A chemiluminescent sandwich ELISA enhancement method using a chromium (III) coordination complex.

Enzyme linked immunosorbent assays (ELISAs) are employed for the detection and quantification of antigens from biological sources such as serum and cell culture media. A sandwich ELISA is dependent on the immobilization of a capture antibody, or antibody fragment, and the effective presentation of its antigen binding sites. Immobilization to common microtitre plates relies on non-specific interactions of the capture protein with a surface that may result in unfavourable orientation and conformation, compromising ELISA signal strength and performance. We have developed a wet chemical surface activation method that utilizes a chromium (III) solution to immobilize native, non-tagged, capture antibodies on commercially available microtitre plates. Antibodies captured by this method had increased antigen binding, particularly from dilute antibody solutions, relative to antibodies adsorbed directly to the plate surface. A variety of monoclonal antibodies with complementary antigen systems were used to demonstrate improvements in ELISA signal and reproducibility. The simplicity and versatility of this method should enable ELISA enhancement in assays where chemiluminescence is used as the detection method.

Mechanisms of Antigen Adsorption Onto an Aluminum-Hydroxide Adjuvant Evaluated by High-Throughput Screening.

The adsorption mechanism of antigen on aluminum adjuvant can affect antigen elution at the injection site and hence the immune response. Our aim was to evaluate adsorption onto aluminum hydroxide (AH) by ligand exchange and electrostatic interactions of model proteins and antigens, bovine serum albumin (BSA), ß-casein, ovalbumin (OVA), hepatitis B surface antigen, and tetanus toxin (TT). A high-throughput screening platform was developed to measure adsorption isotherms in the presence of electrolytes and ligand exchange by a fluorescence-spectroscopy method that detects the catalysis of 6,8-difluoro-4-methylumbelliferyl phosphate by free hydroxyl groups on AH. BSA adsorption depended on predominant electrostatic interactions. Ligand exchange contributes to the adsorption of ß-casein, OVA, hepatitis B surface antigen, and TT onto AH. Based on relative surface phosphophilicity and adsorption isotherms in the presence of phosphate and fluoride, the capacities of the proteins to interact with AH by ligand exchange followed the trend: OVA < ß-casein < BSA < TT. This could be explained by both the content of ligands available in the protein structure for ligand exchange and the antigen's molecular weight. The high-throughput screening platform can be used to better understand the contributions of ligand exchange and electrostatic attractions governing the interactions between an antigen adsorbed onto aluminum-containing adjuvant.

Preparation of Quenchbodies by protein transamination reaction.

Quenchbody (Q-body) is an antibody fragment labeled with fluorescent dye(s), which functions as a biosensor via the antigen-dependent removal of the quenching effect on fluorophores. It is based on the principle that the fluorescence of the dye(s) attached to the antibody N-terminal region is quenched primarily by the tryptophan residues present in the variable regions, and this quenching is released when the antigen binds to the antibody, resulting in increased fluorescence intensity. Hence Q-body is utilized in various immunoassays for the rapid and sensitive detection of analytes. So far, Q-bodies have been prepared by using a cell-free translation system or by combining Escherichia coli expression and post-labeling steps. However, the above methods need antibody gene cloning, and are time-consuming. In this study, we report a novel approach to prepare Q-bodies by protein N-terminal transamination. We used the antigen-binding fragment (Fab) of an antibody against the bone-Gla-protein (BGP), a biomarker for bone diseases, which was expressed in E. coli. The purified Fab was treated with Rapoport's salt to convert the amino group at the N-terminus to a ketone group, which in turn was allowed to react with fluorescent probes that have aminooxy or hydrazide groups, to prepare a Q-body. The Q-body prepared by this method could detect the BGP-C7 antigen at concentrations as low as 10 nM. Since the approach can label the protein N-terminus directly, it could be applied for preparing Q-bodies from natural antibodies and for the rapid screening of high-performance Q-bodies.

