Enzyme Immunoassays and ELISA Techniques

Antibodies

• Antibodies are molecules released by B cells that have been activated and transformed into plasma cells.

• Plasma cells produce antibodies, which consist of:

  • Constant regions: Determine the antibody type (IgM, IgG, etc.).

  • Variable regions: Identify specific antigens.

• Antigens can be bacteria, toxins, fungi, viruses, pollen, cat hair, bee venom, or any foreign molecule.

Enzyme Immunoassays

• Enzyme immunoassays utilize antibodies with enzymes attached to their constant regions.

• Enzymes are chemically conjugated (attached) to the antibodies.

• The enzyme's function is to convert a substrate into a product.

• The substrate is typically colorless or non-fluorescent.

• The enzyme acts on the substrate to produce a colored or fluorescent product, allowing for detection.

ELISA (Enzyme-Linked Immunosorbent Assay)

• ELISA is a type of enzyme immunoassay where either an antigen or antibody is attached to a surface (immunosorbent).

Direct ELISA

• Used to detect antigens directly.

• Steps:

  1. Attach the antigen to the surface of a well in a plate.

  2. Wash away excess, unattached antigen.

  3. Add an antibody conjugated to an enzyme that specifically binds to the antigen.

  4. Wash away excess, unbound antibody.

  5. Add a substrate for the enzyme.

  6. A color change (or fluorescence) indicates the presence of the antigen.

• The antibody is directly attached to the enzyme.

Sandwich ELISA

• Used when the antigen concentration is low or diluted (e.g., urine, blood, saliva).

• Involves two types of antibodies: primary and secondary.

• Steps:

  1. Attach the primary antibody (detects the antigen) to the surface of the well.

  2. Wash away excess, unattached antibody

  3. Add the sample containing the antigen.

  4. Allow incubation for the antibody to capture the antigen (if present).

  5. Wash away any unbound antigen.

  6. Add the secondary antibody, which also recognizes the antigen and is conjugated to an enzyme.

  7. Wash away any unbound secondary antibody.

  8. Add the substrate.

  9. A color change indicates the presence of the antigen.

• The antigen is "sandwiched" between two antibodies.

Indirect ELISA

• Most commonly used in clinics due to cost-effectiveness.

• Detects antibodies from the patient's sample (e.g., blood) to identify infections (HIV, Lyme disease, mononucleosis, etc.).

• Steps:

  1. Attach the antigen to the surface of the well.

  2. Wash away excess antigen.

  3. Add a blocking agent to prevent antibodies from non-specifically attaching to the surface.

  4. Add the patient's blood sample (plasma or serum) containing primary antibodies.

  5. Allow incubation for patient antibodies to bind to the antigen.

  6. Wash away unbound antibodies.

  7. Add a secondary antibody that recognizes and binds to the constant region of the primary antibody, and which is conjugated to an enzyme.

  8. Wash away unbound secondary antibody.

  9. Add the substrate.

  10. A color change indicates the presence of the patient's antibodies, thus presence of infection.

• The secondary antibody binds to the primary antibody, not directly to the antigen.

• Attaching an enzyme to an antibody is a complex chemical reaction that often reduces the number of functional antibodies. E.g. $100$ antibodies will result in $70$ antibodies at the end of conjugation.

  • The other $30$ or more are nonfunctional.