Antibodies against non-structural c100/3 and structural core antigen of hepatitis C virus (HCV) in hemodialysis patients

Two groups of patients undergoing hemodialysis (HD) maintenance were evaluated for their antibody response to non-structural c100/3 protein and structural core protein of hepatitis C virus (HCV). Forty-six patients (Group 1) never presented liver abnormalities during HD treatment, while 52 patients (Group 2) had either current or prior liver enzyme elevations. Prevalence rates of 32.6% and 41.3% were found for anti-c100/3 and anti-HCV core antibodies, respectively, in patients with silent infections (Group 1). The rate of anti-c100/3 in patients of Group 2 was 71.15% and reached 86.5% for anti-HCV core antibodies. The recognition of anti-c100/3 and anti-core antibodies was significantly higher in Group 2 than in Group 1. A line immunoassay composed of structural and non-structural peptides was used as a confirmation assay. HBV infection, measured by the presence of anti-HBc antibodies, was observed in 39.8% of the patients. Six were HBsAg chronic carriers and 13 had naturally acquired anti-HBs antibodies. The duration of HD treatment was correlated with anti-HCV positivity. A high prevalence of 96.7% (Group 2) was found in patients who underwent more than 5 years of treatment. Our results suggest that anti-HCV core ELISA is more accurate for detecting HCV infection than anti-c100/3. Although the risk associated with the duration of HD treatment and blood transfusion was high, additional factors such as a significant non-transfusional spread of HCV seems to play a role as well. The identification of infective patients by more sensitive methods for HCV genome detection should help to control the transmission of HCV in the unit under study.

Malaria serology: performance of six Plasmodium falciparum antigen extracts and of three ways of determining serum titers in IgG and IgM-ELISA

The study evaluated six Plasmodium falciparum antigen extracts to be used in the IgG and IgM enzyme-linked immunosorbent assays (ELISA), for malaria diagnosis and epidemiological studies. Results obtained with eighteen positive and nine negative control sera indicated that there were statistically significant differences among these antigen extracts (Multifactor ANOVA, p< 0.0001). Urea, sodium deoxycholate and Zwittergent antigen extracts performed better than did the three others, their features being very similar for the detection of IgG antibodies. Urea, alkaline and sodium deoxycholate antigen extracts proved to be better than the others for the detection of IgM antibodies. A straight line relationship was found between the optical densities (or their respective log 10) and the log 10 of antibody dilutions, with a very constant slope. Thus serum titers could be determined by direct titration and by two different equations, needing only one serum dilution. For IgM antibody detections, log 10 expression gave results that better correlated with direct titration (95% Bonferroni). For IgG antibody detections, the titer differences were not significant. The reproducibility of antibody titers and antigen batches was also evaluated, giving satisfactory results.

Usefulness of the detection of Toxoplasma gondii antigens in AIDS patients

Toxoplasmic encephalitis (TE) is a mayor cause of central nervous system infection in patients with acquired immunodeficiency syndrome (AIDS). Toxoplasma antibodies were detected in 56 of 79 patients with AIDS (71%), in the present study. Fourteen out of 57 seropositive patients developed TF (25%) and had Toxoplasma gondii antigen detected in their urine. For this, most of them received an effective therapy, with the subsequent disappearance of the symptoms and discontinuity of excretion of the T. gondii antigens. Our results suggest that the monitoring of T. gondii antigen in the urine of AIDS patients may be useful to decide on the proper time for therapy, as well as to avoid the beginning of neurologic signs in these patients.

Dot-ELISA for the detection of IgM and IgG antibodies to Schistosoma mansoni worm and egg antigens, associated with egg excretion by patients

Human schistosomiasis, caused by Schistosoma mansoni, is highly prevalent in Brazil and usually diagnosed by time consuming stool analysis. Serological tests are of limited use in this disease, mainly for epidemiological studies, showing no discrimination between previous contact with the parasite and active infections. In the present study, we standardized and compared a Dot-ELISA for IgM and IgG antibodies against S. mansoni antigens from eggs and worms with a routine IgG and IgM immunofluorescence assay using similar antigens, in the study of sera from 27 patients who had quantified egg stool excretion. The positivity obtained for IgG Dot-ELISA was 96.3% and 88.9% for IgM Dot-ELISA with worm antigen and 92.6% and 90.9% with egg antigen. The IFI presented similar positivities using worm antigen, 92.6% (IgG) and 96.3% (IgM),and lower results with egg antigen, 77.8% (IgG and IgM). The patients studied were divided into two groups according to their egg excretion, with greater positivity of serological tests in higher egg excreters. When comparing the quantitative egg excretion and the serological titers of the patients, we detected a correlation only with IgM Dot-ELISA, with r=0.552 (p=0.0127). These data show that Dot-ELISA can be used for the detection of specific antibodies against S. mansoni in sera from suspected patients or in epidemiological studies and, with further purification of egg antigen and larger samples, IgM Dot-ELISA could be a possible tool for rough estimates of parasite burden in epidemiological studies.

