ELISA Blocking Buffers: Overview & Application

ELISA Blocking Buffers: Overview & Application

ELISA Blocking Buffers: Overview

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:

  • Effectively prevent non-specific binding of assay reactant to the surface of the ELISA plate.
  • Ensure that the components of the assay are not disrupted while they are adsorbed to the well.
  • Prevent denaturation of assay reactant on the solid phase and act as its stabilizer
  • Possess no enzyme activity capable of generating signals from substrates or degrading reactants.
  • Do not cross-react with other assay reactants.
  • Consistently perform across lots.

In this article, we will cover more about the working of the blocking buffer, its types, and applications in a range of industries.

Need new or refurbished lab equipment? Excedr leases.

See our equipment list and browse a sample selection of what we can source. Or, if you’re ready, request an estimate.

How Do ELISA Blocking Buffers Work?

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.

Types of Blocking Buffers

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

lab applications:

  • Normal serum: It carries antibodies that bind to the remaining sites of the solid phase. It’s commonly used at a concentration of 1-5% (w/v). Moreover, the serum has a high concentration of albumin and other proteins that bind to nonspecific protein-binding sites.
  • Bovine serum albumin (BSA): It’s used at a concentration of 1-5% and competes with the antibody to bind to the non-specific binding site of the ELISA plate. However, it can cross-react with antibodies prepared against BSA-hapten conjugates.
  • Non-fat dry milk (NFDM): This is generally used between 1-3% concentration. It’s a common blocking agent used for covalent surfaces because of its amphipathic characteristics and molecular diversity. However, if not prepared and stored properly, it can deteriorate.

What Are ELISA Blocking Buffers Used For?

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:

  • Blocking non-specific binding: Blocking buffers cover the binding sites of the surface of the ELISA plate, which prevent the non-specific binding of proteins and other molecules. It overall rescues background noise, increases signal-to-noise stability, and improves the sensitivity and accuracy of assays.  
  • Diluent for antibodies: In some assays, blocking buffers are used as a diluent for primary antibodies or horseradish peroxidase (HRP)-conjugated secondary antibodies. This ensures the optimum concentration of the reagents in the assay.

What Industries Use Blocking Buffers?

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.

Plant Pathology

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.

Don’t have the budget to purchase lab equipment outright? Consider leasing through Excedr to save your lab time and money. Browse your leasing options today!

Procure Your R&D Lab Needs with Excedr

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.

Acquiring such expensive reagents and equipment can make you break the bank. But, it can be avoided if you choose to lease equipment rather than purchase them.

Excedr’s leasing solution is an effective program for scientists looking for accreditations for their labs or expanding them by making an impact through their work. The program ensures that you get any equipment you desire on lease with the budget in your hand.

There’s no upfront cost involved, and no repair and maintenance cost is needed, as it’s all covered in our program. Moreover, there’s no stress of delivery and installation of the instruments we even handle that as part of our program.

The range of equipment that you can lease from Excedr includes analytical equipment, life sciences and biotech equipment, clinical instruments, and other general biology equipment.

Excedr’s comprehensive leasing solution not only extends your cash runway but saves you more time to focus on your research and contribute your part in making this world better.