Enzyme-Linked Immunosorbent Assay 

Mr. Arvind Kumar
  • Enzyme-Linked Immunosorbent Assay (ELISA) is a popular and widely used biochemical technique designed to measure the concentration of substances, usually antigens or antibodies, in a liquid sample.
  • It is based on the specific binding between an antigen and its corresponding antibody, and it uses an enzyme as a marker to generate a measurable signal, usually colorimetric, in response to the antigen-antibody interaction.

Principle of ELISA

  • ELISA is a highly sensitive immunoassay technique to detect and quantify substances like proteins, peptides, antibodies, hormones, and antigens.
  • The principle of ELISA relies on the specific binding of an antigen to its corresponding antibody.
  • An enzyme is conjugated (linked) to the antibody or antigen in ELISA.
  • The enzyme reacts with a substrate to produce a detectable signal (usually a color change, fluorescence, or luminescence).
  • This signal’s intensity is directly proportional to the concentration of the target substance in the sample.

ELISA assays can be classified into four main types based on the configuration of the antigen-antibody binding:

  1. Direct ELISA:
    • The antigen is immobilized directly on the plate.
    • A specific enzyme-labeled antibody binds to the antigen.
    • The enzyme-substrate is added, and a color change indicates the presence and amount of the antigen.
  2. Indirect ELISA:
    • The antigen is immobilized on the plate.
    • A primary antibody binds to the antigen.
    • An enzyme-linked secondary antibody, which recognizes the primary antibody, binds to it.
    • The enzyme-substrate is then added to detect the antigen-antibody complex.
  3. Sandwich ELISA:
    • A capture antibody is first immobilized on the plate.
    • The antigen is added, and it binds to the capture antibody.
    • A detection antibody, which binds to the antigen but at a different epitope, is added. The detection antibody is enzyme-labeled.
    • A color change is produced after adding the enzyme substrate.
  4. Competitive ELISA:
    • The antigen in the sample competes with a labeled antigen for antibody binding sites.
    • The amount of color produced inversely correlates with the target antigen concentration in the sample.

The enzyme-linked detection system used in ELISA is typically horseradish peroxidase (HRP) or alkaline phosphatase (ALP), which catalyzes reactions that produce measurable signals.

 

Requirements for ELISA

To perform an ELISA, the following materials and reagents are essential:

  • Microplate: A 96-well polystyrene microplate is the most common surface for performing ELISA. Each well holds the reagent solutions that react with the sample.
  • Antigen or Antibody: Depending on the type of ELISA, you need:
    • A capture antibody that specifically binds to the antigen to detect antigens is immobilized on the plate.
    • For detecting antibodies, an antigen is immobilized on the plate, and a secondary antibody is used for detection.
  • Enzyme-Conjugated Antibody or Antigen: An enzyme like horseradish peroxidase (HRP) or alkaline phosphatase (AP) is conjugated to the antibody or antigen for detection. The enzyme catalyzes a substrate reaction to produce a measurable signal.
  • Substrate Solution: Substrates such as tetramethylbenzidine (TMB) for HRP or p-nitrophenyl phosphate (pNPP) for alkaline phosphatase react with the enzyme to produce a color change or fluorescence.
  • Buffers: Solutions like phosphate-buffered saline (PBS) maintain the correct reaction pH and prevent components’ degradation.
  • Blocking Reagents: To prevent non-specific binding, a blocking agent (e.g., Bovine Serum Albumin (BSA) or non-fat milk powder) is used to cover the remaining surface of the plate wells.
  • Washing Solutions: These remove unbound substances, ensuring the final signal is due to specific binding between antigen and antibody.
  • Microplate Reader: A spectrophotometer or other plate reader is required to measure the absorbance or fluorescence intensity of the reaction product.

