2.6.7 ELISA

How ELISA Works:

1. Sample Preparation:

• A sample (e.g., blood or serum) is added to a surface coated with specific antibodies or antigens.

2. Binding:

• If the target molecule (antigen or antibody) is present in the sample, it binds to the antibody or antigen on the surface.

3. Detection:

• A secondary antibody that is linked to an enzyme is added. This secondary antibody binds to the target molecule, forming a complex.

4. Enzyme Reaction:

• A substrate is added that reacts with the enzyme, producing a colour change.
• The intensity of the colour change is proportional to the amount of the target molecule present.

5. Quantification:

• A spectrophotometer is used to measure the intensity of the colour change, providing a quantitative result.

Types of ELISA:

1. Direct ELISA:

• Detects antigens in a sample using a single antibody linked to an enzyme.

2. Indirect ELISA:

• Detects antibodies by using a secondary antibody linked to an enzyme.
• Example: Detecting HIV antibodies in blood.

3. Sandwich ELISA:

• Detects antigens by using two antibodies: one to capture the antigen and another linked to an enzyme for detection.

Uses of ELISA:

1. HIV Testing:

• ELISA is used to detect HIV antibodies in blood, confirming exposure to the virus.

2. Disease Diagnosis:

• Identifies markers for diseases such as hepatitis, COVID-19, and autoimmune disorders.

3. Pregnancy Testing:

• Detects human chorionic gonadotrophin (hCG) in urine.

4. Allergy Testing:

• Measures levels of IgE antibodies associated with allergic reactions.

5. Drug Monitoring:

• Detects specific drugs or hormones in medical testing.

Advantages of ELISA:

• Sensitive and Specific:
  • Can detect small amounts of antigens or antibodies.
• Quantitative:
  • Provides data on the concentration of the target molecule.
• Versatile:
  • Can be adapted for various diagnostic and research applications.

Limitations of ELISA:

1. False Positives:

• Cross-reactivity with non-target molecules can lead to false-positive results.

2. Sample Quality:

• Requires high-quality samples to ensure accurate results.

3. Time and Cost:

• ELISA tests can be time-consuming and expensive.

Example: ELISA for HIV Detection:

1. Purpose:

• Detects the presence of HIV antibodies in blood, indicating exposure to the virus.

2. Process:

• HIV antigens are attached to the test surface.
• A blood sample is added, allowing HIV antibodies (if present) to bind to the antigens.
• A secondary antibody with an enzyme is added, producing a colour change if HIV antibodies are present.