Efeitos de compostos naturais, sintéticos e da fototerapia antifúngica sobre Candida tropicalis resistente ao fluconazol / Effects of natural compounds, synthetic and herbal medicine on Candida tropicalis antifungal fluconazole-resistant

A candidíase é uma infecção oportunista provocada por diversas espécies de fungos do gênero Candida, frequentemente encontrados integrando a microbiota, da superfície cutânea, no trato gastrointestinal e cavidades mucosas do ser humano desde o seu nascimento. A incidência das infecções fúngicas sistêmicas têm aumentado consideravelmente nas últimas décadas em função do grande número de pacientes com SIDA, a grande quantidade de transplantes e condições crônicas como o câncer, a terapia prolongada com imunossupressores e o uso de agentes corticosteroides. Além disso, a exposição prolongada aos antifúngicos azólicos promove a seleção de patógenos resistentes. No presente estudo avaliou-se a atividade antifúngica do complexo Rutênio-pirocatecol (RPC) frente a um isolado clinico de Candida tropicalis resistente ao fluconazol. A metodologia empregada para os testes de susceptibilidade foi de acordo com o documento M27-A3 do National Committee for Clinical Laboratory Standards (NCCLS, 2008). Esplenócitos de camundongos Balb/c foram obtidos de forma asséptica para avaliar a citotoxicidade do composto para células de mamíferos. O estresse oxidativo promovido pelo composto foi avaliado através da reação ao ácido tiobarbitúrico (TBARS) e ensaios de fluorescência com a sonda diclorodihidrofluoresceína diacetato (DCFH2DA). O Calcofluor White foi empregado para avaliar a integridade da parede celular. A análise ultraestrutural foi realizada através da microscopia eletrônica de varredura e transmissão. Os resultados encontrados para os testes de atividade antifúngica foram analisados através do teste estatístico ANOVA e pós-teste Dunnett. Os resultados encontrados para os testes de atividade antifúngica do RPC mostraram uma Concentração Inibitória de 50% (IC50) de 20,3 μM, enquanto em esplesnócitos a concentração efetiva de 50% foi de 325 μM mostrando um índice de seletividade igual a 16...

Candidiasis is an opportunistic infection caused by several species of fungi of the genus Candida, often found is the microbiota, on the skin, gastrointestinal tract and mucous cavities of the human beings birth. The incidence of systemic fungal infections have increased considerably in recent decades due to the large number of AIDS patients, the large number of transplants and chronic conditions such as cancer, prolonged therapy promotes the selection of resistant pathogen with immunosuppressant and corticosteroid agents. Also prolonged exposure azole antifungals to make them strong candidates for patients resistance. In the present study we evaluated the antifungal activity of Ruthenium-pyrocatechol complex (RPC) against a clinical isolate of Candida tropicalis resistant to fluconazole. The methodology for susceptibility testing was in accordance with the M27-A3 document of there National Committee for Clinical Laboratory Standards (NCCLS, 2008). Splenocytes from Balb/c mice were obtained aseptically to evaluate the cytotoxicity of the compound to mammalian cells. Oxidative stress caused by the compound was assessed by reaction to thiobarbituric acid (TBARS) and fluorescence assays with the probe diclorodihidrofluoresceína diacetate (DCFH2DA). The Calcofluor White was used to evaluate the integrity of the cell wall. The ultrastructural analysis was performed by scanning and transmission electron microscopy. The results for the antifungal activity tests were analyzed using ANOVA and pos-test Dunnett test statistic. The results for the tests of antifungal activity of the RPC showed a 50% inhibitory concentration (IC50) of 20.3 μM while in splenocytes the 50% effective concentration was 325 μM showing a selectivity index of 16...

EDTA glycine acid treatment of red blood cells.

IgG dissociation is necessary when a sample is direct antiglobulin test (DAT) positive and antigen testing using blood grouping serum reactive by the antiglobulin test is performed. Exposure of IgG-coated red blood cells (RCBs) to a low pH of 3.0 with EDTA glycine acid successfully dissociates the IgG, rendering the RCBs DAT negative 82 to 85 percent of the time. The procedure takes one minute or less and leaves RBC antigens intact and able to be typed except for those antigens in the Kell blood group system and for the high-prevalence antigen Er(a).

