What is the Difference Between Indirect and Sandwich ELISA

The main difference between indirect and sandwich ELISA is that in indirect ELISA, the microtiter plate is coated with the sample with proteins to be detected whereas this plate is coated with primary antibody in sandwich ELISA. Furthermore, sandwich ELISA is 2-5 times sensitive method than indirect ELISA.

Indirect and sandwich ELISA are two methods of ELISA (enzyme-linked immunosorbent assay) that are solid phase enzyme immunoassays (EIA). They are comparatively sensitive methods, which are also faster and reproducible.

Key Areas Covered

1. What is Indirect ELISA
     – Definition, Procedure, Importance
2. What is Sandwich ELISA
     – Definition, Procedure, Importance
3. What are the Similarities Between Indirect and Sandwich ELISA
     – Outline of Common Features
4. What is the Difference Between Indirect and Sandwich ELISA
     – Comparison of Key Differences

Key Terms

ELISA, Enzyme-Linked Secondary Antibody, Indirect ELISA, Primary Antibody, Sandwich ELISA, Sensitivity

What is Indirect ELISA

Indirect ELISA is a type of two-step ELISA, which uses two types of antibodies for the detection and quantification of a specific protein in a sample. The procedure of the indirect ELISA is as follows.

• Coating the surface of the microtiter plate with the sample;
• Washing to remove unbound proteins from the plate;
• Incubation of the protein-coated microtiter plate with the primary antibody;
• Washing to remove unbound antibodies from the plate;
• Incubation of the microtiter plate with the enzyme-linked secondary antibody;
• Washing to remove unbound antibodies from the plate;
• Addition of the corresponding chromogenic substrate required by the enzymatic reaction;
• Detection of the signals produced from the plate.
  

  Figure 1: Indirect ELISA

In general, indirect ELISA is a good method to diagnose bacterial, viral or parasitic infections by quantifying antibodies produced by the body against the pathogenic antigens. Here, polyclonal antibodies can be used as the secondary antibody in indirect ELISA. Therefore, it is more economical. On the other hand, different types of primary antibodies in different numbers can be used to bind with the target protein. Hence, indirect ELISA is a respectively more flexible and more sensitive method for the detection of a target protein.

What is Sandwich ELISA

Sandwich ELISA is the other type of two-step ELISA, which uses both primary and enzyme-linked secondary antibody for the detection. The main feature of the sandwich ELISA is that the sandwiching of the protein of interest in between primary and secondary antibody. The procedure for the sandwich ELISA is as follows:

• Coating the surface of the microtiter plate with the primary antibody;
• Washing to remove unbound primary antibodies from the plate;
• Incubation of the microtiter plate with the sample;
• Washing to remove unbound proteins from the plate;
• Incubation of the microtiter plate with the enzyme-linked secondary antibody;
• Washing to remove unbound secondary antibodies from the plate;
• Addition of the corresponding chromogenic substrate required by the enzymatic reaction;
• Detection of the signals produced from the plate.
  

  Figure 2: Sandwich ELISA

Significantly, sandwich ELISA is good to detect low-to-high molecular weight proteins. Also, it is more sensitive (2-5 times) than the direct or indirect ELISA. In addition, sandwich ELISA is a more specific technique since it uses two antibodies to capture the target protein.

Similarities Between Indirect and Sandwich ELISA

• Indirect and sandwich ELISA are two types of ELISA methods that help to detect and quantify proteins in a biological sample.
• Both types of ELISA methods consist of a two-step method.
• Moreover, they use both primary antibody and enzyme-linked secondary antibody for the detection of a specific protein including antigens, antibodies, hormones, etc.
• Also, due to the involvement of two types of antibodies in these two types of assays, their specificity is high.
• Besides, both methods are high-throughput methods which are also highly sensitive. They are faster and reproducible assays.

Difference Between Indirect and Sandwich ELISA

Definition

Indirect ELISA refers to a two-step ELISA which involves two binding processes of primary antibody and labeled secondary antibody while sandwich ELISA refers to another two-step ELISA type in which the protein of interest is sandwiched between primary and secondary antibody. Thus, this is the main difference between indirect and sandwich ELISA.

Coating the Microtiter Plate

In indirect ELISA, the coating of the microtiter plate is the sample with proteins while, in sandwich ELISA, the coating is the primary antibody.

Binding of the Secondary Antibody

The secondary antibody binds to the primary antibody in indirect ELISA while the secondary antibody binds to the protein of interest in sandwich ELISA.

Sensitivity

Another difference between indirect and sandwich ELISA is that the indirect ELISA is a sensitive method while sandwich ELISA is 2-5 times sensitive than indirect ELISA.

Applications

While indirect ELISA is better for determining the total antibody concentration in samples, sandwich ELISA is better for the analysis of complex samples. Hence, this is another difference between indirect and sandwich ELISA.

Conclusion

Indirect ELISA is a type of two-step ELISA which is involved in the binding of the primary antibody to the protein of interest on the microtiter plate followed by the detection by an enzyme-linked secondary antibody. On the other hand, sandwich ELISA is the other type of two-step ELISA which is involved in the binding of the protein of interest to the primary antibody, which coats the microtiter plate followed by the detection with the enzyme-linked secondary antibody. Therefore, the main difference between indirect and sandwich ELISA is the sequence of the procedure.

References:

1. “TechNote: What Are the Differences between ELISA Assay Types?” Enzo Life Sciences, 27 July 2018, Available Here

Image Courtesy:

1. “Indirect ELISA” By Cawang – Own work (CC0) via Commons Wikimedia  
2. “Sandwich ELISA” By Cavitri, E_A_S, Tobias Vornholt – Modified version of https://commons.wikimedia.org/wiki/File:ELISA_diretto_e_sandwich.png (CC BY 3.0) via Commons Wikimedia