ELISA Detection: Types, Methods, & More

ELISA Detection: Types, Methods, & More

What Is ELISA?

ELISA, or enzyme-linked immunosorbent assay, is a type of immunoassay used in labs to detect and quantify soluble substances, such as peptides, proteins, and antibodies, in a complex mixture of serum or blood samples. The assay is also known as enzyme immunoassay (EIA).

In this assay, antigens or antibodies are immobilized on the surface of ELISA plates, and the analytes are incubated in the plate for the target binding. The antigen-antibody complex is then eluted by providing specific physiological conditions.

The whole process of the ELISA test can be divided into three stages:

  • Coating/capture: Specific antigens are immobilized on the surface of 96-well plates. Often capture antibodies are immobilized on the surface to capture target antigens.
  • Plate blocking: To avoid background signals and ensure efficient binding between the target antigen and specific antibodies, the remaining surface of the plate is covered using some proteins.
  • Probing/detection: The ELISA plate containing the reaction mixture is incubated with antigen-specific antibodies that bind with the target antigen. The enzyme-conjugated detection antibodies are used to generate a signal that displays the amount of antigen/analyte present in a test sample.

The detection antibodies used in the ELISA assays are either traditional monoclonal antibodies, polyclonal antibodies, or recombinant monoclonal antibodies. Further, one thing to ensure during the assay is that both capture and detection antibodies must identify non-overlapping epitopes.

The enzyme conjugates frequently involved in the ELISA protocols include β-galactosidase, alkaline phosphatase (AP), horseradish peroxidase (HRP), and catalase. They bind with a specific substrate to produce a detectable signal range.

  • Signal measurement: A plate reader machine is used to read the amount of signal generated from the reaction between target antigens and antibodies. The data obtained can be quantitative, semiquantitative, and qualitative. In quantitative studies, concentration results are plotted against a standard curve, which compares optical density to log concentration.

In this article, we will review the four common types of ELISA methods and the detection techniques used in immunology labs for a range of research applications.

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What are the Types of ELISAs?

ELISA assay is mainly of four types:

1. Direct ELISA

The direct ELISA technique involves immobilizing the antigens on the polystyrene microplate, followed by blocking the plate using proteins, such as aprotinin, ovalbumin, BSA, or any other animal protein. Then, the plate is washed and incubated with the primary antibody.

In this assay, the primary antibody binds directly with the substrate, such as HRP or AP, resulting in color change. Because of involving only fewer steps in the process, direct ELISA is a quick technique for lab workflows, which also eliminates secondary antibody cross-reactivity. However, its disadvantage is the low sensitivity and cost of the reaction.

Figure: The steps involved in Direct ELISA.

2. Indirect ELISA

The steps for this technique are similar to direct ELISA. The only difference is the involvement of additional wash steps and the type of antibodies used in the process.

Indirect ELISA involves two types of antibodies in the workflow: primary antibodies and secondary antibodies.  

The primary antibody is added first, and washed, followed by an incubation step with the labeled secondary antibody.

The advantages of indirect ELISA are that it’s less expensive and more sensitive and flexible. However, due to the presence of a secondary antibody, there are chances of cross-reactivity during the assay.  

3. Sandwich ELISA

In this assay, antigens are sandwiched between two antibodies. That’s why the assay is known as sandwich ELISA. The assay starts with coating the surface of the microplate using a capture antibody, blocking the surface with BSA, followed by adding antigens to the plate for around 90 minutes.

After the incubation step, the plate is buffer washed, followed by incubation with a labeled secondary antibody. Then, a substrate is added to study and measure the reaction signal.

Though the sandwich ELISA has the highest sensitivity, its major disadvantage is the time and expense involved in the process. Further, the necessity of finding a perfect antigen-antibody pair is also a limitation of the assay.

Figure: The illustration of steps involved in sandwich ELISA.

4. Competitive ELISA

The assay detects the presence of antigen-specific antibodies in a test sample. It involves the use of two antibodies: an antibody present in the sample and an enzyme-conjugated antibody. Both of them compete for the antigen during the reaction. The absence of color represents positive test results, whereas the presence of it shows a negative test result.

This assay can’t be used for the sample undergoing any level of dilutions and is less sensitive. However, it has many advantages, such as the requirement of less sample purification, less variability, and analysis of a spectrum of antigens in a sample.

Figure: An illustration of the four types of ELISA assay.

What are Common ELISA Detection Methods?

In ELISA, labeled antibodies that bind to the specific antigen are often used. Therefore, when a substrate that can convert the enzyme is added to the reaction mixture, it causes a change in the color of the solution. The signal is detected or analyzed using detection methods.

Based on the enzyme and substrate, the detection methods in ELISA assay vary considerably. Today, many ELISA kits are also available to make your ELISA assays easy. However, it won’t contain generic reagents that also you will need to perform the assay, such as tween-20, phosphate-buffered saline (PBS), blocking buffer, wash buffer, etc.

Many detection methods are available today to study the reaction between antigen-antibody complexes, such as the colorimetric method, fluorescent, chemiluminescent, and chromogenic assay. However, the three most common of them are covered below.

Chemiluminescent Assay

This technique utilizes peroxidase-conjugated detection antibodies, such as HRP and AP. A luminol-based solution is used as a substrate that forms a reaction product, emitting light photons, which are read by using a luminometer.

The technique is ultra-sensitive compared to other detection methods, has a wider dynamic range, and can detect even the smallest amount of the analyte in a given sample.

Chromogenic Assay

The chromogenic assay is one of the most common types of ELISA detection methods. It involves the use of antibodies labeled with horseradish peroxidase (HRP-) or alkaline phosphatase (AP-) in combination with a chromogenic substrate, such as a tetramethylbenzidine (TMB) solution.

The technique exploits the absorbance of the reaction to determine the readout, which is used to compare the samples or detect the concentration of the molecule of interest based on the standard curve.  

Fluorescent Assay

This technique combines peroxidase-conjugated detection antibodies, such as HRP and AP, with fluorescent substrates that fluoresce when exposed to certain light wavelengths. The signal does not get saturated even at the higher concentration of the analyte and provides more accurate results.

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Enzyme-linked immunosorbent assay (ELISA) is a commonly used technique in immunology labs to detect the presence of an antigen or antibody in a given sample. The technique involves immobilizing the antigen or antibody on the surface of a microplate and then incubating it with specific labeled antibodies that pair with the target antigen. Then, an enzyme-substrate reaction is exploited to detect the concentration or presence of the target molecule.

The four types of ELISA used in labs include direct, indirect, sandwich, and competitive. They all have a spectrum of applications as diagnostic tools in plant pathology and medicine, such as detecting SARS-CoV-2 or disease biomarkers. Further, in many industries, such as the food industry, they are used as quality-control checks.

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