Sandwich ELISA: Overview & Applications

Sandwich ELISA: Overview & Applications

Sandwich ELISA: Overview

Enzyme Linked-Immunosorbent Assay, or ELISA, is a commonly used immunoassay in labs for the quantitative and qualitative analysis of biological molecules, such as antibodies, proteins, and peptides. 

During the process, one of the reaction components is adsorbed on a solid phase, such as a microtiter plate or ELISA plate surface. The sample containing the analyte or antigen is then incubated to form a specific antigen-antibody complex, facilitating the identification and separation of a specific biomolecule. 

Based on the bound and free components of the reaction and the procedure, the ELISA assay is of four types:

  • Direct ELISA: This ELISA protocol only involves a few steps compared to other ELISA types. Here, the antigen is immobilized on the plate surface, and then enzyme-conjugated primary antibodies are added for the formation of an antigen-antibody complex. After incubation with a suitable substrate, such as alkaline phosphatase (AP) or horseradish peroxidase (HRP), a change in color is observed due to the formation of an enzyme-substrate complex.
  • Indirect ELISA: It has similar steps as direct ELISA, except it involves the use of two primary antibodies (or detection antibody) and a secondary antibody which adds a few more washing steps in the process. It’s a more sensitive technique than the Direct ELISA assay. 
  • Sandwich ELISA: The assay is known as sandwich ELISA because the antigen is sandwiched between two antibodies: a capture antibody and a detection antibody. It offers the highest sensitivity but does require a suitable antigen-antibody pair to perform the enzymatic reaction.
  • Competitive ELISA: In this process, two antibodies (an enzyme-conjugated antibody and an antibody present in the sample) fight to bind with a single target antigen. Though the technique has less specificity, it can be used to measure many antigens present in a single sample. 
Figure: Four types of ELISA.

In this article, we will cover more about the sandwich enzyme-linked immunosorbent assay, its principle, and applications in the biotech and pharma industries. 

How Does Sandwich ELISA Work?

ELISA works on the principle of antigen-antibody interaction. Antigen-specific antibodies are immobilized on the ELISA plate surface and then incubated with the target antigen-containing samples. The incubation leads to the formation of an antigen-antibody complex that provides a color change or signal (based on the detection method) for the analysis of the target antigen. 

The antibodies used in ELISA assay as capture or detection antibodies are either monoclonal antibodies or polyclonal antibodies. The choice of antibody also determines the sensitivity of an assay. 

The steps involved in Sandwich ELISA include: 

  • Coating the plate with the capture antibody: The microtitre plate is covered with a capture antibody in a carbonate/bicarbonate buffer. Wash the plates with PBS to remove unbound antibodies. 
  • Blocking and adding samples: The blocking buffer (prepared by using reagents such as 5% non-fat dry milk/PBS) is added to the plate to block the remaining protein-binding sites and prevent non-specific binding. Then, the plate is washed, and the sample containing the target antigen is added and then washed again after incubation with PBS to remove unbound antigens. 
  • Secondary antibody incubation: A detection antibody (recognizing a different epitope on antigen than the capture antibody) is added to the reaction plate and the plate is incubated at room temperature for two hours. This is followed by washing the plate with PBS. The two most commonly used conjugates in the assay include Horseradish peroxidase (HRP) and alkaline phosphatase (ALP). 
  • Detection: A suitable substrate, such as P-Nitrophenyl-phosphate (pNPP), TMB (3,3’,5,5’-tetramethylbenzidine), or HRP chromogens, is added based on the conjugate enzyme to visualize the reaction and perform the analysis. The methods used to quantity unbound antigens and antibodies include fluorescent or colorimetric detection techniques. 

A standard curve with a concentration on the X-axis vs absorbance on the Y-axis is plotted using the serial dilution data to determine the concentration of a sample. 

Figure: An illustration of the Sandwich ELISA process.

Advantages & Disadvantages of Sandwich ELISA

Sandwich ELISA has higher specificity and sensitivity compared to other ELISA techniques as it involves the use of two antibodies having different epitopes. Additionally, it can be used with crude or impure samples without affecting the detection of specific antigens. 

The disadvantage of Sandwich ELISA is that it requires the preparation of two antibodies which is a complex, labor-extensive, and expensive process. 

What Is Sandwich ELISA Used For?

Sandwich ELISA is commonly employed in labs for the quantitative and qualitative analysis of a molecule of interest in the given sample. 

Protein Detection

ELISA is widely used to detect a range of proteins in a variety of samples, such as cell lysates, serum, and plasma. It involves the use of two types of antibodies—capture antibodies (made of monoclonal antibodies) and detection antibodies (made using polyclonal antibodies)—for protein detection. The antibody-antigen-antibody complex formation provides better insight into the detection or concentration of target antigen in the given sample. 

Protein Quantification

Protein quantification is measuring the amount of a specific protein in the given sample. The intensity of color change or fluorescence obtained after the detection step of ELISA shows the amount of target protein in the sample. The obtained signals are compared with a standard curve plotted using a known concentration of standard to determine the amount of unknown concentration of the molecule in the givens sample. 

What Industries Use Sandwich ELISA?

In addition to western blotting approaches, the Sandwich ELISA method is an extensively utilized technique in various Life Sciences labs and industries to study different biological molecules and their functional role.


Sandwich ELISA is used by biotechnologists in very research applications to study protein-protein interactions, hormone behavior, signaling pathways, toxins’ effects, and immune response functions.


ELISA is a crucial tool in pharma labs to diagnose many diseases. For example, it’s used to examine serum or blood for antibodies against viruses. Additionally, it is used to determine and quantify biomarkers in other clinical samples as well.

Food and Beverage

ELISA tests are used to detect and quantify various proteins, pathogens, allergens, and contaminants. It’s a crucial tool to assess the quality and authenticity of materials used in food production. Overall, it’s essential to ensure the safety and quality of food products. 

Procure Your R&D Lab Needs with Excedr

Sandwich ELISA is a type of ELISA assay used in biochemistry and biotechnology labs to study characteristics of proteins, including protein-protein interaction, concentration, and function in biochemical pathways. The technique is named so because the antigen is sandwiched between capture and detection antibodies.

To accelerate the ELISA workflow, many companies provide ELISA kits that obtain everything one needs to perform the test. However, scientists usually prefer using individual regents to make their ELISA assay kits suit their requirements. 

You must know the quality of reagents and the equipment used in the process also play a major role in the quality of the results. Therefore, it’s essential to ensure what you use in the process is effective and reliable.

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