Bond elasticity controls molecular recognition specificity in antibody-antigen binding.

Force-spectroscopy experiments make it possible to characterize single ligand-receptor pairs. Here we measure the spectrum of bond strengths and flexibilities in antibody-antigen interactions using optical tweezers. We characterize the mechanical evolution of polyclonal antibodies generated under infection and the ability of a monoclonal antibody to cross-react against different antigens. Our results suggest that bond flexibility plays a major role in remodeling antibody-antigen bonds in order to improve recognition during the maturation of the humoral immune system.

A novel free radical scavenger rescues retinal cells in vivo.

The benzopyran BP (3,4-dihydro-6-hydroxy-7-methoxy-2,2-dimethyl-1(2H)-benzopyran) is a free radical scavenger that is structurally similar to alpha-tocopherol and has provided neuro-protection in a number of disease models where oxidative stress is a causative factor. A novel derivative of BP with improved lipid solubility, which we have designated BP3, was synthesized and its neuro-protective efficacy subsequently analyzed in three mouse models of retinal disease in vivo. In the acute light damage model, balb/c mice received a single intra-peritoneal injection (200 mg/kg) of BP3 one hour prior to phototoxicity, reducing photoreceptor degeneration for up to 48 h post insult. In the rd10/rd10 mouse, a chronic model of inherited retinal degeneration, systemic dosing with BP3 on alternate days between post-natal day 18 and 25 preserved rod photoreceptor numbers and cone photoreceptor morphology. Finally, NMDA induced toxicity in retinal ganglion cells was diminished for at least 72 h after the initial insult by a single dose of BP3. In each disease model, BP3 alleviated cellular oxidative burden as MDA levels were markedly reduced. These results demonstrate that systemically administered BP3 has potent free radical scavenging capacity in the retina and may represent a single therapeutic strategy applicable across several retinopathies.

Quantum dot-based array for sensitive detection of Escherichia coli.

A fluorescent quantum dot-based antibody array, used in sandwich format, has been developed to detect Escherichia coli O157:H7. Numerous parameters such as solid support, optimal concentration of immunoreagents, blocking reagents, and assay time were optimized for array construction. Quantum dot-conjugated anti-IgG was used as the detecting system. The array allows the detection of E. coli O157:H7 at concentrations below 10 CFU mL(-1) without sample enrichment, exhibiting an increase of three orders of magnitude in the limit of detection compared to ELISA. The interference caused by Gram (+) and Gram (-) bacteria was negligible at low concentrations of bacteria.

Multifunctional nanoparticles--properties and prospects for their use in human medicine.

A major aim of medicine has long been the early and accurate diagnosis of clinical conditions, providing an efficient treatment without secondary effects. With the emergence of nanotechnology, the achievement of this goal seems closer than ever. To this end, the development of novel materials and devices operating at the nanoscale range, such as nanoparticles, provides new and powerful tools for imaging, diagnosis and therapy. This review focuses on the significant improvements in performance that nanoparticles offer compared with existing technologies relevant to medicine. Specifically, we address the design of multifunctional nanoparticles as an alternative system for drug and gene delivery, which has great potential for therapy in areas, such as cancer and neuropathologies. Moreover, we discuss the controversy generated by the possible toxic health effects of nanoparticles.

Determination of haloanisols in white wine by immunosorbent solid-phase extraction followed by enzyme-linked immunosorbent assay.

A high through-put screening immunochemical method to control the presence of 2,4,6-trichloroanisol (TCA) and 2,4,6-tribromoanisol (TBA), the main agents responsible for the musty odor in wine samples, has been developed. The method involves a selective (antibody-antigen) solid-phase extraction (SPE), followed by enzyme-linked immunosorbent assay (ELISA) analysis. The sample preparation method established uses for immunosorbents (ISs) prepared by covalently coupling antibodies developed for TCA on a sepharose support. At present, about 200-400 ng L-1 of TBA and TCA can be detected in white wine samples by the IS-SPE-ELISA method described here without any preconcentration step. Simultaneous analyses of many samples are possible with this method. Related chloroanisoles (2,3- and 2,6-dichloroanisols and 2,3,4,5-tetrachloroanisol) and chlorophenols (2,3,4,6-tetrachlorophenol and pentachlorophenol) usually present in contaminated wine samples are also effectively retained by the IS, although only 2,4,6-TCA and 2,4,6-TBA are detected by the ELISA used. The immunopurification procedure developed could also be useful as a selective cleanup method prior to chromatographic analysis.

