Enzyme immunoassays are powerful tools for detecting specific molecules in biological samples. These techniques use enzyme-labeled antibodies or antigens to produce measurable signals, enabling the detection of various substances like viruses, bacteria, and antibodies.

ELISA, a type of enzyme immunoassay, is widely used in medical diagnostics and research. It can be performed in different formats, such as direct, indirect, and sandwich ELISA, each with unique advantages for detecting antigens or antibodies in samples like blood or urine.

Enzyme Immunoassays and ELISA Techniques

EIA vs FEIA vs ELISA techniques

• Enzyme Immunoassay (EIA) uses an enzyme-labeled antibody or antigen to detect the presence of a specific antigen or antibody (HIV, hepatitis)
  • Enzyme catalyzes a reaction producing a detectable signal such as color change (colorimetric), fluorescence (FEIA), or chemiluminescence
  • Includes ELISA and FEIA as specific types of EIA
• Fluorescent Enzyme Immunoassay (FEIA) is a type of EIA that uses an enzyme-labeled antibody or antigen
  • Enzyme catalyzes a reaction producing a fluorescent signal (fluorescein, rhodamine)
  • More sensitive than colorimetric EIA due to the use of fluorescence detection enables detection of lower concentrations of analytes
• Enzyme-Linked Immunosorbent Assay (ELISA) is a type of EIA that uses an enzyme-labeled antibody or antigen
  • Enzyme catalyzes a reaction producing a colorimetric signal (tetramethylbenzidine, o-phenylenediamine)
  • Performed on a solid surface coated with an antigen or antibody (microtiter plate, beads)
  • Widely used for the quantitative detection of antigens or antibodies in a sample (serum, plasma, urine)

Immunohistochemistry vs immunocytochemistry applications

• Immunohistochemistry (IHC) detects antigens in tissue sections preserving tissue architecture
  • Involves fixing and sectioning tissue samples before applying labeled antibodies (paraffin-embedded, frozen)
  • Allows for the localization of antigens within specific cell types or structures (tumor cells, lymphocytes)
  • Applications in immune response analysis include:
    • Identifying immune cell infiltrates in tissues during an immune response (CD4+ T cells, macrophages)
    • Assessing the expression of cytokines, chemokines, or other immune-related molecules in tissue samples (IL-6, TNF-α)
• Immunocytochemistry (ICC) detects antigens in individual cells or cell smears without preserving tissue architecture
  • Involves fixing cells on a slide before applying labeled antibodies (cytospin, smear)
  • Allows for the detection of antigens in specific cell types (lymphocytes, monocytes)
  • Applications in immune response analysis include:
    • Detecting the presence of specific immune cell populations in blood or other fluid samples (B cells, NK cells)
    • Assessing the expression of cell surface markers or intracellular molecules in isolated immune cells (CD3, CD19)

Direct and indirect ELISA methods

• Direct ELISA detects the presence and quantity of a specific antigen in a sample
  • Principles:
    1. Antigen is immobilized on a solid surface (microtiter plate)
    2. Enzyme-labeled primary antibody specific to the antigen is added
    3. Substrate is added, and the enzyme catalyzes a reaction producing a detectable signal
  • Applications include:
    • Detecting the presence of a specific antigen in a sample (viral proteins, bacterial antigens)
    • Quantifying the amount of antigen present in a sample (pg/mL, ng/mL)
• Indirect ELISA detects the presence and quantity of specific antibodies in a sample
  • Principles:
    1. Antigen is immobilized on a solid surface (microtiter plate)
    2. Unlabeled primary antibody specific to the antigen is added
    3. Enzyme-labeled secondary antibody specific to the primary antibody is added
    4. Substrate is added, and the enzyme catalyzes a reaction producing a detectable signal
  • Applications include:
    • Detecting the presence of specific antibodies in a sample (serum antibodies against a pathogen)
    • Quantifying the amount of antibody present in a sample (titer, concentration)
    • Allows for increased sensitivity and specificity compared to direct ELISA due to the use of a secondary antibody amplifies signal

Additional ELISA types and steps

• Sandwich ELISA: A highly specific and sensitive method where the antigen is "sandwiched" between two antibodies
• Competitive ELISA: Used when the antigen is small and has only one epitope, involving competition between sample antigen and added labeled antigen
• Common steps in ELISA protocols:
  • Blocking step: Prevents non-specific binding of antibodies to the plate surface
  • Washing step: Removes unbound reagents between each step of the assay
  • Signal amplification: Enhances the sensitivity of the assay through various methods
  • Enzyme-substrate reaction: The final step where the enzyme converts the substrate into a detectable signal