Entamoeba histolytica: fecal antigen capture immunoassay for the diagnosis of enteric amebiasis by a monoclonal antibody

Amebiasis continues to be of epidemiological importance in underdeveloped countries. Clinical diagnosis and epidemiological setting in a region are based on the fecal microscopic identification of cysts or trophozoites. This procedure requires well trained personnel, is laborious, of low sensitivity and frequently yields false-positives results. The present study was designed to develop an immuno-enzymatic fecal 96 kDa antigen capture test (COPROELISA-Eh) more sensitive and specific than microscopic diagnosis of amebiasis. Triplicates of 177 stool samples processed by the formol-ether concentration method, were defined as positive or negative by three experienced microscopic observers. Another aliquot was submitted to the antigen capture test by a monoclonal antibody against a specific membrane antigen of pathogenic strains of Entamoeba histolytica. Optical densities were interpreted as positive when they exceeded the mean value of negative samples plus two standard deviations. COPROELISA-Eh showed a 94.4% sensitivity, 98.3% specificity, 96.2% positive predictive value and 97.6% negative predictive value for the detection of E. histolytica in feces. COPROELISA-Eh is more sensitive and specific than microscopic examination, does not require specially trained personnel and allows the simultaneous processing of a large number of samples.

Immunoperoxidase for the detection for the detection of antibodies in cerebrospinal fluid in neurocysticercosis: use of Cysticercus cellulosae and Cysticercus longicollis particles fixed on microscopy slides

The ORF strain of Cysticercus longicollis represents an important model for the study of heterologous antigens in the immunodiagnosis of neurocysticercosis (NC). The immunoperoxidase (IP) technique was standardized using a particulate antigen suspension of Cysticercus longicollis (Cl) and Cysticercus cellulosae (Cc). Cerebrospinal fluid (CSF) samples were incubated on the antigen fixed to microscopy slides; the conjugate employed was anti-IgG-peroxidase and the enzymatic reaction was started by covering the slides with chromogen solution (diaminobenzidine/H2O2). After washing with distilled water, the slide was stained with 2% malachite green in water. Of the CSF samples from 21 patients with NC, 19 (90.5%) were positive, whereas the 8 CSF samples from the control group (100%) were negative. The results of the IP-Cl test applied to 127 CSF samples from patients with suspected NC showed 28.3% reactivity as opposed to 29.1 % for the IP-Cc test. The agreement index for the IP test (Cl x Cc) was 94.2%, with no significant difference between the two antigens.

Dot-ELISA for the detection of anti-Cysticercus cellulosae antibodies in cerebrospinal fluid using a new solid phase (resin-treated polyester fabric) and Cysticercus longicollis antigens

A dot-ELISA was developed for the detection of antibodies in CSF in the immunologic diagnosis of human neurocysticercosis, using antigen extracts of the membrane and scolex of Cysticercus cellulosae (M+S-Cc) and, alternately, membrane (M) and vesicular fluid (VF) of Cysticercus longicollis (Cl) covalently bound to a new solid phase consisting of polyester fabric treated with N-methylol-acrylamide resin (dot-RT). The test was performed at room temperature, with reduced incubation times and with no need for special care in the manipulation of the support. The sensitivity rates obtained were 95.1% for antigen Cc and 97.6% for antigen Cl. Specificity was 90.6% when Cc was used, and 96.9% and 100% when M-Cl and VF-Cl were used, respectively. No significant differences in titer were observed between tests carried out with homologous and heterologous antigens. The low cost and easy execution of the dot-RT test using antigen extracts of Cysticercus longicollis indicate the test for use in the immunodiagnosis of human neurocysticercosis.

AN ELISA SUITABLE FOR THE DETECTION OF RABIES VIRUS ANTIBODIES IN SERUM SAMPLES FROM HUMAN VACCINATED WITH EITHER CELL-CULTURE VACCINE OR SUCKLING-MOUSE-BRAIN VACCINE

