An immunohistochemical study of the distribution of biotin in tissues of pigs and chickens

An optimised indirect peroxidase-anti-peroxidase immunohistochemical technique was used to detect endogenous biotin in frozen tissue sections from biotin-supplemented and biotin-depleted pigs and chickens. A monoclonal anti-biotin antibody was used as primary antibody in this technique. Immunoreactive biotin was detected in many tissues of both species including liver, kidney, pancreas, adipose tissue, adrenal gland, testis, brain, choroid plexus, cardiac and skeletal muscle, epithelium of the respiratory and digestive systems, skin and lymphoid tissues. The specificity of immunostaining for biotin was confirmed by the finding of reduced staining intensities in tissues of biotin-depleted animals compared to those of biotin-supplemented animals. The results of this study suggest that biotin has metabolic functions in a wider range of tissues than previously known. They also indicate that endogenous tissue biotin should be considered as a source of false positive staining when immunohistochemical or histochemical techniques which use avidin or streptavidin reagents or anti-biotin antibodies as components of the detection system, are applied to tissue sections.

A strategy for sensitivity and specificity enhancements in prostate specific antigen-[alpha] 1-antichymotrypsin detection based on surface plasmon resonance

A biochip based on surface plasmon resonance was fabricated to detect prostate specific antigen-a1-antichymotrypsin (PSA-ACT complex) in both HBS buffer and human serum. To reduce non-specific binding and steric hindrance effect, the chemical surface of the sensor chips was constructed by using various oligo(ethylene glycol) mixtures of different molar ratios of HS(CH2)11(OCH2CH2)6OCH2COOH and HS(CH2)11(OCH2CH2)3OH. The self-assembled monolayers were biotinylated to facilitate the immobilization of streptavidin. Using the chip surfaces, PSA-ACT complex in HBS buffer and human serum was detected at 20.7 and 47.5 ng/ml by primary immunoresponse, respectively. However, the limit of detection could be simply enhanced by a sandwich strategy to improve the sensitivity and specificity of the immunoassay. An intact PSA polyclonal antibody was used as an amplifying agent in the strategy. As a result, PSA-ACT complex concentrations as low as 10.2 and 18.1 ng/ml were found in the HBS buffer and human serum sample, respectively. The result indicates that this approach could satisfy our goal without modifying the secondary interactant.

Surface plasmon resonance-based inhibition assay for real-time detection of Cryptosporidium parvum oocyst

A surface plasmon resonance (SPR)-based inhibition assay method using a polyclonal anti-mouse IgM arrayed Cryptosporidium sensor chip was developed for the real-time detection of Cryptosporidium parvum oocysts. The Cryptosporidium sensor chip was fabricated by subsequent immobilization of streptavidin and polyclonal anti-mouse IgM (secondary antibody) onto heterogeneous self-assembled monolayers (SAMs). The assay consisted of the immunoreaction step between monoclonal anti-C. parvum oocyst (primary antibody) and oocysts, followed by the binding step of the unbound primary antibody onto the secondary antibody surface. It enhanced not only the immunoreaction yield of the oocysts by batch reaction but also the accessibility of analytes to the chip surface by antibody–antibody interaction. Furthermore, the use of optimum concentration of the primary antibody maximized its binding response on the chip. An inversely linear calibration curve for the oocyst concentration versus SPR signal was obtained in the range of 1×106–1×102 oocysts ml-1. The oocyst detection was also successfully achieved in natural water systems. These results indicate that the SPR-based inhibition assay using the Cryptosporidium sensor chip has high application potential for the real-time analysis of C. parvum oocyst in laboratory and field water monitoring.

Gold-based optical biosensor for single-mismatched DNA detection using salt-induced hybridization

In this study, a gold nanoparticle (Au-NP)-based detection method for sensitive and specific DNA-based diagnostic applications is described. A sandwich format consisting of Au-NPs/DNA/PMP (Streptavidin-coated MagnetSphere Para-Magnetic Particles) was fabricated. PMPs captured and separated target DNA while Au-NPs modified with oligonucleotide detection sequences played a role in recognition and signal production. Due to the much lower stability of mismatched DNA strands caused by unstable duplex structures in solutions of relatively low salt concentration, hybridization efficiency in the presence of different buffers was well investigated, and thus, the optimized salt concentration allowed for discrimination of single-mismatched DNA (MMT) from perfectly matched DNA (PMT). Therefore, quantitative information concerning the target analyte was translated into a colorimetric signal, which could easily and quantitatively measured by low-cost UV–vis spectrophotometric analysis. The results indicated this to be a very simple and economic strategy for detection of single-mismatched DNA strands.

