What is the Difference Between ELISA and ELISpot

The main difference between ELISA and ELISpot is that ELISA determines the total concentration of the secreted signaling protein or antibody, whereas ELISpot detects individual cytokine or antibody-secreting cells.  

ELISA and ELISpot are two analytical biochemical assays used to measure immunogenic cells and substances.       

Key Areas Covered

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

Key Terms 

ELISA, ELISpot

What is ELISA

ELISA (enzyme-linked immunosorbent assay) is a type of solid-phase enzyme immunoassay responsible for detecting specific proteins in biological samples with the help of primary antibodies. In this test, an enzymatic reaction helps to detect 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. Also, there are three types of ELISA: direct ELISA, Indirect ELISA, and sandwich ELISA.

Figure 1: ELISA

Direct ELISA

In direct ELISA, coating the surface of a plate with the sample enables 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.

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 a link with an enzyme. Therefore, adding the chromogenic substrate aids in detecting the specific protein on the plate using color development.

Sandwich ELISA

The first step in the sandwich ELISA is to sandwich the specific protein to be detected from the sample between the primary and the secondary antibody. 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 ELISpot

ELISpot, or enzyme-linked immunosorbent spot, is another type f immunoassay that quantitatively measures the frequency of cytokine secretion in single cells. It involves the immunostaining of protein analytes using antibodies. Here, the protein analyte can be either a chemical or biological substance. Cecil Czerkinsky developed ELISpot in 1983. Also, the mechanism of ELISpot contains seven steps. They include antibody coating, cell incubation, cytokine capture, detection of antibodies, streptavidin-enzyme conjugate, the addition of substrate, and analysis.

Figure 2: ELISpot

Antibody Coating: Coting wells on a plate with cytokine-specific specific monoclonal antibodies.

Cell Incubation: The addition of desired cells with cytokine expression onto the wells. Cells produce cytokines in response to stimuli. 

Cytokine Capture: The cytokines secreted by cells start to attach to the antibodies through specific epitopes. 

Detection of Antibodies: The rinsing of the wells removes cells and other undesirable substances, remaining antibody-bound cytokines. Then, biotinylated cytokine-specific detection antibodies are added to the wells. They also bind to the cytokines in the wells. 

Streptavidin Enzyme Conjugate: The addition of streptavidin enzyme-conjugate. A strong affinity occurs between biotin and cytokine-specific antibodies. 

Addition of Substrate: The addition of the substrate into the wells. It forms an insoluble precipitate creating spots in the wells. Here, the type of substrate added to the wells depends on the type of enzyme used in the assay.

Analysis: An automated ELISpot reader records the results of the forming precipitates on wells. 

Moreover, ELISpot is important for the detection of vaccine development, allergies, cancer development, macrophages/monocytes/dendritic cell characterization, apolipoprotein analysis, and in veterinary research.

Similarities Between ELISA and ELISpot

• ELISA and ELISpot are two analytical biochemical assays that measure immunogenic cells and substances.
• They detect the presence of protein analytes in samples.
• They use antibodies for the detection of the presence of protein analytes.
• These antibodies are specifically bind to the detecting analyte. 

Difference Between ELISA and ELISpot

Definition

ELISA (enzyme-linked immunosorbent assay) refers to a technique to detect the presence of antigens in biological samples. At the same time, ELISpot is a type of assay that quantitatively measures cytokine secretion frequency for a single cell.

Type of Detection

ELISA detects the presence of protein analytes in the sample, while ELISpot detects the presence of cytokines produced by cells in the sample.

Types of Antibodies Used

ELISA uses primary and secondary antibodies to detect the analyte, while ELISpot uses cytokine-specific specific monoclonal antibodies.

Importance

ELISA is important for the detection of infectious diseases, while ELISpot is important for the detection of vaccine development, allergies, cancer development, macrophages/monocytes/dendritic cell characterization, apolipoprotein analysis, and in veterinary research.

Conclusion

In brief, ELISA and ELISpot are two types of immunoassays that detect the presence of protein analytes in samples. ELISA detects protein analyte in the sample using primary and secondary antibodies that specifically bind to the analyte. It is important for the detection of infectious diseases. In comparison, ELISpot is another immunoassay that detects the presence of cytokines in the sample. It uses cytokine-specific specific monoclonal antibodies. Also, it is important in vaccine development, allergies, cancer development, macrophages/monocytes/dendritic cell characterization, apolipoprotein analysis, and in veterinary research. Therefore, the main difference between ELISA and ELISpot is the type of detection.

References:

1. Alhajj M, Zubair M, Farhana A. Enzyme Linked Immunosorbent Assay. [Updated 2023 Apr 23]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2023 Jan-
2. Elispot. ELISPOT – an overview | ScienceDirect Topics. (n.d.). 

Image Courtesy:

1. “ELISA TMB” By Ajpolino – Own work (CC0) via Commons Wikimedia
2. “ELISPOT-en” By DadOfBeanAndBug – Own Work (CC-BY SA 3.0) via Commons Wikimedia