Label-Based Indirect Detection

On the other hand, organic dyes in combination with CNTs are also used in electrode fabrication and hybridization techniques for developing an ultrasensitive, selective, and miniaturized electrochemical DNA biosensor for quick and reliable DNA sequence analysis. CNTs have good biocompatibility which allows them to increase the attached DNA amount on the substrate surface, thanks to their high surface area, an enhanced electronic conductivity, and a high mechanical resistance in electroanalytical chemistry can accelerate the electron-transfer rate between the redox active ssDNA molecule and electrode. 

Li and coauthors [40] have immobilized a chitosan film doped with CNTs onto the graphite electrode and co-immobilized fish sperm DNA for the detection of salmon sperm. They employed MB as a redox-active indicator for the electrochemically quantitative 

An electrochemical DNA biosensor has been reported by Tichoniuk et al. [41] for the discrimination between ss- and dsDNA, and the voltammetric detection of target NA fragments typical of the aerolysin gene for the detection of Aeromonas hydrophila in food. 

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In label-based (indirect) detection, an electroactive hybridisation indicator binds ssDNA and dsDNA with different affinities, resulting in an unequal concentration in electrode surface, resulting in a change of the electrochemical signal. ... 

Common haptens used for labeling DNA probes for BISH assays are biotin, DIG, DNP, FITC, and Texas Red. Based on the size of your DNA targets, you may choose from a direct detection or an indirect detection for BISH assays. In general, an indirect detection system can provide better sensitivity compared to a direct detection system. For an indirect detection, you need to select a combination of two antibodies raised with two different animal species, such as a mouse anti-DIG antibody and a rabbit anti-DNP antibody, so that enzyme-labeled anti-mouse antibody and anti-rabbit antibody can be applied for signal detection. If a direct BISH detection is going to be applied, anti-hapten antibodies raised in the same animal species that are labeled with either AP or HRP enzyme molecules... 

Immunoassays, electrochemical — A quantitative or qualitative assay based on the highly selective antibody-antigen binding and electrochemical detection. Poten-tiometric, capacitive, and voltammetric methods are used to detect the immunoreaction, either directly without a label or indirectly with a label compound. The majority of electrochemical immunoassays are based on -> voltammetry (-> amperometry) and detection of redox-active or enzyme labels of one of the immunochemical reaction partners. The assay formats are competitive and noncompetitive (see also -> ELISA). 

Bound fraction Antigen/competitor present as a complex, attached to the corresponding antibody the fi-action of the reaction mixture which contains the antigen-antibody complex Carrier protein Large non-immunogenic protein that, when coupled to a hapten, induces production of anti-hapten antibodies in an animal Competitive assay Immunoassay based on the principle of competition between the analyte in the sample ( unknown ) and competitor Competitor Antigen derivative that is modified (i.e., labelled, coupled to a large protein) and serves for indirect detection of the analsde in competitive immunoassays... 

A new element for simultaneous indirect detection of C and signals in labelled proteins was proposed by Uhrin et The CT- CHs, VT- N-HSQC sequence combines constant-time carbon evolution with variable delay nitrogen evolution. This is achieved by the variation of the position of a 90° pulse creating transverse coherence within the C constant-time period. The maximum indirect acquisition time for both nuclei is determined by constant-time period set to l/ /(C,C) = 28.6 ms. The method is best suited for detection of CH3 signals due to their slower relaxation. The proposed element was incorporated into NOESY-based experiments resulting in 3D NOESY-CH3NH and 3D HSQC-NOESY-CH3NH sequences. The experiments... 

The requirements for SNP discovery- and SNP typing (or scoring) technologies are quite different. All of the above-mentioned methods for SNP discovery have merits. For large scale SNP scoring projects, however, they will fail to be efficient. The reasons are a lack of reasonable automation and an inherent susceptibility to errors. The first issue is mainly due to gel electrophoresis technology which is cumbersome to automate. Another issue is caused by indirect detection methods based on labels. 

The rapid expansion of lectin-based applications for the detection and quantification of glycoconjugates has been led by the development of commercially available, purified and chemically derivatized lectins, and in some cases, anti-lectin antibodies. Over 50 purified plant lectins are sold commercially by a number of producers and vendors, with this number growing annually. Equally important is the ease by which investigators can obtain lectins labeled with various fluorescent dyes, haptenic moieties, biotin, and radioactive atoms, as well as conjugated to enzymes and solid-phase supports. These derivatized lectins are useful for either direct or indirect detection and quantification techniques, or for the physical separation of particulate-bound or soluble glycoconjugates. Table 4 lists many of the commercially available lectin reagents and sources. 

