Inmunoensayos enzimáticos para detectar agentes infecciosos o sus productos: algunos diseños y aplicaciones / Enzymatic immunoassays for the detection of infectious agents or their products: presentation of some designs and applications

Se hizo una valoración del impacto de los ensayos inmunoenzimáticos en la analítica de base inmunoquímica en las últimas 4 décadas, en la detección de agentes infecciosos o los productos asociado a su presencia y(o) actividad patogénica. Además se hace una incursión en algunos diseños y formatos que han tenido estos inmunoensayos desde los métodos electroquímicos de detección, los ensayos para detectar actividad proteolítica de origen microbiano y sus inhibidores como posibles blancos terapéuticos, los inmunoensayos directos de triple anticuerpo para lograr mayor sensibilidad, reveladores alternativos de la actividad enzimática, ensayos para el estudio de la serología viral con un mínimo de determinaciones, así como ensayos de competencia para evaluar la efectividad de candidatos vacunales basados en combinaciones peptídicas seleccionadas. Se concluyó con una rápida visión del futuro inmediato de este tipo de inmunoensayos a la luz de las tecnologías analíticas emergentes de detección.

This paper assessed the impact of the immunoenzymatic assays on the field of the immunochemistry-based analytics for the last 40 years, and on the detection of infectious agents or the products related to their presence and/or pathogenic activity. It also addressed some designs and formats of these immunoassays from electrochemical methods of detection, assays to determine proteolytic microbial activity and their inhibitors as possible therapeutical targets, more sensitive direct triple antibody systems, alternative enzymatic activity detectors, assays for viral serology of minimal determinations to competitive assays for evaluation of vaccinal candidate effectiveness based on selected peptide combinations. Finally, it provided a rapid overview of the near future of this type of immunoassays in the light of the emerging detection analytical technologies.

Los anticuerpos y su papel como herramientas analíticas en los ensayos inmunoenzimáticos / The antibodies and their role as analytic tools in immunoenzymatic assays

En este trabajo se presentó la historia y evolución, desde el descubrimiento, de los anticuerpos, así como la elucidación de su compleja estructura y función que ha servido de base metodológica para crear paradigmas inimaginables en su momento, como la fina especificidad de reconocimiento; también derrumbar otros, aparentemente inamovibles, como la invariabilidad y universalidad del genoma celular. Se revisó la evolución de los sistemas analíticos basados en la reacción antígeno-anticuerpos para llegar al estado actual y problemática de las enfermedades infecciosas y el determinante papel que desempeñan en su control la detección y el monitoreo de agentes infecciosos. La extraordinaria capacidad de los anticuerpos para discriminar estructuras antigénicamente similares, les permite ser parte fundamental de los inmunoensayos como herramientas básicas de lo que es hoy día una disciplina productiva muy bien establecida: la inmunotecnología.

This paper presented the history and evolution of the antibodies since their discovery. It also elucidated their complex structure and function that have served at a given time as methodological basis for creating unimaginable paradigms such as fine recognition specificity, and also for destroying other apparently immutable ones as invariability and universality of the cellular genome. A review was made of the evolution of antigen-antibody reaction-based analytical systems up to the present, the situation of infectious diseases and the determining role that detection and monitoring of infectious agents play in their control. The extraordinary capability of antibodies to discriminate antigenically similar structures allows them to be fundamental tools in immunoassays and also in a well-established discipline at present, that is, immunotechnology.

Análise imunoistoquímica do ki-67 em 140 pacientes com câncer de mama - correlação com fatores clínico-patológicos e sobrevida / Immunohistochemical analysis of ki-67 in 140 patients with breast cancer- correlation with clinicopathological factors and survival

