Indirect assays

The indirect assays are divided in a system generating the superoxide anion and an indicator reaction. The many sources for as well as the indicator systems are listed in Table 4  

Out of multifarious possible test systems three will be described in short. At present these assays are most frequently used. 

The reduction of ferricytochrome c by superoxide was firstly described by McCord and Fridovich Upon reduction, the extinction of the a-band of cytochrome c at 550 nm is increased. Usually the xanthine oxidase/xanthine system is employai for producing the superoxide anion. Superoxide dismutases compete with cytochrome c for the substrate. The assay is executed in the following way  

If ferricytochrome c is added under these conditions a burst of reduction can be seen at 550 nm. In the presence of superoxide dismutase the burst is diminished. The assay is accomplished in the following manner Ferricytochrome c is added at zero time and after ten minutes to the reaction mixture containing xanthine oxidase and xanthine. Aj, (zerotime) is subtracted from Ajjq (10 minutes). Using this test system, SOD in the picomolar range of concentration can be detected. 

Activity staining for SOD is carried out in the following way After electrophoresis the gels are immediately soacked in 2,45 x 10 M nitroblue tetrazolium for 20 minutes, followed by an immersion for 15 minutes in a solution of 28 mM TEMED (tetramethylethylendiamine) and 2,8 x 10 riboflavin in 36 mM potassium phosphate pH 7.8. Finally the gels are exposed to an UV-light source and illuminated for 15 minutes. Superoxide dismutase bands appear as colourless zones on the blue gels. 

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Nasmyth Cytologically we don t see it associated with chromosomes. From an indirect assay, if we isolate yeast chromosomes there is Espl there. We have a biological assay for that. 

The techniques developed in enzyme immobilization have facilitated the development of enzyme electrodes and of novel enzyme -based, automated, analytical methods (l6,17,l8). Enzyme electrodes have resulted from the combination of an enzyme membrane and an ion-selective electrode they were used successfully to assay directly appropriate substrates. Enzyme columns or enzyme tubes, prepared in a conventional manner, were used as a specific auxiliary component in the indirect assay of substrates in many of the novel automated analytical procedures. 

In this context, one has to consider that these types of indirect assays are subjected to many variables that are difficult to control and interpretation of results is somewhat limited. Artefacts due to biological components like enzymes or cytochrome c within these assays may falsify the observed activities, and other possible... 

Stopped-flow measurements with superoxide in aqueous solution at physiological pH are not possible due to its fast self-dismutation under these conditions. Therefore, the indirect assays such as McCord-Fridovich, adrenalin and nitroblue tetrazolium (NET) assays are widely used in the literature, not only for qualitative but also for quantitative detection of SOD activity of small molecular weight mimetics 52). Not going into details, we just want to stress that the indirect assays have very poor even qualitative reliability, since they can demonstrate the SOD activity of the complexes which does not react with superoxide at all. It has been reported in the literature that this is caused by the interference of hydrogen peroxide 29). We have observed that the direct reaction between complexes and indicator... 

Laboratory methods for detecting marine toxins (Table 7.2) can be characterized into two types of analyses indirect assays and direct measurement analyses. Indirect assays, or bioassays, measure the biological effect of a toxin on a system and can implicate, but not verify, the presence of a particular toxin. By contrast, direct measurements can both confirm and quantify the amount of a specific toxin in a food or biological specimen. 

The mouse bioassay, an indirect assay, historically has been used to evaluate shellfish toxicity (especially for PSP). Other bioassay procedures have been developed but not generally applied for regulatory purposes (Schantz et al., 1958). The mouse bioassay involves intraperitoneal (i.p.)... 

Plot the peak positions against the manufacturer s specified antibody binding capacity for each bead in the mixture to create a standard curve. Figure 3 illustrates curves for Simply Cellular beads stained with a PE-labeled antibody in a direct assay (A), and m an indirect assay with a PE-labeled antimouse IgG (B). The increased sensitivity of the indirect assay is evident from the rightward shift of the curve. 

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

B) INDIRECT ASSAY Measurement of the analyte-receptor interaction usin l racers or coupled reactions... 

As in RIA, high sensitivity RILAs can be obtained through amplification of the binding reaction between the MIP and the target by means of radiolabels which act as tracers. Obviously, as the analyte itself is not radioactive, the measuring principle is based on an indirect assay, typically a competitive one. 

The major problem for the application of absorption measurements to MIP-based direct or indirect assays is the dispersion of light at low wavelengths and the absorption at the same wavelength of other species. For these reason is necessary to develop optical dyes with high absorption in the lowest energy part of the electromagnetic spectrum. 

Nevertheless, the major issue in the miniaturization of this kind of assays continues to be the strong non-selective interactions with the polymers. This problem is often avoided, or at least minimized, on large scale experiments such as chromatographic separations. However, it is not easy to solve in the case of microsensors or microchips and direct or indirect assays, where competition can be strongly affected by unspecific binding. 

