Automation of the Anthrone Assay for Carbohydrate Concentration Determinations

Abstract

Reported is the adaptation of a manual polysaccharide assay applicable for glycoconjugate vaccines such as Prevenar to an automated liquid handling system (LHS) for improved performance. The anthrone assay is used for carbohydrate concentration determinations and was scaled to the microtiter plate format with appropriate mixing, dispensing, and measuring operations. Adaptation and development of the LHS platform was performed with both dextran polysaccharides of various sizes and pneumococcal serotype 6A polysaccharide (PnPs 6A). A standard plate configuration was programmed such that the LHS diluted both calibration standards and a test sample multiple times with six replicate preparations per dilution. This extent of replication minimized the effect of any single deviation or delivery error that might have occurred. Analysis of the dextran polymers ranging in size from 214 kDa to 3.755 MDa showed that regardless of polymer chain length the hydrolysis was complete, as evident by uniform concentration measurements. No plate positional absorbance bias was observed; of 12 plates analyzed to examine positional bias the largest deviation observed was 0.02% percent relative standard deviation (%RSD). The high purity dextran also afforded the opportunity to assess LHS accuracy; nine replicate analyses of dextran yielded a mean accuracy of 101% recovery. As for precision, a total of 22 unique analyses were performed on a single lot of PnPs 6A, and the resulting variability was 2.5% RSD. This work demonstrated the capability of a LHS to perform the anthrone assay consistently and a reduced assay cycle time for greater laboratory capacity.

Transformation of postingestive glucose responses after deletion of sweet taste receptor subunits or gastric bypass surgery

“…A 10% slurry was made by adding 90% vol/wt of phosphate-buffered saline (pH 7.4) to the feces and mixing thoroughly by vortexing. Total carbohydrate concentration was measured using a spectrophotometric method described previously (72). Briefly, the 10% slurry was mixed with Anthrone reagent (1 g Anthrone/500 ml sulfuric acid; Sigma-Aldrich), and the absorption was measured at 650 nm.…”

Multivalent display of minimal Clostridium difficile glycan epitopes mimics antigenic properties of larger glycans

“…Anthrone reactions followed a modified protocol by Turula et al . 65 To 35 μl of a 0.1% (w/v) solution of anthrone reagent (Sigma) in concentrated (98%) sulfuric acid, 5 μl of desalted solutions of 12 , 13 or CRM 197 were added in 96-well round-bottom microtitre plates. Absorbance at 579 nm was determined in a spectrophotometric plate reader and compared with standard curves using equimolar amounts of D -glucose and L -rhamnose.…”

Fermentation of Multigrain Dough – An Approach to Reduce Glycemic Index for Healthy Bread

“…Original Research Article: full paper [Total reducing sugars (TRS)] and total carbohydrates (TC) that were released in dough (0, 3, 5 and 7 days) were estimated [25,26]. An in-vitro dialysis procedure was followed [27], where a 5 g sample was homogenized in 20 mL of 0.1 M potassium phosphate buffer solution (pH 6.9) at 37.8 °C in 100 mL Erlenmeyer flask.…”

“…Each of the so withdrawn dialyzed samples was placed in a boiling water bath for 5 minutes, quickly cooled and kept frozen until further analysis for total reducing sugars and total carbohydrates. Thus, these individually dialyzed samples of sourdough (that were sampled in different periods of being fermented), were analyzed for glucoses (as TRS) [25] and TC [26], using glucose as a standard and buffer (phosphate buffer solution pH 6.9), as a negative control of fermentation. The concentration of glucose (moles/L) vs time (0-180 min) was graphically analyzed for an area under curve (AUC) for each of the fermented samples.…”