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Ready-to-use assay systems for DMPK and ADMET studies

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A fast high throughput assay for measuring the dissociation constant of drugs from human serum albumin (HSA).

The dissociation constant is used in screening to rapidly predict plasma binding or to model plasma binding under various physiological and disease states using our spreadsheet software or Simcyp™.

  • Ready-to-use 96-well microtiter plates carrying TRANSIL beads with albumin immobilized in a random orientation to expose all binding sites. The assay is designed to conveniently measure albumin binding of drugs and when used in combination with TRANSILXL α1 acid glycoprotein (AGP) Binding assay it can accurately predict drug binding to plasma proteins.
  • Includes a spreadsheet, facilitating manipulation of statistical parameters and calculation of final results.
  • Designed for automated liquid handlers or manual pipetting.
  • TRANSIL beads are integrated into 96-well microtiter plates to enable easy handling.
  • One plate can be used to measure HSA binding of up to 12 compounds.
  • Only 12 minutes assay incubation time.
  • Rapid compound quantification due to immobilized albumin (e.g. injection via RapidFire™).
  • Highly reproducible results and robust correlation with conventional dialysis technique.
Background and Technical Information

Human serum albumin (HSA) is the most abundant protein in blood plasma. It is synthesized in the liver and concentrations in healthy subjects normally range between 35 and 60 g/L with an average of 42 g/L. HSA comprises 60% of the total plasma proteins. Its main physiological function is to bind and carry endogenous anions, with long-chain fatty acids. Two high affinity binding sites have been proposed in subdomains IIA (also known as Sudlow’s site I or warfarin site) and IIIA (also known as Site II or benzodiazepine site) of HSA. These comprise highly elongated hydrophobic pockets with charged lysine and arginine residues near the surface that interact with polar ligand parts. In contrast to AGP (?1-acid glycoprotein), HSA blood levels are much more stable. Hyperalbuminemia (HSA > 55 g/L) is seldom seen in the absence of dehydration whereas hypoalbuminemia is the more common condition resulting from malnutrition or liver disease with serum albumin levels dropping to 20-23 g/L.

The resolution of conventional methods (e.g. dialysis, ultrafiltration) is limited, particularly when examining drugs that are highly bound to plasma proteins. These conventional methods require highly sensitive analytical techniques that exhibit a linear range of more than two orders of magnitude or the use of highly pure radiolabeled compounds to resolve plasma protein binding of compounds with fu values less than 0.01. Given those constraints for the determination of plasma protein binding, the TRANSILXL HSA and TRANSILXL AGP Assay kits were developed and validated employing a novel method that overcomes these limitations and addresses the problem of varying HSA and AGP levels. To overcome the analytical limitations, when examining drugs that are highly bound to plasma proteins, the TRANSILXL HSA and TRANSILXL AGP Binding assays allow KD values to be determined by titrating different subphysiological concentrations of HSA and supraphysiological concentrations of AGP against a constant drug concentration.

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