ELISA- Direct, Indirect, Sandwich, and Competitive

Enzyme-linked immunosorbent Assay (ELISA) is a widely used immunological technique designed for the detection and quantification of specific proteins, typically antigens or antibodies, in biological samples. There are several types of ELISA, including Direct ELISA, Indirect ELISA, Sandwich ELISA, and Competitive ELISA, each designed for specific purposes but following the same underlying principle.

Principle of ELISA

The principle behind ELISA is the antigen-antibody interaction. Antigens are foreign substances, such as proteins or polysaccharides, that elicit an immune response in an organism. Antibodies are proteins produced by the immune system in response to the presence of an antigen. These antibodies can bind specifically to their corresponding antigen, forming an antigen-antibody complex. ELISA takes advantage of this specificity by using enzymes linked to either the antigen or antibody, which react with a substrate to produce a detectable signal, such as a colour change.

A. Direct ELISA

In a direct ELISA, the antigen is immobilized directly onto the microplate surface. The primary antibody, which is specific to the antigen of interest, is then added directly to the well. A secondary antibody labelled with an enzyme is used to detect the binding of the primary antibody.

Coating the microplate: The sample containing the target antigen is added to each well of the microtiter plate. The plate is incubated overnight at 4°C to allow the antigen to adsorb to the plate.
Blocking: After incubation, the plate is washed with a wash buffer to remove unbound antigens. A blocking solution, such as bovine serum albumin, is added to prevent nonspecific binding of antibodies on sites unoccupied by the antigens. The plate is then incubated and washed again to remove the blocking solution.
Adding the primary labelled antibody: The primary antibody, labelled with an enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase (AP), is added to each well and incubated to facilitate the binding of the primary antibody to the antigen. Subsequently, the plate is washed to remove any unbound primary antibodies.
Enzyme-substrate reaction: A substrate specific to the enzyme conjugated to the primary antibody is added to the wells. The enzyme-substrate reaction produces a detectable signal, such as a colour change. The intensity of the signal is proportional to the amount of target antigen present in the sample.
Measuring the signal: The optical density (OD) of each well is measured using a microplate reader. The OD values are compared to a standard curve, which is generated by running known concentrations of the target antigen alongside the samples. This allows for the quantification of the target antigens in the samples.

B. Indirect ELISA

In an indirect ELISA, the antigen is immobilized onto the microplate surface, similar to the direct method. Instead of adding the primary antibody directly, a primary antibody specific to the antigen is added, followed by a secondary antibody labelled with an enzyme.

Coating the microplate: The sample containing the target antigen is added to each well of the microtiter plate. The plate is incubated overnight at 4°C to allow the antigen to adsorb to the plate.
Blocking: After incubation, the plate is washed with a wash buffer to remove unbound antigens. A blocking solution, such as bovine serum albumin, is added to prevent nonspecific binding of antibodies on sites unoccupied by the antigens. The plate is then incubated and washed again to remove the blocking solution.
Adding the primary antibody: The primary antibody specific to the target antigen is added to each well and incubated to facilitate the binding of the primary antibody to the antigen. Subsequently, the plate is washed to remove any unbound primary antibodies.
Adding the secondary labelled antibody: Enzyme-conjugated secondary antibody specific to the primary antibodies is added to each well. The plate is then incubated and washed again to remove any unbound secondary antibodies.
Enzyme-substrate reaction: A substrate specific to the enzyme conjugated to the secondary antibody is added to the wells. The enzyme-substrate reaction produces a detectable signal, such as a colour change. The intensity of the signal is proportional to the amount of target antigen present in the sample.
Measuring the signal: The optical density (OD) of each well is measured using a microplate reader. The OD values are compared to a standard curve for quantification of the target antigen in the samples.

C. Sandwich ELISA

In a sandwich ELISA, the antigen is trapped between two layers of antibodies- a capture antibody and a detection antibody. The capture antibody is immobilized on the microplate and binds to the antigen. The detection antibody, labelled with an enzyme, is then added to detect the bound antigen.

