Immunology and Infection Lecture: Use of Antibodies in Diagnostics and Research – ELISAs

ELISAs (Enzyme-Linked Immunosorbent Assays), which have become critical tools in immunological tests and evaluations.

Objectives of the Lecture and Workshop

• Recap antibody structures.

• Recognize the significance of antibodies in research, diagnostics, and therapeutics, especially regarding infectious diseases and autoimmune disorders.

• Understand the principles of various types of ELISAs, including their mechanisms and applications.

• Analyze and interpret data from an ELISA assay using standard curves, enhancing data accuracy and reliability.

• Connect ELISA results to clinical conditions linked with immunological defects such as allergies, autoimmune diseases, and infectious diseases.

Recap: Antibody Structure

Antibodies, also known as immunoglobulins, are composed of heavy (H) and light (L) chains held together by interchain disulfide bonds, which are crucial for their structural integrity and function.

• Heavy Chain (denoted as H): Types of heavy chains include μ (mu), γ (gamma), α (alpha), δ (delta), and ε (epsilon), each defining different immunoglobulin classes.

• Light Chain (denoted as L): Types include kappa (κ) and lambda (λ). An individual antibody can possess either chain type but never both, which allows for specificity in antigen recognition.

• The Hinge region is rich in proline and cysteine, providing flexibility that allows movement of the Fab arms, enhancing antigen binding capabilities.

• Antibodies are glycoproteins; the carbohydrate components enhance solubility, stability, and biological functions, impacting how antibodies function in diverse physiological and pathological contexts.

Variable and Constant Regions of Antibodies

Light Chains

• Variable Region: The first 100-110 residues (VL) are critical for binding specificity, influencing the antibody's interaction with specific antigens.

• Constant Region: Unique sequences lead to two light chain types (κ and λ), which play a role in the functional characteristics of the antibody.

Heavy Chains

• Variable Region: The first 100-110 residues (VH) serve a similar purpose as in the light chains, ensuring the diverse recognition of antigens.

• Constant Regions: Five heavy chain isotypes exist (γ, μ, α, ε, δ), each associated with different immune responses.

Classes of Antibodies

Antibodies can be classified based on their heavy chain constant regions:

• IgA: Predominantly found in mucosal areas, offering protection at mucosal surfaces.

• IgD: Primarily functions as a receptor on B cells, involved in their activation.

• IgE: Associated primarily with allergic reactions and defense against parasitic infections.

• IgG: The most abundant immunoglobulin in serum, crucial for long-term immunity and opsonization.

• IgM: The first antibody produced in response to an infection, effective in agglutinating pathogens.

• Light chains have two isotypes: kappa (κ) and lambda (λ).

Hypervariable Regions

Key Features
Hypervariable regions, also known as Complementarity Determining Regions (CDRs), are segments within the variable domains of both light and heavy chains that exhibit significant variability, crucial for high specificity in binding to antigens. These regions enhance the adaptability of the immune system to recognize an enormous variety of pathogens.

Monoclonal vs Polyclonal Antibodies

• Polyclonal Antibodies: Composed of a mix of antibodies targeting multiple epitopes on an antigen. They form large complexes, which are beneficial in assays like agglutination and neutralization. However, drawbacks include potential cross-reactivity, which can affect the specificity of the assays.

• Monoclonal Antibodies: Produced from a single B cell, they recognize a single epitope and provide consistent results in clinical diagnostics. Monoclonal antibodies are generated through hybridoma technology, which involves fusing B cells with myeloma cells, leading to stable cell lines that can produce large quantities of a specific antibody.

Production of Monoclonal Antibodies

1. Isolate spleen cells: Spleen cells are isolated from immunized mice.

2. Fuse with myeloma cells: Spleen cells are fused with myeloma cells to create hybridomas that can divide indefinitely while producing the desired antibody.

3. Screen and select for desired antibodies: Hybridomas are screened for production of the antibody of interest, which can then be cultured to produce large amounts of the antibody for therapeutic or diagnostic use.

Therapeutic Use of Antibodies: Trastuzumab (Herceptin)

Trastuzumab is a monoclonal antibody used to treat breast cancer by targeting the HER2 protein, which is overexpressed in some tumor cells. This targeted therapy helps inhibit tumor growth and is often used in conjunction with other cancer therapies to improve patient outcomes and survival rates.

Applications of Antibodies in Research: SDS-PAGE and Western Blotting

1. Load SDS-treated protein samples into a gel: SDS (sodium dodecyl sulfate) denatures proteins and imparts a negative charge.

2. Separate proteins via electrophoresis: Proteins are separated based on their molecular weight.

3. Transfer proteins to a PVDF membrane: The separated proteins are transferred for further analysis.

4. Bind target antigens with enzyme-linked antibodies: Specific antibodies that recognize the target antigen are applied to the membrane.

5. Add substrate to visualize results: A substrate is added, producing a measurable signal indicating the presence of the targeted proteins.

Types of ELISAs

ELISAs utilize antibodies linked to enzymes for detection based on substrate conversion:

• Indirect ELISA: Used to measure specific antibodies present in a sample, commonly used in diagnostics for immune status.

• Sandwich ELISA: For measuring specific antigens where the antigen