A pesquisa apresentada aqui foi apoiado por Charles River.

<ol>
	<li>Jacobson, R. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+validation+of+diagnostic+assays+for+infectious+diseases.">Principles of validation of diagnostic assays for infectious diseases.</a> Manual of Standards for Diagnostic Tests and Vaccines. , 8-15 (1996).</li><li>Wright, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=International+standards+for+test+methods+and+reference+sera+for+diagnostic+tests+for+antibody+detection.">International standards for test methods and reference sera for diagnostic tests for antibody detection.</a> Rev. sci. tech. Off. int. Epiz. 17 (2), 527-533 (1998).</li><li>Pepe, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Statistical Evaluation of Medical Tests for Classification and Prediction. , (2003).</li><li>Barr, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+and+interpretation+of+diagnostic+tests+for+feline+immunodeficiency+virus+infection.">Comparison and interpretation of diagnostic tests for feline immunodeficiency virus infection.</a> J. Am. Vet. Med. Assoc. 199, 1377-1381 (1991).</li></ol>

Todo o trabalho de autores por Charles River RADS, um importante fornecedor de serviços de sorologia em animais de testes e reagentes.

O processo de teste MFIA é altamente eficiente, que requer menos equipamento e menores volumes de amostra e de reagente do que os testes tradicionais singleplex. A funcionalidade do sistema de multiplex dá ao utilizador a flexibilidade para rastrear simultaneamente por várias estirpes ou serotipos de agentes comuns em roedores de laboratório (Coronavirus ie, parvovírus, etc.) Isto também permite, de concepção personalizada painéis grânulo com base na área de interesse (ou seja, a família de vírus específico) e é adaptável a outros tipos de triagem de biomoléculas, incluindo citocinas e outros biomarcadores. Além disso, permite-nos incorporar vários ensaios de controlo interno a fim de verificar a adequação do sistema de amostra e e, assim, garantir a precisão dos resultados. Estes incluem o controlo de tecido e ensaio de IgG anti-imunoglobulina de espécies de soro (αIg) conjuntos de esferas revestidas para avaliar a adequação da amostra. Um tecido de controlo do grânulo detecta a ligação não específica de imunoglobulina do soro e a esfera de controlo αIg confirmaque o soro foi adicionado e contém uma concentração de imunoglobulina suficiente. Outra grânulo de controlo, revestidas com imunoglobulina de espécies de soro, demonstra que os reagentes marcados e leitor de ensaio estão a funcionar correctamente. Outros comercialmente disponíveis multiplex ensaios formato sorológicos (microarrays ie, ImmunoComb) pode não oferecer o mesmo nível de confirmação de resultado. A habilidade para diminuir a possível fonte de fracasso ensaio antes de reteste pode ajudar o pesquisador economizar em tempo e materiais. Aspectos críticos do MFIA deve ser confirmada antes de repetir uma amostra falhou. Uma vez que o ensaio é realizado à temperatura ambiente, deve-se verificar que a temperatura de laboratório é de cerca de 27 ° C ± 2 ° C, as temperaturas mais elevadas podem conduzir a uma redução do que esperado para controlos e amostras. Lavagem é, talvez, o passo mais importante no ensaio. Garantindo que a placa de teste é devidamente lavado e ressuspenso blotted pode eliminara grande maioria dos erros de amostragem, devido à baixa contagem de talão (devido esferas de agregados) ou a adição de reagente insuficiente (perdido por capilaridade para fora da parte inferior da placa de ensaio do filtro). Rotineiramente contar 25 pérolas por ensaio (agente) e não encontraram nenhuma diferença estatística nos resultados por contagem maior número de pérolas que também conduzem a um maior tempo de leitura para a placa. Realizamos estudos de validação abrangentes de MFIA em várias espécies de animais de laboratório comumente utilizados (camundongo, rato, hamster, porquinhos e coelho) para demonstrar a precisão diagnóstica, reprodutibilidade e robustez, testando um grande número de conhecidos positivos e negativos amostras de soro, e comparando sua ELISA, IFA e resultados MFIA. Os limites de detecção (isto é, o padrão de titulação de soro imune endpoints) de MFIA foram comparáveis, e em alguns casos ultrapassou, os de ELISA correspondente. Especificidade do diagnóstico, medida com roedores SPF soros, superou 99%, o betwee correspondência geraln ELISA e MFIA realizada em conhecidos soros positivos e negativos conhecidos foi superior a 95%. Em resumo, estes resultados mostraram que MFIA multiplex é um bom substituto para o ELISA singleplex, e é adequada para a sua pretendida utilização, ou seja, em serovigilância rotina de colónias de animais laboratoriais.