Immunochemical determination of 2,4,6-trichloroanisole as the responsible agent for the musty odor in foods. 1. Molecular modeling studies for antibody production.

Nine antisera have been raised against 2,4,6-trichloroanisole (2,4,6-TCA) by immunizing them with three different haptens. With the spacer arm at the meta position, hapten A (3-(2,4,6-trichloro-3-methoxyphenyl)propanoic acid) preserved all of the functional groups of the target analyte. In hapten B (5-(2,4,6-trichlorophenoxy)pentanoic acid), the spacer was placed in the molecule substituting the methoxy group. Finally, hapten C (3-(3,5-dichloro-4-methoxyphenyl)propanoic acid) held the spacer arm at the para position instead of the chlorine atom of the target analyte. Using theoretical models, we have studied how the molecular geometry and the electronic distribution are affected by the introduction of the linker. The evaluation of the avidity of the resulting antibodies demonstrates that the orientation produced by the spacer arm must also be considered an essential aspect. The screening for competitive assays performed after synthesizing a battery of heterologous competitors has provided with these antibodies eight indirect enzyme-linked immunosorbent assays with acceptable properties. From the number of assays obtained, their maximal absorbance, their signal-to-noise ratio, the slope, and the IC(50) values obtained, it can be concluded that hapten C provided the best antibodies.

Immunochemical determination of 2,4,6-trichloroanisole as the responsible agent for the musty odor in foods. 2. Immunoassay evaluation.

Immunoassays for 2,4,6-trichloroanisol (TCA) have been evaluated. The assays were developed after raising antibodies against three different immunizing haptens (1). Lack of reproducibility has been one of the main problems of these assays. Precision was worse on these assays, reaching lower limits of detection. The high lipophilicity of TCA and its, consequently, low water solubility have been found to be the major cause of this problem. A reliable microplate-based enzyme-linked immunosorbent assay (ELISA) has been set after consideration of the TCA physicochemical features and evaluation of important parameters affecting immunoassay performance. The immunoassay uses As78 (developed against hapten B-KLH) and C9-OVA as the coating antigen. The selectivity is high although the brominated analogue 2,4,6-TBA is also recognized. In buffered media containing 7% ethanol, the resulting assay shows a good accuracy with an IC(50) value of 0.53 microgram L(-)(1) and a limit of detection of 0.044 microgram L(-)(1). Red and white wine samples caused important interferences in the immunoassay demonstrating the necessity of a cleanup procedure prior to the ELISA.

Preparation of antibodies and development of an enzyme-linked immunosorbent assay for determination of dealkylated hydroxytriazines.

The development of an indirect competitive enzyme-linked immunosorbent assay (ELISA) for dealkylated hydroxytriazines is reported here for the first time. The assay uses polyclonal antibodies raised against N-(4-amine-6-hydroxy-[1,3,5]triazin-2-yl)-4-aminobutanoic acid (hapten 2g) conjugated to keyhole limpet hemocyanin by the active ester method. The immunizing hapten was synthesized by first introducing the amino group to the triazine ring in a protected form in order to increase its solubility in organic media. Subsequent steps consisted of reacting this compound with an appropriate spacer arm, followed by removal of the protecting group in acidic media. The resulting assay uses a homologous competitor hapten coupled to conalbumin by the mixed anhydride method. Coating antigens prepared using a homologous covalent coupling procedure failed to produce competitive immunoassays. The assay tolerates media with high ionic strength (up to 70 mS cm(-)(1)) and basic pH values (7.5-9.5 units). Under the optimized conditions, this ELISA is specific for dealkylated hydroxytriazines, reaching suitable limits of detection.

