Plasma Protein Binding (In Vitro)
Plasma protein binding (PPB) assay the proportion of unbound (free) compounds in plasma is determined by equilibrium dialysis. Since only compounds in the free state can bind to their targets for activity, compounds bound to plasma proteins may have longer half-lives and clearance rates. Therefore, the determination of plasma protein binding rate is of great reference significance for understanding the activity and tissue distribution of compounds. Plasma protein binding assays are one of the in vitro ADME screening services of BOC Sciences. BOC Sciences provides cost-effective, consistent, high-quality data in a highly automated approach.
Binding of compounds to plasma proteins may affect drug pharmacokinetics, efficacy, and toxicity. Because only free (unbound) drugs can diffuse freely into the target to exert their pharmacological effects. It is then metabolized by the liver and excreted from the body. In addition, PPB information can also be useful in estimating metabolism-related drug interactions (DDIs) if two drugs are used simultaneously. Generally, acidic drugs tend to bind strongly to serum albumin, while basic compounds also bind to alpha-acid glycoprotein (AGP) or lipoprotein.
Rapid equilibrium dialysis (RED) is a general, accurate and reliable method for determining the binding degree of compounds to plasma proteins. The plasma containing the test compound was added to the central chamber of a commercial plate-based RED device. The blank isotonic sodium phosphate buffer was added to the peripheral chamber of the RED device and the plate was incubated at 37 °C for 4 hours. The equilibrium of free compounds is achieved by diffusion of unbound compounds through the dialysis membrane. The buffer and plasma equipartition samples were withdrawn at predetermined time points and the concentrations of free and bound test compounds were determined by LC/MS/MS analysis.
Case Study of Rapid Equilibrium Dialysis
| Assay Design | Analysis | Report: | Quantity of test article required |
| Test articles are assayed in duplicate | LC/MS/MS detection | % compound bound | 1.0 - 2.0 mg |
| Test articles are mixed with human plasma (other species available) | Summary of Assay: Human or specific species of interest plasma in the sample chamber are spiked with test compounds at 100x dilution of stock solution (typically 10 mM in DMSO). The chamber is sealed, and the compound is dialyzed against PBS, pH 7.4 at 37°C for 4 hours. Aliquots from each chamber (plasma and PBS) are collected and the concentrations of compound in each sample are determined by LC/MS/MS. Adjustments are made for non-specific binding. | ||
| One concentration of test article (10 μM, different concentrations available) | |||
| One time point (t = 4 hours at 37°C) | |||
| Positive control: Propranolol (high binding) and Metoprolol (low binding) | |||
| Negative control: No plasma (PBS only) | |||
Our PPB Screening Assays
- Rapid equilibrium dialysis, ultrafiltration and ultracentrifugation
- Simultaneous evaluation of batches of 6-8 compounds
- Positive control at single concentration
- Up to six different types of plasma at a single concentration
- Repeat determination
- LC-MS/MS analysis
- Summary of data including percent unbound (fu ), % recovery (mass balance), percent remaining (plasma stability)
Species and Isolated Proteins Available
- Mouse, rat, rabbit, dog, monkey, hamster, guinea pig, mini pig and human
- Human serum albumin (HSA)
- Human alpha-1-acidglycoprotein (AGP)
- Human sex hormone binding protein
- Globulins
- Lipoproteins
- Fibrinogen
BOC Sciences offers plasma protein binding assays using equilibrium dialysis, ultrafiltration and ultracentrifugation. In addition, we offer plasma stability assays to measure the degradation of your compound in plasma. If you are interested in our plasma protein binding assays services, please contact us for more information.