Academic literature on the topic 'Immunochemistry – Technique'

Although the histology of Dupuytren’s tissue is well-documented, conventional stains do not distinguish between the different types of collagen which biochemistry and immunochemistry suggest are present. Dupuytren’s specimens [nodules ( n = 26), cords ( n = 15) and dermofasciectomies ( n = 6)] were stained with haematoxylin and eosin, Van Gieson’s, Mallory’s, Masson’s, and Herovici’s picropolychrome stain, and examined for both cellularity and collagen staining characteristics. All stains illustrated the marked cellularity of the nodules, contrasting with a paucity of cells within the cords. The first four stains demonstrated uniformity of the collagen staining within the tissues. Herovici’s picropolychrome, however, showed distinct staining patterns for the dermis, nodules and cords, with both purple/red and blue areas. Other studies suggest that those fibres stained purple/red and blue are types I and III collagens respectively. These findings may shed further light on the tissue of origin of Dupuytren’s disease.

Abstract The growing volume of literature concerning immunoassay analysis for trace levels of agrochemicals and other low molecular weight contaminants in various matrixes is indicative of the tremendous interest in and utility of this analytical technique. Most immunoassay methods described in the literature analyze compounds directly, for example, a herbicide in water, or involve solvent exchange of an organic sample extract or dilution of an aqueous-based sample to minimize the matrix effect. As immunoassay for small molecules becomes widely accepted and applied, new challenges involving more complex chemicals in more difficult matrixes arise. The integration of “classical” analytical chemistry with immunochemistry can provide new techniques and approaches useful in discovering the movement, mode of action, and ultimate impact of certain chemicals on humans and the environment.

Abstract: Immunochemistry with anti-vimentin, anti-lysozyme, anti-alpha 1 antitrypsin, anti-CD3 and anti-CD79α antibodies has been used for characterization of primary cell culture in the transmissible venereal tumor (TVT). Samples for primary cell culture and immunohistochemistry assays were taken from eight dogs with cytological and clinical diagnosis of TVT. To validate the immunochemical results in the primary cell culture of TVT, a chromosome count was performed. For the statistical analysis, the Mann-Whitney test with p<0.05 was used. TVT tissues and culture cells showed intense anti-vimentin immunoreactivity, lightly to moderate immunoreactivity for anti-lysozyme, and mild for anti-alpha-antitrypsin. No marking was achieved for CD3 and CD79α. All culture cells showed chromosomes variable number of 56 to 68. This is the first report on the use of immunocytochemical characterization in cell culture of TVT. Significant statistic difference between immunochemistry in tissue and culture cell was not established, what suggests that the use of this technique may provide greater certainty for the confirmation of tumors in the primary culture. This fact is particularly important because in vitro culture of tumor tissues has been increasingly used to provide quick access to drug efficacy and presents relevant information to identify potential response to anticancer medicine; so it is possible to understand the behavior of the tumor.

Abstract Aims: Cutaneous lymphomas (CL) represent a unique group of lymphomas and are the second most frequent extranodal lymphomas. CL probably is the result of a multifactor and multistep process. Firstly an inflammatory reactive process is developing secondary to various chronic stimuli that may be genetic, environmental, infectious and immunologic. The consequences are the negative effects in cell regulation and deregulation of oncogenes and/or suppressor genes later promotes transition from pre-neoplastic conditions to neoplasia. Detailed molecular expression analysis of cutaneous T-cell lymphoma is not available. Some oncogenic alterations have been demonstrated, such as functional inactivation of the Fas receptor, constitutive activity of STAT 3, or the inactivation of the p16 gene via deletion or promoter hypermethylation. Objective: To study the expression of p16, c-myc, Ki67, bcl-2, CD1a, CD123, TCL1, CD68, STAT 3, STAT 4 and MAL1 in an homogeneous group of. CL diagnosed in a general Hospital in order to know more about the expression of these markers in this neoplastic process. Design: retrospective, analytical study in 30 CL diagnosed consecutively as Mycosis Fungoide (14 early and 16 advanced stages) since January 2005 to December 2006. A macrotissue array technique by incubation with monoclonal antibodies and immunohistochemistry protocol to visualize by avidin-biotin peroxidases was applied in all paraffin histological samples. A semi quantitative method was applied in order to categorize the positive cells. Results: In 28 samples (92%) we have observed p16 positive over expression, the two negative samples corresponding to early affectation. In 14 samples from early stages (48%) c-myc was negative. In 29 samples (96%) CD1a was positive in dermal and epidermal layer. CD123 (interleukine 3 receptor) was negative in 16 samples (52%) and TCL1 was positive in 12 cases (39%) in small cells with oval and cleaved nucleus and scarce cytoplasm. STAT3 and STAT4 were positive in 29 cases (97%), Over expression of MAL1 (48%) was observed in advanced patients with aggressive CL. Conclusions: In our study an over expression of p16 is observed in the majority of cases and high c-myc expression in advanced stages. Probably dendritic plasmacytic cells are involved in the pathogenesis of skin lymphoproliferative disorders with cutaneous T cell infiltration. MAL1 could be a predictor of aggressive CL but it is necessary more studies and more cases in order to confirm it.

