In the development of immunoassay reagents, buffer is typically the second most critical factor after core materials. It can affect various fundamental properties of the reagents, such as stability, sensitivity, and specificity. In previous articles, we have mentioned the roles of various buffer components several times. Because it is so crucial, today I would like to summarize it again: What are the typical components of immunoassay buffers, what materials are commonly used for each component, for everyone’s reference in the process of reagent development and optimization.
In vitro diagnostic reagent buffers typically consist of six components: buffer agents, salt ions, stabilizers, surfactants, blockers, and preservatives. Below, we’ll elaborate on the functions of each component and common ingredients used.
Buffer agents
Buffer agents resist the influence of external acids or bases on solution pH, maintaining stability within a certain range. pH is a critical factor for many biochemical and enzyme-catalyzed reactions. Thus, buffer agents not only affect the stability of various proteins during storage but also ensure relatively stable pH values during reagent testing, ensuring accurate and reproducible results. Additionally, buffer agents provide a certain degree of ionic strength, promoting immune reactions and reducing nonspecific binding.
Common buffer agents include neutral PB buffer, HEPES buffer, slightly acidic MES buffer, citrate buffer, slightly alkaline Tris buffer, CB buffer, etc.
Chemicals used for buffer at BOC Sciences
| Name | CAS | Description |
| Tromethamine | 77-86-1 | Tris(hydroxymethyl)aminomethane is a component of buffer solution such as TAE and TBE buffers. It has been used in nucleic acid studies. |
| HEPES | 7365-45-9 | HEPES, a very effective N-substituted aminosulfonic acid biological buffer, is an important flexible exfoliating component that reorganizes the stratum corneum and promotes keratinocyte metabolism. |
| MOPS | 1132-61-2 | MOPS acts as a multi-purpose buffering agent used in various biological research. |
| Citric acid | 77-92-9 | Citric acid is mainly used as an acidifier, flavoring agent and chelating agent. |
| Sodium 2-Morpholinoethanesulfonate | 71119-23-8 | Buffering agent. Working pH range in aqueous solution: 9.0-10.5. |
| PIPES | 5625-37-6 | PIPES is a frequently used buffering agent in biochemistry. |
Salt ions
Salt ions in reagent buffers typically serve three functions. Firstly, they promote reaction rates. In some cases, salt ions can act as catalysts or co-factors, accelerating chemical reactions in the reagents, thereby enhancing detection sensitivity and speed. Secondly, they stabilize molecules. Salt ions can help stabilize the three-dimensional structure of proteins and other biomacromolecules through salt bridging, preventing denaturation or degradation. Lastly, they suppress nonspecific binding. In certain situations, salt ions can reduce nonspecific electrostatic interactions through charge shielding, thereby reducing false-positive results.
Common salt ions include monovalent ions such as sodium chloride, potassium chloride, and divalent ions such as magnesium chloride, calcium chloride, etc.
Stabilizers
Stabilizers, as the name suggests, maintain the stability of reagents during storage, transportation, and usage. They are typically divided into several categories. Firstly, the most common are protein stabilizers, which prevent protein and enzyme degradation and nonspecific adsorption. Secondly, sugar stabilizers. Sugars can increase stability by forming hydrogen bonds with proteins or other biological molecules, preventing denaturation or degradation. This stability is particularly important for maintaining the activity of enzymes and other biological catalysts. Lastly, other types such as glycerol, DTT, etc. Glycerol can increase solution viscosity, reducing the movement speed of protein molecules and collision frequency between molecules, which helps reduce protein denaturation. Additionally, glycerol molecules contain multiple hydroxyl groups, which can form hydrogen bonds with amino acid residues on the protein surface, stabilizing the protein’s three-dimensional structure and preventing denaturation. Glycerol can also form hydrogen bonds with water molecules, increasing effective water content in the solution, providing a stable hydration layer for proteins, thus preventing protein aggregation and precipitation. DDT is a reducing agent that can act as an antioxidant in individual projects.
Common stabilizers include protein stabilizers like BSA, BGG, casein, FSG, animal sera, sugar stabilizers like sucrose, trehalose, and others like glycerol, DTT, etc.
Surfactants
Surfactants firstly have a solubilizing effect on proteins, helping proteins maintain a stable, non-aggregated state in the buffer, which is crucial for maintaining reagent stability. Secondly, they can reduce liquid surface tension, increasing permeability, which is essential in tests where rapid penetration of reagents into samples is required. Thirdly, surfactants can assist in dispersing and suspending solid particles such as enzymes, cells, or other particles, thereby improving reagent uniformity and reaction efficiency. Additionally, surfactants can prevent nonspecific protein adsorption to container or detection device surfaces, stabilizing reagent performance. Lastly, surfactants can help improve reagent stability during storage, extending their shelf life.
Common surfactants include non-ionic surfactants like Tween, Brij-35, Triton X-100, etc.
Non-ionic surfactants at BOC Sciences
| Name | CAS | Description |
| Brij30 | 9002-92-0 | Brij-30 is a non-ionic surfactant, mainly used as an emulsifier for water-in-oil and oil-in-water emulsions, as well as a stabilizer for microemulsions and multiple emulsions. |
| Brij52 | 9004-95-9 | Brij-52 can be used as a solubilizing agent for essential oils, perfumery chemicals, vitamin oils and low water-soluble drugs |
| Triton X-100 | 9002-93-1 | Triton X-100 is a non-ionic surfactant commonly used in biochemistry and molecular biology. It is often utilized for lysing cells to extract proteins or organelles, for permeabilizing cells in immunofluorescence staining protocols, and as a detergent in various laboratory applications. |
Blockers
Blockers are divided into active and passive types, mainly aiming to reduce nonspecific binding, thereby improving reagent sensitivity and specificity. They achieve this by covering surface sites that may cause nonspecific binding, reducing background signals, making detection signals mainly derived from specific target molecules. Additionally, by reducing nonspecific adsorption, blockers can significantly improve the signal-to-noise ratio of detection signals, making the results clearer and more reliable.
Common blockers include protein blockers, polymer blockers, small molecule blockers, etc. Their main functions include anti-HAMA, anti-complement, anti-RF factor, anti-biotin interference, etc.
Preservatives
Preservatives primarily inhibit microbial growth, thereby extending product shelf life and maintaining product effectiveness. In addition to prolonging product shelf life, preservatives achieve various other objectives. For instance, microbial growth may affect reagent activity and other performance indicators. The addition of preservatives helps maintain the original performance of the reagents. By extending the shelf life of reagents, preservatives help reduce economic losses due to premature reagent disposal and increase economic benefits by increasing reagent batch size or reducing batch production.
Common preservatives include proclin, thiomersal, sodium azide, antibiotics, etc.