Identification, cloning and expression of core protein gene of hepatitis C genotype 4A

Although Egypt has very high rates of HCV, not much is known about genotype 4a which is the most predominant genotype in Egypt. In the present study, core [C_ED43] gene of the Egyptian strain ED43 of HCV genotype 4a was first analyzed using PC/GENE program. Computer analysis of Core region of the isolate ED43 revealed that the Egyptian genotype 4a is different from those isolated from Europe and Central Africa and that it is closely related to genotype Ib. The DNA region coding for the Core was amplified from HCV_ED43/PUC19 plasmid. The PCR product was then cloned and expressed in E. coli M15 using pQE-30 vector. The expression and antigenicity of the core [Core_4a] protein in E. coli was confirmed by SDS-PAGE and western blotting, which will make it useful for developing assay systems for detecting anti-HCV antibodies and HCV antigen, respectively and which might help in the design of a vaccine against the Egyptian genotype 4a

Characterization, cloning and expression of NS3 protein gene of hepatitis c genotype 4A

Clone and express NS3 gene of the Egyptian strain ED43 of HCV genotype 4a in E. coli was studied. Gene and protein sequences of NS3 gene of the ED43 strain were first analyzed using PC/GENE program. DNA homology was 89% the homologies and that of the protein was 78.8% indicating that NS3 gene of the genotype 4a is different from those isolated from other strains. DNA of NS3 region of genotype 4a was amplified from HCV_ED43/PUC19 plasmid. The PCR product was cloned and expressed in E. coli M15 using pQE-30 vector. Fusion protein containing the peptides coded by HCV NS3 [NS3_4a] was expressed by Escherichia coli. The specific HCV antigenicity of the NS3_4a fusion protein was identified by western blotting

Application and evaluation of the immunofiltration assay in the diagnosis of human schistosomiasis

In a trial to reach fast, simple and efficient diagnostic assay for schistosomiasis, Dot Dye Immunofiltration assay using protein A conjugated gold colloid was applied and evaluated in comparison to Dipstick assay and Enzyme Immunoelectrotransfer Blot Assay [EITB], Both soluble egg antigen [SEA] of Schistosorna mansoni and Schistosoma haematobium were used as capture antigens in each assay. Dot Dye Immunofiltration assay, Dipstick assay and EITB were found to be more efficient by using SEA of S. mansoni, than by using SEA of S. haeinatobium. Using SEA of S. mansoni, Dipstick assay was found to be the most efficient [82.2%] among all performed assays followed by Dot Immunofiltration assay and EITB [80.5%] for each. On the other hand Dot Dye Immunofiltration assay was found to be simpler and faster than Dipstick assay and EITB

Cloning and sequence analysis of genes encoding Fasciola hepatica immunodominant antigens

An immuno-screening of fusion protein produced in lambda plaques was done using IPAb. Two clones were isolated and identified as Fh lambda 400 and Fh lambda 800. Both clones were sequenced, Fh lambda 400 contained 305 translated bases encoding 11.509 kDa and designated as SFh12, while Fh lambda 800 contained 311 translated bases encoding protein of 11.058 kDa designated as SFh11. The DNA sequence homology search of Fh lambda 400 revealed a relatively high degree of identity with F. hepatica amoebapore-like protein mRNA. However, Fh lambda 800 revealed the highest similarity with F. hepatica tegumental antigen [T1] mRNA. The protein homology search of SFh12 gave 100% identity with amoebapore-like protein [APLP], while SFh11 showed 75% identity with F. hepatica tegumental antigen [TA]. The biochemical analysis of the deduced proteins was identified. In addition, the predicted T- and B- cell epitopes were also evaluated. However, a histological localization of the identified antigens was achieved using the IPAb in an indirect immunofluorescent antibody assay [FA]. The results revealed that the IPAb labeled the outer glycocalyx in a characteristic pattern, which proved that the identified antigens were tegumental in origin and the infected fasciola subjects induced antibodies directed mainly against tegumental components

Cellular and humoral immune responses to recombinant Smp17.7 Schistosoma mansoni antigen

To determine the immunological responses to S. Mansoni antigen rSmp 17.7, a total of 184 subjects [174 patients from a schistosomiasis endemic area and 10 controls] was used. Proliferation, cytokine profile in culture supernatants from antigen-stimulated peripheral blood mononuclear cells and specific IgG1, IgG3, IgG4, IgA, IgM and IgE levels were assessed. The highest stimulation index to rSmp 17.7 was detected in S. mansoni patients. The evaluation of the cytokine profile [IL-2, IL-4 and IFN-gamma] in response to this antigen showed a significant increase as demonstrated by enzyme linked immunosorbent assay [ELISA]. Specifically, IFN-gamma and IL-2 were significantly detected by flow cytometry. IgG1 and IgM were significantly increased. These results highlighted the importance of rSmp 17.7 as a candidate vaccine for schistosomiasis. The results facilitate to understand the mechanism of schistosome vaccine efficacy

