Significance of isolated hepatitis B core antibody in blood donors from São Paulo

The clinical significance of isolated anti-HBc is still a challenge. To elucidate the real importance of this finding in our blood donors, an investigation algorithm was tested. One hundred and twelve isolated anti-HBc seropositive blood donors underwent clinical evaluation and retesting of HBV markers. Those who presented repeatedly reactive isolated anti-HBc, received a single dose of hepatitis B recombinant vaccine to verify anti-HBs early response. A HBV-DNA determination by PCR was done for those who did not test positive to anti-HBs after vaccine. The level of anti-HBc was recorded as a ratio of the sample-to-cut-off values (S:C ratio) in 57 candidates at donation. Comparing true and false-positive anti-HBc results, the different S:C ratios of them were statistically significant and when less than 2, implying in a false-positive result probability over 80%. A high percent of false-positive results (16.07%) was verified after anti-HBc retesting. HBV immunity was characterized in 49.11%, either by anti-HBs detection in retesting (15.18%), or after a single dose HBV vaccination (33.93%). HBV-DNA was negative in all tested donors. In conclusion, this algorithm was useful to clarify the meaning of isolated anti-HBc in most of our blood donors.

Human polyclonal anti-hepatitis B surface antigen immunoglobulin reduces the frequency of acute rejection after liver transplantation for chronic hepatitis B

BACKGROUND: Use of polyclonal anti-hepatitis B surface antigen immunoglobulin (HBIg) has been shown to reduce hepatitis B virus (HBV) recurrence after liver transplantation (LT) and to decrease the frequency of acute cellular rejection (ACR). However, the protective role of HBIg against ACR remains controversial, since HBV infection has been also associated with a lower incidence of ACR. AIM: To assess the relationship between HBIg immunoprophylaxis and the incidence of rejection after LT. METHODS: 260 patients (158 males, 43 ± 14 years old) submitted to LT were retrospectively evaluated and divided into three groups, according to the presence of HBsAg and the use of HBIg. Group I was comprised of HBsAg-positive patients (n = 12) that received HBIg for more than 6 months. Group II was comprised of HBsAg-positive patients that historically have not received HBIg or have been treated irregularly for less than 3 months (n = 10). Group III was composed of 238 HBsAg-negative subjects that have not received HBIg. RESULTS: HBIg-treated patients (group I) had significantly less ACR episodes, when compared to group II and III. No differences between groups II and III were observed. CONCLUSIONS: Long-term HBIg administration contributes independently to reduce the number of ACR episodes after LT.

Evaluation of hemostasis disorders and anticardiolipin antibody in patients with severe leptospirosis

A prospective study was designed to evaluate disorders of hemostasis and levels of anticardiolipin antibodies (ACL) in 30 patients with severe leptospirosis and acute renal failure (ARF) (ARF was defined as serum creatinine > or = 1.5 mg/dL). The patients had been admitted to the Walter Cantídio University Hospital, São José Infectious Diseases Hospital and General Hospital of Fortaleza, Ceará, from August 1999 to July 2001. They all were male, with a mean age of 32 ± 14 years and with clinical and laboratory diagnoses of ARF leptospirosis. The time elapsed between onset of symptoms and the first hemorrhagic manifestation was 9 ± 4 days. Bleeding was observed in 86% of the patients. Laboratory tests showed significantly high levels of urea (181 ±95 mg/dl), fibrinogen, (515 ± 220 mg/dl), prothrombin time (13.3 ± 0.9 seconds) and low platelet counts (69 ± 65x10³/mm³) on admission. There was no elevation in activated partial thromboplastin time or thrombin time. Levels of IgM and IgG ACL concentrations were significantly increased (p < 0.05) in leptospirosis patients when compared to control patients (28.5 ± 32.4 vs. 11.5 ± 7.9MPL U/ml and 36.7 ± 36.1 vs. 6.5 ± 2.5 GPL U/ml), respectively. Vasculitis, thrombocytopenia and uremia should be considered important factors for the pathogenesis of hemorrhagic disturbances and the main cause of death in severe leptospirosis.