The assays were carried out using monoclonal antibodies in the direct and indirect formats. OTA working range, /50 and LODs were 0.05-2.5 pg/L and 0.1-7.5 pg/L, 0.35 ( 0.04) pg/L and 0.93 ( 0.10) pg/L, 60 pg/L and 120 pg/L in the direct and indirect assay formats, respectively. The immunosensor in the direct format was selected for the determination of OTA in wheat. Samples were extracted with aqueous acetonitrile and the extract analyzed directly by the assay without clean-up. The I50 in real samples was 0.2 pg/L corresponding to 1.6 pg/ kg in the wheat sample with an LOD of 0.4 pg/kg (calculated as blank signal—3er). Within- and between-assay variability were less than 5%... 

On the other hand, the main types of immunoassays that can be performed by using labelled antibodies or antigens are direct sandwich, competitive and indirect assays. The labels can be enzymes (alkaline phosphatase, peroxidise or glucose oxidase) metal NPs (gold) fluorescent or electrochemiluminescent probes. 

Another possible approach, which is broadly used, is to use high-purity substances (indirectly assayed) as standards. For use at the highest level, this approach requires the determination of all important impurities in the sample. This means not only metallic impurities, commonly stated in the manufacturers certificates, but also non-metals as oxygen, carbon, etc. The content of impurities is not always known in advance. If the total content of impurities is very low, the uncertainty of their determination does not affect the required uncertainty of the sample assay. Some other problems are discussed in Ref. [5], The need of determining the molar weight may equally apply here. 

In this indirect assay system, 05 is generated continuously by xanthine and xanthine oxidase, since 05 disproportionates spontaneously in the absence of a reactant for 05. In... 

In the so-called direct assay format, a biotinylated component binds to an antibody directly coupled to acceptor beads or to a protein captured by this antibody (Figure 8.4A). In the indirect assay format, the antibody used for capturing the biomolecule is in turn bound by a secondary antibody or by Protein A conjugated to the acceptor beads. In principle, any method capable of capturing the interaction partners to donor and acceptor beads, respectively, is suitable for setting up an AlphaScreen assay. 

The CUps-O epoxide 33-41 and related substrates are currently the only available selective fluorogenic substrates for EH. On the other hand various indirect assays have been reported to detect either the unreacted epoxide [46] or the carbonyl product resulting from periodate cleavage of the 1,2-diol product [26,47, 48], These assays are suitable for fluorescence or colorimetric assays for the hydrolysis of any epoxide of interest... 

In addition to the ability to work with very small amounts of reagent antibody, the indirect assay principle has the advantage that one labeled antibody may be utilized for a wide variety of assays provided only that the reagent antibody for all the different assays is raised in the same species. The materials used for the production of the labeled antibody are easy to prepare in large quantities (e.g., rabbit IgG and, say, a sheep antiserum to this) and can be carefully characterized at each stage before committing scarce materials to the assay itself. If the two-site assay procedure is used, this also avoids the use of an immunoadsorbent separation step with consequent economy in the use of the primary antigen. 

Transhydrogenase was also purified somewhat later and independently by a more complicated procedure which, however, also produced the 115000 molecular weight polypeptide [102]. Subsequently, additional improved methods have been published, which involve the use of immobilized antibodies [103] and affinity chromatography on immobilized NAD" [104] or NADP" [105]. With all preparations reconstituted transhydrogenase was shown to be a proton pump by both an indirect assay using 9-aminoacridine as pH probe for the interior space of the vesicles [106], and a direct assay using a pH electrode [107]. 

An indirect chemiluminescence immunoassay is an assay, with another component than the primary chemiluminescent emitter coupled to the antigen or antibody. This can be a cofactor or a catalyst or even a molecule capable of converting a non-chemiluminescent precursor to a chemiluminescent or potentially chemiluminescent species. Most indirect assays are enzyme mediated. 

Numerous methods have been developed for assessing the concentrations of FT4 and FT3 in serum. These methods include direct assays that currently serve as reference methods and indirect assays that are more widely available for general laboratory use. The following section describes the principles of these methods and offers some guidelines for their use. The theoretical basis, analytical validity, and clinical utility of these methods have been discussed. Special reports from the Nomenclature Committee of the American Thyroid Association, the National Academy of Clinical Biochemistry, and the NCCLS also review some of the issues and concerns regarding free thyroid hormone measurements. 

Fig. 14.2. Solid-phase EIA (indirect assay) with purified 5-aminosalicylic acid as H-donor and different concentrations of substrate. Best results are obtained with 0.0025% H2O2. Courtesy Archives of Virology.

Indirect assays Competitive assays, in which a non-labeled product of an enzymatic reaction competes with a fluorescent labeled tracer in binding to a product-specific antibody or streptavidin [80-82]. An example of an indirect FP is illustrated in Fig. 11. 

A PF3 indirect assay using a synthetic substrate was developed in 1979 by Sandberg and Anderson (S2). This test monitored thrombin production with the substrate S-2238 and claimed to be 10 times more sensitive than the above assay and to be free of EDTA interference. Later Harsialvi and coworkers claimed to improve the technique by adding soybean trypsin inhibitor to better control the reaction (H2). These workers believe this assay can be used for diagnosis of platelet disorders. 

Because the enzyme substrate and the products are unstable, it is difficult to measure the disappearance of substrate or the formation of products as is usual in enzymatic assays. Routine assays for SOD usually employ an indirect assay in which one unit of enzyme activity is defined as the amount of enzyme that inhibits the reaction of 02 with the indicator by 50%. The most frequently used method for measuring SOD activity employs the xanthine/xanthine oxidase reaction for... 

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