multiplexed fluorometric immunoassay testing methodology and troubleshooting

Para assegurar a qualidade dos modelos animais utilizados em pesquisa biomédica, temos desenvolvido um número de estratégias de ensaio e métodos de diagnóstico para determinar se os animais foram expostos a agentes infecciosos adventícias (vírus, micoplasmas e outros microorganismos aeróbios). Infecções de animais imunocompetentes são geralmente transitórios, mas as respostas de anticorpos séricos a infecção, muitas vezes pode ser detectado dentro de dias a semanas e persistir durante toda a vida do hospedeiro. Serologia é a metodologia de diagnóstico primário pelo qual os animais de laboratório são monitorizados. Historicamente, o ensaio com enzima ligada indirecto (ELISA) foi o principal método de rastreio para serovigilância. O ELISA é realizado como uma singleplex, em que uma reacção antigénio-anticorpo microbiana é medida por poço. Em comparação a MFIA é realizado como um ensaio multiplex. Uma vez que as micro-esferas entram em 100 conjuntos de cores distintas, até 100 diferentes ensaios podem ser realizados Simultaneously numa única microplaca bem. Esta inovação diminui a quantidade de soro, os reagentes e produtos descartáveis ​​requeridos para testes de rotina, enquanto que aumentando a quantidade de informação obtida a partir de um único teste bem. Além disso, são capazes de incorporar múltiplas esferas de controlo internos para verificação da amostra e adequação do sistema e, assim, garantir a precisão dos resultados. Estes incluem o controlo de tecido e ensaio de IgG anti-imunoglobulina de espécies de soro (αIg) conjuntos de esferas revestidas para avaliar a adequação da amostra. Tal como no ELISA e IFA, o controlo de tecido detecta a ligação não específica de imunoglobulina no soro. O controle αIg (controle de Soro) confirma que o soro tenha sido adicionado e contém uma concentração de imunoglobulinas suficiente enquanto o IgG esfera de controlo (controle de Adequação do Sistema), revestido com imunoglobulina espécie de soro, demonstra que os reagentes marcados e Luminex leitor estão funcionando corretamente.

Watch this Scientific Journal Video about Multiplexed Fluorometric ImmunoAssay Testing Methodology and Troubleshooting at JoVE.com

Multiplexed Fluorometric ImmunoAssay Testing Methodology and Troubleshooting

Usando a tecnologia Luminex Corporation microesfera xMAP, desenvolvemos o imunoensaio multiplexado fluorométrico (MFIA) para serovigilância de várias espécies de animais de laboratório. O MFIA é um microarray de suspensão onde o antigénio controlo de tecido, ou imunoglobulinas são ligados covalentemente a um código de cores de poliestireno microesferas. O método de ensaio MFIA, bem como vários tópicos de resolução de problemas é abordada.

Multiplexados Metodologia de Testes de imunoensaio fluorimétrico e solução de problemas

To ensure the quality of animal models used in biomedical research we have developed a number of diagnostic testing strategies and methods to determine if ...

multiplexed-fluorometric-immunoassay-testing-methodology

Nanyang Technological University

Research

JoVE Journal

Biology

28.3K visualizações.  Charles River. Usando a tecnologia Luminex Corporation microesfera xMAP, desenvolvemos o imunoensaio multiplexado fluorométrico (MFIA) para serovigilância de várias espécies de animais de laboratório. O MFIA é um microarray de suspensão onde o antigénio controlo de tecido, ou imunoglobulinas são ligados covalentemente a um código de cores de poliestireno microesferas. O método de ensaio MFIA, bem como vários tópicos de resolução de problemas é abordada.

Vídeo: Multiplexed Fluorometric ImmunoAssay Testing Methodology and Troubleshooting

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Teste de sensibilidade visual para a velocidade e direção do movimento em Lagartos

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Biologia

Olfactory Behavioral Testing in the Adult Mouse

The rodent olfactory system is of increasing interest to scientists, studied, in part, in systems biology because of its stereotyped, yet accessible circuitry. In addition, this area's unique ability to generate new neurons throughout an organism's lifetime makes it an attractive system for developmental and regenerative biologists alike. Such interest necessitates a means for a quick, yet reliable assessment of olfactory function. Many tests of olfactory ability are complex, variable or not specifically designed for mice. Also, some tests are sensitive to memory deficits as well as defects in olfactory abilities, confounding obtained results.
Here, we describe a simple battery of tests designed to identify defects in olfactory sensitivity and preference. First, an initial general health assessment allows for the identification of animals suitable for further testing. Second, mice are exposed to various dilutions of scents to ascertain whether there is a threshold difference. Third, mice are presented with various scents, both attractive and aversive, that allow for the assessment of olfactory preference. These simple studies should make the initial characterization of olfactory behavior accessible for labs of varied resources and expertise.