Highly sensitive nano-porous lattice biosensor based on localized surface plasmon resonance and interference.

We propose a design for a highly sensitive biosensor based on nanostructured anodized aluminum oxide (AAO) substrates. A gold-deposited AAO substrate exhibits both optical interference and localized surface plasmon resonance (LSPR). In our sensor, application of these disparate optical properties overcomes problems of limited sensitivity, selectivity, and dynamic range seen in similar biosensors. We fabricated uniform periodic nanopore lattice AAO templates by two-step anodizing and assessed their suitability for application in biosensors by characterizing the change in optical response on addition of biomolecules to the AAO template. To determine the suitability of such structures for biosensing applications, we immobilized a layer of C-reactive protein (CRP) antibody on a gold coating atop an AAO template. We then applied a CRP antigen (Ag) atop the immobilized antibody (Ab) layer. The shift in reflectance is interpreted as being caused by the change in refractive index with membrane thickness. Our results confirm that our proposed AAO-based biosensor is highly selective toward detection of CRP antigen, and can measure a change in CRP antigen concentration of 1 fg/ml. This method can provide a simple, fast, and sensitive analysis for protein detection in real-time.

Serological detection and immunogold localization of cross-reactive antigens shared by Camellia sinensis and Exobasidium vexans.

AIMS: Pathogenicity of Exobasidium vexans, causal agent of blister blight of tea, was studied in 30 commercially cultivated tea varieties by analysing the antigenic patterns of host and pathogen using immunological techniques. METHODS AND RESULTS: Whole plant inoculation of tea varieties with E. vexans showed that T-78 and T-17/1/54 were most susceptible and most resistant respectively. Antigen preparations from tea varieties, pathogen, nonpathogen (Fusarium oxysporum) and of nonhosts (Glycine max, Leucaena leucocephala and Oryza sativa) were compared by indirect enzyme-linked immunosorbent assay and dot-immunobinding assay using polyclonal antibodies raised against the pathogen, nonpathogen, susceptible and resistant tea varieties. Cross-reactive antigens (CRA) were found among susceptible varieties and E. vexans isolates but not in resistant varieties, nonhosts or nonpathogen. Indirect staining of antibodies using fluorescein isothiocyanate indicated CRA were concentrated mainly around epidermal and mesophyll cells in compatible host (T-78). This was substantiated by ultrastructural studies using gold-labelled antibodies through transmission electron microscopy which showed specific localization in the chloroplasts and host cytoplasm. CONCLUSION: Pathogenicity of E. vexans to different tea varieties is therefore related to the level of antigenic similarity between host and pathogen. SIGNIFICANCE AND IMPACT OF THE STUDY: Immunological methods proved to be valuable in screening commercially cultivated tea varieties against E. vexans.

A quantitative analysis of cedar pollen-specific immunoglobulins in nasal lavage supported the local production of specific IgE, not of specific IgG.

Many studies have proved the relevance of local immune responses, rather than systemic immunity, to the pathogenesis of allergic rhinitis. Indeed, allergen-specific B lymphocyte undergoes class switching to IgE in situ. However, the relative contribution of in situ production to the amount in serum is still ambiguous. Here, a quantitative comparison of the local concentration of allergen-specific IgE with the systemic concentration was explored for the estimation. Among seasonal rhinitis patients, total and Japanese cedar pollen (JCP)-specific IgE, IgA and IgG antibodies were quantified in nasal lavage fluid (NLF) and serum with the time-resolved fluorescence immunosorbent assay. Although the total amounts of IgE and IgG classes in the NLF, which were apparently passive discharge from the mucosal tissue, were smaller and variable, the relative proportions of JCP-specific antibodies could be quantitatively compared between NLF and serum or between subjects. The proportions of specific IgE in the NLF were remarkably higher than in serum (average 13.2-fold) in most subjects, which strongly supported the predominant in situ production of the specific IgE and subsequent dilutions in the systemic circulations. Similar but smaller values were obtained for IgA (average 3.7-fold). In contrast, the specific proportions of IgG in the NLF were surprisingly consistent with serum (average 1.0-fold), suggesting that the specific IgG was mostly produced in the downstream lymphoid organs. The local productions of specific IgE would encourage the topical therapies and the usage of the NLF for the diagnosis of allergic rhinitis.