O antígeno Ki-67 é utilizado para avaliar a atividade proliferativa em vários tumores. No entanto, o papel do Ki-67 como fator prognóstico em câncer de mama é ainda indefinido. O objetivo do trabalho foi analisar a expressão do antígeno Ki-67 correlacionando com fatores clínico-patológicos e sobrevida em pacientes com câncer de mama. Realizou-se a análise imunoistoquímica utilizando anticorpos monoclonais MIB-1 em 140 amostras de câncer de mama de mulheres com idade entre 27 e 90 anos, atendidas no Hospital Araújo Jorge da Associação de Combate ao Câncer de Goiás. O ponto de corte para a análise foi de 25%. A sobrevida global em cinco anos foi de 77,1%. Houve sobrevida significativamente pior nos casos com linfonodos positivos (p < 0,0001), tumores maiores (p < 0,0001), estádios mais avançados (p < 0,0001) e pacientes com receptores hormonais negativos (p = 0,019). Pacientes com imunodetecção de Ki-67 em mais de 25% das células tiveram pior sobrevida quando comparadas àquelas que expressaram Ki-67 em menos de 25% das células examinadas (70,8% x 82,7%), porém essa diferença não foi estatisticamente significativa (p = 0,094). A hiperexpressão do Ki-67 não se correlacionou com pior sobrevida no grupo de pacientes analisadas.

The Ki-67 antigen is used to evaluate proliferative activity in several tumors. However the role of the Ki-67 as a prognostic factor in breast cancer is still undefined. The aim of present study was to analyze the expression of Ki-67 antigen correlating with clinicopathological factors and survival in patients with breast cancer. We carried out an immunohistochemical analysis using MIB-1 monoclonal antibody in 140 specimen of breast cancer of women with age between 27 and 90 years. The cut-off point was 25%. The five year overall survival was 77.1%. There was a significant worse survival of patients with node positive (p < 0.0001), larger tumors (p < 0.0001), more advanced stages (p < 0.0001) and with negative hormonal receptors (p = 0.019). Patients with Ki-67 immunodetection in more than 25% of the tumors cells showed a poorer outcome when compared with those that express Ki-67 in less than 25% of the cells examined (70.8% x 82.7%), however this difference was not statistically significative (p = 0,094). The overexpression of Ki-67 did not correlate with a worse survival in the group of patients analyzed.

Imunoensaios: uma alternativa para a detecção de fungos toxigênicos em alimentos / alimentosImmunoassays: an alternative for toxigenicfungi detection in foods

O monitoramento constante da contaminação fúngica é imprescindível para assegurar a qualidade e segurança dos alimentos, reduzindo as perdas econômicas, assim como os riscos à saúde humana e animal. Os métodos tradicionais de identificação e detecção de fungos (cultivo em diversos meios, exame microscópico e análises bioquímicas) geralmente consomem muito tempo e exigem pessoal com experiência. Os imunoensaios, particularmente os ensaios imunoenzimáticos, constituem uma alternativa promissora aos métodos tradicionais devido à alta sensibilidade, especificidade, reprodutibilidade e potencial como método rápido de controle de qualidade. Dentre os ensaios imunoenzimáticos, aqueles baseados em exoantígenos são os mais empregados na resolução de problemas taxonômicos, detecção e identificação de fungos toxigênicos. Nesta revisão serão abordados conceitos básicos de imunoensaios, métodos de detecção de fungos, assim como diversos ensaios imunoenzimáticos para a detecção de fungos toxigênicos em alimentos.

Constant monitoring of mould contamination is essential in order to assure the food quality and safetyand reduce the economic losses, as well as to minimize the potential hazards to human and animal health.The traditional methods for mould identification and detection (culture in several media, microscopicexamination and chemical analysis) are usually time-consuming and require trained staff. Immunoassays,particularly enzyme-linked immunosorbent assay (ELISA) could be a promising alternative to the traditionalmethods due to high sensitivity, specificity, reproducibility and potential for use in rapid quality control.Among ELISAs, those based on exoantigens are the most employed in the resolution of taxonomicproblems, detection and identification of toxigenic fungi. This review discusses the basic principles ofimmunoassays, methods of mould detection and the several ELISAs developed for toxigenic fungidetection in food

Produção e caracterização de exoantígenos de Fusarium verticillioides / Production and characterization of Fusarium verticillioides exoantigens

