Two-color assay

Table 3-2. Enantiodiscrimination of selected library members using the two-color assay with proline derivatives.

There are times when two color assays may be required. This can be accomplished by altering the enzyme system used for one of the assays or the chromogen used for one of the assays if the enzymes are not altered. Perhaps the easiest method is to alter the color product of the DAB chromogen for one of the assays. This can be done be adding a heavy metal compound such as cobalt or nickel to the dilute solution (9). The resultant precipitate will be blue to black, not brown. It is helpful in this circumstance to use a Methyl Green counterstain, which will enhance the black precipitate and avoid the confusion of color seen with some other counter stains. 

Eu3+, Tb3+, Sm3+, Dy3+ complexes have different emission wavelengths with sharp peak profiles, which are suitable for multi-component immunoassay. Several Eu-Sm two-color time-resolved immunoassays have been reported. Since the sensitivity of Sm3+ chelates is not high compared to Eu3+ and Tb3+, Eu-Sm two-color assays are used for the simultaneous determination of a low concentration component (Eu) and a relatively high concentration component (Sm) in serum the assayed combinations are lutropin and follitropin, myoglobin and carbonic dehydratase, AFP and free (3-subunit of human chorionic gonadotrophin (Hemmila et al., 1987). Use of Eu-Tb is reported by Eriksson et al. (2000) for the simultaneous determination of human serum free and total PSA. 

Fig. 3. Fluorescence profiles of 2, 7 -dichlorofluorescin-loaded cells assayed in whole blood. (A) Compares the fluorescence histograms of unstimulated, control cells (shaded curve) with granulocytes exposed to opsonized S. aureus (open curve). (B) illustrates the two-color analysis profde of the granulocytes that were exposed to Texas Red-labeled S. aureus. Red fluorescence is the result of particle association with each granulocyte, whereas green fluorescence is the result of the oxidation of 2, 7 -dichlorofluorescin to 2, 7 -dichlorofluorescein (DCF). The red and green fluorescence analyses were performed with log-scale detection amplification for each fluorochrome.

S. O. Obare and C. J. Murphy, A Two-Color Fluorescent Lithium Ion Sensor, Inorg. Chem. 2001,40, 6080 L. Fabbrizzi, N. Marcotte, F. Stomeo, and A. Taglietti, Pyrophosphate Detection in Water by Fluorescence Competition Assays, Angew. Chem. Int. Ed 2002,41, 3811. 

Schutz E, von Ahsen N, Oellerich M. Genotyping of eight thiopurine methyltransferase mutations three-color multiplexing, two-color/shared anchor, and fluorescence-quenching hybridization probe assays based on thermodynamic nearest-neighbor probe design. Clin Chem 2000 46 1728-1737. 

Deveaux et al. [21] reported that mefenamic acid, being an N-aryl derivative of anthranilic acid, can be characterized by two color reactions. The color reactions, negative with N-aryl derivatives of aminonicotinic acid, are associated with the diphenylamine structure. For the first color reaction, add to a test tube approximately 0.5 g of oxalic acid dihydrate and at least 1 mg of the test material. Place the tube into an oil bath at 180-200°C for 4-5 min. After cooling, dissolve the residue in 10 mL of 95% ethanol to obtain a stable, intense blue solution (absorption maximum at 586-590 nm). To use the reaction for capsule formulations, extract the active ingredient with acetone and filter prior to the assay. For the second color reaction, add mefenamic acid (100-800 pg) in 1 mL of H0Ac-H2S04 (98 2, v/v), 5 mL of HOAc-HCl (50 50, v/v) and 1 mL of 0.10% (w/w) aqueous levulose. Heat the mixture for 25 min at 100°C, and after cooling, measure the absorbance at 597 nm. 

These assays resemble a hybrid of an immunohistochemistry assay and ELISA. Whole cells are fixed, for example with 3.7% formaldehyde, to MTPs permeabilized by repetitive washing with 0.1% Triton X-100, blocked with a protein solution, probed with primary antibodies (phospho-spe-cific, and non-phospho-specific), washed, and subsequently the secondary antibodies labeled with infrared fluorescent tags are added. After washing, these assays are read in a reader (such as the Odyssey or Aerius) designed for high sensitivity detection of two colors. The two colors are useful because one color can be used to accommodate a stain assigned as a total protein or cell number control or as an antibody to total protein, which allows for normalization. These assays may only require a single antibody versus the dual antibody sandwich required for ELISA. 

The Folin-Ciocalteau Assay of Protein Concentration. The Folin-Ciocalteau assay is one of the most sensitive and most commonly used assays to determine protein concentration (sensitive to about 10 /rg/m I protein). This procedure employs two color-forming reactions to assay protein concentration photometrically. In the first reaction (a biuret reaction), compounds with two or more peptide bonds form a dark blue-purple color in the presence of alkaline copper salts. In the second reaction, tryptophan and tyrosine side chains react with the Folin solution to produce cuprous ions. This reaction is most efficient under basic condi-... 

