronic hepatic disease in Egypt. Adhesion molecules especially ICAM-1 play an important role in inflammatory and immunological responses of chronic liver disease. Cytokeratin 18 [CK-18], although normally expressed in hepatic tissue, yet it is altered during chronic inflammatory hepatic lesions. This work was planned to study ICAM-1 as expressed in hepatic tissue in the different grades of the disease activity, in relation to its circulating levels in patients sera, and to evaluate the level of CK-18 expression in relation to the different grades of chronic inflammation and stages of fibrosis in the examined liver biopsies. The material for this study comprised 33 patients [17 ch.HCV and 16 MHL]. Seven cases, that proved to have nearly normal serological data and insignificant histopathological hepatic features, were considered as controls. All patients were assessed for HCV serological markers as well as serum levels of soluble ICAM-1 [sICAM-1] by the Enzyme Linked Immunosorbent. Assay [ELISA]. Liver needle biopsy specimens were processed and assessed for the histopathological grade of the disease activity and stage of fibrosis of the hepatic lesion. Tissue expression of ICAM-1 and CK-18 was detected using immunohistochemical techniques. Our results revealed significantly higher levels of serum sICAM-1 in both ch.HCV and MHL groups compared to controls [p<0.01]. Meanwhile, higher levels of sICAM-1 were recorded in the MHL cases relative to ch.HCV cases. ICAM-1 expression was not detected in any of the control cases, while it was positively expressed in all ch. HCV and MHL cases, with a higher score recorded in the later group [P>0.001 compared to the control group]. ICAM-1 expression was detected mainly within the sinusoidal cells [endothelial and Kupffer cells], hepatocytes, mononuclear inflammatory cells and vascular endothehail cells in portal areas. On classifying patients according to their grades of active inflammation and stages of fibrosis, higher scores of ICAM-1 expression within the hepatic tissue were recorded in cases with more active inflammatory grades and higher fibrotic stages. On the other hand, serum of ICAM-1 levels though were significantly elevated in patients with higher grades of inflammatory activity yet, they were decreased in patients with higher fibrotic stages. CK18 expression was mainly detected within hepatocytes of the periportal areas [in a combined membranous and intracytoplasmic pattern], as well as within the bile ducts epithelium in the portal areas. Over expression of CKI 8 was detected in both the ch.HCV and MHL groups [p<0.001 relative to controls], but the expression scores were higher in the MHL group. From these results we may conclude that MHL is a more aggressive and active chronic inflammatory hepatic disease than ch.HCV alone. Also, serum levels of sICAM-1 as well as hepatic expression of both ICAM-1 and CK-18 are related to the degree of disease activity, which may point out to the possibility of using serum sICAM-1 levels as well as the expression scores of hepalic ICAM-1 and CK-18 as efficient tools for monitoring the disease activity in ch. HCV and MHL patients. While ICAM-1 expression in tissue could be used as indicator for the stage of fibrosis in those patients also recommend that both ICAM-1 in serum andhepatic tissue could be used for monitoring the effect of therapy on the studied pattern of chronic hepatitis C

Detection of circulating schistosome antigens in serum and urine of schistosomiasis patients and assessment of cure by monoclonal antibody

From a panel of monoclonal antibodies [MAb], an IgM monoclonal antibody [7F1/6B] reactive with repetitive epitopes on S. Mansoni soluble egg antigen was selected. This MAb was employed both as antigen capture and detection antibody in a sandwich ELISA and had a detection limit <1 ng S. mansoni SEA/mi. Serum and urine samples were collected from rural students who had S. mansoni [169 subjects] or mixed S. mansoni and S. hematobium [64 subjects] infections. Samples were collected before and at 4, 8 and 12 weeks after praziquantel therapy. Circulating schistosome antigens [CSA] were demonstrated in 90% of sera and 97% of urine samples of S. mansoni group and in 91% of sera and 100% of urine samples of mixed infection group. All sera from 29 uninfected individuals, 30 patients with other parasites and 70% of 55 S. hematobium-infected subjects were negative in this assay. CSA level in serum and urine samples correlated positively with the number of S. mansoni eggs/g stool in both groups. A significant reduction in CSA level was observed in serum and urine samples after praziquantel therapy. By 12 weeks post-treatments, negativity was 98% in sera and 97% in urine of S. mansoni-infected group and 98% in sera and 91% in urine of mixed infection group. The data demonstrated that the use of MAb 7F1/6B for the detection of CSA provides a sensitive method of immunodiagnosis of schistosomiasis and monitoring of cure