Fundamental, yet unique properties of the rodent olfactory system have led to its increasing study among biologists. A relatively simple assessment of its function is then also needed. Here we describe sensitive tests for the characterization of mouse olfactory sensitivity and preference.

Teste Comportamental olfativo no rato adulto

The rodent olfactory system is of increasing interest to scientists, studied, in part, in systems biology because of its stereotyped, yet accessible ...

Induction and Testing of Hypoxia in Cell Culture

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc&#x2026; Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7. 
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.

Indução de hipóxia e Testes em Cultura de Células

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...

Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies

In the biological sciences there have been technological advances that catapult the discipline into golden ages of discovery. For example, the field of microbiology was transformed with the advent of Anton van Leeuwenhoek&#39;s microscope, which allowed scientists to visualize prokaryotes for the first time. The development of the polymerase chain reaction (PCR) is one of those innovations that changed the course of molecular science with its impact spanning countless subdisciplines in biology. The theoretical process was outlined by Keppe and coworkers in 1971; however, it was another 14 years until the complete PCR procedure was described and experimentally applied by Kary Mullis while at Cetus Corporation in 1985. Automation and refinement of this technique progressed with the introduction of a thermal stable DNA polymerase from the bacterium Thermus aquaticus, consequently the name Taq DNA polymerase.
PCR is a powerful amplification technique that can generate an ample supply of a specific segment of DNA (i.e., an amplicon) from only a small amount of starting material (i.e., DNA template or target sequence). While straightforward and generally trouble-free, there are pitfalls that complicate the reaction producing spurious results. When PCR fails it can lead to many non-specific DNA products of varying sizes that appear as a ladder or smear of bands on agarose gels. Sometimes no products form at all. Another potential problem occurs when mutations are unintentionally introduced in the amplicons, resulting in a heterogeneous population of PCR products. PCR failures can become frustrating unless patience and careful troubleshooting are employed to sort out and solve the problem(s). This protocol outlines the basic principles of PCR, provides a methodology that will result in amplification of most target sequences, and presents strategies for optimizing a reaction. By following this PCR guide, students should be able to: 
 
&#9679; Set up reactions and thermal cycling conditions for a conventional PCR experiment 
&#9679; Understand the function of various reaction components and their overall effect on a PCR experiment 
&#9679; Design and optimize a PCR experiment for any DNA template 
&#9679; Troubleshoot failed PCR experiments

PCR has emerged as a common technique in many molecular biology laboratories. Provided here is a quick guide to several conventional PCR protocols. Because each reaction is a unique experiment, optimal conditions required to generate a product vary. Understanding the variables in a reaction will greatly enhance troubleshooting efficiency, thereby increasing the chance to obtain the desired result.

Reação em Cadeia da Polimerase: Protocolo Basic Plus Estratégias de solução de problemas e otimização

In the biological sciences there have been technological advances that catapult the discipline into golden ages of discovery. For example, the field of ...

Solid Phase Synthesis of a Functionalized Bis-Peptide Using "Safety Catch" Methodology

In 1962, R.B. Merrifield published the first procedure using solid-phase peptide synthesis as a novel route to efficiently synthesize peptides. This technique quickly proved advantageous over its solution-phase predecessor in both time and labor. Improvements concerning the nature of solid support, the protecting groups employed and the coupling methods employed over the last five decades have only increased the usefulness of Merrifield's original system. Today, use of a Boc-based protection and base/nucleophile cleavable resin strategy or Fmoc-based protection and acidic cleavable resin strategy, pioneered by R.C. Sheppard, are most commonly used for the synthesis of peptides1.
Inspired by Merrifield's solid supported strategy, we have developed a Boc/tert-butyl solid-phase synthesis strategy for the assembly of functionalized bis-peptides2, which is described herein. The use of solid-phase synthesis compared to solution-phase methodology is not only advantageous in both time and labor as described by Merrifield1, but also allows greater ease in the synthesis of bis-peptide libraries. The synthesis that we demonstrate here incorporates a final cleavage stage that uses a two-step "safety catch" mechanism to release the functionalized bis-peptide from the resin by diketopiperazine formation.
Bis-peptides are rigid, spiro-ladder oligomers of bis-amino acids that are able to position functionality in a predictable and designable way, controlled by the type and stereochemistry of the monomeric units and the connectivity between each monomer. Each bis-amino acid is a stereochemically pure, cyclic scaffold that contains two amino acids (a carboxylic acid with an &alpha;-amine)3,4. Our laboratory is currently investigating the potential of functional bis-peptides across a wide variety of fields including catalysis, protein-protein interactions and nanomaterials.