Identification of toxocara canis antigens by Western blot in experimentally infected rabbits

Toxocariasis is a frequent helminthiasis that can cause visceral and ocular damage in humans specially in children. The identification of specific antigens of Toxocara canis is important in order to develop better diagnostic techniques. Ten rabbits were infected orally with a dose of 5000 Toxocara canis embryonated eggs. Rabbits were bled periodically and an ELISA assay was performed to determine levels of specific Toxocara IgG antibodies. ELISA detected antibodies at day 15 after infection. Western blot (WB) assay was performed using excretory/secretory antigens (E/S) of T. canis second stage larvae. Different antigen concentrations were evaluated: 150, 200, 250 and 300 µg/mL. The concentration of 250 µg/mL was retained for analysis. Rabbit sera were diluted 1:100. Secondary antibody was used at a dilution of 1:1000. Results of WB indicated that in the first month after infection specific antibodies against the 200 KDa, 116 KDa, 92 KDa and 35 KDa antigens were detected; antibodies against the 92 KDa, 80 KDa, 66 KDa, 45 KDa, 31 KDa and 28 KDa antigens appeared later. All positive sera in the ELISA test were also positive in WB. Two antigen bands, 92 KDa and 35 KDa, were identified since the beginning and throughout the course of infection. These antigens merit further evaluation as candidates for use in diagnosis.

Haemoglobin smart plastic antibody material tailored with charged binding sites on silica nanoparticles: its application as an ionophore in potentiometric transduction

This work uses surface imprinting to design a novel smart plastic antibodymaterial (SPAM) for Haemoglobin (Hb). Charged binding sites are described here for the first time to tailor plastic antibody nanostructures for a large size protein such as Hb. Its application to design small, portable and low cost potentiometric devices is presented. The SPAM material was obtained by linking Hb to silica nanoparticles and allowing its ionic interaction with charged vinyl monomers. A neutral polymeric matrix was created around these and the imprinted protein removed. Additional materials were designed in parallel acting as a control: a neutral imprinted material (NSPAM), obtained by removing the charged monomers from the procedure, and the Non-Imprinted (NI) versions of SPAM and NSPAM by removing the template. SEM analysis confirmed the surface modification of the silica nanoparticles. All materials were mixed with PVC/plasticizer and applied as selective membranes in potentiometric transduction. Electromotive force (emf) variations were detected only for selective membranes having a lipophilic anionic additive in the membrane. The presence of Hb inside these membranes was evident and confirmed by FTIR, optical microscopy and Raman spectroscopy. The best performance was found for SPAM-based selective membranes with an anionic lipophilic additive, at pH 5. The limits of detection were 43.8 mg mL 1 and linear responses were obtained down to 83.8 mg mL 1, with an average cationic slope of +40 mV per decade. Good selectivity was also observed against other coexisting biomolecules. The analytical application was conducted successfully, showing accurate and precise results.

Disposable immunosensor with simple antibody orientation for label-free real-time detection of a cancer biomarker

This work proposes a novel approach for a suitable orientation of antibodies (Ab) on an immunosensing platform, applied here to the determination of 8-hydroxy-2’-deoxyguanosine (8OHdG), a biomarker of oxidative stress that has been associated to chronic diseases, such as cancer. The Anti-8OHdG was bound to an amine modified gold support through its Fc region after activation of its carboxylic functions. Non-oriented approaches of Ab binding to the platform were tested in parallel, in order to show that the presented proposal favored Ab/Ag affinity. The immunosensor design was evaluated by Quartz-Crystal microbalance with Dissipation, Atomic Force Microscopy, Electrochemical Impedance Spectroscopy (EIS) and Square-Wave Voltammetry. EIS was also a suitable technique to follow the analytical behavior of the device against 8OHdG. The affinity binding between 8OHdG and the antibody immobilized in the gold modified platform increased the charged transfer resistance across the electrochemical sep-up. The observed behavior was linear from 0.02 to 7.0 ng/mL of 8OHdG concentrations. The interference from Glucose, Urea and Creatinine was found negligible. An attempt of application to synthetic samples was also successfully conducted. Overall, the presented approach enabled the production of suitably oriented Abs over a gold platform by means of a much simpler process than other oriented-Ab binding approaches described in the literature, as far as we know, and was successful in terms of analytical features and sample application.

