Entamoeba histolytica: detection of coproantigens by purified antibody in the capture sandwich ELISA

A sensitive and specific Capture Sandwich ELISA (CSE) was developed using polyclonal purified rabbit antibodies against three different axenic strains of Entamoeba histolytica: CSP from Brazil and HM1 - IMSS from Mexico, for the detection of coproantigens in fecal samples. Immunoglobulin G (IgG) againstis E. histolytica was isolated from rabbits immunized with throphozoites whole extract in two stages: affinity chromatography in a column containing E. histolytica antigens bound to Sepharose 4B was followed by another chromatography in Sepharose antibodies 4B-Protein A. A Capture Sandwich ELISA using purified antibodies was able to detect 70ng of amebae protein, showing a sensitivity of 93% and specificity of 94%. The combination of microscopic examination and CSE gave a concordance and discordance of 93.25% and 6.75%, respectively. It was concluded that CSE is highly specific for the detection of coproantigens of E. histolytica in feces of infected patients, is quicker to perform, easier and more sensitive than microscopic examination.

Dietary patterns in pregnancy and birth weight

OBJECTIVE To analyze if dietary patterns during the third gestational trimester are associated with birth weight.METHODS Longitudinal study conducted in the cities of Petropolis and Queimados, Rio de Janeiro (RJ), Southeastern Brazil, between 2007 and 2008. We analyzed data from the first and second follow-up wave of a prospective cohort. Food consumption of 1,298 pregnant women was assessed using a semi-quantitative questionnaire about food frequency. Dietary patterns were obtained by exploratory factor analysis, using the Varimax rotation method. We also applied the multivariate linear regression model to estimate the association between food consumption patterns and birth weight.RESULTS Four patterns of consumption – which explain 36.4% of the variability – were identified and divided as follows: (1) prudent pattern (milk, yogurt, cheese, fruit and fresh-fruit juice, cracker, and chicken/beef/fish/liver), which explained 14.9% of the consumption; (2) traditional pattern, consisting of beans, rice, vegetables, breads, butter/margarine and sugar, which explained 8.8% of the variation in consumption; (3) Western pattern (potato/cassava/yams, macaroni, flour/farofa/grits, pizza/hamburger/deep fried pastries, soft drinks/cool drinks and pork/sausages/egg), which accounts for 6.9% of the variance; and (4) snack pattern (sandwich cookie, salty snacks, chocolate, and chocolate drink mix), which explains 5.7% of the consumption variability. The snack dietary pattern was positively associated with birth weight (β = 56.64; p = 0.04) in pregnant adolescents.CONCLUSIONS For pregnant adolescents, the greater the adherence to snack pattern during pregnancy, the greater the baby’s birth weight.

An ELISA method using serum derived HDAg for the sorological detection of HDV antigens and antibodies

One of the main difficulties related to the detection of the Hepatitis Delta Virus (HDV) antigen and antibody has been the source of the needed HD antigen since HDV containing human and animal livers are very difficult to obtain and since yield is low. This fact prompted us to try to use the serum of patients in the acute phase of HDV infection as a source of HDAg and turn to enzyme immunoassays (EIA) instead of RIA for the sake of easiness and economy in the amount of HDAg needed. The antigen for EIA was obtained from patients during the acute phase of HDV infection and the antibody from patients who have been carriers for many years. For the detection of the antigen, a sandwich type method was employed, whereas for the antibody a competition assay was developed. In order to assess the relative specificity and sensibility of the test, the antibody assay was compared to a commercial RIA (C. RIA, Abbott) and to a non-commercial RIA (NC RIA). Forty-two sera were tested by the two methods and only in two cases discrepant results were obtained. Its is concluded that: 1) sera from patients in the acute and chronic phases of HDV infection can be used as source of both antigen and antibody, for immunoassays; 2) EIA and RIA have comparable relative specificity and sensibility and 3) EIA is easier to perform, cheaper, non-hazardous, has a longer shelf-life and saves scarce HDAg.