Cell staining is a necessary and useful technique for visualizing cell morphology and structure under a microscope. This technique has been used in many areas such as cytology, hematology, oncology, histology, virology, serology, microbiology, cell biology, and immunochemistry. One of the key pieces of equipment for preparing a slide for cell staining is cytology centrifuge (cytocentrifuge) such as cytospin. However, many small labs do not have this expensive equipment and its accessory, cytoclips (also expensive relatively), which makes them difficult to study cell cytology. Here we present an alternative method for preparing a slide and cell staining in the absence of a cytocentrifuge (and cytoclips). This method is based on the principle that a regular cell centrifuge can be used to concentrate cells harvested from cell culture and then deposit the concentrated cell suspension to a slide evenly by using a cell spreader, followed by cell staining. The method presented is simple, rapid, economic, and efficient. This method may also avoid a possible change in cell morphology induced by cytocentrifuge.

Three-dimensional (3D) in vitro microphysiological cultures, such as spheroids and organoids, promise increased patient relevance and therapeutic predictivity compared with reductionist cell monolayers. However, high-throughput characterization techniques for 3D models are currently limited to simplistic live/dead assays. By sectioning and staining in vitro microtissues, researchers can examine their structure; detect DNA, RNA, and protein targets; and visualize them at the level of single cells. The morphological examination and immunochemistry staining for in vitro cultures has historically been done in a laborious manner involving testing one set of cultures at a time. We have developed a technology to rapidly screen spheroid phenotype and protein expression by arranging 66 spheroids in a gel array for paraffin embedding, sectioning, and immunohistochemsitry. The process is quick, mostly automatable, and uses 11 times less reagents than conventional techniques. Here we showcase the capabilities of the technique in an array made up of 11 different cell lines stained in conventional hematoxylin and eosin (H&E) staining, as well as immunohistochemistry staining for estrogen (ER), progesterone (PR), and human epidermal growth factor (Her-2) receptors, and TP53. This new methodology can be used in optimizing stem cell–based models of disease and development, for tissue engineering, safety screening, and efficacy screens in cancer research.

A large number of studies have been conducted to explore the mechanism of Back-Shu and Front-Mu points. While several lines of evidence addressed the acupuncture information of Shu acupoints and Mu acupoints gathering in the spinal cord, whether the convergence is extended to the high centre still remains unclear. The study selected gastric Mu points (RN12) and gastric Shu points (BL21) regulating gastric motility and its central neural mechanisms as the breakthrough point, using the technique of immunochemistry, nuclei lesion, electrophysiology, and nerve transection. Here, we report that gastric motility regulation of gastric Shu and Mu acupoints and their synergistic effect and the signals induced by electroacupuncture (EA) stimulation of acupoints RN12 and RN12 gather in the dorsal vagal complex (DVC), increasing the levels of gastrointestinal hormones in the DVC to regulate gastric motility through the vagus. In sum, our data demonstrate an important role of DVC and vagus in the regulation of gastric motility by EA at gastric Shu and Mu points.

