Solid phase microtiter plates

Another commonly used ELISA format is the immobilized antibody assay or direct competitive assay (Eigure 3). The primary anti-analyte antibody is immobilized on the solid phase and the analyte competes with a known amount of enzyme-labeled hapten for binding sites on the immobilized antibody. Eirst, the anti-analyte antibody is adsorbed on the microtiter plate wells. In the competition step, the analyte and enzyme-labeled hapten are added to microtiter plate wells and unbound materials are subsequently washed out. The enzyme substrate is then added for color production. Similarly to indirect competitive immunoassay, absorption is inversely proportional to the concentration of analyte. The direct competitive ELISA format is commonly used in commercial immunoassay test kits. 

The deprotected lactosides were evaluated as inhibitors against lectin binding in a solid-phase inhibition assay with immobilized ASF on the surface of microtiter plate wells, mimicking cell-surface presentation, while mammalian galectins-1, -3, and -5 were in solution. Strong multivalency effects and selectivity were observed for the... 

The group of Grieco has presented a method for efficiently performing macrocy-clizations on a solid phase (Scheme 7.31) [48]. The preparation of the macrocyclic peptides required several standard transformations, which are not described in detail herein. The final intramolecular nucleophilic aromatic substitution step was carried out under microwave irradiation at 50 °C in a dedicated CombiCHEM system (see Fig. 3.9) utilizing microtiter plates in a multimode batch reactor. The cycli-zation product was obtained in good yield after a reaction time of 10 min and sub-... 

In our laboratories, a cycle time of 90 sec can be achieved with a dilution factor of 1 25 for a given sample concentration, allowing the purity and identity control of two and a half 384-well microtiter plates per day. The online dilution eliminated an external step in the workflow and reduced the risks of decomposition of samples in the solvent mixture (weakly acidic aqueous solvent) required for analysis. Mao et al.23 described an example in which parallel sample preparation reduced steps in the workflow. They described a 2-min cycle time for the analysis of nefazodone and its metabolites for pharmacokinetic studies. The cycle time included complete solid phase extraction of neat samples, chromatographic separation, and LC/MS/MS analysis. The method was fully validated and proved rugged for high-throughput analysis of more than 5000 human plasma samples. Many papers published about this topic describe different methods of sample preparation. Hyotylainen24 has written a recent review. 

Classic solid phase substrates used in biotesting, such as microtiter plates, membrane filters or microscope slides, have been the first supports used for NA immobilization in array fabrication [27]. Desired attributes of any DNA array substrate include (i) chemical homogeneity (ii) thermal and chemical stability (iii) ability to control surface chemical properties such as polarity or hydrophobicity (iv) ability to be activated with a wide range of chemical functionalities (v) reproducibihty of the surface modification processes involved (vi) inert with respect to enzymatic activity especially ones involved in DNA manipulation and (vii) ultra-low intrinsic fluorescence. 

Direct competition. The solid phase (a microtiter plate) is coated with an antibody specific for the antigen being assayed. The sample and enzyme-labelled antigen (antibiotic) are added. There is a competition for the antibody between the labelled and unlabelled antigen (antibiotic). Substrate is added and the color produced by the enzymatic hydrolyse is inversely proportioned to the concentration of antigen in the sample... 

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... 

Fiehn, O., L. Vigelahn, G. Kalnowski, T. Reemtsma, and M. Jekel. 1997. Toxicity-directed fractionation of tannery wastewater using solid-phase extraction and luminescence inhibition in Microtiter plates. Acta Hydrochim. Hydrobiol. 25 11-16. 

Chemical activities in the field of mass screening are often related to combinatorial chemistry [51,52]. One major goal, especially in the field of solid phase chemistry involving polymers like DNA or peptides, aims at the increase in the number of compounds per reactor volume and time. Commercially available microtiter plates are established as reactors in this case whereby robotic feed systems fit perfectly to their dimensions. A drastic reduction of reaction volume and increase in number of reaction vessels ( wells ) leads to the so-called nanotiter plates (e.g. with 3456 wells). Microfabrication methods such as the LIGA process are ideal means for the cost effective fabrication of nano-titer plates in polymeric materials by embossing or injection molding techniques so that inexpensive one-way tools are realized. 

Some particular features of the analysis of products obtained by combinatorial methods have impaired the use of NMR spectroscopy in the initial phase of the development of this technique. Combinatorial chemistry produces large number of compounds in a very short period of time, in small quantities and instead of using traditional glassware for synthesis employs 96-well microtiter plates to store, transport and sometimes even to synthesize the compounds of interest. Another issue is the need to characterize solution and solid samples, since solid phase synthesis is extensively used in combichem. In this context, the need of an efficient and universal sample analysis remains a challenge. Actually, most combichem programs obtain mass spectrometry and UV (photodiode-array detection) data on their samples but clearly the use of NMR spectroscopy provides a structural characterization unparalleled by the aforementioned techniques. In the last years an increasing number of new NMR methods opened the possibility for the utilization of this analytical technique for monitoring combinatorial chemistry reactions. The first part of this chapter will focus on the recent developments introduced in NMR spectroscopy to overcome these difficulties. 

SOPHAS M (Figure 13.10) is an automatic modular solid-phase synthesizer based on a robotic system. Synthesis can be carried out in a variety of reaction vessels, such as 96-well microtiter plates, tubes, or vials. The vessels are mowed on the 1- or 1.2-m-length workbench in aluminum carriers (12 mm x 86 mm) by a robotic arm. The content of the reaction vessels is isolated from the atmosphere by a pierceable double seal. There are four independent pipetting probes on the synthesizer. Each probe has three independent channels. The channels allow the synthesizer to simultaneously aspirate and add washing solvents and nitrogen. 

Take off an aliquot of each fraction for bioassays, solid phase ELISA, or SDS-PAGE analysis using a microtiter plate. 

Modem methods employ solid phases (e.g. tubes, particles, microtiter plates). 

A known amount of antibody is bound to a solid phase (e.g., PVC test tubes or wells of a microtiter plate). 

In a conventional microtiter plate assay, a 1.5-mm movement would be necessary for the most distandy located antibody molecule to react with the antigen fixed on the surface of the well, since the liquid depth was 1.5 mm. On the other hand, the liquid phase of the microchannel filled with polystyrene beads was much smaller. The longest distance from an antibody molecule to the reaction-solid surface may be less than 20 pm. Diffusion time is proportional to the squares of the diffusion distance, so the diffusion time of the antibody molecule to the antigen in the microchip would be more than 5600 times shorter than the conventional method. 

Combinatorial Chemistry. The application of high-throughput, parallel methods to the synthesis, analysis, screening, and testing of materials. This approach relies on robotics and computer-assisted methods to generate and analyze the results. Synthesis, analysis, and testing of samples occurs in the wells of microtiter plates, which may contain as few as 96 samples or as many as a few thousand. Solid-phase and solution methods are used, and samples may be one-bead-one-com-pound" or they may contain mixtures, which require "deconvolution" to determine which component is responsible for observed activity. 

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