Microtiter plate

EIAs can be used per se or with a spectrophotometer. Traditionally, EIAs have been developed in 96-weU microtiter plates which provide the immobilization support for the assay, the reaction vessel, and, when linked to a spectrophotometer-based reader, a rapid means to detect and quantify the color resulting from interaction of a substrate with the antibody—antigen—enzyme complex. Automated immunoassay analyzers targeted primarily for use in the clinical laboratory have taken automation one step further, utilizing robotics to carry out all reagent additions, washings, and final quantification including report preparation. 

Eig. 5. Determination of the for the binding of DAS to its polyclonal antibody raised in mice. A fixed amount of immobilized antibody in a microtiter plate is reacted with increasing amounts of DAS and the amount of DAS bound at each concentration is deterrnined using an EIA based on alkaline... 

This same experimental approach can be used to determine the appHcabiUty of the aDAS—AP to a competitive assay for DAS. As shown in Eigure 6, increasing amounts of free DAS were used to define the 50% inhibition level (ID q) of DAS for binding of two aDAS—AP conjugates to immobilized DAS. This approach was also used to determine the sensitivity of an EIA, as well as the specificity of the assay, as shown in Table 2. Increasing amounts of trichothecene mycotoxins closely related to DAS were added to microtiter plate wells containing a constant amount of prereacted DAS—aDAS—AP. After 30 min, excess toxin and any free toxin—aDAS—AP were washed out, and substrate was added. Quantification of the color produced was directly related to the abihty of the added toxin to displace aDAS—AP from the immobilized DAS, which is an indication that the aDAS also has an avidity for that toxin. 

Stanley, P. E. (1992). A survey of more than 90 commercially available luminometers and imaging devices for low-light measurements of chemiluminescence and bioluminescence, including instruments for manual, automatic and specialized operation, for HPLC, LC, GLC and microtiter plates. Part I descriptions. T. Biolumin. Chemilumin. 7 77-108. 

In each cycle, the library of mutated genes is first inserted in a standard bacterial host such as Escherichia coli or Bacillus subtilis. Subsequently, bacterial colonies are plated out on agar plates and harvested individually by a colony picker. Each colony is placed in a separate well of a microtiter plate containing nutrient broth, so that the bacteria grow and produce the protein of interest. Because each colony originates... 

The sensitivity of enzyme assays can also be exploited to detect proteins that lack catalytic activity. Enzyme-linked immunoassays (ELlSAs) use antibodies covalently finked to a reporter enzyme such as alkafine phosphatase or horseradish peroxidase, enzymes whose products are readily detected. When serum or other samples to be tested are placed in a plastic microtiter plate, the proteins adhere to the plastic surface and are immobilized. Any remaining absorbing areas of the well are then blocked by adding a nonantigenic protein such as bovine serum albumin. A solution of antibody covalently linked to a reporter enzyme is then added. The antibodies adhere to the immobilized antigen and these are themselves immobilized. Excess free antibody molecules are then removed by washing. The presence and quantity of bound antibody are then determined by adding the substrate for the reporter enzyme. 

Bevan, C. D., Uoyd, R. S. A high-throughput screening method for the determination of aqueous drug solubility using laser nephelometry in microtiter plates. Anal. Chem. 2000, 72,1781-1787. 

Avdeef and Bucher [24] investigated the use of universal buffers in potentiomet-ric titrations. Recently, such a buffer system, formulated with several of the Good components, has been designed specifically for robotic applications, where automated pH control in 96-well microtiter plates is required, with minimal interference to the UV measurement [48]. This universal buffer has a nearly perfectly linear pH response to additions of standard titrant in the pH 3-10 region [8, 48]. 

Figure 3.2(a) shows a plot of log S versus pH for naproxen, based on re-analysis (unpublished) of the shake-flask [49, 77] and microtiter plate [20] data reported in the literature. The dashed curves in Fig. 3.2 were calculated with the simple Henderson-Hasselbalch equations. For pH pKa, the function reduces to the horizontal line log S = log Sq. For pH pXi, log S is a straight line as a function of pH, exhibiting a slope of 1 (and an intercept of log So-pKj). Where the slope is 0.5, the pH equals to the pKj. 

The following is a generic description of the immobilized antigen ELISA (Figure 2), commonly termed indirect competitive immunoassay, on a microtiter plate. 

Preparation of microtiter plates. A constant amount of the coating antigen is bound to the surface of polystyrene microtiter plate wells by passive adsorption. After a predetermined incubation time, the plate is washed to remove unbound coating antigen. 

Competitive inhibition. A constant amount of anti-analyte antibody (primary antibody) and a series of solutions containing increasing amounts of analyte are added to the prepared microtiter plate wells. During incubation, the free analyte and bound... 

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. 

Figure 3 Immobilized antibody ELISA. Primary antibody (Y) is passively adsorbed to the surface of a polystyrene microtiter plate. Analyte (free H) and an enzyme-labeled hapten (H-E) compete for the fixed number of primary antibody binding sites. Following a wash step (dotted line), the substrate for the enzyme is added O) and a colored product formed ( ). The amount of product is inversely proportional to the amount of analyte present...