The indiscriminate and/or improper use of veterinary drugs is the cause of potential adverse health effects due to the risk of entering into the food chain and the appearance of residues in food products of animal origin. Moreover, in the case of antibiotics, this fact has been identified as one of the causes for the appearance of antimicrobial resistance mechanisms in bacteria causing human diseases, which is the cause of a big concern within the health authorities, distinct governmental agencies and the society in general. There is an increased need to ensure safety and quality of the foodstuff and customers have also started to become more exigent forcing the industry to introduce consumer worries in their daily procedures, in terms of getting more natural, ecological and healthy products with a clear traceable origin of the ingredients. Nowadays, food control is performed on centralized laboratories that in most cases use very reliable procedures, but involving expensive equipment, specialized personnel and time consuming sample treatment procedures. In order to drastically improve this situation, the European Commission (EC) and the member state agencies are strongly supporting research activities aimed to increase the efficiency of the actual analytical methods. An strategy is to take advantage of the latest bio-micro-nanotechnological advances and of the complementary skills of multidisciplinary research teams to develop more rapid, sensitive and specific methodologies capable of detecting a wide variety of chemical, biological or any other health risk associated to the agrofood industry and along to the whole food chain. This thesis describes the research performed in respect to the possibility to develop improved alternatives for food residue analysis based on the combination of selective receptors and novel micro/nanotechnologies. Particularly, the final objective of this thesis was addressed to the development of a multiplexed sensor device to detect inappropriate farm practices and or the contamination of food products by antibiotic residues, mainly milk. In this respect, production of selective receptors with a broad recognition of the most important antibiotic families used in the veterinary field has been one of the most important aims of this research work. Thus, we report here the investigation made regarding development of synthetic receptors for sulfonamide antibiotics (SAs), particularly molecular imprinted polymers (MIPs), and their evaluation of a rational approach. Moreover, production of generic (or class-selective) antibodies for SAs and tetracycline antibiotics (TCs) has also been approached through the rational design and synthesis of appropriate haptens. Evaluation of the features of the antibodies produced has been accomplished through the development of microplate-based ELISAs (enzymelinked immunosorbent assays). The results show that although it has been possible to obtain class-selective antibodies for SAs (up to 11 congeners can be detected), the approach followed for the case of the TCs afforded antibodies with a high selectivity versus TCs possessing an alkyl/alkene group at position 6 of the C-ring, but lacking the hydroxyl group at this position. The necessary protocols to apply these immunochemical procedures to the analysis of milk and hair samples have been established showing that determination of these antibiotics according to the EC regulations was possible for the case of milk. For the case of hair, no regulations do exist at the moment. However this matrix holds great value regarding their potential use to trace inappropriate treatments through the life of the farm animals. The immunoreagents and immunochemical procedures established have been implemented on an optical sensor device developed by the Centre Suisse for Electronics and Microelectronics Inc. (CSEM, Neuchâtel - Switzerland). This device is based on the evanescent wave principle using a particular technological design based on waveguide grating couplers and it is very sensitive to the changes in the refractive index produced at the surface of the transducer. Moreover, the chip developed by CSEM has 24 sensing pads which allow multiple measurements with the same transducer. As a consequence of this collaboration it has been possible to develop a biosensor device able to detect SA residues in milk samples in compliance with the EC regulations. Further investigation, has led to the development of a multiplexed biosensor device by combining immunoreagents (SAs and fluoroquinolone antibiotics (FQs)) produced at the Applied Molecular Receptors group (AMRg) of the CSIC, with bioreceptors (ß lactam antibiotics (BLs) and TCs) provided by UNISENSOR S.A. (Liege - Belgium). The results obtained were very good. About 34 antibiotics from four different antibiotic families can be detected in milk samples following all the EC regulations with the device developed. Before, a multianalyte microplate-based ELISA had been developed combining the same bioreagents to evaluate performance and to establish the most appropriate immunochemical procedures. The immunochemical methods developed within this thesis, including immunoassays and immunosensors, have been preliminarily evaluated in collaboration with the Nestlé Research Centre (NRC), at Lausanne in Switzerland, in order to demonstrate performance in real milk samples.
El uso indiscriminado y/o inadecuado de medicamentos veterinarios es la causa de posibles efectos adversos para la salud por el riesgo que entren en la cadena alimentaria y la aparición de sus residuos en los productos alimenticios de origen animal. Además, en el caso de los antibióticos, este hecho ha sido identificado como una de las causas de la aparición de mecanismos de resistencia a los antimicrobianos en las bacterias que causan enfermedades humanas, lo cual es causa de una gran preocupación en las autoridades sanitarias, distintos organismos gubernamentales y de la sociedad en general. Actualmente, existe una creciente necesidad de garantizar la seguridad y calidad de los productos alimenticios y los consumidores también han comenzado a ser más exigentes obligando a la industria a introducir sus preocupaciones en sus procedimientos, en términos de conseguir los productos más naturales, ecológicos y saludables con un origen de los ingredientes rastreable. Hoy en día, el control de alimentos se realiza en laboratorios centralizados que en la mayoría de casos utilizan procedimientos muy fiables, pero necesitan equipos caros, personal especializado y elevados tiempos de tratamiento de muestra. Con el fin de mejorar drásticamente esta situación, la Comisión Europea (CE) y los organismos de los Estados Miembros apoyan firmemente actividades de investigación orientadas a aumentar la eficiencia de los métodos de análisis actuales. Una estrategia es tomar ventaja de los últimos avances en las biomicro-nanotecnologías y de las capacidades complementarias de los equipos de investigación multidisciplinarios para desarrollar metodologías más rápidas, sensibles y específicas capaces de detectar una amplia variedad de sustancias químicas, biológicas o cualquier otro riesgo para la salud asociado a la industria agroalimentaria, a lo largo de toda la cadena alimentaria. En esta tesis se describe la investigación llevada a cabo en relación a la posibilidad de desarrollar alternativas más adecuadas para el análisis de residuos de alimentos basados en la combinación de receptores selectivos y las nuevas micro/nanotecnologías. En particular, el objetivo final de esta tesis fue dirigido hacia el desarrollo de un dispositivo sensor multiplexado para detectar prácticas agrícolas inadecuadas y/o la contaminación de alimentos, principalmente la leche, por residuos de antibióticos. A este respecto, la producción de receptores selectivos con un amplio reconocimiento de las familias de antibióticos más importantes que se utilizan en el sector veterinario ha sido uno de los objetivos más importantes de este trabajo de investigación. Así, lo constata la investigación realizada en relación con el desarrollo de receptores sintéticos para antibióticos de sulfonamida (SA), polímeros de huella molecular (MIPs), y su evaluación como enfoque racional para otros contaminantes. Además, la producción de anticuerpos genéricos (o selectivos de clase) para SAs y antibióticos de tetraciclina (TC) también ha sido abordada mediante el diseño racional y síntesis de haptenos. La evaluación de las características de los anticuerpos producidos se ha logrado mediante el desarrollo de ELISAs (Enzyme-Linked ImmunoSorbent Assay - ensayo por inmunoabsorción ligado a enzimas) en microplacas. Los resultados muestran que a pesar de que ha sido posible obtener anticuerpos selectivos de clase para las SA (hasta 11 congéneres pueden ser detectados), el enfoque adoptado para el caso de los anticuerpos TCs ofrece una alta selectividad frente a TC que poseen un grupo alquilo/alqueno en la posición 6 del anillo C, pero que carecen del grupo hidroxilo en esta posición. Los protocolos necesarios para aplicar estos procedimientos inmunoquímicos para el análisis de muestras de leche y el cabello han sido establecidos mostrando que fue posible la determinación de estos antibióticos en leche de acuerdo con la normativa de la CE. Para el caso del cabello, no existen regulaciones por el momento. Sin embargo, esta matriz tiene un gran valor en cuanto a su uso potencial para seguir tratamientos inadecuados a lo largo de la vida de los animales de granja. Los inmunoreactivos y procedimientos inmunoquímicos establecidos se han implementado en un dispositivo sensor óptico desarrollado por el Centre Suisse de Electrónica y Microelectrónica Inc. (CSEM, Neuchâtel - Suiza). Este dispositivo se basa en el principio de onda evanescente usando un diseño tecnológico particular basado en acopladores de rejilla de guía de ondas que es muy sensible a los cambios en el índice de refracción producida en la superficie del transductor. Además, el chip desarrollado por CSEM tiene 24 zonas de detección que permiten múltiples mediciones con el mismo transductor. Como consecuencia de esta colaboración ha sido posible desarrollar un dispositivo biosensor capaz de detectar residuos de SA en muestras de leche en el cumplimiento de la normativa CE. Investigaciones posteriores, han llevado al desarrollo de un dispositivo biosensor multiplexado que combina inmunoreactivos (antibióticos SAS y fluoroquinolonas (FQs)) producidos en el grupo de Receptores Moleculares Aplicados (AMRg) del CSIC, con bioreceptores (ß-lactámicos (BLs) y TCs) proporcionados por Unisensor SA (Lieja - Bélgica). Los resultados obtenidos fueron muy buenos. Alrededor de 34 antibióticos de cuatro familias diferentes de antibióticos pueden ser detectados en muestras de leche siguiendo todas las normativas de la CE con el dispositivo desarrollado. Anteriormente, se desarrolló un ELISA multianalito (en microplacas) mediante la combinación de los mismos bioreactivos para evaluar el sus características y establecer los procedimientos inmunoquímicos más adecuados. Los métodos inmunoquímicos desarrollados en esta tesis, incluyendo los inmunoensayos e inmunosensores, han sido evaluados en colaboración con el Centro de Investigación de Nestlé (NRC), en Lausana, en Suiza, con el fin de demostrar su rendimiento en las muestras de leche real.