 

Procedure of ELISA

The general procedure for ELISA consists of the following steps:

  1. Coating the Plate with Antigen or Antibody:
    • In sandwich ELISA, the capture antibody is added to the microplate wells and incubated to allow it to adsorb onto the surface.
    • In direct ELISA, the antigen itself is adsorbed to the surface of the well.
  2. Blocking the Plate:
    • After the antigen or antibody is adsorbed, the uncoated areas of the plate are blocked with a blocking solution (e.g., BSA or milk powder). This step is essential to prevent non-specific binding of antibodies in subsequent steps.
  3. Adding the Sample:
    • The sample, which may contain the antigen (in sandwich or competitive ELISA) or antibody (in indirect or competitive ELISA), is added to the wells and incubated for a specific period to allow binding.
  4. Washing:
    • Unbound components (unbound antigen or antibody) are removed by washing the wells with an appropriate buffer solution.
  5. Adding the Enzyme-Conjugated Antibody:
    • For sandwich and indirect ELISA, an enzyme-linked secondary antibody is added. This antibody binds to the antigen-antibody complex (sandwich) or the primary antibody (indirect ELISA).
  6. Washing:
    • After incubation, the wells are washed again to remove unbound conjugated antibodies.
  7. Adding the Substrate:
    • A substrate specific to the enzyme (HRP or alkaline phosphatase) is added. The enzyme catalyzes the conversion of the substrate into a detectable product (often a color change).
  8. Measurement of Signal:
    • The intensity of the color change (or fluorescence or luminescence) is measured using a microplate reader. The optical density (OD) is directly proportional to the sample’s target antigen or antibody amount.
  9. Data Analysis:
    • The measured absorbance is compared to a standard curve generated from known concentrations of the target antigen or antibody, allowing quantification of the sample.
  1. Results of ELISA

The results of an ELISA are typically expressed as:

  • Positive Results: If the color change or signal is present, it indicates that the target antigen or antibody is present in the sample. The intensity of the signal can be quantitatively measured and is directly related to the concentration of the target analyte.
  • Negative Results: No color change or very low signal means the target is absent or in a very low concentration.
  • Quantification: The absorbance values obtained from the plate reader are compared against the standard curve (generated from known concentrations of the antigen or antibody) to determine the analyte concentration in the sample.

 

Uses of ELISA

ELISA is used extensively in various fields:

  • Medical Diagnostics:
    • Detection of infectious diseases (e.g., HIV, Hepatitis B, COVID-19, Malaria).
    • In pregnancy tests, hormonal assays include measuring insulin, thyroid hormones, or human chorionic gonadotropin (hCG).
    • Detection of autoimmune diseases, such as rheumatoid factor in rheumatoid arthritis or antibodies to DNA in lupus.
    • Cancer markers such as prostate-specific antigen (PSA) for prostate cancer and alpha-fetoprotein (AFP) for liver cancer.
  • Research:
    • Study of protein-protein interactions.
    • Quantification of cytokines and other biomarkers in immunology and cell biology.
  • Environmental Testing:
    • Detection of toxic chemicals and pollutants such as pesticides or heavy metals (e.g., lead, cadmium).
  • Food Safety and Quality Control:
    • Detecting food allergens (e.g., peanuts, gluten).
    • Detection of foodborne pathogens like Salmonella or E. coli.
  • Drug Monitoring:
    • Therapeutic drug monitoring, such as measuring blood concentrations of phenytoin, digoxin, or theophylline.

 

Advantages of ELISA

  • Highly Sensitive: ELISA can detect antigens and antibodies at low concentrations (nanogram to picogram levels).
  • Quantitative: ELISA provides precise and reproducible quantitative results.
  • No Radioactivity: Unlike radioimmunoassay (RIA), ELISA does not involve using radioactive materials, making it safer.
  • Easy to Perform: The procedure is straightforward and can be completed in a few hours.
  • Multiplexing Capability: It can be adapted for high-throughput screening using automated systems for processing large samples.
  • Wide Application: ELISA can detect various analytes (hormones, drugs, proteins, cytokines, pathogens) across different fields.

 

Disadvantages of ELISA

  • Cross-Reactivity: Antibodies may sometimes bind to similar, non-target substances, leading to false positives.
  • Sensitivity Limitations: While ELISA is sensitive, it may not detect extremely low concentrations of analytes compared to other methods like PCR.
  • Interference: Substances in the sample, such as high levels of lipids or hemoglobin, can interfere with the assay and result in inaccurate results.
  • Time-Consuming: ELISA can take several hours depending on the assay type, particularly if multiple incubation steps are involved.
  • Complexity: Some ELISA formats (e.g., sandwich ELISA) require careful optimization to avoid non-specific binding, requiring more time and effort.

 

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