An indirect ELISA for determination of post-vaccination rabies antibody was applied. Purified rabies virus was used as antigen to coat plates, and staphylococcal protein A linked with horseradish peroxidase was used for detecting IgG antibody in human sera. Sera from humans, vaccinated with cell-culture vaccine or suckling-mouse-brain vaccine, were examined. ELISA results were compared to those obtained from the virus neutralization test. The mean and standard deviation of OD were determined for 126 negative sera (pre-vaccination) and for 73 sera from vaccinated persons showing antibody titers lower than 0.5 IU/ml. Results were defined as ELISA -positive, -negative or -doubtful. Establishment of a doubtful region reduced the number of sera otherwise classified as positive (false-positive sera). In this way, the sensitivity, specificity and agreement values were respectively 87.5%, 92.4% and 88.5%. No significant differences were observed in these values when the group vaccinated with cell-culture vaccine and the group vaccinated with suckling-mouse-brain vaccine were compared. It was shown that much of the disagreement between the values obtained by neutralization test and ELISA occurred in sera obtained at the beginning of the immunization process, and was probably due to the presence of IgM in the serum samples, detected only by the former test. This ELISA method can be used as a screening test in rabies laboratories regardless of the kind of vaccine used for immunization.

Novel Prostate Specific Antigen plastic antibody designed withcharged binding sites for an improved protein binding and itsapplication in a biosensor of potentiometric transduction

This work shows that the synthesis of protein plastic antibodies tailored with selected charged monomersaround the binding site enhances protein binding. These charged receptor sites are placed over a neutralpolymeric matrix, thus inducing a suitable orientation the protein reception to its site. This is confirmed bypreparing control materials with neutral monomers and also with non-imprinted template. This concepthas been applied here to Prostate Specific Antigen (PSA), the protein of choice for screening prostate can-cer throughout the population, with serum levels >10 ng/mL pointing out a high probability of associatedcancer.Protein Imprinted Materials with charged binding sites (C/PIM) have been produced by surfaceimprinting over graphene layers to which the protein was first covalently attached. Vinylben-zyl(trimethylammonium chloride) and vinyl benzoate were introduced as charged monomers labellingthe binding site and were allowed to self-organize around the protein. The subsequent polymerizationwas made by radical polymerization of vinylbenzene. Neutral PIM (N/PIM) prepared without orientedcharges and non imprinted materials (NIM) obtained without template were used as controls.These materials were used to develop simple and inexpensive potentiometric sensor for PSA. Theywere included as ionophores in plasticized PVC membranes, and tested over electrodes of solid or liq-uid conductive contacts, made of conductive carbon over a syringe or of inner reference solution overmicropipette tips. The electrodes with charged monomers showed a more stable and sensitive response,with an average slope of -44.2 mV/decade and a detection limit of 5.8 × 10−11mol/L (2 ng/mL). The cor-responding non-imprinted sensors showed lower sensitivity, with average slopes of -24.8 mV/decade.The best sensors were successfully applied to the analysis of serum, with recoveries ranging from 96.9to 106.1% and relative errors of 6.8%.

Disposable immunosensor using a simple method for oriented antibody immobilization for label-free real-time detection of an oxidative stress biomarker implicated in cancer diseases

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 methodology favored Ab/Ag affinity and immunodetection of the antigen. 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 charge transfer resistance across the electrochemical set-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.

Immunoprecipitation techniques and Elisa in the detection of anti-Fonsecaea pedrosoi antibodies in chromoblastomycosis

Chromoblastomycosis (CBM) is a chronic subcutaneous infection caused by several dematiaceous fungi. The most commonly etiological agent found in Brazil is Fonsecaea pedrosoi, which appears as thick walled, brownish colored cells with transverse and longitudinal division in the lesions, called "muriform cells". This disease is found worldwide but countries like Madagascar and Brazil have highest incidence. Diagnosis is made by clinical, direct and histopathologic examination and culture of specimens. Serological tests have been used to identify specific antibodies against Fonsecaea pedrosoi antigens, as well as immunotechniques have been used for CBM serological identification and diagnosis. In the present study double immunodiffusion (DID), counterimmunoelectrophoresis (CIE) and immunoenzymatic test (ELISA) have been used to evaluate humoral immune response in patients with CBM caused by F. pedrosoi. Metabolic antigen was used for immunoprecipitation tests (DID and CIE) while somatic antigen for ELISA. Our results demonstrated 53% sensitivity and 96% specificity for DID, while CIE presented 68% sensitivity and 90.5% specificity. ELISA demonstrated 78% sensibility and 83% specificity. Serological tests can be a useful tool to study different aspects of CBM, such as helping differential diagnosis, when culture of the pathogenic agent is impossible.