Historically, the causative agent of infectious coryza has been identified as the NAD requiring bacterium Haemophilus paragallinarum and the implementation of an intensive vaccination program led to the effective control of this contagious upper respiratory infection. More recently, however, a decline in the protective capacity of a vaccine conditioned immune response was noted, with a number of contributing factors, including the emergence of a fast-growing NAD-independent bacterium, which has largely replaced the traditional NAD-dependent variety. As such, accurate, reproducible methods for determining and continually monitoring the type of infecting bacteria was necessitated. To address this need, strains of H. paragallinarum were evaluated according to both their phenotypic and their genotypic properties, in a combination serodiagnostic approach. A data bank of NAD-dependent H. paragallinarum reference strain and field isolate serovar-specific fingerprints was established on both a whole cell and outer membrane protein level. Visual comparative analysis of the qualitatively and quantitatively similar outer membrane protein patterns of all strains of NAD independency studied with the formulated data bank, indicate that the NAD-independent strains displayed profiles typical of serovar C-3. The outer membrane proteins have been identified as putative virulence determinants and, as such, were characterised according to their surface location, susceptibility to heat modification, functional role as endotoxins, sequence homology to structural membrane counterparts, and finally, their ability to induce an immune response. These studies represent novel efforts and form the foundation for identifying those antigens responsible for maintaining an infection in the host milieu. Ribotype analysis served as an adjunct to phenotypic observations, with the local NAD-independent field isolates being identified as serotype A. These contradictory outcomes call for the creation of a set of reference strains specific for NAD-independent isolates. The identification of restriction fragment length polymorphisms in the conserved 16S rRNA gene sequences indicate the potential application of this method for type assignment, requiring the recognition of a battery of versatile restriction enzymes to generate serovar-specific polymorphic profiles. The complexity of serotype allocation demands that a combination approach in which genotypic analyses complement phenotypic-based methods of haemagglutination inhibition and outer membrane protein profiling. The groundwork for implementation of such a system has been accomplished.
Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1998.

