ELISA Lecture Notes

Learning Objectives

  • Demonstrate knowledge of basic immunity principles.

  • Describe immunodiagnostic techniques.

  • Describe ELISA principles.

  • Discuss ELISA applications in veterinary medicine.

  • Evaluate ELISA applications and potential problems.

Key Definitions

  • Antibodies: Proteins produced by B cells in response to pathogens.

  • Antigen: A substance the body recognizes as non-self, triggering an immune response.

  • Immuno: Related to an immune response.

  • Assay: A test.

  • Immunoassay: A test using antibody-antigen complexes to generate a measurable response.

  • Immunocomplex: Antibodies bound to antigens.

  • Enzyme Immunoassay: A technique using enzymes to measure antibody-antigen complexes; ELISA is an example.

  • Sensitivity: Proportion of diseased subjects with positive test results (true positives).

  • Specificity: Proportion of non-diseased subjects with negative test results (true negatives).

Diagnostics

  • Laboratory diagnostics include:

    • Hematology.

    • Pathology (blood, urine).

    • Tissue analysis.

  • Used to identify pathogens, assess organ function, and monitor treatment response.

Immune System Recap

  • First Line of Defense:

    • Mechanical barriers (e.g., skin, mucus, stomach acid).

    • Non-specific.

  • Second Line of Defense:

    • Inflammation, fever.

    • Innate immune response.

    • Non-specific; involves phagocytosis by neutrophils.

  • Third Line of Defense:

    • Acquired/adaptive immunity (humoral and cell-mediated).

    • Specific; involves antibody (B cells) and T cell responses.

Immunodiagnostic Techniques (Serology)

  • Study of antigen-antibody interactions for diagnostic purposes.

  • Types:

    • In vivo: In the animal (e.g., tuberculin test).

    • Secondary binding tests: In vitro, measure antibody-antigen reactions.

    • Primary binding tests: Directly measure antibody-antigen reactions using labeled reactants (fluorescent dyes, colloidal metals, radioisotopes, enzymes).

Enzyme Immunoassays

  • Homogeneous: Enzyme inactivated when bound; no washing steps; easy to use but expensive and low sensitivity.

  • Heterogeneous: Antigen and antibody-specific; use ELISA; detect antibodies and antigens in soluble solution.

ELISA Principles

  • Quantitative method.

  • Measures antigen-antibody reaction using absorbance (color change).

  • Requires positive, negative, and blank controls, and a standard curve.

  • High specificity.

  • Can measure substances at low concentrations.

  • Adapted for various veterinary diagnostic tests.

  • Wells are coated with specific antigen or antibody.

ELISA Step-by-Step

  1. Microplates (usually 96 wells) absorb antibody or antigen.

  2. Add serum or test sample.

  3. Antibodies bind to the antigen if present.

  4. Wash step to remove unbound antibodies (prevent false positives).

  5. Add labeled anti-globulin (secondary antibody) that binds to the antibodies in the sample.

  6. Wash step again.

  7. Add enzyme substrate (e.g., horseradish peroxidase) to produce a color change.

  8. Read results in a spectrophotometer to measure absorbance.

Interpreting ELISA Results

  • Color intensity is proportional to the amount of enzyme-linked anti-globulin.

  • Anti-globulin is proportionate to the antibody.

  • The antibody is proportionate to the antigen number.

  • Each sample usually run in duplicate.

Types of ELISAs

  • Direct ELISA: Direct binding event; primary antibody is conjugated to an enzyme substrate.

  • Indirect ELISA: Uses two antibodies; a primary antibody binds to the antigen, and a secondary enzyme-linked antibody binds to the primary antibody.

  • Sandwich ELISA: Antigen is sandwiched between two antibodies (capture and detection antibody).

  • Competitive ELISA: Unlabeled antigen competes with the sample's antigen for binding to the capture antibody.

ELISA Types Breakdown

  • Direct ELISA

    • Shortest protocol, easiest to perform, no cross-reactivity.

    • High background, lower signal amplification, inflexible.

  • Indirect ELISA

    • Signal amplification, flexible.

    • Longer protocol, potential cross-reactivity.

  • Sandwich ELISA

    • High specificity, suitable for complex samples, very flexible.

    • Demanding design (finding matched antibody pairs).

  • Competitive ELISA

    • Depends on the specific ELISA selected.

    • Good for small antigens, minimal sample processing.

    • Can be time-consuming/expensive.