Definition of ELISA:

ELISA (Enzyme-Linked Immunosorbent Assay) is a widely used technique in immunological and biomedical research for detecting and quantifying specific proteins or antibodies in a sample.

Principle of ELISA:

ELISA is based on the binding of an antigen (or hapten) to a specific antibody, followed by detection of the bound complex using a second, “detection” antibody that is linked to an enzyme. The enzyme catalyzes a chemical reaction that produces a measurable signal, such as a color change, which can be quantified.

Types of ELISA

There are several different types of it, including:

• Direct ELISA: In this method, the antigen is directly coated onto the plate and the sample is added.
• Indirect ELISA: In this method, the sample is added to the plate first, and the antigen is then detected using a labeled (usually biotinylated) detection antibody.
• Sandwich ELISA: In this method, the antigen is captured between two antibodies, one that is coated onto the plate and one that is used as the detection antibody. This is the most sensitive and specific type of ELISA.

Reagents and Materials

• Antigen: The protein or hapten of interest that will be detected in the sample.
• Antibodies: Primary and secondary antibodies are used in ELISA. The primary antibody is specific to the antigen and is used to capture it on the plate or in the sample. The secondary antibody is used to detect the bound primary antibody and is usually linked to an enzyme.
• Enzyme substrate: A chemical reagent that is used to produce a measurable signal when it reacts with the enzyme.
• Plates: ELISA plates are typically made of polystyrene and have 96 or 384 wells.
• Other materials: Wash buffer, blocking buffer, sample diluent, and standards may also be used in ELISA.

Procedure

• Coating the plate: The plate is coated with the primary antibody or the antigen.
• Blocking: The plate is blocked to prevent non-specific binding.
• Sample addition: The sample is added to the plate and allowed to bind to the primary antibody or antigen.
• Wash: The plate is washed to remove unbound sample.
• Detection: The detection antibody is added to the plate and allowed to bind to the bound primary antibody or antigen.
• Wash: The plate is washed again to remove unbound detection antibody.
• Substrate addition: The substrate is added to the plate and allowed to react with the bound detection antibody-enzyme complex.
• Readout: The signal is quantified using a plate reader or by visually inspecting the plate.

Data Analysis

• The data obtained from ELISA can be analyzed using various methods, including:
• Comparison to a standard curve: A standard curve is generated by running a series of known concentrations of the antigen or antibody in parallel with the sample. The sample’s concentration can then be determined by interpolating from the standard curve.
• Comparison to a positive and negative control: A positive control containing the antigen or antibody of interest and a negative control containing only buffer are run in parallel with the sample. The sample’s results are then compared to the controls to determine if it is positive or negative for the antigen or antibody.
• Data interpretation: The data obtained from ELISA is usually presented as an optical density (OD) value, which is a measure of the intensity of the signal produced by the enzyme. The OD value is then compared to the standard curve or controls to determine the concentration of the antigen or antibody in the sample.

Advantages

• Sensitivity: ELISA is a highly sensitive technique and can detect very low concentrations of proteins or antibodies in a sample.
• Specificity: ELISA is specific to the antigen or antibody being detected, and non-specific binding is usually minimal.
• Versatility: ELISA can be used to detect a wide variety of proteins and antibodies, and can be adapted to detect multiple analytes in a single sample.
• High throughput: ELISA can be easily automated and performed in large numbers of samples, making it a cost-effective method for large-scale studies.

Limitations

• Cross-reactivity: ELISA can be affected by cross-reactivity, which occurs when non-specific binding of the primary or detection antibody to other proteins or antibodies in the sample occurs, leading to false-positive results.
• Interferences: Certain substances in the sample can interfere with the ELISA, leading to inaccurate results.
• Quantitation limitations: ELISA is not always able to accurately quantitate the amount of antigen or antibody in a sample, particularly at low concentrations.

Applications

• ELISA is widely used in various fields including medical, veterinary, pharmaceuticals and food industries.
• ELISA has been used to detect and quantify specific proteins, such as enzymes, hormones, and growth factors, as well as antibodies in a wide variety of samples, including serum, plasma, saliva, and urine.
• ELISA can be used to detect and quantify a wide variety of pathogens, including bacteria, viruses, and parasites, and is commonly used in diagnostic applications.

Conclusion

ELISA is a powerful technique that is widely used in immunological and biomedical research. It is a highly sensitive and specific method for detecting and quantifying proteins and antibodies in a sample. However, it is not without limitations and interference must be carefully controlled to obtain accurate results. Despite its limitations, it is a widely accepted tool for quantifying proteins and antibodies in different samples, and its versatility and high throughput make it a cost-effective method for large-scale studies.