Effects of Gold Nanoparticle and Enzyme Precipitation on Enhancement of Surface Plasmon Resonance Immunoassay: A Super Sensitive Measurement of Anti- Glutamic Acid Decarboxylase Antibody

Introduction: In this study, colloidal gold nanoparticle and precipitation of an insoluble product formed by HRP-biocatalyzed oxidation of 3,3'-diaminobenzidine (DAB) in the presence of H2O2 were used to enhance the signal obtained from the surface plasmon resonance biosensor.

Methods: The colloidal gold nanoparticle was synthesized as described by Turkevitch et al., and their surface was firstly functionalized with HS(CH2)11(OCH2CH2)3COOH (OEG3¬-COOH) by self assembling technique. Thereafter, those OEG3-COOH functionalized nanoparticles were covalently conjugated with horseradish peroxidase (HRP) and anti-IgG antibody (specific to the Fc portion of all human IgG subclasses) to form an enzyme-immunogold complex. Characterization was performed by several methods: UV-Vis absorption, dynamic light scattering (DLS), transmission electron microscopy (TEM) and FTIR. The as-prepared enzyme-immunogold complex has been applied in enhancement of SPR immunoassay. A sensor chip used in the experiment was constructed by using 1:10 molar ratio of HS(CH2)11(OCH2CH2)6COOH and HS(CH2)11(OCH2CH2)3OH. The capture protein, GAD65 (autoantigen) which is recognized by anti-GAD antibody (autoantibody) in the sera of insulin-dependent diabetes mellitus patients, was immobilized onto the 1:10 surface via biotin-streptavidin interaction.

Results and conclusions: In the research, we reported the influences of gold nanoparticle and enzyme precipitation on the enhancement of SPR signal. Gold nanoparticle showed its enhancement as being consistent with other previous studies, while the enzyme precipitation using DAB substrate was applied for the first time and greatly amplified the SPR detection. As the results, anti-GAD antibody could be detected at pg/ml level which is far higher than that of commercial ELISA detection kit. This study indicates another way to enhance SPR measurement, and it is generally applicable to other SPR-based immunoassays.

Bacteria microarrays as sensitive tools for exploring pathogen surface epitopes and recognition by host receptors

We describe a protocol for the generation and validation of bacteria microarrays and their application to the study of specific features of the pathogen's surface and interactions with host receptors. Bacteria were directly printed on nitrocellulose-coated glass slides, using either manual or robotic arrayers, and printing quality, immobilization efficiency and stability of the arrays were rigorously controlled by incorporating a fluorescent dye into the bacteria. A panel of wild type and mutant strains of the human pathogen Klebsiella pneumoniae, responsible for nosocomial and community-acquired infections, was selected as model bacteria, and SYTO-13 was used as dye. Fluorescence signals of the printed bacteria were found to exhibit a linear concentration-dependence in the range of 1 x 10(8) to 1 x 10(9) bacteria per ml. Similar results were obtained with Pseudomonas aeruginosa and Acinetobacter baumannii, two other human pathogens. Successful validation of the quality and applicability of the established microarrays was accomplished by testing the capacity of the bacteria array to detect recognition by anti-Klebsiella antibodies and by the complement subcomponent C1q, which binds K. pneumoniae in an antibody-independent manner. The biotin/AlexaFluor-647-streptavidin system was used for monitoring binding, yielding strain-and dose-dependent signals, distinctive for each protein. Furthermore, the potential of the bacteria microarray for investigating specific features, e.g. glycosylation patterns, of the cell surface was confirmed by examining the binding behaviour of a panel of plant lectins with diverse carbohydrate-binding specificities. This and other possible applications of the newly developed arrays, as e.g. screening/evaluation of compounds to identify inhibitors of host-pathogen interactions, make bacteria microarrays a useful and sensitive tool for both basic and applied research in microbiology, biomedicine and biotechnology.

