Student manual BioRAD ELISA

Page 1: Introduction to ELISA and Immune Response

Experiment Overview

  • Simulated sharing of “body fluids” with classmates.

  • Conduct an enzyme-linked immunosorbent assay (ELISA) to check for exposure to a contagious “disease.”

  • ELISA detects disease agents (viruses, bacteria, parasites) through antibodies in body fluids.

Immune Response Basics

  • Antigens: Molecules triggering an immune response (from infectious agents).

  • Antibodies: Proteins produced in response to antigens, circulating in the bloodstream to target and flag invaders for destruction.

  • Approx. 10^6 to 10^11 different antibodies present in blood, with antibodies making up 15% of blood serum protein.

  • Each antibody is specific to a single antigen.

Immunology and Antibody Production

  • Immunology: Study of the immune system.

  • Animals can be injected with antigens to produce relevant antibodies for diagnostic tests.

  • Primary antibodies directly recognize specific antigens.

Antibody Structure

  • IgG Structure: Includes heavy chains and light chains with disulfide bonds.

Page 2: Secondary Antibodies and ELISA Application

Secondary Antibodies

  • Recognize and bind to primary antibodies from another species.

  • Made by injecting species antibodies into another species (e.g., human primary antibodies injected into rabbits).

  • Conjugated with horseradish peroxidase (HRP) to produce detectable signals (blue color).

Real-World Applications of ELISA

  • Used in:

    • Pregnancy tests

    • Disease detection in humans and animals

    • Drug testing

    • Food labeling accuracy

  • Rapid results critical for emerging diseases (e.g., SARS).

Home Pregnancy Tests Explained

  • Detect hCG hormone via capillary action on dipstick tests creating visual results:

    1. Wick coated with anti-hCG antibody.

    2. Binding of hCG to complex.

    3. Migration and visualization of results.

Page 3: Importance of Controls in ELISA

Role of Controls

  • Positive and negative controls are essential for validating ELISA results.

  • Positive control: Known to contain the disease agent.

  • Negative control: Known to be free of the disease agent.

Experiment Protocol Overview

  • Share simulated body fluid with classmates.

  • Use provided control samples and test for disease agent presence.

Page 4: ELISA Experimental Protocol

Procedure Steps

  1. Share body fluid samples randomly.

  2. Add samples and controls to microplate wells and incubate (5 min).

  3. Add anti-disease primary antibody and incubate.

  4. Add HRP-labeled secondary antibody to detect primary antibodies.

  5. Add enzyme substrate and observe color change:

    • Blue = positive result.

    • Colorless = negative result.

Page 5: Pre-Lab Focus Questions

Questions to Consider

  1. How does the immune system protect against disease?

  2. Doctor's role in immune response protection?

  3. How do diseases spread?

  4. Examples of diseases affecting the immune system.

  5. Issues that can weaken the immune system.

  6. Importance of immunosuppressants in transplants.

  7. Significance of rapid disease detection.

  8. ELISA acronym and enzyme use rationale.

  9. Importance of control samples in assays.

Page 6: Laboratory Guide Checklist

Checklist for Workstations

  • Yellow tubes: Student samples (4 total).

  • Violet tube: Positive control (1).

  • Blue tube: Negative control (1).

  • Green tube: Primary antibody (1).

  • Orange tube: Secondary antibody (1).

  • Brown tube: Enzyme substrate (1).

  • Microplate strips (2).

  • Micropipets and tips.

  • Required buffers and cleaning items.

Page 7: Sharing Samples and ELISA Steps

Sharing Procedure

  1. Label yellow tubes with initials.

  2. Share samples with 3 classmates, recording partners in order.

  3. Perform ELISA by labeling and adding controls/samples to microplate wells.

Binding Antigen to Wells

  1. Add controls and samples to wells (50 µl each).

  2. Incubate for 5 minutes.

  3. Wash unbound samples out.

Page 8: Washing and Antibody Steps

Washing Steps

  1. Wash wells multiple times to remove unbound proteins.

Adding Antibodies

  1. Add primary antibody (50 µl) to all wells.

  2. Wait 5 min, then wash unbound antibody.

  3. Add secondary antibody (50 µl).

Page 9: Final Steps and Observations

Final Experimental Steps

  1. Wait for secondary antibody binding.

  2. Wash out unbound secondary antibodies.

  3. Add enzyme substrate (50 µl) and observe results (5 min).

Result Evaluation

  • Determine positive/negative based on color change:

    • Record results next to shared fluid partners.

Page 10: Class Results Tracking

Class Tracking Table

  • Each student records their exposure test results alongside their sharing partners.

Page 11: Post-Lab Focus Questions

Reflection Questions

  1. Protein behavior in wells with/without antigen.

  2. Purpose of washing wells.

  3. Outcomes of primary antibody addition based on antigen presence.

  4. Outcomes of secondary antibody addition based on antigen presence.

  5. Implications of negative results.

  6. Importance of running samples in triplicate.

  7. Common antibody-based tests available over the counter.

  8. Analyzing disease transmissibility through class results.