Sol-Gel Chemistry in Biosensing Devices of Electrical Transduction: Application to CEA Cancer Biomarker

Sol-gel chemistry allows the immobilization of organic molecules of biological origin on suibtable solid supports, permitting their integration into biosensing devices widening the possibility of local applications. The present work is an application of this principle, where the link between electrical receptor platform and the antibody acting as biorecognition element is made by sol-gel chemistry. The immunosensor design was targeted for carcinoembryonic antigen (CEA), an important biomarker for screening the colorectal cancer, by electrochemical techniques, namely electrochemical impedance spectroscopy (EIS) and square wave voltammetry (SVW). The device displayed linear behavior to CEA in EIS and in SWV assays ranging from 0.50 to 1.5ng/mL, and 0.25 to 1.5ng/mL, respectively. The corresponding detection limits were 0.42 and 0.043 ng/mL. Raman spectroscopy was used to characterize the surface modifications on the conductive platform (FTO glass). Overall, simple sol-gel chemistry was effective at the biosensing design and the presented approach can be a potential method for screening CEA in point-of-care, due to the simplicity of fabrication, short response time and low cost. - See more at: http://www.eurekaselect.com/127192/article#sthash.m1AWhINx.dpuf

Backside-surface imprinting as a new strategy to generate specific plastic antibody materials

A backside protein-surface imprinting process is presented herein as a novel way to generate specific synthetic antibody materials. The template is covalently bonded to a carboxylated-PVC supporting film previously cast on gold, let to interact with charged monomers and surrounded next by another thick polymer. This polymer is then covalently attached to a transducing element and the backside of this structure (supporting film plus template) is removed as a regular “tape”. The new sensing layer is exposed after the full template removal, showing a high density of re-binding positions, as evidenced by SEM. To ensure that the templates have been efficiently removed, this re-binding layer was cleaned further with a proteolytic enzyme and solution washout. The final material was named MAPS, as in the back-side reading of SPAM, because it acts as a back-side imprinting of this recent approach. It was able to generate, for the first time, a specific response to a complex biomolecule from a synthetic material. Non-imprinted materials (NIMs) were also produced as blank and were used as a control of the imprinting process. All chemical modifications were followed by electrochemical techniques. This was done on a supporting film and transducing element of both MAPS and NIM. Only the MAPS-based device responded to oxLDL and the sensing layer was insensitive to other serum proteins, such as myoglobin and haemoglobin. Linear behaviour between log(C, μg mL−1) versus charged tranfer resistance (RCT, Ω) was observed by electrochemical impedance spectroscopy (EIS). Calibrations made in Fetal Calf Serum (FCS) were linear from 2.5 to 12.5 μg mL−1 (RCT = 946.12 × log C + 1590.7) with an R-squared of 0.9966. Overall, these were promising results towards the design of materials acting close to the natural antibodies and applied to practical use of clinical interest.

Smart Plastic Antibody Material (SPAM) tailored on disposable screen printed electrodes for protein recognition: application to Myoglobin detection

This work introduces two major changes to the conventional protocol for designing plastic antibodies: (i) the imprinted sites were created with charged monomers while the surrounding environment was tailored using neutral material; and (ii) the protein was removed from its imprinted site by means of a protease, aiming at preserving the polymeric network of the plastic antibody. To our knowledge, these approaches were never presented before and the resulting material was named here as smart plastic antibody material (SPAM). As proof of concept, SPAM was tailored on top of disposable gold-screen printed electrodes (Au-SPE), following a bottom-up approach, for targeting myoglobin (Myo) in a point-of-care context. The existence of imprinted sites was checked by comparing a SPAM modified surface to a negative control, consisting of similar material where the template was omitted from the procedure and called non-imprinted materials (NIMs). All stages of the creation of the SPAM and NIM on the Au layer were followed by both electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). AFM imaging was also performed to characterize the topography of the surface. There are two major reasons supporting the fact that plastic antibodies were effectively designed by the above approach: (i) they were visualized for the first time by AFM, being present only in the SPAM network; and (ii) only the SPAM material was able to rebind to the target protein and produce a linear electrical response against EIS and square wave voltammetry (SWV) assays, with NIMs showing a similar-to-random behavior. The SPAM/Au-SPE devices displayed linear responses to Myo in EIS and SWV assays down to 3.5 μg/mL and 0.58 μg/mL, respectively, with detection limits of 1.5 and 0.28 μg/mL. SPAM materials also showed negligible interference from troponin T (TnT), bovine serum albumin (BSA) and urea under SWV assays, showing promising results for point-of-care applications when applied to spiked biological fluids.

