7 ELISA,Virus neutralization reaction,Western blot,PCR.

Immunity and Immune Reactions Overview

  • Nature of Immunoassays

    • Depend on antibody (Ab) recognizing and binding a specific macromolecule (Antigen - Ag).

    • Produces measurable signals upon binding.

    • Often involve chemically linking antibodies or antigens with detectable labels.

    • Labels enable detection via various methods: radiation emission, color change, fluorescence, etc.

Types of Labels in Immunoassays

  • Common Labels Utilized:

    1. Enzymes

      • Most popular in immunoassays.

    2. Radioactive Isotopes

      • Employed in radioimmunoassays (RIA).

    3. Fluorogenic Reporters

      • Utilizes fluorochromes like Fluorescein and Rhodamine.

    4. DNA Reporters

      • Combines reverse transcription quantitative PCR (RT-qPCR) with immunoassay techniques.

    5. Electrochemiluminescent Tags

      • Emit detectable light in response to electrical current.

Enzyme Immunoassays

  • Overview

    • Technique to identify and quantify specific antigens or antibodies in samples through Ag-Ab binding.

    • Types of Enzyme Immunoassays:

      • Competitive:

        • Analyte competes with labeled analyte for limited antibody.

      • Noncompetitive:

        • Sample interacts with an excess of antibody.

      • Heterogeneous (Separational):

        • Separation of bound from unbound antigen post-reaction.

      • Homogeneous (Nonseparational):

        • Binding monitored without separation step; signal modulated through reaction.

The ELISA Technique

  • History

    • Developed by Perlmann & Engvall (1971) in Sweden and Schuurs & Weemen (Netherlands).

    • Traditional ELISA involves chromogenic reporters for observable color changes to indicate presence of antigen or antibody.

    • New methods apply fluorogenic, electrochemiluminescent, and quantitative PCR reporters for improved sensitivity and multiplexing.

  • Types of Antibodies Used in ELISA:

    1. Monoclonal Antibodies:

      • Derived from hybridomas; specific to a unique epitope.

    2. Polyclonal Antibodies:

      • Pool of antibodies capable of binding to multiple epitopes from animal sera.

  • Sensitivity and Detection

    • Enhancements in sensitivity linked to characteristics of antibody-antigen interactions and optimized substrates.

ELISA Variants

  1. Direct ELISA:

    • Analyte is directly bound and detected.

  2. Indirect ELISA:

    • Involves using a secondary antibody to enhance the signal.

  3. Sandwich ELISA:

    • Capture and detection antibodies utilized for quantitation of antigens.

  4. Competitive ELISA:

    • Involves competition between sample antigen and labeled antigen, inversely correlating the signal with the amount of antigen in the sample.

Virus Neutralization

  • Definition

    • Neutralizing antibody (NAb) inhibits or neutralizes the biological effects of pathogens.

    • Essential for blocking viral infections via various mechanisms: interferes with receptor binding, blocks uptake into cells, prevents uncoating, leads to viral particle aggregation.

  • Neutralization Process

    • Antibody production occurs in response to infection; specific antibodies neutralize virus infectivity.

Diagnostic Applications

  • Western Blot Technique

    • Analytical method for detecting specific proteins in samples; often used to confirm ELISA results for HIV.

    • Proteins transferred and captured by antibodies to visualize immune response.

    • Various detection methods: colorimetric, chemiluminescent, radioactive, and fluorescent.

  • PCR (Polymerase Chain Reaction)

    • Routine diagnostic tool for DNA amplification, allowing for significant product increases in target sequences.

    • Characterized by cycles of denaturation, annealing, and extension to produce amplified DNA.

Overview of Molecular Detection Techniques

  • Molecular Methods in Diagnosis:

    • Include Southern Blot (DNA), Northern Blot (RNA), Southwestern Blot (protein-DNA binding).

    • PCR, LCR, NASBA, and bDNA are newer techniques enhancing diagnosis via nucleic acid amplification.