For comparative studies, two-color binding assays are often used, wherein two different sets of fluorescent-labeled target molecules from two separate populations are made, and interacted with a microarray. The relative level in a specific target (e.g., a protein, or luRNA transcripts for each gene) is measured by the fluorescent ratio [50]. 

Tunel (Terminal deoxynucleotide transferase dUTP nick end labeUng) assay In a modified version of the Tunel assay, apoptotic ceUs can be measured in a two-color system using flow cytometry and microscopy. In this assay, DNA breaks are labeled by deoxynucleotidyl transferase using bromodeoxyuridine triphosphate (BrdUTP) and visualized by anti-BrdU antibody. Propidium iodide is used to counterstain the total ceUular DNA. 

This assay is used to measure cell viability. It is a two-color fluorescence assay that simultaneously determines live (viable) and dead (nonviable) cells Live cells have intracellular esterases that convert nonfluorescent, cell-permeable fluorescein di-O-acetate to the intensely fluorescent fluorescein (green). Cleaved fluorescein is retained within cells. Dead cells have damaged membranes propidium iodide enters damaged cells and is fluorescent when bound to nucleic acids. It produces a bright red fluorescence in damaged or dead cells. 

W. Gregory Cox, M.P. Beaudet, J.Y. Agnew, J.L. Ruth, Possible sources of dye-related signal correlation bias in two-color DNA microarray assays, Anal. Biochem. 2004, 331, 243-254. 

Two-color primer extension assay with real time monitoring of fluorescent polarisation Mass spectrosopy - (MS)... 

Dragan, A. I., Golberg, K., Elbaz, A., Marks, R., Zhang, Y., Geddes, C. D. (2011). Two-color, 30 second microwave-accelerated Metal-Enhanced Fluorescence DNA assays A new Rapid Catch and Signal (RCS) technology. /. Immunohg. Meth, 366, pp. 1-7, ISSN 0022-1759... 

Two general assay methods are now available. One is colorimetric it depends on the isolation of ALA from an incubated mixture, its separation from aminoacetone by resin chromatography, its conversion to a pyrrole by condensation with acetylacetone, and the production of a colored derivative of the pyrrole with Ehrlich s reagent containing Hg " to remove interfering SH groups [Mauzerall and Granick, 49,52]. 

Wronski, R. Golob, N. Grygar, E. Windisch, M. Two-color, fluorescence-based microplate assay for apoptosis detection. BioTechniques 2002, 32, 666-668. 

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. 

The general sales specification under which maleic anhydride is sold ia the United States specifies a white fused mass or briquettes of 99.5% minimum assay and 52.5°C minimum crystallisation poiat. The melt color specification is 20 APHA maximum with a maximum APHA color of 40 after two hours of heating at 140°C. Four grams of maleic anhydride ia 10 milliliters of water are to be completely soluble. The resulting solution is to be colodess. The acidity resulting from maleic acid is allowed to be a maximum of 0.2%. 

A 250-mL, two-necked, round-bottomed flask equipped with a magnetic stirbar, thermometer, and a reflux condenser fitted with a rubber septum and balloon of argon is charged with a solution of methyltrioxorhenium (MTO) (0.013 g, 0.05 mmol, 0.1% mol equiv) in 100 mL of methanol (Note 1). Urea hydrogen peroxide (UHP) (14.3 g, 152 mmol) is added (Notes 1, 2, 3, 4), the flask is cooled in an ice bath, and dibenzylamine (9.7 mL, 50.7 mmol) is then added dropwise via syringe over 10 min (Notes 1, 5). After completion of the addition, the ice bath is removed and the mixture is stirred at room temperature (Note 6). A white precipitate forms after approximately 5 min (Note 7) and the yellow color disappears within 20 min (Note 8). Another four portions of MTO (0.1% mol equiv, 0.013 g each) are added at 30-min intervals (2.5 hr total reaction time). After each addition, the reaction mixture develops a yellow color, which then disappears only after the last addition does the mixture remain pale yellow (Note 9). The reaction flask is cooled in an ice bath and solid sodium thiosulfate pentahydrate (12.6 g, 50.7 mmol) is added in portions over 20 min in order to destroy excess hydrogen peroxide (Note 10). The cooled solution is stirred for 1 hr further, at which point a KI paper assay indicates that the excess oxidant has been completely consumed. The solution is decanted into a 500-mL flask to remove small amounts of undissolved thiosulfate. The solid is washed with 50 mL of MeOH and the methanol extract is added to the reaction solution which is then concentrated under reduced pressure by rotary evaporation. Dichloromethane (250 mL) is added to the residue and the urea is removed by filtration through cotton and celite. Concentration of the filtrate affords 10.3 g (97%) of the nitrone as a yellow solid (Note 11). 

B) at the indicated stages, and the PGl, PG2, and total PG activities were determined by differential heat inactivation. Each time point is the average of at least two separate extractions assayed in duplicate. The developmental stages are MG, mature green Br, breaker stage, (time of first external color development) and +2, +5, +7, and +10 are days after breaker. 

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