Development of an immunoassay for terbutryn: study of the influence of the immunization protocol.

Heterologous immunization has proven to be useful to enhance the selectivity and specificity of catalytic antibodies. However, in the field of immunoassays, few studies have been done to establish how the immunization protocol influences the antibody characteristics. In the present study, we have developed an enzyme-linked immunosorbent assay (ELISA) for the detection of the pesticide terbutryn following a homologous and a heterologous immunization strategy. No significant differences have been observed between the immunization procedures regarding immunoassay sensitivity and selectivity. Thus, immunoassays with a limit of detection below the 25 ng/l established by current European regulations have been obtained with both immunization protocols. Initial studies have been performed to assess the applicability of these ELISAs to the analysis of real water matrixes.

Development of a cellular biosensor for the detection of 2,4,6-trichloroanisole (TCA).

2,4,6-trichloroanisole (TCA) is a microbial metabolite formed from chlorophenols through the activity of several natural fungal strains present on the cork oak bark. TCA is the primary compound responsible for the mousty/mould off-odour known as "cork taint" present in cork stoppers, wine, water and alcoholic beverages. Chromatographic and electrochemical methods are currently used for the determination of TCA, however its detection at low concentrations remains a technical challenge. The aim of this study was the development of a rapid novel biosensor system based on the Bioelectric Recognition Assay (BERA). The sensor measured the electric response of cultured membrane-engineered fibroblast cells suspended in an alginate gel matrix due to the change of their membrane potential in the presence of the analyte. Membrane-engineered cells were prepared by osmotic insertion of 0.5 µg/l of specific TCA antibodies into the membrane of the cells. The BERA-based sensor was able to detect TCA in a few minutes (3-5 min) at extremely low concentrations (10(-1)ppt), thus demonstrating higher sensitivity than the human sensory threshold. In addition, the assay was quite selective against other haloanisoles and halophenols structurally related to or co-occurring with TCA. Finally the sensor was tested against real white wine samples from cork soaks. At this real test, the BERA sensor was able to detect TCA from cork soaks rapidly (3-5 min) at very low concentrations (1.02-12 ng/l), covering the whole range for the detection threshold for wines (1.4-10 ng/l). Therefore, this novel biosensor offers new perspectives for ultra-rapid, ultra-sensitive and low-cost monitoring of TCA presence in cork and wine and possibly also other food commodities.

Development of an enzyme-linked immunosorbent assay for determination of the miticide bromopropylate.

This paper reports for the first time the development of an immunoassay for the analysis of the miticide bromopropylate (BP). The chemical structure of the immunizing haptens was designed to maximize the recognition of the bis-bromophenyl group of BP. Thus, the assay uses polyclonal antibodies raised against 2,2-bis(4-bromophenyl)-N-2-hydroxyacetamide-butanoic acid (hapten 2) conjugated to keyhole limpet hemocyanin from horseshoe crab. A heterologous indirect competitive enzyme-linked immunosorbent assay (ELISA) has been developed that can detect BP down to 0.14 microg L(-1). The assay has been proven to tolerate a wide range of ionic strengths and pH values. Studies on the selectivity of this immunoassay have demonstrated a high recognition of related pesticides that contain a bis-halophenyl group in their structure. Other pesticides do not interfere in the analysis of BP using this immunochemical technique. Preliminary experiments have shown that BP can be directly analyzed in white wine samples down to 0.16 microg L(-1) without the necessity of a cleanup procedure prior to the ELISA.

Ceramide is the key mediator of oxidative stress-induced apoptosis in retinal photoreceptor cells.

