Solid-phase assays

Eisner, H.I., and Mouritsen, S. (1994) Use of psoralens for covalent immobilization of biomolecules in solid phase assays. Bioconjug Chem. 5(5), 463M67. 

F. Emmrich, Reduction of non-specific human-IgM binding in solid-phase assays. J. Immunol. Methods 72, 501-503 (1984). 

A direct contact test has been developed for solid samples. A solid-phase assay eliminates the need for soil extracts and utilizes whole sediments and soils. In the current procedure the solid sample is suspended in 2% NaCl. Dilutions of the stock suspension are measured to determine the EC50 and ECjo at 5 and 15 minute contact times. For this the homogenized sample and photobacterial suspension mixture are incubated. The suspended solid material is then centrifuged out and hght emission of the supernatant determined [24-26,32]. 

Figure 10.8. Left The sequence of RRE IIB, a fragment used to study RRE-Ugand binding. Right The components of our solid-phase assay that utilize the native RRE sequence. See color plates.

While the binding of aminoglycosides to the RRE provides a proof of principle, their affinity and, in particular, selectivity traits need to be improved for true therapeutic utility. To facilitate the discovery of potent and selective RRE binders, we developed a solid-phase assay. The components of this assembly include (a) insoluble agarose beads (or microtiter plates) covalently modified with streptavidin, (b) a biotinylated RRE fragment, and (c) a fluorescein-labeled Rev fragment (RevFl). Assembly of the three components generates an immobilized ternary complex whereby the biotinylated RRE binds to the beaded... 

Mock, D. M. (1990) Sequential solid-phase assay for biotin based on 125I-labeled avidin Methods Enzymol 184,224-233... 

Antibody Assays Monoclonal antibodies can be analyzed by the solid-phase assay techniques such as enzyme linked immunosorbent assays (ELISA) or radioimmuno assays (RIA). The typical assay procedures are as follows (Figure 5.9)... 

Chial, B.Z., Persoone, G. and Blaise, C. (2003) Cyst-based toxicity tests. XVIII. Application of ostracodtoxkit microbiotest in a bioremediation project of oil-contaminated sediments Sensitivity comparison with Hyalella azteca solid-phase assay, Environmental Toxicology 18 (5), 279 - 283. 

Bitton, G., Garland, E., Kong, I.-C., Morel, J.L. and Koopman, B. (1996) A Direct solid phase assay for heavy metal toxicity. I. Methodology, Journal of Soil Contamination 5, 385-394. 

SPT Solid-Phase Test. b ASPA Algal Solid-Phase Assay. 

The demonstration that MTs from a wide variety of fish species are recognized by an antiserum raised against one piscine MT has enabled the development of immunotechniques based on ELISA143 and radioimmunoassay (RIA) procedures144 for the quantification of these compounds. A competitive solid-phase assay based on dissociation-enhanced lanthanide fluoroimmuno-detection (DELFI A) of anti-MT monoclonal antibody bound to a solid phase has been reported.145 An electrochemical determination of MTs by square wave cathodic stripping voltammetry has also been developed and optimized.146... 

Fig. 5. Results of the solid-phase assay for Pt-DNA-HMGl binding. A 19-bp DNA duplex was modified with cis- or trans-DDP and covalently linked to a nylon membrane. The membrane was incubated with HMGl-GFPuv and washed extensively. Bound protein was determined by measuring fluorescence retained on the membrane.

After library synthesis and solid-phase assay of 100,000 beads on a red-labeled synthetic receptor [23], 55 deep staining beads were selected and their code was released via photolysis at 350 nM the released alcohols were silylated with N, O-bis(trimethylsilyl) acetamide and injected into a capillary GC with EC detection decoding 52 different structures, which are shown in Figure 9.7. 

Standard radio-immunoassay procedures are applied, nowadays mainly solid phase assays which can be rapidly performed and evaluated, they are also several enzyme-linked immunoassay (ELISA) procedures from commercial suppliers. It is recommended to perform an internet check for the most appropriate method at the time of the study (for example DSL 2005). Methods have been described for triiodothyronine (Nejad et al. 1975, Chopra etal. 1972a, Larsen 1972a, 1972,) and for thyroxin (Chopra etal. 1971, Chopra 1972). The use of assays based on thyroxin binding globulin (Chopra et al. 1972b) is no longer recommended and cannot be applied to the rat, because the rat does not have this binding protein. However, for the human, measurement... 

The RIA-gnost Ferritin kit (no more commercially available) from formerly Behringwerke AG, Radiochemical Laboratory described below uses the principle of an immunoradiometric assay (IRMA). It is a two-site solid phase assay of the sandwich type, based on a plastic bead as solid phase to which the antiferritin antibody adheres. The antibody-solid phase is incubated with standards or serum samples containing ferritin and in this process the ferritin in the solution is bound quantitatively to the solid phase via the antibody. The amount of ferritin bound to the solid phase is then determined by a reaction with 125I-labeled anti-ferritin antibody. An antibody-ferritin-125I-antibody complex is thus formed. 

Wang, G., Marquardt, R. R., Xiao, H., and Zhang, Z., Development of a 96-well enzyme-linked solid-phase assay for beta- glucanase and xylanase. JAgric Food Chem 1999, 47 (3), 1262-7. 

Liquid-phase and solid-phase reactions Antibody or antigen reactions can occur where both components are in the same liquid phase (in solution), or one component is in the solid phase and the other is in the liquid phase (solid-liquid interface). The former liquid phase assays were the first type used but have since largely been superseded by various forms of solid-phase immunoassay. Solid-phase assays... 

Zatta PE, Nyame K, Cormier Ml, Mattox SA, Prieto PA, Smith DF, Cummings RD. A solid-phase assay for beta-1,4-gaIactosyI-transferase activity in human serum using recombinant aequorin. Anal. Biochem. 1991 194 185-191. 

NaCN effectively inactivates the enzyme without affecting the color, but it is poisonous and smells bad. However, it has been difficult to find a more suitable reagent that would inactivate the enzyme without affecting the color. Lowering the pH by addition of HCl or H4SO4 increases the color development nonspecifically, whereas elevating the pH by addition of NaOH or inactivation of the enzyme with NaNs similarly reduces the color. In solid phase assays, where the enzyme activity on the solid phase is measured, the best way to stop further color development is to separate the substrate solution from the enzyme. ... 

Juppner H, Mohr H, Hesch RD. Adsorption of parathyrin Pitfall for solid phase assays using radio-labelled antibodies J Chn Chem Clin Biochem 1980 18 585-90. 

The kinetic features of POase in solid-phase assays may be masked by diffusion or partitioning phenomena (Section 9.2.2). Most probably, an incorrect H2O2 concentration is the most important single factor in interassay variations and enzyme inactivation. 

Enzymes modified with carbohydrates (neoglycoenzymes) can be used in cytochemistry as described above or in biochemical detection of lectins in solid-phase assays to gain greater sensitivity in analysis. For example, bacterial 3-galactosidase modified with p-aminophenyl a-D-mannopyranoside via amide linkage was useful in determination of Con A immobilized on plastic microtiter plates, and lactose-modified P-galactosidase was effective in histochemical detection of galactoside-specific lectins [63]. Other enzymes frequently used for these applications are alkaline phosphatase and horse radish peroxidase. There are a number of colorimetric, fluorometric, and chemiluminescent substrates available for these enzymes. 

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