High prevalence of hepatitis B virus among female sex workers in Nigeria

Hepatitis B virus (HBV) infection is endemic in Nigeria and constitutes a public health menace. The prevalence of HBV infection in many professional groups has been described in Nigeria. However, literature on HBV infection among female sex workers (FSW) in Nigeria is scanty. FSW in Nigeria are not subjected to a preventive control of HBV infection. This study assesses the extent of spread of HBV among FSW in Nigeria. Seven hundred and twenty (n = 720) FSW (mean age = 26.7 years) were tested for hepatitis B surface antigen (HBsAg) by a double antibody sandwich ELISA method. The overall HBV prevalence among the FSW was 17.1%. FSWs between the ages of 31-35 year (20.5%) and those with 'age-at-first-sex' below 10 years of age (28%) were most affected. This high prevalence of a vaccine preventable disease is unacceptable, therefore, vaccination of this high risk HBV reservoir group should be considered worthwhile.

Circulating Antigens Levels in Different Clinical Forms of the Schistosoma mansoni Infection

With the aim to evaluate the circulating cathodic antigen (CCA) levels in relation to the different clinical phases of Schistosoma sp. infection a sandwich ELISA using monoclonal antibody 5H11 was performed. The sera of three groups of 25 Brazilian patients with acute, intestinal and hepatosplenic forms of S. mansoni infection were tested and compared to a non-infected control group. Patients and control groups were matched for age and sex and the number of eggs per gram of feces was equally distributed among the three patient groups. Sensitivity of 100%, 72%, 52% of the assay was observed for the intestinal, hepatosplenic and acute toxemic groups respectively. The specificity was 100%. Intestinal and hepatosplenic groups presented CCA levels significantly higher in comparison to those observed for acute patients (F-ratio = 2,524; p = 0.000 and F-ratio = 6,314; p = 0.015 respectively). There was no significant difference of CCA serum levels between hepatosplenic and intestinal groups (F-ratio = 1,026; p = 0.316).

Improved Methods for the Diagnosis of African Trypanosomosis

The diagnosis of trypanosomosis in animals with low parasitaemia is hampered by low diagnostic sensitivity of traditional detection methods. An immunodiagnostic method based on a direct sandwich enzyme-linked immunosorbent assay (ELISA), using monoclonal antibodies, has been examined in a number of African laboratories for its suitability for monitoring tsetse control and eradication programmes. Generally, the direct sandwich ELISAs for the detection of trypanosomal antigens in serum samples have proved to be unsatisfactory with respect to diagnostic sensitivity when compared with traditional parasitological methods such as the dark ground/phase contrast buffy-coat technique. Consequently, antigen-detection systems exploiting various other direct, indirect and sandwich ELISA systems and sets of reagents are being developed to improve diagnosis. In addition, an existing indirect ELISA for the detection of antibodies has been improved and is being evaluated in the field in order to detect cattle that are or have been recently infected with trypanosomes. Developments and advantages of other diagnostic techniques, such as dip-stick assay and tests based on the polymerase chain reaction are also considered.

IgG Anti-IgE Autoantibodies in Visceral Leishmaniasis

Procedures for IgG depletion in visceral leishmaniasis (VL) and schistosomiasis sera using Sepharose-protein G beads also deplete IgE. In this study, the presence of IgG anti-IgE autoantibodies in sera from patients with VL (n = 10), and hepatic-intestinal schistosomiasis (n = 10) and from healthy individuals (n = 10) was investigated. A sandwich ELISA using goat IgG anti-human IgE to capture serum IgE and goat anti-human IgG peroxidase conjugate to demonstrate the binding of IgG to the IgE captured was performed. VL sera had higher titers (p < 0.05) of IgG anti-IgE autoantibodies (OD = 2.01 ± 0.43) than sera from healthy individuals (OD = 1.35 ± 0.16) or persons infected with Schistosoma mansoni (OD = 1.34 ± 0.18). The immunoblotting carried out with eluates from Sepharose-protein G beads used to deplete IgG from these sera and goat anti-human IgE peroxidase conjugate, showed a similar pattern of bands, predominating the 75 kDa epsilon-heavy chain and also polypeptides resulting from physiological enzymatic digestion of IgE. A frequent additional band immediately above 75 kDa was observed only in VL sera.

