What is the Difference Between ELISA and Dot ELISA

The main difference between ELISA and Dot ELISA is that ELISA helps in the detection and quantification of antibodies, hormones, peptides or other proteins in a biological sample whereas, in Dot-ELISA, the chromogenic substrate only precipitates into the area of enzyme activity. Furthermore, the three main types of ELISA are direct ELISA, indirect ELISA, and immunometric or sandwich ELISA while dot-ELISA is a type of solid phase enzyme immunoassays, same as the common ELISA.

ELISA and dot-ELISA are two types of immunological tools used to detect and quantify the amount of proteins especially, antigens. They are faster, flexible, reproducible, highly-sensitive, and high-throughput assays.

Key Areas Covered

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

Key Terms

Direct ELISA, Dot ELISA, ELISA, Immunological Assays, Indirect ELISA, Sandwich ELISA

What is ELISA

ELISA (enzyme-linked immunosorbent assay) is a type of solid phase enzyme immunoassay responsible for the detection of specific proteins in biological samples with the help of primary antibodies. In this test, an enzymatic reaction helps in detecting the bound primary antibodies. Here, the enzyme uses a chromogenic substrate to develop a color. Moreover, the solid phase used by the ELISA is mainly a microtiter plate.

Figure 1: Microtiter Plate

Furthermore, there are three main types of ELISA based on the methodology.

Direct ELISA

In direct ELISA, coating the surface of a plate with the sample enable the enzyme-linked antibodies to bind with the specific protein on the plate. Then, adding the chromogenic substrate aids in detecting the protein-bound antibodies.

Figure 2: Direct ELISA

Indirect ELISA

Indirect ELISA involves the detection of a specific protein in a sample with a two-step binding process. Here, the first step is coating a plate with the sample and incubating it with a specific type of primary antibody. Then, the next step is incubating this plate with a secondary antibody, which binds to the primary antibody. Further, this secondary antibody has link with an enzyme. Therefore, adding the chromogenic substrate aids in detecting the specific protein on the plate using the color development.

Immunometric/Sandwich ELISA

In the sandwich ELISA, the first step is to sandwich the specific protein to be detected from the sample in between the primary and the secondary antibody. In here, the first process is to coat the plate with the primary antibody, but not with the sample. Then, the next process is to incubate the sample with this plate. After that, the specific protein bound to the primary antibody can be detected with the help of a secondary antibody.

What is Dot ELISA

Dot-ELISA is a type of solid phase enzyme immunoassays. The most significant feature of dot-ELISA is the precipitation of the chromogenic substrate only into the area with the enzymatic activity. Here, the solid phase is a nitrocellulose membrane, which allows the binding of proteins to the surface. After incubating the sample with the nitrocellulose membrane, the excess sample is washed away, and the unbound surface of the nitrocellulose membrane is blocked.

Figure 3: Dot ELISA

Then, the primary antibody is added to the nitrocellulose membrane. Finally, the addition of a secondary antibody linked to an enzyme aids in the detection of the protein-bound secondary antibodies. Thus, this allows the protein-antibody complex to appear as a clear dot on the nitrocellulose membrane.

What are the Similarities Between ELISA and Dot ELISA

• ELISA and dot ELISA are two types of immunological assays used to detect and quantify proteins in biological samples.
• They are solid-phase enzyme immunoassays (EIAs).
• Also, both methods use enzymes such as alkaline phosphatase (AP) and horseradish peroxidase (HRP) linked to the secondary antibody. With the addition of the appropriate chromogenic substrate, which develops a color through the enzymatic action, the specific protein in the sample can be detected.
• Besides, both methods are high-sensitive and high-throughput. Also, they are faster and reproducible as well.

What is the Difference Between ELISA and Dot ELISA

Definition

ELISA (enzyme-linked immunosorbent assay) refers to a rapid immunochemical test which involves an enzyme used for measuring a wide variety of tests of body fluids while dot ELISA refers to a solid-phase immunoassay that can be used to detect either antigen or antibody. Thus, this is the main difference between ELISA and dot ELISA.

Types

The three types of ELISA are direct ELISA, indirect ELISA, and sandwich ELISA while dot ELISA is a type of sandwich ELISA.

Method

ELISA involves detecting a specific protein with the help of an antibody and detecting bound antibodies through an enzymatic reaction while in dot ELISA, the chromogenic substrate only binds to the area with enzymatic activity. Hence, this is another difference between ELISA and dot ELISA.

Solid Phase

One more difference between ELISA and dot ELISA is that ELISA mainly uses microtiter plates as the solid phase while dot-ELISA uses nitrocellulose membranes as the solid phase.

Conclusion

ELISA is a type of immunological assay which involves the detection of a specific protein in a biological sample with the help of an antibody. Here, a color development through an enzymatic reaction is involved in the detection of the bound antibodies to the protein. In addition, dot ELISA is a type of sandwich ELISA. Here, the chromogenic substrate precipitates in the areas with enzymatic activity. Therefore, the main difference between ELISA and dot ELISA is the method of detection.

Reference:

1. “TechNote: What Are the Differences between ELISA Assay Types?” Enzo Life Sciences, 27 July 2018, Available Here
2. Z, Svobodova. “Dot-ELISA Affinity Test: An Easy, Low-Cost Method to Estimate Binding Activity of Monoclonal Antibodies.” Journal of Analytical & Bioanalytical Techniques, vol. 04, no. 03, 2013, doi:10.4172/2155-9872.1000168.

Image Courtesy:

1. “Microtiter plate” By Jeffrey M. Vinocur – Own work (CC BY 2.5) via Commons Wikimedia  
2. “ELISA diagram” By Cavitri – Own work (CC BY 3.0) via Commons Wikimedia  
3. “Dot-Elisa” By MrPotatoDeluxe – Own work (CC BY-SA 3.0) via Commons Wikimedia