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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


  • Why can results from the TRANSIL HSA Binding kit differ from literature values?

    Most literature values were obtained by dialysis experiments. Usually, dialysis experiments use pooled plasma that is not controlled for its exact protein composition. However, even minor changes in both HSA and AGP concentration can have major effects on the fraction unbound of some drug candidates. Also, most literature values date back to times when there was no awareness of the influence of pH shifts during the dialysis experiment on the results. And only recently has it been recognized that plasticizers in plasma collection bags can have an equally dramatic effect on plasma protein folding and thus binding of drugs. Thus, differences between results from TRANSIL and dialysis experiments are to be expected due to artefacts that can arise and uncontrolled variables in dialysis.

  • What are dissociation constants of marketed drugs?

    The table below lists dissociation constants for some marketed drugs.

    Compound KD(HSA) in μM KD(AGP) in μM
    Propranolol 430.0 5.2
    Imatinib 51.8 1.3
    Vincristine 123.0 3.9
    Paclitaxel 62.0 58.6
    Docetaxel 15.0 3.3
    Verapamil 357.0 6.1
    Sulfasalazine 0.6 27.0
    Ketoprofen 1.7 290.0
    Warfarin 6.4 8.8
    Chlorpromazine 38.0 0.8
    Progesterone 8.5 10.0
  • Can I use the TRANSIL HSA Binding Kit for compounds with plasma instability?

    When a compound is unstable in plasma equilibrium dialysis fails to report adequate results. However, the TRANSIL HSA binding assay performs well under these conditions, because incubation time is 15 minutes or less and there are no enzymes present in the assay system that would degrade the compound.

  • Can I use the TRANSIL HSA Binding Kit for compounds that are unstable?

    When a compound is unstable and degrades within hours during equilibrium dialysis results will not be useful. However, the TRANSIL HSA binding assay performs well under these conditions, because incubation time can be reduced to only 2 minutes.

  • Will it be necessary to run the TRANSIL HSA Binding assay in a controlled CO2 atmosphere like it is required for dialysis?

    No, there are two reason why this is not needed. Remember, the CO2 atmosphere is needed for dialysis because there is only a low buffer capacity in the system which makes it susceptible to pH changes by diffusion of CO2. That pH change then results in biased plasma protein binding estimates.

    This doesn’t happen in the TRANSIL HSA Binding Kit as comes with a strong phosphate buffer, thus diffusion of CO2 will not alter the pH. Also, the kit is only incubated for 12 minutes instead of several hours which minimizes the potential impact of CO2-concentration changes.

  • Why is plasma protein binding important?

    The extent of binding to plasma influences the way in which a drug distributes into tissues in the body. If a compound is highly bound, then it is retained in the plasma, which results in a low volume of distribution. This may impact on the therapeutic effects of the compound by limiting the amount of free compound which is available to act at the target molecule. Extensive plasma protein binding also limits the amount of free compound available to be metabolized which can, in turn, reduce the clearance of the compound.

  • Why is it useful measuring the dissociation constant KD instead of the fraction unbound?

    Healthy humans have a plasma composition that breaks down into 60% albumin and 40% globulins. The latter break down into 4% α1 globulins, of which α1 acid glycoprotein is only a small part, 8% α2 globulins (e.g. haptoglobulin, macrogobulin etc), 12% β globulins, and 16% γ globulins (i.e. our antibodies). However, these are textbook number referring to healthy humans. The plasma composition changes with age, race, sex, and disease condition and the unbound fraction of a drug can change accordingly (Figure 3). In fact, it has been shown that, for example, a 2-fold change in the AGP (AAG) concentration can lead to a 3-fold reduction in survival time of NSCLC patients treated with docetaxel. Thus, measuring the dissociation constant KD of your discovery compound to the major plasma proteins HSA and AGP can be critical in many indication areas.


    Figure 2: Illustration of the reduction of the unbound fraction in response to an increasing AGP (AAG) content of human plasma.
  • Which compounds can I use as positive and negative controls in the TRANSIL HSA Binding Kit?

    For instance, as a positive control you can use warfarin (KD=6.4 µM), and as a negative control compound you can use propranolol (KD=430 µM).

  • What are the main quality control measures applied in TRANSIL assays?

    The TRANSIL Quality Index (TQI) is based on independent measures derived from the data analysis.

    • Overall fit of the data to the standard protein binding model
    • Recovery: does the model derived compound concentration equal the true concentration?
    • Data consistency: does membrane binding increase proportionally with the increasing TRANSIL bead content in each well?
    • Data consistency: are the estimated reference concentrations in alignment with the compound concentration used?
    • Missing data and outliers.
  • How long does it take to run the assay?

    One assay plate can be used for 12 compounds. Thus, you’ll need to pipette 15 µl of test item to each of 8 wells and repeat this for all 12 compounds. This takes less than 10 minutes even with manual pipetting. After compound addition, the plate is ready for incubation. You can do this with an electronic 8 or 12 channel pipette by aspirating and dispensing a volume of 120 µl for 15 times. That takes just over a minute for each column or row. In total, that makes 8 to 15 minutes depending on your pipette. When using a pipetting robot with a 96 well head this time decreases to 2 minutes. After incubation, the plate needs to be spun in a plate centrifuge for 10 minutes. The supernatants are then ready for quantification by LC/MS/MS, UV, fluorescence or any other method of your choice.

    Thus, the total time the start and end of the experiment varies between 7 and 25 minutes depending upon your equipment.

  • How many compounds can be analyzed with one plate?

    One assay plate can be used for 12 compounds. A special feature of the 96 well plates used for these kits is that each of the 12 columns can be separated from the plate. Thus, it is possible to use the plate for one compound at a time.

  • Are TRANSIL assay kits supplied in low-binding plates?

    The TRANSIL assay kits utilize Micronic 96 well plates with ultra-low-binding tubes. Standard polypropylene tubes have 41x higher non-specific binding and low-binding tubes from other vendors have 2.6x higher non-specific binding.