Assessing protein stability of the dimeric DNA-binding domain of E2 human papillomavirus 18 with molecular dynamics

The objective of this study is to understand the structural flexibility and curvature of the E2 protein of human papillomavirus type 18 using molecular dynamics (6 ns). E2 is required for viral DNA replication and its disruption could be an anti-viral strategy. E2 is a dimer, with each monomer folding into a stable open-faced β-sandwich. We calculated the mobility of the E2 dimer and found that it was asymmetric. These different mobilities of E2 monomers suggest that drugs or vaccines could be targeted to the interface between the two monomers.

Schistosoma mansoni antigens alter the cytokine response in vitro during cutaneous leishmaniasis

Schistosoma mansoni infection or associated products are able to down-modulate the type 1 CD4+ T cell inflammatory response characteristic of autoimmune diseases. In this study, we evaluated how S. mansoni antigens altered the immune response that was induced by the soluble Leishmania antigen (SLA) from cutaneous leishmaniasis (CL) patients. Cytokines were measured from the supernatants of peripheral blood mononuclear cell cultures stimulated with SLA. This was performed using the sandwich enzyme linked immunosorbent assay technique in the presence or absence of S. mansoni recombinant antigens Sm29, SmTSP-2 and PIII. The addition of S. mansoni antigens to the cultures resulted in the reduction of interferon gamma (IFN-γ) levels in 37-50% of patients. Although to a lesser extent, the antigens were also able to decrease the production of tumour necrosis factor-alpha (TNF-α). We compared patients that either had or did not have reduction in IFN-γ and TNF-α production in cultures stimulated with SLA in the presence of S. mansoni antigens. We found that there was no significant difference in the levels of interleukin (IL)-10 and IL-5 in response to S. mansoni antigens between the groups. The antigens used in this study down-modulated the in vitro proinflammatory response induced by SLA in a group of CL patients through a currently undefined mechanism.

Derivados organometálicos de estanho(II) - Parte 1. Compostos ciclopentadienílicos

This paper provides a brief review of the chemistry of cyclopentadienyl Sn(II) derivatives which includes the preparation, the molecular structure and reactivity associated with such bis-sandwich tin(II) species. It is compared structural and spectroscopic results and it is also discussed how the nature of the cyclopentadienyl ring bonded to the Sn centre plays an important role in the structural and stability features of the derivatives. Bulk rings such as C5HPr i4- , C5Bz5-, C5Me4SiMe2Bu t- and C5Ph5- render air-stable and parallel ring-bonded compounds.

Sistema de análises em fluxo polivalente para a determinação espectrofotométrica de fármacos

A flow system based on the sandwich technique is proposed for the sequential determination of ascorbic acid, dipyrone, acetylcysteine, captopril and paracetamol. The procedure is based on the reduction of Cu(II) by the analytes followed by the spectrophotometric measurement of the complex of Cu(I) with 2,2'-biquinoline 4,4'-dicarboxylic acid. Linear responses were achieved in the µmol L-1 range, with coefficients of variation better than 1.7%. Sampling rate was estimated as 60 determinations per hour, consuming 230 µg of BQA and generating 2.5 mL of waste per determination. Results for commercial samples agreed with those obtained by procedures recommended by the American and European pharmacopeias at the 95% confidence level.

