The instability of IVD reagents is caused by these reasons!

In the field of in vitro diagnostic reagents, antibodies, antigens, enzymes, and other proteins are crucial key materials. Their specific binding and catalytic reaction functions make them very sensitive to potential harmful effects during operations such as preprocessing, storage, and use. While storing in a sealed and dry environment helps ensure long-term stability, the drying process may lead to severe denaturation of proteins unless they receive proper protection.

In a solution state, the potential harmful factors increase. For instance, a simple change in pH values or buffer solution can significantly affect the stability of proteins in a solution. Methods that stabilize a certain solution may not necessarily be effective for another. Other factors during the production process, including raw material sources, dilution and timing errors, inappropriate temperature and humidity, exposure, and container materials, can also influence the performance of the product. To ensure the effectiveness and accuracy of diagnostic testing, these protein materials must remain stable and effective. Let's explore the factors that affect protein stability from three aspects, hoping to assist you.

Factors Affecting Stability of Solid-State Reagents

Although most proteins function in their natural conformation in a solution state, drying or freezing is still the preferred method for stable storage. In these relatively stable states, negative-acting solvents like proteinases and oxidants have the least impact due to the reduced frequency of collisions between them and protein molecules. However, removing solvents from protein molecules through drying or other phase transition methods, such as precipitation and freeze-drying, may damage the functional structure of proteins. These phase transitions may expose hydrophobic amino acids usually folded inside the molecule to the molecular surface, leading to the binding of protein molecules through hydrophobic surfaces with other molecules, resulting in protein denaturation. Drying methods include natural air drying or freeze-drying, such as drying bound substances in reagent bottles, and drying antibodies on plastic plates or membrane materials.

Adding appropriate solutes to the solution to prevent protein denaturation during drying is a very effective method to maintain protein stability. These solutes typically have hydrophilic groups, which stabilize the functional structure of proteins by masking hydrophobic amino acids. Therefore, it is crucial to add protective agents before drying, as solvent removal during drying promotes interactions between proteins, especially the interaction of proteins with the solid-phase surface.

Regardless of the drying method or storage condition, maintaining stability is demanding. When applying proteins to membranes, rapid drying or freeze-drying is often required to preserve the optimal performance and vitality of proteins. For protein-coated plates, test tubes, or beads, ensuring thorough drying is more important than rapid drying. To maximize shelf life, all dried products need to be stored in airtight containers with desiccants.

Factors Affecting Stability of Liquid-State Reagents

Although antibodies, antigens, enzymes, and other diagnostic proteins are in their natural functional state in water solutions, it is not their most stable state. In solution, various factors affect the activity of proteins:

Protein molecules become more flexible and prone to conformational changes.

The collision frequency between protein molecules and other molecules, as well as container walls, increases.

The likelihood of microbial contamination increases.

Sensitivity to the effects of temperature rise and protein concentration decrease becomes more pronounced.

Proteins are more prone to oxidation.

Generally, dissolved protein molecules are most stable at lower temperatures and higher protein concentrations. Just above the freezing point, protein molecules have less kinetic energy, which is necessary for proteins to "escape" their lowest energy functional structures. In solution, some loss of protein activity is caused by adsorption of proteins to container walls, oligomerization of enzymes, and trace contaminants such as oxidants, hydrolases, and microorganisms. The proportion of lost protein activity to total protein activity is lower at higher protein concentrations (in the range of milligrams per milliliter or higher).

Other Factors Affecting Reagent Stability

The source of proteins, purification methods, and methods of chemical conjugation can all affect the activity and stability of protein reagents. For example, the source of alkaline phosphatase has a significant impact on the stability of the complex. Regarding stable storage in solution, it is essential to separate the required proteins from harmful enzymes (such as hydrolases and oxidases) in the raw materials. To ensure solution stability, it is crucial to remove or inactivate these harmful enzymes and microorganisms. In the synthesis of complexes, many chemical reaction steps have been shown to form covalent bonds between enzyme and antibody molecules. Some of these methods have a lower negative impact on the stability of certain enzyme-antibody complexes. If one method results in the loss of enzymatic detection activity but maintains enzymatic activity, choosing another chemical conjugation method might address this stability issue.

Sometimes, what is perceived as a stability issue is actually caused by an insufficient amount of protein. Perhaps it's just a straightforward dilution error. Manufacturers must discard materials such as untreated polystyrene, polysulfone, polycarbonate, or glass, which may absorb proteins or inhibit protein activity. Polyethylene and polypropylene are preferable container materials. Protein solutions containing colored enzymes and other proteins with oxidizable metal ions should not be exposed to sunlight.

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