Enzyme-linked immunosorbent assay, or ELISA, is a commonly performed biochemical assay that analyzes the presence of a biomolecule in a given sample. The immunoassay involves the immobilization of antigens or antibodies on the surface of a microplate and then the use of antigen-specific antibodies to study the test molecule.
Out of many factors, the passive binding of the solid phase is also the one that affects the ELISA process. Because in the absence of appropriate blocking, the detection antibodies will bind to the surface of the plate, resulting in false-positive results or high background signals.
That’s why a blocking buffer is needed to cover the free binding space of the ELISA plate surface, eliminating the possibility of non-specific binding. It also greatly improves the signal-to-noise ratio.
By definition, a blocking buffer is a solution of a mixture of proteins, irrelevant proteins, or any other compound that adsorbs on the free-binding site of the plate surface. In an ELISA assay, it’s used as a reagent that ensures that the primary or secondary antibody binds only to the target molecule.
A variety of blocking solutions are available based on the plate type, detection system, and assay format. The goal of each of them is to reduce background noise while maintaining specificity for antibodies.
Consider the following characteristics to decide on an ideal blocking buffer for your application:
In this article, we will cover more about the working of the blocking buffer, its types, and applications in a range of industries.
The primary role of ELISA-blocking reagents is to prevent the non-specific binding of biomolecules or antibodies to the solid phase, which could interfere with the sensitivity and accuracy of the assay. By reducing the background noise, the buffer improves the signal-to-noise ratio of the assay.
The blocking agents are proteins or other compounds that can effectively block plate sites. ome of the commonly used proteins for the assay include bovine serum albumin (BSA), casein, and non-fat dry milk. They are typically needed in 1-5% concentrations based on the protocol of the assay.
Often, the blocking solution also contains detergents, such as Tween-20, that disrupt the hydrophobic bonds between molecules and expose more non-binding sites for blocking. Some other components of the blocking buffer include a pH stabilizer that maintains a stable pH and a protein stabilizer that prevents coating protein denaturation.
The excessive use of blockers may result in masking the antigen-antibody interactions and inhibition of the marker enzyme. It can result in reduced signal and false outcomes.
A variety of blocking buffers are used in ELISA or western blot assays. A majority of them are a formulation of either a mixture of proteins or made of a single protein having no interaction with the antigens or detection antibodies. Typically, they are either diluted in phosphate-buffered saline (PBS) or Tris-buffered saline (TBS) with or without detergents, such as Tween-20 (used in 0.05%-0.2%).
Below are some types of blocking buffers for research use in life sciences and immunology
Blocking buffers have a spectrum of applications in different types of immunoassays, such as ELISA, immunohistochemistry, western blotting, and immunofluorescence.
Two primary roles played by blocking solutions in the assays include:
ELISA is widely used in life sciences and immunology labs to analyze the concentration of certain biomolecules and detect the presence or absence of an antigen or antibody in a sample, or study the interaction between molecules. In all the applications of ELISA, a blocking buffer is needed to reduce the background noise by preventing the non-specific binding of molecules and increasing the sensitivity of the assay.
Blocking buffers are used in many lab applications, ranging from western blotting, protein arrays, immunoprecipitation, and ELISA. Some most commonly used buffers include the ones made of nonfat dry milk (NFDM), bovine serum albumin (BSA), and casein.
ELISA blocking buffers are used in medical areas to obtain proper ELISA results while quantifying molecules, such as hormones, proteins, and peptides, or detecting antigens/antibodies in a given sample.
In plant pathology, ELISA blocking buffers assist in producing accurate ELISA results while detecting plant viruses in seeds or vegetative materials. ELISA is a common diagnostic tool in plant pathology to study and detect quality antigens and antibodies in different samples.
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ELISA blocking buffer is a commonly used reagent in ELISA assay to block the surface of the ELISA, preventing the non-specific binding of molecules. It ensures increased sensitivity of the assay and reduced background noise.
Some common types of blocking agents include bovine serum albumin (BSA), non-fat dry milk (NFDM), and casein. Often, in addition to blocking non-specific binding, they are also used as a diluent of antibodies in a variety of immunoassays.
While performing such high-throughput assays, it’s essential to use high-quality reagents paired with advanced equipment to obtain accurate data. That’s why it’s recommended to check the safety data sheet (SDS) that comes with the reagent to learn about its quality, ingredients, processing, and safety.
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