Design, synthesis and validation of an inhibitor of CGRP degradation

Introduction: In addition to their afferent role in detection and signalling noxious stimuli, neuropeptide-containing sensory nerves may initiate and maintain chronic inflammation in diseases such as periodontitis by an efferent process known as neurogenic inflammation. Neuropeptides are susceptible to cleavage by peptidases, and therefore, the exact location and level of expression of peptidases are major determinants of neuropeptide action. Previous studies in our laboratory showed that enzyme components of gingival crevicular fluid (GCF) from periodontitis sites selectively inactivated the neuropeptide calcitonin gene-related peptide (CGRP), known to have a role in inhibiting osteoclastic bone resorption. Objectives: The aim of this study was to design and synthesise a specific inhibitor to prevent the degradation of CGRP by components of GCF. Methods: A hydroxamate-based inhibitor with a biotinylated tag was designed to ensure selectivity for CGRP and ease of use for future purification strategies. The biotinylated peptide hydroxamate contained the P1-P4 amino acid sequence of the potential CGRP cleavage site and was synthesised by solid-phase methods using standard Fmoc chemistry. Inhibition of CGRP metabolism by GCF was determined by MALDI-mass spectrometry (MALDI-MS) using pooled GCF samples from periodontitis patients as a crude source of the CGRP-degrading enzyme. Results: MALDI-MS analysis of CGRP degradation showed almost complete inhibition in the presence of the biotinylated inhibitor. Our results showed that the rate-limiting step in the cleavage of CGRP is endopeptidase cleavage, followed by carboxypeptidase attack. Conclusion: This study demonstrates that the enzyme component of GCF responsible for the degradation of CGRP can be inhibited by a biotinylated hydroxamate modelled on a potential endopeptidase cleavage site. The biotin tag on the inhibitor will facilitate our future purification of the CGRP-cleavage enzyme using a streptavidin-agarose column.

Optimisation of an aptamer-biosensor to detect C-reactive protein

Dissertação de Mestrado, Engenharia Biológica, Faculdade de Engenharia de Recursos Naturais, Universidade do Algarve, 2009

Imunossensores Eletroquímicos para o Diagnóstico Precoce e Descentralizado do Cancro da Mama

O cancro é uma das principais causas de morte em todo o mundo. Entre as mulheres, o cancro da mama é o mais frequente. A deteção precoce do cancro é de extrema importância na medida em que pode aumentar as possibilidades de cura dos pacientes e contribuir para a diminuição da taxa de mortalidade desta doença. Um método que tem contribuído para a deteção precoce do cancro é a análise de biomarcadores. Biomarcadores associados ao cancro da mama, como o Recetor 2 do Fator de Crescimento Epidérmico Humano (HER2) e o Antigénio Carbohidratado 15-3 (CA 15-3), podem ser detetados através de dispositivos como os biossensores. Neste trabalho foram desenvolvidos dois imunossensores eletroquímicos para a análise de HER2 e CA 15-3. Para ambos os sensores foram utilizados, como transdutores, elétrodos serigrafados de carbono. A superfície destes transdutores foi nanoestruturada com nanopartículas de ouro. Foram realizados imunoensaios não-competitivos (do tipo sandwich) em ambos os imunossensores, cuja estratégia consistiu na (i) imobilização do respetivo anticorpo de captura na superfície nanoestruturada dos elétrodos, (ii) bloqueio da superfície com caseína, (iii) incubação com uma mistura do analito (HER2 ou CA 15-3) e o respetivo anticorpo de deteção biotinilado, (iv) adição de estreptavidina conjugada com fosfatase alcalina (S-AP; a AP foi utilizada como marcador enzimático), (v) adição de uma mistura do substrato enzimático (3-indoxil fosfato) e nitrato de prata, e (vi) deteção do sinal analítico através da redissolução anódica, por voltametria de varrimento linear, da prata depositada enzimaticamente. Com as condições experimentais otimizadas, foi estabelecida a curva de calibração para a análise de HER2 em soro, entre 15 e 100 ng/mL, obtendo-se um limite de deteção de 4,4 ng/mL. Para o CA 15-3 a curva de calibração (em solução aquosa) foi estabelecida entre 15 e 250 U/mL, obtendo-se um limite de deteção de 37,5 U/mL. Tendo em conta o valor limite (cutoff value) estabelecido para o HER2 (15 ng/mL) pode-se comprovar a possível utilidade do imunossensor desenvolvido para o diagnóstico precoce e descentralizado do cancro da mama. No caso do CA 15-3 serão necessários estudos adicionais para se poder avaliar a utilidade do imunossensor para o diagnóstico do cancro da mama.

Direct binding of peptides to MHC class I molecules on living cells. Analysis at the single cell level.