Fusarium verticillioides Sacc. Niremberg (=F. moniliforme Sheldon) é um patógeno primário de milho e principal produtor de fumonisinas. Este fungo pode causar perdas econômicas significativas para produtores e processadores de grãos, criadores de animais, além de representar sérios riscos á saúde humana e animal. Diversos métodos para a detecção de fungos têm sido utilizados, porém a maioria demanda tempo e pessoal treinado. Por outro lado, os métodos imunológicos, particularmente os ensaios imunoenzimáticos, apresentam diversas vantagens para o emprego rápido em controle de qualidade. Neste trabalho, foram obtidos exoantígenos de 8 isolados de F. verticillioides para a produção de anticorpos policlonais. O perfil eletroforético dos antígenos apresentou bandas com massas moleculares aparentes variando entre 17 e 170 kDa. Os antígenos de 3 isolados (97K, 113F e 162A), tendo como base a concentração de proteínas e número de bandas, foram escolhidos para a produção de anticorpos policlonais. O soro imune anti-97K, com maior título no ELISA indireto (1:12.800), apresenta potencial para a imunodetecção de Fusarium verticillioides.

Fusarium verticillioides Sacc. Niremberg (=F. moniliforme Sheldon) is a primary corn pathogen and themain fumonisin producer. This fungus can cause significant economical losses for the farmers, grainprocessors, animal producers and risk for human and animal health. Several methods for mould detectionhave been used, however most of these are time-consuming and require trained staff. Otherwise,immunoassays (particularly enzyme-linked immunosorbent assay, or ELISA) provide several advantagesand potential for use in rapid quality control. In this work exoantigens from eight F. verticillioidesisolates were obtained for further production of polyclonal antibodies. The electrophoretic profile ofthese antigens showed protein bands with molecular mass ranging from 17 to 170 kDa. The antigens from3 isolates (97K, 113F and 162A) were selected for polyclonal antibodies production, based on proteinconcentration and number of bands. Antiserum against F. verticillioides 97K exoantigens, which showed thehighest titre in indirect ELISA (1:12.800), has potential for the immunodetection of F. verticillioides

Identification of C5+ extraband of butyrylcholinesterase and two protein bands cathodic to it.

Electrophoresis of human plasma yields 4 butyrylcholinesterase (BChE) protein bands, i.e. C1, C2, C3, C4 and in some individuals also an extraband C5+. In addition to that other protein bands called "S" bands are also invariably detected. In order to know whether the C5+ and the "S" bands are related to the BChE protein, we have carried out immunological and peptide mapping studies on these proteins. The immunology approach was done by raising polyclonal antibodies against each protein bands (S1, S2, C4 and C5+) and reacted to the plasma protein bands transferred on nitrocellulose papers. Individual raised antibodies recognized all protein bands studied including the C4, an isozyme of BChE, indicating that the protein bands contain similar epitopes. Several protein bands cathodic to S1 also reacted with the antibodies, suggesting that they are probably fractions of the BChE protein, as well. When individual protein bands were digested with S. aureus V8 toxin and α-chymotrypsin, they revealed a striking similarity in peptide pattern among each other. These studies indicate that the S1, S2 and C5+ protein bands belong to the BChE protein.

Effective Production and Clinical Application of Anti-Interleukin-6 Monoclonal Antibodies / 대한면역학회지

Highly specific and sensitive immunoassay method for soluble human recombinant interleukin-6 (hu rlL-6) was established by two different immunization methods. One is conventional method by Freund's adjuvant method and the other is special method which is directly injected to mouse spleen. Among seven established monoclonal antibodies (mAbs), two typical monoclonal antibodies, designated YB3 (IgG1) and NY2 (IgM), were further characterized. These mAbs highly bound to IL-6, however did not show cross reactivity with IL-1B and IL-2. As the results of ELISA inhibition assay and western blotting method, it was further identified that YB3 and NY2 had high binding specificity with IL-6. And the limiting detection amount of rlL-6 for YB3 was 5 ng/ml and for NY2 was 0.5 ng/ml. Furthermore, N-glycosylated human rlL-6 was also bound to YB3 on ELISA. On the other hand YB-3 furtherly recognized N-glycosylated human rlL-6 by sandwich ELISA method. These mAbs may be of use to diagnose the gynecopathy which contains abortion and preterm labor.

Study on the contents of serum HDL subclasses in type 2 diabetic patients / 中国糖尿病杂志

Objective To study the changes of the contents of serum HDL subclasses in type 2 diabetic patients. Methods The contents of serum HDL subclasses in healthy controls (n=38) and patients (n=38) were determined by two-dimensional gel electrophoresis associated with immunodetection method. Results The contents of pre-?1, HDL(P