TRANSIL High Sensitivity Binding Kit
TRANSIL High Sensitivity Binding Kit
Compounds binding strongly to plasma proteins frequently result in overly small compound concentrations that are difficult to measure accurately. Also, time to equilibrium increases the stronger compounds bind to plasma. The TRANSIL High sensitivity binding kit solves this by introducing another binding matrix and analyzing the binding equilibrium between this solid-phase matrix (natural membranes) and the liquid phase including plasma.
Highly lipophilic and sticky compounds also create notorious difficulties in plasma protein binding assays. The TRANSIL High sensitivity binding kit addresses this by providing plasma in all assay compartments.
Thus, the TRANSIL High Sensitivity Binding Kit accurately determines the unbound fraction of drugs that are tightly bound to plasma proteins - even when the unbound fraction is well below 1%. Also, the TRANSIL assay yields high recovery for drugs that exhibit too high unspecific binding in other assay systems, or precipitate because of low solubility. The kit determines the fraction of drug bound to plasma indirectly by measuring the partitioning of drug between the plasma proteins and artificial cell membranes.
Internal quality controls provide easy assessment of recovery, experiment and data quality.
The kit consists of silanized glass vials pre-filled with buffer and membrane beads.
The assay requires only 6 steps: (i) addition of plasma, (ii) addition of drug candidate, (iii) incubation for 30 minutes while mixing, (iv) removal of beads by centrifugation, (v) precipitation of plasma, and (vi) quantification of compounds.Figure 1: Comparison of fraction unbound measurements obtained via dialysis and the TRANSIL High Sensitivity Binding kit. Marketed drugs having average physchem properties yield comparable results in both assays. Figure 2: Comparison of fraction unbound measurements obtained via erythrocyte partitioning and the TRANSIL High Sensitivity Binding kit. Early discovery compounds binding strongly to plasma proteins yield comparable results in both assays. Figure 3: Comparison of fraction unbound measurements at 4, 6, and 24 hours incubation in dialysis versus 30 minutes incubation in the TRANSIL High Sensitivity Binding kit (dotted line). The compound is a lipophilic acid with a cLogP of 6.1 and a molecular weight of 503. Fraction unbound estimates from the longer incubation times of dialysis converge towards the result of the TRANSIL assay. Figure 4: Reproducibility of the TRANSIL High Sensitivity Binding kit showing results from 5 independent fraction unbound measurements of diclofenac.
Determine PPB of drugs with fu<1%
- Tightly bound fu<0.01% or less
- Highly sticky
- Poorly soluble
- Can be used with any plasma
Fast and easy to use assay kit
- No preparation required
- Build-in quality control
- Suitable for high throughput screening
- Ready-to-use data analysis spreadsheet
The Transil High Sensitivity Binding (HSB) kit is different from all our other screening kits, in that it is designed to measure plasma binding of compounds that are otherwise very difficult – if not impossible – to handle in dialysis or ultracentrifugation. This kit measures plasma binding of only one compound. It does that by establishing 25 different equilibrium binding states. They are characterized by 5 different plasma dilutions crossed by 5 different immobilized membrane surface areas that create a competitive binding substrate for your test items. There is an exact formula for describing this competitive binding situation and the supplied spreadsheet uses it to numerically derive the plasma binding parameters. This approach allows you to have plasma in every assay compartment, so that the solubility is increased, and non-specific binding decreased. The assay kit supplies everything needed for the measurement, except plasma.
The Transil High Sensitivity Binding (HSB) kit measures plasma binding of only one compound. It does that by establishing 25 different equilibrium binding states. They are characterized by 5 different plasma dilutions crossed by 5 different immobilized membrane surface areas that create a competitive binding substrate for your test items. There is an exact formula for describing this competitive binding situation and the supplied spreadsheet uses it to numerically derive the plasma binding parameters. This approach allows you to have plasma in every assay compartment, so that the solubility is increased, and non-specific binding decreased.
Those two kits are very different. The TRANSIL PPB Binding Kit is a fast screening kit for plasma protein binding. It contains both HSA and AGP coated beads, mixed at a physiological ratio. While that kit comes in very high quality polypropylene (made my micronic) with very low non-specific binding, there is still a measurable difference to the non-specific binding in the silanized glass vials of the TRANSIL High Sensitivity Binding (HSB) Kit.
The beads used in the HSB kit are coated with phosphatidylcholine membranes and the assay is utilizes our equilibrium shift assay principle based on competitive binding to plasma (in solution) and membranes (immobilized). This kit is our specialty solution for all compounds that either (i) bind too strongly to plasma that it’s difficult to quantify, (ii) have low solubility in pure buffer and thus precipitate in normal test kits that have assay compartments without plasma, (iii) exhibit high non-specific binding, or any combination of that. The price per compound of the TRANSIL High Sensitivity Binding Kit is substantially higher than alternative means, however, they get you results when the compounds are difficult.
Why is the solubility and recover in the TRANSIL High Sensitivity Binding Kit higher than in any other assay system?
All assay compartments of the Transil High Sensitivity Binding (HSB) kit contain plasma which increases compounds’ solubility and decreases non-specific binding. Also, the HSB kit comes is silanized glass vials that further suppress non-specific binding. As some compounds (e.g. peptides) bind strongly to silanized glass, this kit is also available in low-binding polypropylene wells.
Yes, we offer a variant of the kit in 96-well format with low-binding polypropylene tubes.
The compounds are incubated at room temperature. This reduces esterase activity. And since the incubation time is only 30 minutes, there is only minimal compound degradation due to esterases. In fact, the incubation time can be reduced to 10 minutes when compounds have normal on and off rates.