Direct-indirect detection systems

Detection systems may be divided into radioactive/non-radioactive (Table I) and direct/indirect detection systems. 

Direct/Indirect Detection Systems. Direct detection of molecular probes requires physical attachment of the detection system component to the probe, such that binding of the probe to its target molecule results in immediate attachment of the detection system to the target-probe complex. In contrast, indirect detection requires that further separate steps be performed to identify target-probe complexes after they are formed. Direct and indirect target-probe-... 

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... 

While enzymes may be covalently attached directly to primary probe molecules, as noted above for reasons of reagent versatility, steric factors, and potential signal amplification, indirect detection systems appear to be the more popular. Consequently, enzyme-probe conjugates are typically complexes of a desired enzyme marker and a secondary level probe that is, a probe molecule that can specifically identify a primary level probe molecule, such as an alkaline phosphatase-streptavidin conjugate can identify a biotinylated nucleic acid probe by virtue of the binding affinity between streptavidin and biotin. Other examples of enzyme-probe systems are given in the preceding section on direct and indirect detection systems. 

In recent years, CE has been successfully applied in the field of biochemical and analytical chemistry. It has been found to be attractive for pharmaceutical analysis because of its advantages related to excellent separation efficiency, high mass sensitivity, minimal use of samples and solvents, and the possibility of using different direct and indirect detection systems. This review focuses on analytical assays for barbiturates by CE. 

Many transition metal complexes have been considered as synzymes for superoxide anion dismutation and activity as SOD mimics. The stability and toxicity of any metal complex intended for pharmaceutical application is of paramount concern, and the complex must also be determined to be truly catalytic for superoxide ion dismutation. Because the catalytic activity of SOD1, for instance, is essentially diffusion-controlled with rates of 2 x 1 () M 1 s 1, fast analytic techniques must be used to directly measure the decay of superoxide anion in testing complexes as SOD mimics. One needs to distinguish between the uncatalyzed stoichiometric decay of the superoxide anion (second-order kinetic behavior) and true catalytic SOD dismutation (first-order behavior with [O ] [synzyme] and many turnovers of SOD mimic catalytic behavior). Indirect detection methods such as those in which a steady-state concentration of superoxide anion is generated from a xanthine/xanthine oxidase system will not measure catalytic synzyme behavior but instead will evaluate the potential SOD mimic as a stoichiometric superoxide scavenger. Two methodologies, stopped-flow kinetic analysis and pulse radiolysis, are fast methods that will measure SOD mimic catalytic behavior. These methods are briefly described in reference 11 and in Section 3.7.2 of Chapter 3. 

As was mentioned previously, photometric detection is the most frequently applied detection technique. Most of the commercial CE-systems are equipped with at least a UV detector. Some compounds, such as low molecular weight organic and inorganic ions [57-60], do not produce a direct analytical signal. In such cases indirect detection, by indirect UV or fluorescence [59-64] is applied. Besides photometric detection, an application of indirect amperometric [65] detection was also reported. When the analytical signal results from a decrease in... 

Detection systems most often used to analyse LAS and their metabolites or by-products included direct or indirect UV and FL. Kikuchi et al. [32] found that in LAS determination, the chromatograms... 

For the cationic surfactants, the available HPLC detection methods involve direct UV (for cationics with chromophores, such as benzylalkyl-dimethyl ammonium salts) or for compounds that lack UV absorbance, indirect photometry in conjunction with a post-column addition of bromophenol blue or other anionic dye [49], refractive index [50,51], conductivity detection [47,52] and fluorescence combined with postcolumn addition of the ion-pair [53] were used. These modes of detection, limited to isocratic elution, are not totally satisfactory for the separation of quaternary compounds with a wide range of molecular weights. Thus, to overcome the limitation of other detection systems, the ELS detector has been introduced as a universal detector compatible with gradient elution [45]. 

As previously mentioned, the principles of staining are identical to those used in paraffin sections. The techniques used may be direct or indirect as described in Chapters 15-18. Indirect techniques are generally more sensitive and, therefore, preferable. Indirect techniques can be broken down into three steps. In the first step, an antibody directed against the antigen of interest is applied to the tissue section. In the second step, a labeled secondary antibody directed against the first antibody is applied. The last step consists of a detection step that is composed of linking the secondary antibody to a detection system and... 

Besides the commonly used direct LIF detection, indirect LIF detection on the microchip has also been reported. This method has been employed to detect explosives in spiked soil samples (see Figure 7.9) [620]. In contrast to a capillary-based system, an increase in E from 185-370 V/cm for MEKC separation did not result in an unstable background fluorescence due to excessive loule heating. This was probably because of the effective heat dissipation in the glass chip. However, upon multiple injection, it was found that the detection sensitivity decreased, which might be caused by the degradation of the visualizing dye (Cy7) [620]. Indirect LIF also allows the detection of unlabeled amino acids [683]. 

If a solute of interest does not contain a chromophore, it may be detected by indirect UV detection. Indirect detection is a technically simple and sensitive method for the detection of compounds with little inherent detector response. Indirect UV detection is a nondestructive technique, in which the analyte is characterized in native form. Indirect detection is a universal detection mode, with few requirements as to the exact nature of the analyte. The properties of indirect detection have been reviewed by Yeung.22 Indirect detection is particularly attractive for the analysis of biological compounds. Optical systems are the same for direct or indirect detection the only difference is that, in indirect detection, the mobile phase, rather than the analyte, contains a UV chromophore. 

Multi-step technique (3) This is an indirect/direct method combining unlabeled primary antibodies with directly-conjugated antibodies. The method starts with staining the unlabeled antibody/antibodies with the appropriate detection system, but without performing the final enzymatic staining reaction. The tissue is blocked with normal serum from the host of the first primary antibody before the second, directly-labeled primary antibody is added. The staining ends with the two enzymatic reactions being performed sequentially. 

Indirect detection does require more steps, but oftentimes yields amplified signals relative to direct methods because layering of bridging molecules may increase the number of detector molecules per probe molecule. It is probably this bridging/amplification technique that has allowed current enzyme detection systems to approach the sensitivity of radiolabeled systems. The use of these indirect methods reduces steric problems that might arise from having enzyme molecules directly bound to probe molecules. 

A summary of various clashes of radicals has been given above. Obviously many radicals have not been directly specified but, hopefully, those of biological importance have largely been covered. In the following chapters many points raised herein are elaborated. In Chapter 2, we cover different ways in which radicals are generated, and ways in which biological systems protect themselves from radical induced damage. In Chapter 3, methods of direct and indirect detection of radical intermediates are outlined. 

In response to the need for rapid and simple methods for the detection of short-chain organic acids in complex matrices, a technique was developed that very well suited the specific purpose of CE. This method employed indirect detection and a surfactant to reverse electro-osmotic flow. Direct UV detection provides higher sensitivity and precision and the ability to detect UV-absorbing organic acids, such as oxalic, fumaric, or pyroglutamic acids, which cannot be detected with the indirect system (Galli and Barbas, 2004). 

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