Introduction
ELISA (Enzyme-Linked Immunosorbent Assay) is one of the most widely used immunological techniques for detecting and measuring specific antigens (Ag) or antibodies (Ab) in biological samples. Due to its high sensitivity, specificity, rapid results, and cost-effectiveness, the ELISA test is extensively used in clinical diagnostics, medical laboratories, biotechnology, pharmaceutical research, and disease surveillance. It plays a crucial role in the diagnosis of infectious diseases such as dengue, HIV, hepatitis as well as in hormone analysis, vaccine development, and immunological research.
Basic Principle of ELISA
- ELISA is based on the specific binding between an antigen and its corresponding antibody.
- An enzyme is chemically linked (conjugated) to an antibody.
- When the enzyme reacts with its substrate, it produces a colored product, which can be measured spectrophotometrically to indicate a positive or negative result.
Microtiter Plates: The Foundation of ELISA
- Structure: Microtiter plates have flat-bottomed wells, allowing uniform light absorbance readings.
- Material: Made of polystyrene plastic, which allows passive attachment of antigens or antibodies to the well surface.
- Function: These multi-well plates are the standard platform for running ELISA assays, allowing multiple samples to be tested simultaneously.
Washing Step: A Critical Quality-Control Point
- A washing step is performed after every sequential step in the ELISA protocol.
- Washing Agent: PBS (Phosphate-Buffered Saline) buffer is used to remove unbound reagents.
Types of ELISA
There are four major types of ELISA, each suited to different diagnostic needs:
- Direct ELISA
- Indirect ELISA
- Sandwich ELISA
- Competitive ELISA
Direct ELISA
Objective: It is used to detect and quantify antigens (Ag) directly using an enzyme-labelled antibody.
Procedure:
Sample Addition: The antigen-containing sample is added to the wells and binds to the polystyrene plate.
Washing: Unbound antigens are washed away using PBS buffer.
Blocking: The remaining unoccupied sites are blocked using Bovine Serum Albumin (BSA) or non-fat milk to prevent non-specific binding.
Antibody Binding: An enzyme-labelled primary antibody specific to the antigen binds directly to it (no secondary antibody involved).
Substrate Addition: A substrate like TMB (3,3',5,5'-Tetramethylbenzidine) reacts with the enzyme, producing a color change that confirms a positive result.
Indirect ELISA
Objective: It is primarily used for antibody (Ab) detection ideal for detecting a patient's immune response to an infection.
Procedure:
Antigen Coating: Known antigens are pre-coated onto the microtiter wells.
Sample Addition: Patient serum containing un-labelled primary antibodies is added and allowed to bind the antigen.
Washing & Blocking: Unbound components are washed off; the plate is blocked with BSA.
Secondary Antibody Addition: An enzyme-labelled secondary antibody (targeting the Fc region of the primary antibody) is added.
Substrate Addition: TMB substrate produces a color change, confirming the presence of the target antibody.
Sandwich ELISA
Objective: A highly sensitive method for antigen (Ag) detection, ideal for low-concentration samples.
Procedure:
Capture Antibody: Wells are coated with an un-labelled capture antibody specific to the target antigen.
Sample Addition: The antigen is added and gets "sandwiched" between the capture antibody and a detection antibody.
Detection Antibody: A biotinylated (or enzyme-linked) secondary detection antibody binds the captured antigen.
Streptavidin-HRP Conjugation: If biotin is used, Streptavidin-HRP (Horseradish Peroxidase) is added for high-affinity binding.
Substrate Reaction: TMB substrate produces a measurable colored product.
Competitive ELISA
Objective: Used for Ag or Ab detection when the sample is available in a very low quantity.
Procedure:
Sample & Reference Mixing: The low-quantity antigen sample is mixed with a known quantity of reference antibodies.
Pouring into Plate: This mixture is added to wells pre-coated with antigen, creating competition between sample and reference antigen for antibody binding.
Washing: Unbound components are washed off. Free antibodies bind to the plate-fixed antigen.
Enzyme Conjugation & Detection: An enzyme-linked secondary antibody is added, producing a color reaction that is inversely proportional to the antigen concentration.
Key Inverse Relationship (High-Yield for Exams)
|
Antigen Concentration |
Free Antibody Available |
Colour Intensity |
|
Low Ag (Ag ↓) |
High |
High Colour (Colour ↑) |
|
High Ag (Ag ↑) |
Low |
Low Colour (Colour ↓) |
Application Example: ELISA in Dengue Diagnosis
ELISA is widely used in clinical diagnostics, and one of its most important applications is the Dengue test.
Sample: Blood is drawn from the patient to test for Dengue infection.
Antibody Detection Scenario: High levels of antibodies (Ab) against the Dengue antigen (Ag) result in a positive (+ve) dengue test.
Antigen Detection Scenario: High levels of the Dengue antigen (Ag) itself (e.g., NS1 antigen) also result in a positive (+ve) dengue test.
Conclusion
- ELISA (Enzyme-Linked Immunosorbent Assay) is a highly sensitive, specific, and reliable immunological technique used to detect and quantify antigens or antibodies in biological samples.
- Its simple procedure, rapid results, and wide range of applications make it one of the most important diagnostic tools in clinical laboratories, biomedical research, biotechnology, and pharmaceutical industries. Among the four types of ELISA—Direct, Indirect, Sandwich, and Competitive ELISA.
- Each serves a specific purpose depending on the target molecule and diagnostic requirement.
References
- Murphy, K., & Weaver, C. Janeway's Immunobiology. 10th Edition. Garland Science.