Levels of common antigens in determining pathogenicity of Curvularia eragrostidis in different tea varieties.

AIMS: Pathogenicity of Curvularia eragrostidis, a foliar fungal pathogen of tea was studied in 24 commercially cultivated tea varieties by analysing the antigenic patterns of host and pathogen with the help of immunoserological techniques. METHODS AND RESULTS: Initial testing by cut shoot inoculation technique followed by whole plant inoculation technique showed that among the varieties tested, TV12 was the most susceptible and TV25 most resistant. Antigen preparations from tea varieties, fungal pathogens (C. eragrostidis and Lasiodiplodia theobromae) and a nonpathogen (Gliocladium virens) were compared by immunodiffusion, immunoelectrophoresis and indirect ELISA to detect common antigens shared by host and pathogen. Common antigens were detected by immunodiffusion and immunoelectrophoresis only among susceptible varieties and the pathogens. Such antigens were not found between the pathogens and the resistant varieties and also between nonpathogens and tea varieties. However, ELISA revealed the presence of low level of common antigens between all combinations. A certain minimum level of antigens was present for compatible host-pathogen interaction. Indirect labelling of antibodies with fluorescein isothiocyanate (FITC) showed that cross-reactive antigens were found to be concentrated mainly in the epidermal cells and also spread throughout the cortical cells. CONCLUSION: Pathogenicity of C. eragrostidis to different varieties of tea was found to be related to the level of common antigens present between host and pathogen. SIGNIFICANCE AND THE IMPACT OF THE STUDY: Indirect ELISA proved to be valuable in screening commercially cultivated varieties of tea for their susceptibility to C. eragrostidis.

Valor diagnóstico de segundas biopsias prostáticas en varones de riesgo. Estudio estratificado por valor de PSA / Diagnostic value of the second prostate biopsies in males of risk. Study stratified by value of PSA

OBJETIVO: Valorar el rendimiento diagnóstico de la segunda biopsia prostática (BP). PACIENTES Y MÉTODOS: Un total de 116 varones con BP previa de benignidad fueron sometidos a 2 o más BP por sextantes guiadas con ultrasonidos (US). Los criterios de inclusión fueron: BP previa sospechosa (PIN), PSA elevado, TR o US sospechoso.RESULTADOS: El tiempo medio transcurrido entre la primera y siguiente biopsia fue de 13 ± 11 meses. Se obtuvieron 35 malignas y 4 premalignas en las segundas biopsias realizadas, lo que da un rendimiento diagnóstico global del 33,6%. Cuando estratificamos por valor de PSA, obtenemos que con PSA 10 ng/ml, de 34,6%. CONCLUSIÓN: La repetición de la biopsia seriada de próstata en pacientes de riesgo, mejora el rendimiento diagnóstico y elimina los falsos negativos de carcinoma. prostático

OBJETIVE: To value the diagnostic yield of the second prostate biopsy (BP). PATIENTS AND METHODS: To 116 males with BP previous to kindliness surrendered to 2 or more BP for sextants guided with ultrasounds (US). The criteria of inclusion were: BP previous suspicious (PIN), high PSA, TR or suspicious US. RESULTS: The average time passed between the first and following biopsy was 13 ± 11 months. 35 malignant and 4 premalignant ones were obtained in the second realized biopsies, which gives a diagnostic global yield of 33.6%. When we stratify for value of PSA, we obtain that with PSA 10 ng/ml of 34,6%. CONCLUSION: The repetition of the serial biopsy of prostate in patients of risk, improves the diagnostic yield and eliminates the false negatives of prostate carcinoma

Studies on experimental iodine allergy: 1. Antigen recognition of guinea pig anti-iodine antibody.

