Student manual BioRAD ELISA (1)

Introduction to ELISA Experiment

  • You will conduct an experiment simulating sharing of "body fluids" with classmates.

  • The goal is to perform an Enzyme-Linked Immunosorbent Assay (ELISA) to check for exposure to a contagious disease.

  • ELISA utilizes antibodies to test for disease agents (viruses, bacteria, parasites) in body fluids.

Immune Response and Antibodies

  • Antigens: Molecules that trigger immune response, from infectious agents like bacteria and viruses.

  • Upon exposure, the body produces antibodies—proteins that recognize and bind to specific antigens, aiding in their destruction.

  • Antibodies can range between 10^6 and 10^11 different types, specific to single antigens.

Antibody Structure

  • IgG Structure (Harris et al. 1998): Heavy and light chains connected by disulfide bonds.

  • Antibodies can be represented graphically with antigen bindings.

Production of Antibodies

  • Immunology: Study of the immune system.

  • Animals (chickens, goats, rabbits) can be injected with antigens to produce specific antibodies in their serum.

  • Primary antibodies specifically identify disease antigens in diagnostic tests.

Secondary Antibodies

  • Secondary antibodies recognize primary antibodies, developed by injecting antibodies of one species into another.

  • Example: Human antibodies into rabbits to create anti-human antibodies, used in assays.

  • These antibodies are typically conjugated with enzymes, like horseradish peroxidase (HRP), that produce measurable signals (blue color) upon reaction with substrates (like TMB).

Applications of ELISA

  • Versatile Use: Medical diagnoses (pregnancy tests, disease detection), agricultural testing, drug use detection, air quality testing, and food labeling verification.

  • Rapid testing—results can be available in minutes, crucial for emerging infectious diseases.

  • Example of pregnancy test: Detects hCG hormone through similar ELISA principles with visual indicators.

Importance of Controls in ELISA

  • Controls: Essential for validating test accuracy—positive controls (known infected samples) and negative controls (known non-infected samples) ensure proper assay function.

Experimental Overall Procedure

  1. Share simulated body fluid samples with peers.

  2. Add these samples to microplate wells for binding.

  3. Incubate to allow protein binding via hydrophobic interactions.

  4. Introduce primary antibody to recognize any present antigens.

  5. Use HRP-labeled secondary antibody to bind to primary antibodies.

  6. Apply enzyme substrate for color development indicating reaction.

Pre-Lab Focus Questions

  • Understand how the immune system works, the role of doctors in disease protection, disease transmission, examples of immune-targeting diseases, and the need for controls in experiments.

Laboratory Workstation Checklist

  • Prepare and label items needed for the experiment: student samples, controls, antibodies, enzyme substrates, and tools for mixing and transferring samples.

  • Engage in sharing and combining samples with designated partners and follow exact procedural steps to complete ELISA.

Post-Lab Focus Questions

  • Reflect on sample testing outcomes based on antigen presence and the necessity of washing steps.

  • Consider implications of negative results in diagnosing disease and potential reasons for such outcomes.

  • Discuss practical applications of antibody tests available in pharmacies.