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Plasma Protein Binding Assay

Plasma Protein Binding Assay

The important physiological and pharmacological functions of proteins require the participation of small drug molecules. Small drug molecules can promote or inhibit the realization of protein functions through interaction with proteins, especially the combination of drugs with plasma proteins will affect the biological activity of drugs. Drugs bind to plasma proteins to varying degrees in the plasma, and the degree of binding can affect the absorption, distribution, metabolism, and excretion of the drug in the body, and then affect the pharmacodynamic behavior of the drug.  Therefore, the determination of drug plasma protein binding rate is important for drug development.

Common drug plasma protein binding assays include: equilibrium dialysis, ultrafiltration, partition equilibrium, stable isotope-GC-MS, gel filtration, and spectroscopic techniques. Among them, the equilibrium dialysis method is the most commonly used method to determine the free concentration of drugs based on the principle of drug-binding equilibrium, and it is also a classic method for studying the plasma protein binding rate of drugs.

The Principle of Equilibrium Dialysis :

The protein is placed in a compartment and separated by a semi-permeable membrane. Large molecules such as protein cannot pass through the semi-permeable membrane, while free ligands in the system can pass freely. When equilibrium is reached, the free ligand concentrations on both sides are equal. If the total amount of free ligands in the system is known, the amount of free ligands bound to the protein can be calculated by measuring the concentration of free ligands in the protein-free compartment.

Protocal of Equilibrium Dialysis:

  1.  Put the plasma into the semi-permeable membrane bag and tighten the pocket, put the semi-permeable membrane in a wide-mouth bottle containing dialysate, adjust the liquid level inside and outside the bag to the same level, and make a control at the same time (dialysis in the semi-permeable membrane bag liquid)
  2. Add the item to be tested (150μl-250μl, small volume to avoid affecting the composition of the dialysate) in the dialysate outside each bag, and begin balanced dialysis.
  3. Use LC-MS / MS method to quantitatively analyze and calculate the content of the test item and the rate of binding of the test item to the plasma protein.
  4. Rate of plasma protein binding= (Cdrug in the bag - Cdrug outside the bag/Cdrug in the bag)*100%


  1. Simple operation, easy temperature control, adjustable PH value and low equipment cost;
  2. Equilibrium Dialysis method is stable and reliable without obvious concentration dependence and species differences;
  3. Using the equilibrium dialysis method, the accuracy, precision, extraction recovery, and stability of the test samples meet the requirements of biological sample determination, and there is no interference of endogenous impurities, which is the most classic method.

As a leading pharmaceutical enterprise in the world, Profacgen has extensive experience in in vitro studies of pharmacokinetics such as determination of plasma protein binding rate. Our professional engineers can make different plans according to different needs of customers. Please contact us if you need.

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