A novel antibody-like material for breast cancer antigen CA15-3, used to track breast cancer by potentiometric transduction

This work presents the development of a low cost sensor device for the diagnosis of breast cancer in point-of-care, made with new synthetic biomimetic materials inside plasticized poly(vinyl chloride), PVC, membranes, for subsequent potentiometric detection. This concept was applied to target a conventional biomarker in breast cancer: Breast Cancer Antigen (CA15-3). The new biomimetic material was obtained by molecularly-imprinted technology. In this, a plastic antibody was obtained by polymerizing around the biomarker that acted as an obstacle to the growth of the polymeric matrix. The imprinted polymer was specifically synthetized by electropolymerization on an FTO conductive glass, by using cyclic voltammetry, including 40 cycles within -0.2 and 1.0 V. The reaction used for the polymerization included monomer (pyrrol, 5.0×10-3 mol/L) and protein (CA15-3, 100U/mL), all prepared in phosphate buffer saline (PBS), with a pH of 7.2 and 1% of ethylene glycol. The biomarker was removed from the imprinted sites by proteolytic action of proteinase K. The biomimetic material was employed in the construction of potentiometric sensors and tested with regard to its affinity and selectivity for binding CA15-3, by checking the analytical performance of the obtained electrodes. For this purpose, the biomimetic material was dispersed in plasticized PVC membranes, including or not a lipophilic ionic additive, and applied on a solid conductive support of graphite. The analytical behaviour was evaluated in buffer and in synthetic serum, with regard to linear range, limit of detection, repeatability, and reproducibility. This antibody-like material was tested in synthetic serum, and good results were obtained. The best devices were able to detect 5 times less CA15-3 than that required in clinical use. Selectivity assays were also performed, showing that the various serum components did not interfere with this biomarker. Overall, the potentiometric-based methods showed several advantages compared to other methods reported in the literature. The analytical process was simple, providing fast responses for a reduced amount of analyte, with low cost and feasible miniaturization. It also allowed the detection of a wide range of concentrations, diminishing the required efforts in previous sample pre-treating stages.

Sensitivity of an immunoenzymatic test for detection of ant-L. brasiliensis antibodies compared to other tests used for the diagnosis of American cutaneous leishmaniasis

The diagnosis of American cutaneous leishmaniasis (ACL) is frequently based on clinical and epidemiological data associated with the results of laboratory tests. Some laboratory methods are currently being applied for the diagnosis of ACL, among them the indirect immunofluorescence reaction (IIFR), the Montenegro skin test (MST), histopathological examination, and the polymerase chain reaction (PCR). The performance of these methods varies in a considerable proportion of patients. After the standardization of an immunoenzymatic test (ELISA) for the detection of IgG in the serum of patients with ACL using a crude Leishmania braziliensis antigen, the results obtained were compared to those of other tests routinely used for the diagnosis. The tests revealed the following sensitivity, when analyzed separately: 85% for ELISA IgG, 81% for PCR, 64.4% for MST, 58.1% for IIFR, and 34% for the presence of parasites in the biopsy. ELISA was positive in 75% of patients with ACL presenting a negative MST, in 84.8% of ACL patients with negative skin or mucous biopsies for the presence of the parasite, and in 100% of cases with a negative PCR. Thus, ELISA presented a higher sensitivity than the other tests and was useful as a complementary method for the diagnosis of ACL.

Detection of the peanut allergen Ara h 6 in foodstuffs using a voltammetric biosensing approach

A voltammetric biosensor for Ara h 6 (a peanut allergen) detection in food samples was developed. Gold nanoparticle-modified screen-printed carbon electrodes were used to develop a sandwich-type immunoassay using two-monoclonal antibodies. The antibody-antigen interaction was detected through the electrochemical detection of enzymatically deposited silver. The immunosensor presented a linear range between 1 and 100 ng/ml, as well as high precision (inter-day RSD ≤9.8 %) and accuracy (recoveries ≥96.7 %). The detection and quantification limits were 0.27 and 0.88 ng/ml, respectively. It was possible to detect small levels of Ara h 6 in complex food matrices.

Detection of Cysticercus antigens and antibodies in cerbrospinal fluid of patients with chronic meningitis

Chronic meningitism is a less frequent manifestation of neurocysticercosis caused by Taenia solium cysticerci. In the present study we used Co-agglutination (Co-A), a simple and rapid slide agglutination test to detect specific Cysticercus antigen in the 67 cerebrospinal fluid (CSF) samples from patients with chronic meningitis of unknown etiology. The results were compared with that of ELISA for detection of antibodies. Among these samples four (5.97%) were positive for Cysticercus antigen by Co-A test and six (8.95%) were positive for antibodies by ELISA. Two samples were positive by both Co-A and ELISA, two were positive only by Co-A and four were positive only by ELISA. In the present study, although Cysticercus antigen and antibodies were present in CSF samples from eight (11.94%) patients, we cannot affirm that all the cases of chronic meningitis are due to cysticercosis, but for any case of chronic meningitis of unknown origin, it would be useful to consider the possibility of cysticercal meningitis.