Direct detection biosensors utilize direct measurement of the biological interaction. Such detectors typically measure physical changes (e.g., changes in optical, mechanical, or electrical properties) induced by the biological interaction, and they do not require labeling (i.e., label free) for detection. Direct biosensors can also be used in an indirect mode, typically to increase their sensitivity. Direct detection systems include optical-based systems (most common being surface plasmon resonance) and mechanical systems such as quartz crystal resonators. 

A novel approach based on the application of solid-state NMR spectroscopy has been reported that permits the rapid determination of 3D molecular structure with a single uniformly isotope labelled sample. Analogy with the solution NMR spectroscopic investigations is used, which rely on the detection of short distances between hydrogen atoms providing the principal source of information about the 3D fold of the protein. Since the 2D H, H-correlation methods are of limited use for solid-state NMR spectroscopy due to the restricted spectral resolution, the indirect detection and structural analysis of interactions via C, C-correlation spectroscopy is proposed. It has been shown that combined with dihedral-angle constraints, which can be derived from conformation-dependent chemical shifts, the characterisation of the 3D molecular structure from a single protein sample becomes possible. The new approach has been demonstrated on kaliotoxin, a 38-residue peptide. 

Chromatographic (or flow-injection) immimoassays represent another important mode of lAC. This technique utilizes immobilized antibodies, or antigens, in a column to perform various competitive or noncompetitive immunoassays. Detection in these methods involves the use of a labeled antibody or labeled analyte analog (known as the label) to indirectly measure the amount of a target in a sample, as demonstrated in Figure 1.2. The detection format may be based on absorbance. 

Broadly speaking, CLEM can be obtained with two main types oflabeling approaches the combinatorial and the non-combinatorial. Combinatorial labeling approaches use markers that are directly visible across two or more different types of microscopies, e.g., FluoroNanogold , LY or GFP+photo-oxidation, quantum dots, etc. Non-combinatorial approaches are based on an indirect cross-correlation between the information obtained at LM level by, e.g., IMF, fluorescent dyes, or RFPs and EM data [146, 147]. An example of the non-combinatorial approach is the use of anti-LY or anti-GFP antibodies to localize by EM ICC the sites of sub-cellular distribution of LY or GFP instead of using photo-oxidation to precipitate an electron-dense DAB label for subsequent detection by TEM [148]. 

Pork is the product of a very complex process. All the various characteristics of pork quality cannot be assessed directly in each carcass because these measurements and assessments would be too expensive. Therefore, previous scientific quality assessment of meat is primarily an indirect approach based on a few easily detectable quantitative traits and on the prescription of minimal standards in relation to the product in terms of size or composition and in relation to the production process. The prescriptions and the exclusion criteria vary between countries or between labelling programmes. The most encompassing prescriptions are enshrined in the EC regulation on organic livestock production (EEC No. 2092/91). Owing to this approach, extreme deviations in quality traits and deleterious effects are prevented. However, there is still space left within these framework conditions for huge variability in pork quality. 

Methods for the Detection of Antigens/Antibodies Equilibrium and kinetic inhibition assays based upon fluorescence polarization, 70, 3 fluorescence excitation transfer immunoassay (FETI), 70, 28 indirect quenching fluoroimmunoassay, 70, 60 the homogeneous substrate-labeled fluorescent immunoassay, 70, 79 fluorescence immunoassays using plane surface solid phases (FIAPS), 70, 87. 

Indirect methods require the use of labeled compounds (tracers) or coupled reactions to detect binding events. The first indirect assays were based on... 

Like in RILAs, an advantage of fluorescence detection is the possibility of developing homogeneous FILAs using direct or indirect (competitive or displacement) approaches. The fluorescence polarization immunoassay (PFIA) and their homolog fluorescence polarization immuno-like assay (PFILA) are two of the most widely used procedures in homogeneous fluoroassays. Both are based on the principle that fluorescence polarization gives a direct measure of the bound/free ratio of the labeled analyte (tracer) without the need for their separation [23, 28]. 

Although multistep affinity assays with redox-labeled targets have been described (Wang et al. [117]), most of the assays use enzyme-labeled species in conventional indirect formats (competitive, non-competitive). Direct EILAs based on multistep electrochemical affinity assays have also been developed with excellent results. In all these cases the MIP is used to extract the analyte from the sample and, after elution, the analyte is carried on to the electrochemical flow-through cell for being detected. 

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