To directly assess the binding of exogenous peptides to cell surface-associated MHC class I molecules at the single cell level, we examined the possibility of combining the use of biotinylated peptide derivatives with an immunofluorescence detection system based on flow cytometry. Various biotinylated derivatives of the adenovirus 5 early region 1A peptide 234-243, an antigenic peptide recognized by CTL in the context of H-2Db, were first screened in functional assays for their ability to bind efficiently to Db molecules on living cells. Suitable peptide derivatives were then tested for their ability to generate positive fluorescence signals upon addition of phycoerythrin-labeled streptavidin to peptide derivative-bearing cells. Strong fluorescent staining of Db-expressing cells was achieved after incubation with a peptide derivative containing a biotin group at the C-terminus. Competition experiments using the unmodified parental peptide as well as unrelated peptides known to bind to Kd, Kb, or Db, respectively, established that binding of the biotinylated peptide to living cells was Db-specific. By using Con A blasts derived from different H-2 congenic mouse strains, it could be shown that the biotinylated peptide bound only to Db among > 20 class I alleles tested. Moreover, binding of the biotinylated peptide to cells expressing the Dbm13 and Dbm14 mutant molecules was drastically reduced compared to Db. Binding of the biotinylated peptide to freshly isolated Db+ cells was readily detectable, allowing direct assessment of the relative amount of peptide bound to distinct lymphocyte subpopulations by three-color flow cytometry. While minor differences between peripheral T and B cells could be documented, thymocytes were found to differ widely in their peptide binding activity. In all cases, these differences correlated positively with the differential expression of Db at the cell surface. Finally, kinetic studies at different temperatures strongly suggested that the biotinylated peptide first associated with Db molecules available constitutively at the cell surface and then with newly arrived Db molecules.

Influence de l’agrégation érythrocytaire sur la migration axiale de microparticules simulant des plaquettes sanguines

Lors du phénomène d’hémostase primaire ou de thrombose vasculaire, les plaquettes sanguines doivent adhérer aux parois afin de remplir leur fonction réparatrice ou pathologique. Pour ce faire, certains facteurs rhéologiques et hémodynamiques tels que l’hématocrite, le taux de cisaillement local et les contraintes de cisaillement pariétal, entrent en jeu afin d’exclure les plaquettes sanguines de l’écoulement principal et de les transporter vers le site endommagé ou enflammé. Cette exclusion pourrait aussi être influencée par l’agrégation de globules rouges qui est un phénomène naturel présent dans tout le système cardiovasculaire selon les conditions d’écoulement. La dérive de ces agrégats de globules rouges vers le centre des vaisseaux provoque la formation de réseaux d’agrégats dont la taille et la complexité varient en fonction de l’hématocrite et des conditions de cisaillement présentes. Il en résulte un écoulement bi-phasique avec un écoulement central composé d’agrégats de globules rouges avoisinés par une région moins dense en particules où l’on peut trouver des globules rouges singuliers, des petits rouleaux de globules rouges et une importante concentration en plaquettes et globules blancs. De ce fait, il est raisonnable de penser que plus la taille des agrégats qui occupent le centre du vaisseau augmente, plus il y aura de plaquettes expulsées vers les parois vasculaires. L'objectif du projet est de quantifier, in vitro, la migration des plaquettes sanguines en fonction du niveau d’agrégation érythrocytaire présent, en faisant varier l’hématocrite, le taux de cisaillement et en promouvant l’agrégation par l’ajout d’agents tels que le dextran à poids moléculaire élevé. Cependant, le comportement non Newtonien du sang dans un écoulement tubulaire peut être vu comme un facteur confondant à cause de son impact sur l’organisation spatiale des agrégats de globules rouges. De ce fait, les études ont été réalisées dans un appareil permettant de moduler, de façon homogène, la taille et la structure de ces agrégats et de quantifier ainsi leur effet sur la migration axiale des plaquettes. Du sang de porc anti coagulé a été ajusté à différents taux d’hématocrite et insérer dans un appareil à écoulement de Couette, à température ambiante. Les plaquettes sanguines, difficilement isolables in vitro sans en activer certains ligands membranaires, ont été remplacées par des fantômes en polystyrène ayant un revêtement de biotine. La quantification de la migration de ces fantômes de plaquettes a été réalisée grâce à l’utilisation de membranes biologiques fixées sur les parois internes de l’entrefer du rhéomètre de Couette. Ces membranes ont un revêtement de streptavidine assurant une très forte affinité d’adhésion avec les microparticules biotynilées. À 40% d’hématocrite, à un cisaillement de 2 s-1, 566 ± 53 microparticules ont été comptées pour un protocole préétabli avec du sang non agrégeant, comparativement à 1077 ± 229 pour du sang normal et 1568 ± 131 pour du sang hyper agrégeant. Les résultats obtenus suggèrent une nette participation de l’agrégation érythrocytaire sur le transport des fantômes de plaquettes puisque l’adhésion de ces derniers à la paroi du rhéomètre de Couette augmente de façon quasi exponentielle selon le niveau d’agrégation présent.

