IVDT_In Vitro Diagnostics Technology

IVD Technology, Spring 2013

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PROCESSING TECHNOLOGIES 4. Replace Hazardous Stop Solutions In immunoassays, when the desired intensity of color is achieved, the kinetic reaction is terminated by the addition of a stop solution. Te formulations of most stop solutions often are proprietary, but they are commonly known to be acid-based. Acid stop solutions change the pH, leaving a more intense and stable color for analyte measurement. Tese acids are considered hazardous materials as defned by the International Carriage of Dangerous Goods by Rail (RID) and by Road (ADR), International Maritime Dangerous Goods Code (IMDG) and International Air Transport Association (IATA). Sulfuric acid, as an example, is commonly used in ELISA assays in concentrations greater than the hazardous classifcation limit of 0.51M (5% w/w). Te choice of a TMB substrate can reduce the concentration of acid required for efcient and stable termination of the enzymatic reaction. Product Name Dual purpose blocker and stabilizer Traditional blocking and stabilization method 1.5 OD (650 nm) this allows for high quality and active substrates, it is not practical when running a large number of assays because it adds to preparation and, thus, extends test duration. Trough the use of good manufacturing practices and careful handling, ecosensible reagent manufacturers ofer solvent-free aqueous-based substrate formulations that are very active and have long shelf lives, up to four years at 4°C (Table II). Again, most diagnostic ELISA assay kits have a 6- to 12-month shelf life. Given their long stability, the usage of eco-sensible TMB substrates in a diagnostic kit will not be a limiting factor in kit production or in the determination of fnal kit shelf-life stability. 1 0.5 0 Measured activity Figure 3. Effect of a novel dual-purpose reagent used for plate blocking and stabilization on the signal detection of an ELISA. The graph shows the improvement in optical density (OD) detected in an ELISA when using a novel dualpurpose plate blocker and stabilizer with respect to a traditional method (activity was measured using TMB PLUS2). Some TMBs have been found to achieve 60 minutes of stop stability with sulfuric acid concentrations below 0.5M. Using a lower concentration of acid has a two-fold advantage: it lowers production cost because less acid is required and facilitates worldwide transportation because of the nonhazardous classifcation of the lower acid concentration. 5. Reduce Water Wastage in ELISA Production Te ELISA production process generates a signifcant amount of water waste, caused mostly by the consumption of water during the wash steps. Washing is necessary to remove excess nonbound reagents Stability TMB ONE and Pre-stained TMB ONE 4 years at 4°C TMB PLUS2 and Pre-stained TMB PLUS2 4 years at 4°C TMB X-tra and Pre-stained TMB X-tra 4 years at 4°C TMB SENS and Pre-stained TMB SENS 2.5 years at 4°C Table II. Commercially available ready-to-use aqueous TMB formulations that are solvent-free. 20 IVD TEC HNOLO G Y | SP RIN G 2013 magenta cyan yellow black and, thus, reduce background signal and increase the signal-to-noise ratio. Washing is performed by flling the wells entirely with water-based buffers. A typical sandwich ELISA on a 96-well plate requires about 30 mL of washing solution per washing step. Since any given ELISA can have three to four washing steps, in which washing is repeated three to fve times, upwards of 600 mL of washing bufer could be required per 96-well plate. Tere are two ways to reduce water waste in ELISAs: one option is to reduce the number of washing steps in the assay; another is to combine ELISA steps wherever possible. An efective technology that achieves both of these goals is a new generation of novel dual-purpose reagents that act simultaneously as microwell plate blockers and stabilizers. Tese novel reagents maintain the biological activity of biomolecules on microwell surfaces by preventing degradation, denaturation, and leaching. At the i v d t e c hnol ogy. com ES237410_IV1305_020.pgs 04.25.2013 04:17 UBM

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