It has generally been thought that iodine allergy is cross-sensitive to various iodine-containing chemicals. However, this concept seems to deviate from the immunological principle that immune recognition is specific. To solve this contradiction, we hypothesize that iodine allergy is an immunological reaction to iodinated autologous proteins produced in vivo by iodination reaction from various iodine-containing chemicals. Antisera to iodine were obtained from guinea pigs immunized subcutaneously with iodine-potassium iodide solution emulsified in complete Freund's adjuvant (CFA). The specificity of guinea pig anti-iodine antiserum was determined by enzyme-linked immunosorbent assay (ELISA) inhibition experiments using microplates coated with iodinated guinea pig serum albumin (I-GSA). Antibody activities were inhibited by I-GSA, diiodo-L-tyrosine, and thyroxine, but not by potassium iodide, monoiodo-L-tyrosine, 3,5,3'-triiodothyronine, monoiodo-L-histidine, or diiodo-L-histidine, or by ionic or non-ionic iodinated contrast media. The results that antigen recognition of anti-iodine antibody is specific to iodinated protein support our hypothesis. While protein iodination usually takes place both at histidine residues as well as at tyrosine residues, only iodinated tyrosine acted as an antigenic determinant and no antibody activities to iodinated histidine were detected in our experimental iodine allergy model.

Genetically modified soybeans: false-positive detection in fermented natural soybean (tempe).

Tempe was prepared using mixtures of natural soybean and genetically modified Roundup Ready (RUR) soybean fermented with natural Rhizopus sp. The amount of RUR soybean was quantified using an ELISA plate test. The RUR signal decreased during fermentation. In the control experiments on fermentation of non-RUR soybean, the tempe gave a false-positive RUR signal. The cross-reacting substance was generated only in non-RUR soybean during fermentation by Rhizopus sp., Rhizopus oligosporus, R. oryzae, Mucor rouxii and Aspergillus awamori.

Pollen from different olive tree cultivars contains varying amounts of the major allergen Ole e 1.

BACKGROUND: Commercial olive pollen from uncertain cultivar origin is the common material used for clinical and biological studies. We aimed to assess the putative heterogeneity of olive cultivars with regard to the presence of the major pollen allergen Ole e 1 and to determine whether these differences have clinical relevance. METHODS: The Ole e 1 content of several cultivars was determined by immunoblotting and ultrastructural immunocytochemistry and compared to that of a commercially available olive pollen extract designed for diagnosis. Reverse transcription-polymerase chain reaction analysis of Ole e 1 transcripts was also performed. Crude protein extracts were used to carry out skin prick tests (SPTs) on 30 allergic patients in order to evaluate the clinical importance of such differences. RESULTS: Ole e 1 was present in all cultivars, although significant quantitative differences were detected. Ole e 1 transcripts positively correlated with the amount of the allergen. Significant variations in the average reactivity of allergic patients to SPTs were observed depending on the cultivar considered. CONCLUSIONS: The presence of the Ole e 1 allergen in all the cultivars suggests that this allergen may play an essential biological role. The expression of the allergen is controlled at the transcriptional level. The significant differences in the Ole e 1 content are likely responsible for the different average reactivity exhibited by patients to the cultivars studied, although the role of other allergens cannot be excluded. Our results suggest that the use of the commercial pollen mixtures currently available may lead to mistakes in allergy diagnosis and to limited success in immunotherapy. Therefore, further standardization is strongly recommended.

Distinct neuronal populations specified to form corticocortical and corticothalamic projections from layer VI of developing cerebral cortex.