Nitric oxide and reactive oxygen species play a critical role in photoreceptor apoptosis. However, the exact molecular mechanisms triggered by oxidative stress in photoreceptor cell death remain undefined. Here, we demonstrate that the sphingolipid ceramide is the key mediator of oxidative stress-induced apoptosis in 661W retinal photoreceptor cells. Treatment of 661W cells with the nitric oxide donor, sodium nitroprusside, activates acid sphingomyelinase. As a result, sphingomyelin is hydrolysed, which leads to an increase in the concentration of ceramide. We also show that ceramide is responsible for the activation of the mitochondrial apoptotic pathway in 661W photoreceptor cells and subsequent activation of the caspase cascade. Furthermore, we show for the first time that ceramide is responsible for the increased Ca2+ levels in the mitochondria and cytosol that precedes activation of the calpain-mediated apoptotic pathway. Additionally, we provide evidence that ceramide also activates the endolysosomal protease cathepsin D pathway. In summary, our findings show that ceramide controls the cell death decisions in photoreceptor cells and highlight the relevance of acid sphingomyelinase as a potential therapeutic target for the treatment of retinal pathologies.

Multiplex analysis with peptide-encoded beads.

Paramagnetic beads have considerable potential as identification tags in biological analysis. For example, magnetic sensor-based arrays using the magnetic field generated by paramagnetic beads to test hybridization between interacting molecules have attracted widespread interest in recent years. However, application of paramagnetic beads as identification tags is still limited, since they do not permit differentiation between samples for multiplex analysis. Here, we report the application of a novel encoding of paramagnetic beads with peptide sequences. This strategy allows DNA samples labeled with peptide-encoded paramagnetic beads to be identified by the selective enzymatic cleavage of each peptide cross-linker.

The radical scavenger CR-6 protects SH-SY5Y neuroblastoma cells from oxidative stress-induced apoptosis: effect on survival pathways.

Reactive oxygen species (ROS) and oxidative stress have long been linked to cell death of neurons in many neurodegenerative conditions. However, the exact molecular mechanisms triggered by oxidative stress in neurodegeneration are at present unclear. In the current work we have used the human neuroblastoma SH-SY5Y cell line as a model for studying the molecular events occurring after inducing apoptosis with H2O2. We show that treatment of SH-SY5Y cells with H2O2 up-regulates survival pathways during early stages of apoptosis. Subsequently, the decline of anti-apoptotic protein levels leads to the activation of the calcium-dependent proteases calpains and the cysteine proteases caspases. Additionally, we demonstrate that CR-6 (3,4-dihydro-6-hydroxy-7-methoxy-2,2-dimethyl-1(2H)-benzopyran) acts as a scavenger of ROS and prevents apoptosis by enhancing and prolonging up-regulation of survival pathways. Furthermore, we show that pre-treatment of SH-SY5Y cells with a cocktail containing CR-6, the pan-caspase inhibitor zVAD-fmk (zVal-Ala-Asp-fluoro-methylketone) and the calpain inhibitor SJA6017 confers almost total protection against apoptosis. In summary, the present work characterizes the molecular mechanisms involved in oxidative stress-induced apoptosis in SH-SY5Y cells. Our findings highlight the relevance of CR-6, alone or in combination with other drugs, as potential therapeutic strategy for the treatment of neurodegenerative diseases.

Oxidative stress-induced apoptosis in retinal photoreceptor cells is mediated by calpains and caspases and blocked by the oxygen radical scavenger CR-6.

A critical role for reactive oxygen species (ROS) in photoreceptor apoptosis has been established. However, the exact molecular mechanisms triggered by oxidative stress in photoreceptor cell death remain undefined. This study delineates the molecular events that occur after treatment of the photoreceptor cell line 661W with the nitric oxide donor sodium nitroprusside (SNP). Cytosolic calcium levels increased during photoreceptor apoptosis, leading to activation of the calcium-dependent proteases calpains. Furthermore, caspase activation also occurred following SNP insult. However, although treatment with the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone inhibited caspase activity per se in SNP-treated 661W cells, it did not prevent apoptosis. On the other hand, CR-6 (3,4-dihydro-6-hydroxy-7-methoxy-2,2-dimethyl-1(2H)-benzopyran) acted as a scavenger of ROS and reduced 661W photoreceptor apoptosis induced by SNP by preventing the activation of a pathway in which calpains have a key role. In summary, we report for the first time that both caspases and calpains are involved in 661W photoreceptor apoptosis and that calpain activation can be prevented by the ROS scavenger CR-6.