Epidemiology and immunodiagnosis of schistosomiasis haematobium in low endemic area in Egypt

A survey was performed in Behbeet Village in Giza Governorate including 370 individuals [172 males and 198 females] representing 10% of the house holds. Clinical, stool, urine and serological tests accompanied by a questionnaire were applied to all participants to find out the prevalence, intensity of infection of S. Hematobium, underlying sociodemographic factors, morbidity indicators and the awareness and treatment status among the infected population. It was revealed that the overall prevalence of S. Hematobium based on the detection of eggs in urine was 18.1%, while the prevalence of antibodies to S. Hematobium species specific microsomal antigen was 57.6% detected by enzyme-linked immuno-transfer blot [EITB]. The highest age specific prevalence and intensity of infection were detected among school children in the early teenage. Males were at a higher risk of contracting infection than females with a sex ratio of 2.5: 1. Occupational and recreational water contact were significantly more frequent among the egg positives than the negative ones. Present history of hematuria and microhematuria detected by reagent strips had the strongest association with S. Hematobium infection followed by leucocyturia and dysuria

Application of schistosome microsomal antigens in immunodiagnostic assays

The target of this study is to assess and evaluate the use of S. mansoni and S. haematobium microsomal antigens [MAMA and HAMA] in FAST-ELISA and EITB for immunodiagnosis of schistosomiasis and to compare their findings with those of the dipstick [DS] assay as a practical screening test for field sites. Screening of the S.mansoni-infected sera [Kafr El-Sheikh site] with FAST-MAMA and EITB-MAMA, showed sensitivity levels of 98.3% and 100%, respectively. Meanwhile, screening of these sera with DS assay, resulted in the same sensitivity as the EITB assay. Screening of S. haematobium-infected sera [Giza site] with FAS-TMAMA showed a low sensitivity [80.5%] as compared to the FAST-HAMA where the sensitivity was much better, reaching 95%. About 97% of sera from parasitologically S. haematobium positive subjects sera, which were FAST-HAMA positive, recognized Gp23 in both EITB and DS assays. All participants from Giza site were parasitologically negative for S. mansoni. Sixty one percent of them were positive in FAST-MAMA and 45%-23% of them recognized Gp30 in EITB and DS assays, respectively. This lower percentage in DS assay could be a much more accurate estimation of S.mansoni antibodies in such S. haematobium endemic site. On comparing the EITB findings with that of the DS assay, the results showed that all negative sera in MAMA and HAMA blots were also negative in the DS assay. Ninety six percent of the positive HAMA blots were also positive in DS assay indicating nearly the same sensitivities of the two assays for Gp23 reactivity. About fifty two percent of the mixed infection sera, which weakly recognized Gp30 in MAMA blots, were totally negative in DS assay thus indicating the purity and specificity of the DS-assay antigen is much higher than that used in EITB. DS assay is sensitive, specific, reproducible and highly effective in screening patients with either schistosome infections. Also, DS assay is economic, rapid and lends itself for use under field conditions

Immunoaffinity isolation of Schistosoma mansoni species specific molecules using monoclonal antibodies

Traditional diagnosis of schistosomiasis by stool and urine examination techniques is both insensitive and labor intensive; many efforts are directed towards the development of alternate sensitive and specific assays for diagnosis of schistosomiasis. With the development of hybridoma technology, monoclonal antibodies [MoAb] with high specificity to diagnostic antigens could be produced. In the present study, a species-specific fraction namely, Gp30 prepared from S. mansoni microsomal antigen [MAMA] was purified by a simple, easy and cheap apparatus [491-prep cell] [Bio-Rad]. Balb/c mice were immunized with the isolated glycoprotein [Gp] to produce a panel of MoAbs, which can be used to immunoaffinity purify target antigen. Among the produced panel, 6B3-1B432 MoAb of IgG2a isotope was able to detect 10-20 ng of Gp30 antigen in ELISA [O.D[650] = 1.985] and dot blot. In immunoblotting, 6B3-1B432 recognized only one band in the MAMA antigens, namely Gp30. This MoAb was purified with protein G Suprose using FPLC and PD-10 column and consequently used to immunoaffinity purify target antigen. The specificity and sensitivity of the immunoaffinity purified Gp30 [IP Gp30] was determined before using it in the dipstick assay. Our results indicated the absolute specificity of IP Gp30 and sensitivity results, 98%, was comparable to that obtained by the electroeluted GP30. The simplicity of the purification and the efficacy of the dipstick assay may render this diagnostic method for widespread use in the field of schistosomiasis