Microtiter plate assays

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. 

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... 

Kimball, S. R., Horetsky, R. L., and Jefferson, L. S. (2004). A microtiter plate assay for assessing the interaction of eukaryotic initiation factor eIF4E with eIF4G and eIF4E binding protein-1. Anal. Biochem. 325, 364—368. 

Crespi, C.L., Miller, V.P., and Penman, B.W. 1997. Microtiter plate assays for inhibition of human, drug-metabolizing cytochromes P450. Anal. Biochem. 248 188. 

Naritomi, Y. et al. Utility of microtiter plate assays for human cytochrome P450 inhibition studies in drug discovery. Drug Metab. Pharmacokin. 19 55. 

It was carried out according to Schwikkard et al. [9] and Actinomycin D was used as a positive control [ 16]. Cytotoxicity was determined against A2780 human ovarian cancer cells, using a microtiter plate assay. The plates were seeded with cells and... 

The sensitivity of the microfluidic system was determined by measuring calibration curves of four cathepsin B inhibitors. The inhibitors caused negative peaks in the product mass chromatograms by inhibiting cathepsin B and thus the substrate turnover. The measured order of afEnities of the four inhibitors is in agreement with the affinities determined in microtiter plate assays and the macro-scale system. 

Alternate Protocol 4 Microtiter Plate Assay for BCA Measurement... 

ALTERNATE MICROTITER PLATE ASSAY FOR BCA MEASUREMENT OF TOTAL PROTOCOL 4 PROTEIN... 

This microtiter plate assay has a working range of 1 to 25 pg/ml. 

Alternate Protocol 3 Microtiter Plate Assays of DPOs C4.1.8... 

B. M. van den Hoogen, P. R. van Weeren, and M. Lopes-Cardozo, A microtiter plate assay for the determination of uronic acids, Anal. Biochem., 257 (1998) 107-111. 

Ryan D, Nuccie B, Arvan D. Non-PCR-dependent detection of the factor V Leiden mutation from genomic DNA using a homogeneous invader microtiter plate assay. Mol Diagn 1999 4(2) 135-144. 

Yamamoto T, Suzuki A, Kohno Y. Application of microtiter plate assay to evaluate inhibitory effects of various compounds on nine cytochrome P450 isoforms and to estimate their inhibition patterns. Drug Metab Pharmacokinet 2002 17 437M48. 

Many assays that have been bead-captured and gel-based have been converted to 96-well polystyrene microtiter plates to standardize the format for automation and higher throughput. In many ways, plate formats are a bridge to the higher-density microarray formats and remain quite useful until full conversion to microarray or microfluidic formats can be made. Microtiter plate assays or activated microbeads (i.e., Luminex) for panels of cytokines are widely available because of their use in clinical medicine. 

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. 

Palamanda, J.R. Eavreau, L. Lin, C.C. Nomeir, A.A. Validation of a rapid microtiter plate assay to conduct cytochrome P450 2D6 enzyme inhibition studies. Drug Discov. Today 1998, 5, 466-470. 

The flavoenzyme UDP-galactop)ranose mutase (UGM) plays a key role in the cell wall biosynthesis of many pathogens, including Mycobacterium tuberculosis. McNeil and co-workers developed a microtiter plate assay for UGM (O Scheme 14) [157]. The assay is based on the release of tritiated formaldehyde from UDP-galactofuranose but not UDP-galactopyranose by periodate and was used to identify a uridine-based enzyme inhibitor from a chemical library. The potent inhibitor 320KAW73 (IC50 = 6 pM) was identified. 

Vandeputte, C., Guizon, I., Genestie-Denis, I., Vannier, B., and Lorenzon, G. (1994) A microtiter plate assay for total glutathione and glutathione disulfide contents in cultured/isolated cells performance study of a new miniaturized protocol. Cell Biol. Toxicol. 10,415-521... 

Assays based on sandwich-hybridization are available in several platforms, such as sequential injection analysis (55), microtiter plate assays (61), and microfluidic devices (62). The LFA biosensor assays described in this chapter rely on the sandwich-hybridization of a nucleic acid sequence based amplified (NASBA) RNA target between a membrane immobilized capture probe and SRB-encapsulating liposome conjugated reporter probe. NASBA uses the enzymes avian myeloblastosis virus reverse transcriptase (AMV-RT), RNaseH, and T7 DNA dependent RNA polymerase in the presence of deoxyribonucleoside triphosphates and appropriate primers to amplify relatively few copies of target RNA into... 

A subsequent report describes the evolution of the same enzyme for the hydrolysis of 3-phenylbutyric acid resorufin ester using both a mutator strain and error-prone PCr(2°7] Mutants were screened for improved enantioselectivity based on a microtiter plate assay using the optically pure R- or S-esters. Both mutagenesis methods generated first-generation mutants with higher enantioselectivity ( =6.6 and 5.8 compared to wild-type E=3.5). 

The checkerboard approach is the common way a microtiter plate assay is developed and optimized. In terms of commercial kits, this approach could be used to change a kit component or, in the case of development kits (e.g., R + D systems Duo Sets ), to optimize the assay range for the specific requirements of the program. 

The application of acridinium esters to microtiter plate assays has been recently developed, and assays for TSH and AFP were presented (W24). Since the emission from these labels is transient, i.e., completed in about 1 sec, this represents a novel development in microtiter plate detection. In a comparison between luminol and acridinium ester, a 96-well plate was processed in 15 min for the former label and in 4 min for the latter (VI). 

Li, L. and Singh, B.R., Isolation of synaptotagmin as a receptor for types A and E botulinum neurotoxin and analysis of their comparative binding using a new microtiter plate assay, J. Nat. Toxins, 7, 215, 1998. 

Llewellyn, L.E., Doyle, J., Negri, A.P., 1998. A high-through-put, microtiter plate assay for paralytic shellfish poison using the saxitoxin-specific receptor, saxiphilin. Anal. Biochem. 261 51-56. 

Chien, H.R. Hsu, W.-H., Rapid and sensitive detection of D-hydantoinase producing microorganisms by using microtiter plate assay. Biotechnol. Techniques, 10 (1996) 879-882. 

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