Solution assays

Jones LJ, Yue ST, Cheung C-Y, Singer VL. 1998. RNA quantitation by fluorescence-based solution assay ribogreen reagent characterization. Anal Biochem 265 368-374. 

A nonlinear relationship between enzyme concentration and measured activity is indicative of a more complex reaction system. Complications of this nature may arise from such things as changes in the composition of the reaction mixture (e.g., pH due to the addition of increasing amounts of enzyme solution), assay limitations (e.g., insufficient substrate), limited coupling-enzyme (where assays are based on coupled enzyme systems), the presence of inhibitors, and enzyme-cofactor or enzyme-enzyme dissociation phenomena. Nonlinear relationships may also be an inherent... 

The coupons were contacted with granite groundwater or brine containing six radionuclides for 28 d. The coupons were removed and the solutions assayed to determine the amount of sorbed radionuclides. 

Fixed activity was determined by direct count of cell all other inventories are based on solution assays. 

Silver sulfadiazine is dissolved in dilute hydrochloric acid and determined by titration with sodium nitrite solution (assay of the primary aromatic amine function). The endpoint detection is commonly biamperometric (27). 

Extrinsic. Many of the traditional bioassays are based on optical measurements, but these are solution assays without immobilised reagents. Optical biosensors are concerned with interrogation of reactions at surfaces, but often the reagents are the same the optical fibres acting, in the extrinsic mode as light guides to transmit information to and from a remote target reaction at its terminus. 

Internal Standard Solution, Standard Solution, Assay Preparation, and Chromatographic System Proceed as directed under Assay (above). 

Sulfuric Acid, 95% Add a quantity of sulfuric acid of known concentration to sufficient water to adjust the final concentration to between 94.5% and 95.5% of H2SO4. Because the acid concentration may change upon standing or upon intermittent use, check the concentration frequently and either adjust solutions assaying more than 95.5% or less than 94.5% by adding either diluted or fuming sulfuric acid, as required, or discard them... 

A precondition for miniatiuization of the assay volume to a few microliter is the availability of reliable and versatile liquid-handling tools for precise metering and delivery of microliter and nanoliter aliquots of compound solutions, assay reagents, and biological materials. Low-volume liquid handlers in the life sciences utilize a variety of basic microfluidic methods for precise dosage of the aliquot volume being transferred, including ... 

The combination of fluorophores and suspended colloid particles could be used in metal-enhanced solution assays. Scheme 8.1 depicts the use of fluorophores and suspended colloid particles. Previous studies on fluorescence intensity enhancement between fluorophores and suspended particles in terms of metal core of nanoparticles, fluorophore type, and spacer used are summarized in Table 8.2. 

Many existing solution assays can be applied to the peptide libraries made by this approach. As mentioned above, multistep synthesis and screening that identify only one, not necessarily the best, solution motif are performed. 

The everted intestinal ring model also suffers from serious limitations. Assurance of viability during the course of the experiment is a concern. Deterioration of the epithelial membrane can occur for incubation periods exceeding 10—15 min. Therefore, poorly permeable markers such as mannitol, inulin, or PEG 4000 are routinely employed to assess barrier integrity. Additional complications in the interpretation of data include delineation of uptake versus nonspecific adsorption and elimination of intestinal metabolism as a confounding variable. Low drug accumulation is another concern that necessitates the use of a sensitive, specific analytical methodology in order to obtain reliable quantitation of cellular uptake. If radiolabeled compound is unavailable, solute assay may be difficult. 

These requirements have lead to the following analytical solutions. Assays for low amounts of compounds must typically have highly sensitive detection schemes. Rapid throughput often relies on robotic methods. The large number of compounds has led to parallel high-density analysis formats, such as 96-well plates. To handle the large amounts of data, special software, databases, and visualization methods have been developed. 

The three symmetrically positioned electrophilic centers of trichlorotriazine enable the selective introduction of points of diversity through successive substitution of the chlorine atoms. This feature of trichlorotriazine is well known and widely utilized, e. g., in the chemistry of dyestuffs. The principle has been applied for the preparation of a 12000-membered library [126] for use in both solid-phase and solution assays, -with a selectively cleavable linker stable to the TFA treatment necessary for the deprotection of functional groups present. Methionine was used as a suitable linker between the library and the resin. This type of linkage to the support has been used previously for release of the compounds for mass spectro-metric analysis [127]. Both the first and second chlorine atoms of the triazine scaffold can be substituted at room temperature, but the kinetics of the two reac-... 

Beside solution assays, AMPPD and AMPGD can be used for detection of nucleic acids on membranes since the AMP D anion is hydrophobic and will attach to the membrane in some fashion in the direct vicinity upon formation. This allows its application in assays where resolution is required such as Northern and Southern hybridization. 

A luminescent reaction utilized for visualization of antibodies labeled with AP in immunoblotring techniques appeared very attractive for application to solution assays of TNAP. The reaction utilizes CDP-star substrate and requires alkaline pH in the assay. 

Competition-in-solution assays can quantify the effect of library components on the interaction between components of a protein complex. These assays include traditional biochemical assays that measure interactions through antibody- or tag-mediated pull-down or label displacement assays, along with biophysical methods that measure changes in the property of a component upon binding to its partner protein, such as fluorescence spectroscopic assays and SPR. It is essential for assay development that the requirements (affinity and binding site) for assembly of the interaction partners be at least partially defined such that a tractable model system for the target interaction can be developed, as was the case for MDM2/p53, IL-2/IL2-aR and Bcl/Bak [138-140],... 

Assay Buffers and Solutions Besides critical reagents and microtiter plates, the third major component of an ELISA method comprises assay buffers and solutions, including coating buffer, blocking solution, assay diluents, wash buffer, enzyme and substrate, and stop solution. 

Biosensor instruments such as Biacore (General Electric) exploit the sensitivity of a surface plasmon resonance response to the mass localised near the surface of a sensor chip. Various approaches can be used for kinases. Inhibition in solution assays involve immobilisation of a target definition compound (TDQ on the sensor surface.13,30 A buffer containing the kinase is flowed over the surface so that the protein is able to bind to the immobilised TDC, giving a signal. When test compounds are included in the buffer, they can compete for the TDC-kinase interaction, allowing estimation of Kd. 

The photocleavable linker allows detachment of the cyclic peptide from the resin and to assess biological activity in homogeneous solution assays. 

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