The efficient solid-phase peptide synthesis of a functionalized bis-peptide trimer utilizing a "safety catch" cleavage procedure from HMBA resin is described.

Síntese em Fase Sólida de um funcionalizada Bis Peptide-Usando o &quot;Safety Catch&quot; Metodologia

In 1962, R.B. Merrifield published the first procedure using solid-phase peptide synthesis as a novel route to efficiently synthesize peptides. This ...

Design and Use of Multiplexed Chemostat Arrays

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is constrained by limiting specific nutrients.1,2 Data from chemostats are highly reproducible for the measurement of quantitative phenotypes as they provide a constant growth rate and environment at steady state. For these reasons, chemostats have become useful tools for fine-scale characterization of physiology through analysis of gene expression3-6 and other characteristics of cultures at steady-state equilibrium.7 Long-term experiments in chemostats can highlight specific trajectories that microbial populations adopt during adaptive evolution in a controlled environment. In fact, chemostats have been used for experimental evolution since their invention.8 A common result in evolution experiments is for each biological replicate to acquire a unique repertoire of mutations.9-13 This diversity suggests that there is much left to be discovered by performing evolution experiments with far greater throughput. 
We present here the design and operation of a relatively simple, low cost array of miniature chemostats&mdash;or ministats&mdash;and validate their use in determination of physiology and in evolution experiments with yeast. This approach entails growth of tens of chemostats run off a single multiplexed peristaltic pump. The cultures are maintained at a 20 ml working volume, which is practical for a variety of applications. It is our hope that increasing throughput, decreasing expense, and providing detailed building and operation instructions may also motivate research and industrial application of this design as a general platform for functionally characterizing large numbers of strains, species, and growth parameters, as well as genetic or drug libraries.

We developed and validated a small-footprint array of miniature chemostats built from readily available parts for low cost. Physiological and experimental evolution results were similar to larger volume chemostats. The ministat array provides a compact, inexpensive, and accessible platform for traditional chemostat experiments, functional genomics, and chemical screening applications.

Concepção e utilização de matrizes quemostato Multiplexados

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is ...

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins within complex biological samples. Since then, western blotting methodology has become a common component of the molecular biologists experimental repertoire. A cursory search of PubMed using the term &#8220;western blot&#8221; suggests that in excess of two hundred and twenty thousand published manuscripts have made use of this technique by the year 2014. Importantly, the last ten years have seen technical imaging advances coupled with the development of sensitive fluorescent labels which have improved sensitivity and yielded even greater ranges of linear detection. The result is a now truly Quantifiable Fluorescence based Western Blot (QFWB) that allows biologists to carry out comparative expression analysis with greater sensitivity and accuracy than ever before. Many &#8220;optimized&#8221; western blotting methodologies exist and are utilized in different laboratories. These often prove difficult to implement due to the requirement of subtle but undocumented procedural amendments. This protocol provides a comprehensive description of an established and robust QFWB method, complete with troubleshooting strategies.

The advancement of western blotting using fluorescence has allowed detection of subtle changes in protein expression enabling quantitative analyses. Here we describe a robust methodology for detection of a range of proteins across a variety of species and tissue types. A strategy to overcome common technical problems is also provided.

A Guide to Modern quantitativa fluorescente Western Blotting com estratégias de resolução de problemas

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins ...

Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery of fluorescent proteins and their genes have enabled the engineering of protein fusions for localization, the analysis of transcriptional activation and translation of proteins of interest, or the general tracking of individual cells and cell populations. The use of fluorescent protein genes in combination with retroviral technology has further allowed the expression of these proteins in mammalian cells in a stable and reliable manner. Shown here is how one can utilize these genes to give cells within a population of cells their own biosignature. As the biosignature is achieved with retroviral technology, cells are barcoded &#180;indefinitely&#180;. As such, they can be individually tracked within a mixture of barcoded cells and utilized in more complex biological applications. The tracking of distinct populations in a mixture of cells is ideal for multiplexed applications such as discovery of drugs against a multitude of targets or the activation profile of different promoters. The protocol describes how to elegantly develop and amplify barcoded mammalian cells with distinct genetic fluorescent markers, and how to use several markers at once or one marker at different intensities. Finally, the protocol describes how the cells can be further utilized in combination with cell-based assays to increase the power of analysis through multiplexing.

Since the discovery of the green fluorescent protein gene, fluorescent proteins have impacted molecular cell biology. This protocol describes how expression of distinct fluorescent proteins through genetic engineering is used for barcoding individual cells. The procedure enables tracking distinct populations in a cell mixture, which is ideal for multiplexed applications.

Barcoding Genetic com proteínas fluorescentes para Aplicações Multiplexados

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery ...

Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within tissues. Many semi-quantitative systems have been developed for scoring expression using immunohistochemistry, but inherent subjectivity limits reproducibility and accuracy of results. Furthermore, the investigation of spatially overlapping biomarkers such as nuclear transcription factors is difficult with current immunohistochemistry techniques. We have developed and optimized a system for simultaneous investigation of multiple proteins using high throughput methods of multiplexed immunohistochemistry and multispectral imaging. Multiplexed immunohistochemistry is performed by sequential application of primary antibodies with secondary antibodies conjugated to horseradish peroxidase or alkaline phosphatase. Different chromogens are used to detect each protein of interest. Stained slides are loaded into an automated slide scanner and a protocol is created for automated image acquisition. A spectral library is created by staining a set of slides with a single chromogen on each. A subset of representative stained images are imported into multispectral imaging software and an algorithm for distinguishing tissue type is created by defining tissue compartments on images. Subcellular compartments are segmented by using hematoxylin counterstain and adjusting the intrinsic algorithm. Thresholding is applied to determine positivity and protein co-localization. The final algorithm is then applied to the entire set of tissues. Resulting data allows the user to evaluate protein expression based on tissue type (ex. epithelia vs. stroma) and subcellular compartment (nucleus vs. cytoplasm vs. plasma membrane). Co-localization analysis allows for investigation of double-positive, double-negative, and single-positive cell types. Combining multispectral imaging with multiplexed immunohistochemistry and automated image acquisition is an objective, high-throughput method for investigation of biomarkers within tissues.

Immunohistochemistry is a powerful lab technique for evaluating protein localization and expression within tissues. Current semi-automated methods for quantitation introduce subjectivity and often create irreproducible results. Herein, we describe methods for multiplexed immunohistochemistry and objective quantitation of protein expression and co-localization using multispectral imaging.

A quantificação da expressão da proteína e co-localização Usando multiplexada Imuno-histoquímica Coloração e Multispectral Imagiologia

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within ...

Procedure and Key Optimization Strategies for an Automated Capillary Electrophoretic-based Immunoassay Method

New technologies that utilize capillary-based immunoassays promise faster and more quantitative protein assessment compared to traditional immunoassays. However, similar to other antibody-based protein assays, optimization of capillary-based immunoassay parameters, such as protein concentration, antibody dilution, and exposure time is an important prerequisite to the generation of meaningful and reliable data. Measurements must fall within the linear range of the assay where changes in signal are directly proportional to changes in lysate concentration. The process of choosing appropriate lysate concentrations, antibody dilutions, and exposure times in the human bronchial epithelial cell line, BEAS-2B, is demonstrated here. Assay linearity is shown over a range of whole cell extract protein concentrations with p53 and &#945;-tubulin antibodies. An example of signal burnout is seen at the highest concentrations with long exposure times, and an &#945;-tubulin antibody dilution curve is shown demonstrating saturation. In addition, example experimental results are reported for doxorubicin-treated cells using optimized parameters.

A capillary-based immunoassay using a commercial platform to measure target proteins from total protein preparations is demonstrated. In addition, assay parameters of exposure time, protein concentration, and antibody dilution are optimized for a cell culture model system.

Procedimento e estratégias de otimização de chave para um método automatizado capilar imunoensaio eletroforética com base em

New technologies that utilize capillary-based immunoassays promise faster and more quantitative protein assessment compared to traditional immunoassays. ...