The tagged microarray marker (TAM) method for high-throughput detection of single nucleotide and indel polymorphisms

The tagged microarray marker (TAM) method allows high-throughput differentiation between predicted alternative PCR products. Typically, the method is used as a molecular marker approach to determining the allelic states of single nucleotide polymorphisms (SNPs) or insertion-deletion (indel) alleles at genomic loci in multiple individuals. Biotin-labeled PCR products are spotted, unpurified, onto a streptavidin-coated glass slide and the alternative products are differentiated by hybridization to fluorescent detector oligonucleotides that recognize corresponding allele-specific tags on the PCR primers. The main attractions of this method are its high throughput (thousands of PCRs are analyzed per slide), flexibility of scoring (any combination, from a single marker in thousands of samples to thousands of markers in a single sample, can be analyzed) and flexibility of scale (any experimental scale, from a small lab setting up to a large project). This protocol describes an experiment involving 3,072 PCRs scored on a slide. The whole process from the start of PCR setup to receiving the data spreadsheet takes 2 d.

Multimetallic ruthenium(II) complexes as electrochemiluminescent labels

Two homometallic complexes containing two and three ruthenium polypyridyl units linked by amino acid lysine (Lys) and the related dipeptide (LysLys) were synthesized and their electrochemical, spectroscopic, and electrochemiluminescence (ECL) properties were investigated. The electrochemical and photophysical data indicate that the two metal complexes largely retain the electronic properties of the reference compound for the separate ruthenium moieties in the two bridged complexes, [4-carboxypropyl-4'-methyl-2,2'-bipyridine]bis(2,2'-bipyridine)ruthenium(II) complex. The ECL studies, performed in aqueous media in the presence of tri-n-propylamine as co-reactant, show that the ECL intensity increases by 30% for the dinuclear and trinuclear complexes compared to the reference. Heterogeneous ECL immunoassay studies, performed on larger dendritic complexes containing up to eight ruthenium units, demonstrate that limitations due to the slow diffusion can easily be overcome by means of nanoparticle technology. In this case, the ECL signal is proportional to the number of ruthenium units. Multimetallic systems with several ruthenium centers may, however, undergo nonspecific bonding,to streptavidin-coated particles or to antibodies, thereby increasing the background ECL intensity and lowering the sensitivity of the immunoassay.

Optimisation of recombinant production of active human cardiac SERCA2a ATPase

Methods for recombinant production of eukaryotic membrane proteins, yielding sufficient quantity and quality of protein for structural biology, remain a challenge. We describe here, expression and purification optimisation of the human SERCA2a cardiac isoform of Ca2+ translocating ATPase, using Saccharomyces cerevisiae as the heterologous expression system of choice. Two different expression vectors were utilised, allowing expression of C-terminal fusion proteins with a biotinylation domain or a GFP- His8 tag. Solubilised membrane fractions containing the protein of interest were purified onto Streptavidin-Sepharose, Ni-NTA or Talon resin, depending on the fusion tag present. Biotinylated protein was detected using specific antibody directed against SERCA2 and, advantageously, GFP-His8 fusion protein was easily traced during the purification steps using in-gel fluorescence. Importantly, talon resin affinity purification proved more specific than Ni-NTA resin for the GFP-His8 tagged protein, providing better separation of oligomers present, during size exclusion chromatography. The optimised method for expression and purification of human cardiac SERCA2a reported herein, yields purified protein (> 90%) that displays a calcium-dependent thapsigargin-sensitive activity and is suitable for further biophysical, structural and physiological studies. This work provides support for the use of Saccharomyces cerevisiae as a suitable expression system for recombinant production of multi-domain eukaryotic membrane proteins.

Probing the functionalization of gold surfaces and protein adsorption by PM-IRRAS

The control of morphology and coating of metal surfaces is essential for a number of organic electronic devices including photovoltaic cells and sensors. In this study, we monitor the functionalization of gold surfaces with 11-mercaptoundecanoic acid (MUA, HS(CH(2))(10)CO(2)H) and cysteamine, aiming at passivating the surfaces for application in surface plasmon resonance (SPR) biosensors. Using polarization-modulated infrared reflection-absorption spectroscopy (PM-IRRAS), cyclic voltammetry, atomic force microscopy and quartz crystal microbalance, we observed a time-dependent organization process of the adsorbed MUA monolayer with alkyl chains perpendicular to the gold surface. Such optimized condition for surface passivation was obtained with a systematic search for experimental parameters leading to the lowest electrochemical signal of the functionalized gold electrode. The ability to build supramolecular architectures was also confirmed by detecting with PM-IRRAS the adsorption of streptavidin on the MUA-functionalized gold. As the approaches used for surface functionalization and its verification with PM-IRRAS are generic, one may now envisage monitoring the fabrication of tailored electrodes for a variety of applications.