Heterologous antigen extract in ELISA for the detection of human IgE anti-Strongyloides stercoralis

Strongyloides ratti larval extract was used for the standardization of ELISA to detect genus-specific IgE in human strongyloidiasis. Forty serum samples from monoinfected patients shedding S. stercoralis larvae (Group I), 40 from patients with other intestinal parasites (Group II), and 40 from copronegative healthy subjects (Group III) were analyzed. Genus-specific IgE levels (ELISA Index: EI) were significantly higher in the group I (EI = 1.43) than groups II (EI = 0.70) and III (EI = 0.71), showing positivity rates of 55%, 2.5% and 0%, respectively. Similarly, sera from copropositive patients had significantly higher levels of total IgE (866 IU/mL) as compared to those from group II (302 IU/mL) and III (143 IU/mL). A significant positive correlation was found between levels of Strongyloides specific-IgE and total IgE in sera from patients with strongyloidiasis. In conclusion, S. ratti heterologous extract showed to be a useful tool for detecting genus-specific IgE by ELISA, contributing for a better characterization of the immune response profile in human strongyloidiasis.

Synthesis of an antibody-like material for the detection of Albumin

6th Graduate Student Symposium on Molecular Imprinting

Sol-gel biomimetic material designed to target CEA cancer biomarker

1st ASPIC International Congress

A disposable glass-based immunosensor for monitoring the cancer biomarker CEA in urine

NanoPT 2014 International Conference, in Portugal, on February 12-14. Poster presentation based on topic Nanobio/Nanomedicine

Immunogenicity of three recombinant hepatitis B vaccines administered to students in three doses containing half the antigen amount routinely used for adult vaccination

We evaluated the immunogenicity of three recombinant hepatitis B vaccines, one Brazilian (Butang, Instituto Butantan) and two Korean vaccines (Euvax-B, LG Chemical Ltd. and Hepavax-Gene, Greencross Vaccine Corp.), administered intramuscularly to students aged 17 to 19 years in three 10-µg doses (corresponding to half the amount of antigen routinely used for adult vaccination) at intervals of one month between the first and second dose, and of four months between the second and third dose. A total of 316 students non-reactive for any serological marker of hepatitis B virus infection were vaccinated: 77 (24.4%) with the Butang vaccine, 71 (22.5%) with Euvax-B, 85 (26.9%) with Hepavax-Gene and, for comparison, 83 (26.2%) with Engerix-B (GlaxoSmithKline), whose efficacy in young adults at the dose used here has been confirmed in previous studies. Similar seroconversion rates (anti-HBs > 10 mIU/mL about one month after application of the third dose) were obtained for the Butang, Euvax-B, Hepavax-Gene and Engerix-B vaccines (96.2%, 98.6%, 96.5% and 97.6%, respectively). The frequency of good responders (anti-HBs > 100 mIU/mL) was also similar among students receiving the four vaccines (85.8%, 91.6%, 89.4% and 89.2%, respectively). The geometric mean titers (GMT) of anti-HBs about one month after the third dose obtained with these vaccines were 727.78 ± 6.46 mIU/mL, 2009.09 ± 7.16 mIU/mL, 1729.82 ± 8.85 mIU/mL and 2070.14 ± 11.69 mIU/mL, respectively. The GMT of anti-HBs induced by the Euvax-B and Engerix-B vaccines were higher than those obtained with the Butang vaccine (p < 0.05); this difference was not significant when comparing the other vaccines two-by-two. No spontaneous adverse effects attributable to the application of any dose of the four vaccines were reported.

H antigen presence in an Ascaris lumbricoides extract

Previous experiences have demonstrated the same ABO system and P system antigens in A. lumbricoides extracts and in their hosts. The aim was to show the behavior of an A. lumbricoides extract from an O Group patient against monoclonal antibodies of different specificities. Agglutination Inhibition Tests were carried out facing the extract against monoclonal antibodies (anti A 2.23; anti B 2.54; anti B 2.62; anti AB 2.39 and anti H 2.72) in optimal concentrations. Suspensions of O Group fresh red cells were used as revealing system. The extract only inhibited the agglutination of anti H 2.72 with O erythrocytes. The semiquantitative Agglutination Inhibition Test of the extract was made against two series of anti H 2.72 dilutions by using O Group fresh red cells as revealing system. A difference of five dilutions between the titers of both series has been observed and the presence of H Antigen in the extract has been significantly confirmed. The fact that the extract did not inhibit the agglutination against anti A, anti B and anti AB has corroborated our previous observations about absence of A and B epitopes in A. lumbricoides extracts from O Group patients. The results of the preceding studies and this experience have demonstrated the membrane glycoconjugated importance in A. lumbricoides. They could be involved in molecular mimicry for this parasite.