TRANSIL HSA Binding Kit
TRANSIL HSA Binding Kit
The TRANSIL HSA Binding Kit estimates the binding of drugs to human serum albumin (HSA) and predicts the plasma protein binding. The assay kit measures the dissociation constant (KD) of drugs to albumin and hence allows the calculation of albumin binding even under disease and physiological states that alter the albumin content of human serum. In combination with our TRANSIL AGP Binding Kit it is possible to obtain accurate prediction of plasma protein binding in a highly controlled and reproducible assay environment. Internal quality controls provide easy assessment of recovery, experiment and data quality.
TRANSIL protein binding kits measure the dissociation constant (KD) of the test item in 6 intelligent replicates with varying protein concentration. This allows not only accurate KD determination, but also provides for detailed assay quality control, so that there is no need to perform additional experiments to assess recovery and concentration dependency. We also provide a spreadsheet for the data analysis and comprehensive quality control. The automatically calculates the test items’ dissociation constants and the fraction unbound according to this formula:
When results from TRANSIL HSA Binding and AGP Binding kits shall be combined, the spreadsheet calculates the unbound fraction according to the full binding equation:
The kit consists of ready-to-use 96 well microtiter plates. One plate can be used for measuring HSA binding of up to 12 compounds.
The assay protocol has only 5 steps:
- addition of drug candidate,
- mixing and incubation for up to 12 minutes,
- removal of beads by centrifugation,
- sampling of supernatant,
- quantification of drug candidate.
The assay kits comes in 3 variants. The standard kit is for medium and high albumin binders. The high albumin content kit is used for compounds with low albumin affinity. And the kit variant for peptide is ideal for compounds similar to liraglutide or semaglutide.
Plasma protein binding estimates obtained with TRANSIL kits are comparable to literature data and measurements obtained with dialysis (Figure 2). For most compounds even the measurements from the TRANSIL HSA Binding kit suffice for an accurate fraction unbound estimate. Only drugs with weak albumin binding and strong AGP binding, such as propranolol, exhibit strong change in the estimate of the unbound fraction when including the results from the TRANSIL AGP Binding kit (Figure 3).
Figure 1: Comparison of plasma protein binding measurements obtained with the TRANSIL assay kits and dialysis as well as comparison with literature data (Goodman and Gilman 1996: The Pharmacological Basis of Therapeutics).For most compounds AGP binding makes a small contribution to the overall unbound fraction because AGP is much less abundant than albumin in the plasma (30 times less, in fact). This means that when a compound strongly attaches to albumin, there's not much additional binding to AGP because albumin competes strongly for binding. However, when a compound is a weak binder to albumin and a strong binder to AGP, then AGP has a significant effect on the free fraction in plasma