Layer VI of the cerebral cortex contains heterogeneous populations of pyramidal neurons whose axons project either cortically or subcortically. It has been shown that a subset of layer VI neurons expressing latexin projects ipsilaterally to other cortical areas but does not contribute to the corticothalamic projections. Taking advantage of the connectional specificity of latexin-expressing neurons, we here determine whether corticocortical and corticothalamic neurons are generated at different times, and at which stage the connectional distinction develops in corticogenesis. Our experimental findings indicate that: (1) thalamic-projecting neurons in layer VI of the rat secondary somatosensory cortex (SII) are born at embryonic day 14 or before while latexin-expressing neurons in the same layer are generated at embryonic day 15 or later; (2) axonal invasion by SII neurons into ipsilateral cortical areas and into the posterior dorsal thalamus mainly takes place early in the postnatal period; (3) latexin-expressing neurons never project toward the dorsal thalamus in normal development; (4) presumptive latexin-expressing neurons in the neonatal SII are able to grow into a cortical slice in vitro, but do not invade a thalamic slice even transiently; (5) thalamic-projecting neurons, on the other hand, fail to simultaneously establish connections with a cortical slice. Taken together, our findings suggest that the time frame in which presumptive corticocortical and corticothalamic neurons are generated differs, and that the two populations are restricted in connectional fate potential by the perinatal period prior to target innervation.

Immunoelectrodes in protein detection: comparison between glassy carbon and a semimetallic Ni/P thin film as binding support. Biological applications.

Though immunoelectrodes can allow direct detection of very low protein amounts (about 0.1 pmol) in vitro and in vivo, they are not yet widely used because they need quality improvement. Based on a few works devoted to the basic electrochemical phenomenon occurring when antibodies are linked onto a solid support and during antigen/antibody complex formation, we have coated two different supports with antibodies: the classical glassy carbon fiber or an epoxy plate covered with an amorphous semimetallic (nickel/phosphorus) thin film obtained by means of an electrochemical deposit. The antibody/antigen complex formation induces direct and/or indirect ionic movements and a current flow through the conductive support toward a very low-noise and high-sensitivity preamplifier stage in an I/V configuration. The proposed electrochemical treatment (hydrophilization), applied to both carbon and Ni/P electrodes, improves antibody binding and reliability of the response to antigens. The Ni/P probes present several advantages when compared to carbon fiber: better conductivity, possibility of surface quality control, and semimetallic nature, making them unbreakable. Several applications were proposed: somatostatin-14 detection with both carbon fiber and Ni/P plate electrodes, and histamine detection in simple and complex fluid media. Dose-response curves and analysis of the results lead us to conclude that the obtained currents are directly related to the quantity of antigen.

Changes in immunostaining of inner ears after antigen challenge into the scala tympani.

To study the mechanisms of immune responses and immune injuries in inner ears, labyrinthitis was induced by inoculation of keyhole limpet hemocyanin (KLH) into the scala tympani of systemically sensitized guinea pigs. Inner ears were then immunostained for KLH, immunoglobulin G (IgG), albumin, connexin26 (Cx26), and sodium-potassium adenosine triphosphate (Na,K-ATPase). Inflammatory cells containing KLH were observed in the scala tympani and in the collecting venule of the spiral modiolar vein (SMV). Spiral ligament, spiral limbus, and blood vessels including the SMV were diffusely positive for IgG and albumin. Immunoreactivity for Cx26 and Na,K-ATPase was decreased compared with the normal ears in the fibrocytes of the spiral ligament. These results suggest that inflammatory cells and blood constituents could extravasate into the cochlea from blood vessels and that fibrocyte damage in the spiral ligament could cause cochlear dysfunction.

Practical environmental modifications for the inhalant allergy patient.

Environmental management of inhalant allergens is an important part of a comprehensive allergy management program and is the most controllable aspect by the patient. The safest and most effective way to eliminate an allergic reaction is to eliminate exposure to the antigen that provokes the response. The basic control principles for all inhalant allergens are to (1) remove the source of the allergen if possible, (2) remove accumulated allergen, and (3) prevent the return of the allergen. This article examines the published evidence for environmental control measures in terms of effectiveness, cost, and ease of implementation.