Coating the microplate: The capture antibody, specific to the target antigen, is immobilized onto the microplate surface. The plate is incubated to allow the capture antibody to bind to the plate.
Blocking: After incubation, the plate is washed with a wash buffer to remove unbound capture antibodies. A blocking solution, such as bovine serum albumin, is added to prevent nonspecific binding of antibodies on sites unoccupied by the capture antibodies. The plate is incubated and washed again to remove the blocking solution.
Adding the sample: The sample containing the target antigen is added to each well. The plate is incubated, allowing the capture antibody to bind the target antigen in the sample.
Adding the detection antibody: The detection antibody, labelled with an enzyme (such as HRP or AP), is added to each well. The plate is incubated, facilitating the binding of the detection antibody to the target antigen, forming a “sandwich” complex. The plate is washed to remove any unbound detection antibodies.
Enzyme-substrate reaction: A substrate specific to the enzyme conjugated to the detection antibody is added to the wells. The enzyme-substrate reaction produces a detectable signal, such as a colour change. The signal intensity is proportional to the amount of target antigen present in the sample.
Measuring the signal: The optical density (OD) of each well is measured using a microplate reader. The OD values are compared to a standard curve for quantification of the target antigen in the samples.

D. Competitive ELISA

In competitive ELISA, a labelled antigen competes with the sample antigen for binding to a limited amount of immobilized antibodies. The amount of labelled antigen bound is inversely proportional to the concentration of the antigen in the sample.

*Figure: Procedure for Competitive ELISA*

Coating the microplate: The antibody specific to the target antigen is immobilized onto the microplate surface. The plate is incubated to allow the antibody to bind to the plate.
Blocking: After incubation, the plate is washed with a wash buffer to remove unbound capture antibodies. A blocking solution, such as bovine serum albumin, is added to prevent nonspecific binding of antibodies on sites unoccupied by the capture antibodies. The plate is incubated and washed again to remove the blocking solution.
Adding the labelled antigen and the sample: A known amount of labelled antigen and the sample containing the target antigen are simultaneously added to each well. The plate is incubated, allowing competition between the sample antigen and labelled antigen for binding to the immobilized antibody.
Enzyme-substrate reaction: A substrate specific to the enzyme conjugated to the antigen is added to the wells. The enzyme-substrate reaction produces a detectable signal, such as a colour change. The signal intensity is proportional to the amount of target antigen present in the sample.
Measuring the signal: The optical density (OD) of each well is measured using a microplate reader. The OD values are compared to a standard curve for quantification of the target antigen in the samples.

Applications 👩🏻‍🔬

Disease Diagnosis: ELISA is widely used in clinical laboratories for the diagnosis of various diseases, including infectious diseases, autoimmune disorders, and cancer.
Hormone and Protein Quantification: ELISA is used to quantify hormones, cytokines, and proteins in biological samples, providing valuable information about their concentrations.
Blood Typing: ELISA is utilized in blood typing and crossmatching, assisting in blood transfusion compatibility testing.
Allergen Detection: ELISA is employed to detect and quantify allergens in food products, helping in food safety assessments.
Research Applications: ELISA is widely used in research to study protein-protein interactions, signal transduction pathways, and cellular processes.

Advantages of ELISA 👍🏼

Sensitivity: ELISA is highly sensitive, allowing the detection of low concentrations of antigens or antibodies.
Specificity: ELISA can provide highly specific results due to the use of antigen-antibody interactions.
Quantification: ELISA allows for the quantitative measurement of antigens or antibodies, providing valuable information about sample concentrations.
High Throughput: ELISA can be adapted for high-throughput screening, making it suitable for processing a large number of samples simultaneously.
Versatility: ELISA can be modified for various applications, including direct, indirect, sandwich, competitive, and capture formats.

Limitations of ELISA 👎🏼

False-positive and false-negative reactions: Cross-reactivity and interference may lead to false-positive or false-negative results.
Limited dynamic range: ELISA may have a limited dynamic range, making it challenging to accurately quantify samples with extremely high or low concentrations.
Time-consuming: Some ELISA procedures, especially those with multiple steps, can be labour-intensive and time-consuming.
High Cost: ELISA kits or reagents can be relatively expensive, especially for large-scale studies.
Limited Multiplexing: ELISA is typically limited to the detection of a single analyte at a time, limiting its ability to analyze multiple targets simultaneously.