This chapter covers the basic techniques used in immunochemistry, including electrophoresis, immunoelectrophoresis, immunofixation, isoelectric focusing, and immunoblotting. It then specifies the units, normal ranges, test principles, and indications for over 50 immunological tests.

Techniques α‎₁-acid glycoprotein α‎₁-antichymotrypsin α‎₁-antitrypsin (α‎₁-AT) α‎₁-antitrypsin (α‎₁-AT) genotype (PI typing) α‎₂-macroglobulin Acute-phase proteins (CRP, ESR, SAA) Amyloid proteins Avian precipitins Bacterial and viral antibodies (specific antibodies; functional antibodies)...

Evaluation of engineered tissue is often limited to endpoint analyses, such as characterizing histology, gene expression and solutes [1]. Most of these applied analysis approaches are based on immunochemistry procedures that require excision, fixation and sectioning of the tissue as well as cell membrane perforation and labeling of proteins [2]. These analyses are time-consuming, do not facilitate high throughput processing and do not allow for online monitoring of engineered tissue. Because of these limitations, there is a need for online, high throughput monitoring techniques to evaluate engineered tissue. In this work, we introduce an approach for microscopic imaging and online analysis of living engineered tissue. The approach is based on application of a non-toxic dye specific for the extracellular space and subsequent interrogation by in vivo confocal microcopy. We hypothesized that the approach will allow for online characterization of cell structure.