Label-independent detection

Label-independent methods such as surface plasmon resonance (SPR) can overcome variations caused by inconsistencies in labeling chemistries that are often seen in label-dependent detection systems. SPR has been used to measure affinities... 

Tosar, J.P., Keel, K. and Laiz, J. (2009) Two independent label-free detection methods in one electrochemical DNA sensor. Biosens. Bioelectron., 24 (10), 3036-3042. 

The ability of SPR to probe both kinetic and thermodynamic processes, as well as to provide micro-structural information, make it a very important component of the experimental methodology available to probe molecular interactions occurring at surfaces. Furthermore, it allows some of the limitations of other techniques to be overcome. For example, other methods often require one of the partners to be labelled in some way in order to allow it to be detected. Fluorescent probes, radioactive labels, and attachment of independently detectable molecules (e.g. enzymes) have all been used for this purpose. These suffer from the drawback that they may interfere with the binding of the labelled partner to the unlabelled one, or cause unwanted structural perturbations. SPR observations can be based solely on the dielectric properties of molecules, or their intrinsic light absorption characteristics, and thus require no specific labelling. 

Ethanolamine ammonia lyase has a molecular weight of 520,000 and consists of 8 or 10 subunits. Two 5 -deoxyadenosylcobalamin molecular bind per enzyme molecule, and recent kinetic studies by Babior show that these two molecules carry out catalysis independently. Evidence is available that this enzyme functions by a radical mechanism since both spin labeling and Co(II) esr experiments indicate that Co(II) is an intermediate during H-transfer. Also, 5 -deoxyadenosine has been detected as a product of oxygenation of the enzyme-substrate complex (99—101). 

Biomolecular MS and in particular MALDI-TOF-MS (see Sections 2.1.22 and 2.2.1) permit the routine analysis of oligonucleotides up to 70-mers, intact nucleic acids, and the direct detection of DNA products with no primer labels with an increase in analysis speed and mass accuracy especially in contrast to traditional DNA separation techniques such as slab gels or capillary electrophoresis. Applications focus on the characterization of single nucleotide polymorphisms (SNPs) and short tandem repeats (STRs). Precise and accurate gene expression measurements show relative and absolute numbers of target molecules determined independently of the number of PCR cycles. DNA methylation can be studied quantitatively. 

The binding of the antigen and antibody can be affected by several factors, including the conjugated label chosen for detection and the method used to conjugate the label, as well as the assay format itself. The selectivity of the ELISA can be affected by the assay format. In an ELISA with a two-site sandwich format, independent epitopes are bound by different antibodies.26 The specificity comes from multiple site recognition. Polyclonal antibodies can react with many epitopes on a complex antigen surface.24... 

Photons with detectable energy differences that are emitted by various radionuclides can be quantified simultaneously, but independently from each other. This allows the use of dual-labeling approaches (4). These experiments will reveal information regarding both the liposomal carrier—labeled with one radionuclide—and the encapsulated compound—labeled with a different radionuclide—after a single injection in the same animal. However,... 

Considerable international effort has since been directed towards these goals and substantial progress has been made in a number of directions. The development of accepted detection procedures not only gives enforcement authorities the ability to check that products are correctly labelled but also gives the consumer confidence that adequate independent controls are available. Perhaps the detection method that is, to date, the most internationally accepted is Electron Paramagnetic Resonance (EPR) spectroscopy which can also be referred to as Electron Spin Resonance (ESR) spectroscopy. 

How may we distinguish between pathways that involve external attack by hydroxide and those that involve co-ordinated hydroxide There is a considerable accumulation of data that suggest the two latter pathways are the most important (i.e., attack of external hydroxide upon monodentate amino acid ester is not greatly accelerated). The attack by external hydroxide may be studied independently and accurate rate constants may be determined for insertion in the composite rate equation with the two competing processes. In some cases it is possible to detect the five-co-ordinate and the other intermediates. Finally, some elegant labelling studies have provided very strong evidence for the exi-... 

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