RT-PCR-ELISA for detection of Apple stem grooving virus in apple trees

A method to detect Apple stem grooving virus (ASGV) based on reverse transcription polymerase chain reaction (RT-PCR) was developed using primers ASGV4F-ASGV4R targeting the viral replicase gene, followed by a sandwich hybridisation, in microtiter plates, for colorimetric detection of the PCR products. The RT-PCR was performed with the Titan™ RT-PCR system, using AMV and diluted crude extracts of apple (Malus domestica) leaf or bark for the first strand synthesis and a mixture of Taq and PWO DNA polymerase for the PCR step. The RT-PCR products is hybridised with both a biotin-labelled capture probe linked to a streptavidin-coated microtiter plate and a digoxigenin (DIG)-labelled detection probe. The complex was detected with an anti-DIG conjugate labelled with alkaline phosphatase. When purified ASGV was added to extracts of plant tissue, as little as 400 fg of the virus was detected with this method. The assay with ASGV4F-ASGV4R primers specifically detected the virus in ASGV-infected apple trees from different origins, whereas no signal was observed with amplification products obtained with primers targeting the coat protein region of the ASGV genome or with primers specific for Apple chlorotic leaf spot virus (ACLSV) and Apple stem pitting virus (ASPV). The technique combines the power of PCR to increase the number of copies of the targeted gene, the specificity of DNA hybridization, and the ease of colorimetric detection and sample handling in microplates.

Self-assembly of supermolecular species directed by hydrogen bonding and aromatic π-π stacking interactions

A new Cu(II) trimers, [Cu3(dcp)2(H2O)8]. 4DMF, with the ligand 3,5-pyrazoledicarboxylic acid monohydrate (H3dcp) has been prepared by solvent method. Its solid-state structure has been characterized by elemental analysis, thermal analysis (TGA and DSC), and single crystal X-ray diffraction. X-ray crystallographic studies reveal that this complex has extended 1-D,2-D and 3-D supramolecular architectures directed by weak interactions (hydrogen bond and aromatic π-π stacking interaction) leading to a sandwich solid-state structure.

Coupling of palmitate to ovalbumin inhibits the induction of oral tolerance

Oral tolerance is a phenomenon that may occur in animals exposed to protein antigens for the first time by the oral route. They become unable to produce immune responses at the levels normally observed when they are immunized parenterally with antigen in the presence of adjuvants. Lipids have been used as adjuvants for both parenteral and oral immunization. In the present study we coupled ovalbumin with palmitate residues by incubating the protein with the N-hydroxysuccinimide palmitate ester and tested the preparation for its ability to induce oral tolerance. This was performed by giving 20 mg of antigen to mice by the oral route 7 days prior to parenteral immunization in the presence of Al(OH)3. Mice were bled one week after receiving a booster that was given 2 weeks after primary immunization. Specific antibodies were detected by ELISA. Despite the fact that the conjugates are as immunogenic as the unmodified protein when parenterally injected in mice, they failed to induce oral tolerance. This discrepancy could be explained by differences in the intestinal absorption of the two forms of the antigen. In fact, when compared to the non-conjugated ovalbumin, a fast and high absorption of the lipid-conjugated form of ovalbumin was observed by "sandwich" ELISA.

Serum cytokine profile in the subclinical form of visceral leishmaniasis

The factors determining the development or not of visceral leishmaniasis (VL) have not been completely identified, but a Leishmania-specific cellular immune response seems to play a fundamental role in the final control of infection. Few studies are available regarding the production of cytokines in the subclinical form of VL, with only the production of IFN-g and TNF-a known. The aim of the present study was to identify immunological markers for the oligosymptomatic or subclinical form of VL. A prospective cohort study was conducted on 784 children aged 0 to 5 years from an endemic area in the State of Maranhão, Brazil, between January 1998 and December 2001. During 30 consecutive months of follow-up, 33 children developed the oligosymptomatic form of the disease and 12 the acute form. During the clinical manifestations, serum cytokine levels were determined in 27 oligosymptomatic children and in nine patients with the acute form using a quantitative sandwich enzyme immunoassay. In the subclinical form of VL, variable levels of IL-2 were detected in 52.3% of the children, IL-12 in 85.2%, IFN-g in 48.1%, IL-10 in 88.9%, and TNF-a in 100.0%, with the last two cytokines showing significantly lower levels than in the acute form. IL-4 was not detected in oligosymptomatic individuals. Multiple discriminant analysis used to determine the profile or combination of cytokines predominating in the subclinical form revealed both a Leishmania resistance (Th1) and susceptibility (Th2) profile. The detection of both Th1 and Th2 cytokine profiles explains the self-limited evolution accompanied by the discrete alterations observed for the subclinical form of VL.