EIAs are more desirable for the measurement of agrochemicals than enzyme-linked immunosorbent assays (ELISAs) for several reasons. EIAs are easier to run, require minimal liquid transfers, and are completed in brief time frames, approximately 40 min for tube assays to 2.5 h for microtiter plate assays. In contrast, ELISAs are more complex, have many steps involving transfer of reagents, and require 6-8 h to complete. Most commercially available immunoassays utilize the EIA format. 

Maintaining a moderate, consistent pipetting rhythm is the best way to ensure that all samples and standards are treated equally. This is easy to accomplish with tube assays, because relatively few samples can be analyzed per set. Microtiter plates present more of a challenge, because up to 96 wells may be utilized at the same time. One solution developed in this laboratory involves the use of a microtiter plate not coated with reagent - the reservoir plate." An excess of all samples and standards is loaded into the reservoir plate. If 0.10 mL is needed for the inhibition step, for example, 0.15 or 0.20 mL of each solution is added to a pre-determined position in the reservoir plate the excess amount simplifies the next pipetting step. The location of each sample and standard is identified on a plate layout sheet, a map of the reservoir plate previously completed by the analyst (Figure 3). When the reservoir... 

An additional requirement not noted in Table 1 is compliance with GLP7 These practices establish a paper trail for all procedures involved in the determination of residues. With regard to immunoassays, GLPs require calibration of measurement devices such as adjustable pipettors and dedicated spectrophotometers. Computer software output, as noted above, must be verified prior to use. This process can be simplified by limiting the application of specialized software to the operation of an instrument and carrying out the residue calculations in a broadly available spreadsheet such as Excel. On a positive note, in recent years, the software accompanying most microtiter plate readers has become generally easier to use and usually incorporates internal spreadsheets that are compatible with external systems. 

Probes that mediate capture of the target nucleic acid are termed capture extenders. These probes are approximately 50 bases, one portion (20 to 40 bases) of which is complementary to the target, while the second portion (approximately 20 bases) binds the probe-target complex to a capture probe that is coupled to the surface of a microtiter plate well. 

Ishihama et al. [147] have describe a rapid screening method for determining pK values of pharmaceutical samples by pressure-assisted CE, coupled with a photodiode array detector. Each CE run was completed in less than 1 min, so a 96-well microtiter plate could be measured in one day. Determinations of the pKa values of 82 drugs illustrated this interesting new method. 

A high-throughput method using a 96-well microtiter plate format and plate UV spectrophotometry has been described [26]. Solubilities at a single pH, or at <12 pH values can be determined, using one of two methods. 

In the commercial version of the PAMPA assay, a sandwich (Fig. 7.9) is formed from a specially-designed 96-well microtiter plate [pION] and a 96-well microfilter plate [several sources], such that each composite well is divided into two chambers donor at the bottom and acceptor at the top, separated by a 125-pm-thick microfilter disk (0.45 pm pores, 70% porosity, 0.3 cm2 cross-sectional area), coated with a 10% wt/vol dodecane solution of egg lecithin (a mixed lipid containing mainly PC, PE, a slight amount of PI, and cholesterol), under conditions that multilamellar bilayers are expected to form inside the filter channels when the system contacts an aqueous buffer solution [543]. 

Four neutral lipid models were explored at pH 7.4 (1) 2% wt/vol DOPC in dode-cane, (2) olive oil, (3) octanol, and (4) dodecane. Table 7.5 lists the effective permeabilities Pe, standard deviations (SDs), and membrane retentions of the 32 probe molecules (Table 7.4). The units of Pe and SD are 10 6 cm/s. Retentions are expressed as mole percentages. Figure 7.22a is a plot of log Pe versus log Kd (octanol-water apparent partition coefficients, pH 7.4) for filters loaded with 2% wt/vol DOPC in dodecane (model 1.0, hlled-circle symbols) and with phospholipid-free dodecane (model 4.0, open-circle symbols). The dashed line in the plot was calculated assuming a UWL permeability (see Section 7.7.6) Pu, 16 x 10-6 cm/s (a typical value in an unstirred 96-well microtiter plate assay), and Pe of 0.8 x 10-6 cm/s... 

An infrequently used method (in pharmaceutical research) for determining the UWL permeability involves measuring transport of molecules across a high-porosity microfilter that is not coated by a lipid. The molecules are able to diffuse freely in the water channels of the microfilter. The filter barrier prevents convective mixing between the donor and acceptor sides, and an UWL forms on each sides of the microfilter. Camenisch et al. [546] measured the effective permeabilities of a series of drug molecules in 96-well microtiter plate-filterplate (Millipore GVHP mixed cellulose ester, 0.22 pm pore) sandwich where the filters were not coated by a lipid. The permeabilities were nearly the same for all the molecules, as shown in Fig. 7.8a. Our analysis of their data, Fig. 7.8b, indicates / aq = 460 pm (sandwich stirred at 150 rpm). We have been able to confirm similar results in our laboratory with different microfilters, using the lipid-free method. 

The errors mentioned above represent the reproducibility obtained on the same microtiter plate when the sample molecule is assayed in several different wells. When the reproducibility of Pe measurement is assessed on the basis of assays performed at different times over a long period of time, more systematic sources of errors show up, and the reproducibility can be about 2-3 times worse. Figure 7.56 shows reproducibility of standard compounds taken over a period of about 12 months. Carbamazepine show a long-term reproducibility error of 15%. The other compounds show somewhat higher errors. 

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