VET 2003 - Lab session 3- Serology Testing II

Overview of Veterinary Pathology Lab Manual

  • Course: Bachelor of Veterinary Science

  • Division: Health Sciences

  • Issued by: Dr. Mahmoud Mohamadin, Clinical Instructor, Veterinary Science Program

Lab Sessions on Immunodiagnostic Techniques

Session 2: Immunodiagnostic Techniques (Serology Lab)

  • Focus: Serologic tests

    • Definition: Blood tests searching for antibodies produced by the immune system.

    • Application: Diagnose various disease conditions; techniques vary but focus on immune proteins.

Types of Serologic Tests

  • ELISA (Enzyme-linked immunosorbent assay)

    • Uses solid-phase enzyme immunoassay to detect ligands (proteins) in a liquid sample.

  • Complement-fixation test

    • Tests for specific antibody or antigen via complement fixation.

Additional Techniques

  • Fluorescent antibodies technique

    • Directly tests for antigens using tagged antibodies.

  • Agglutination assay

    • Determines if antibodies cause clumping upon exposure to specific antigens.

  • Precipitation test

    • Assesses similarity of antigens based on antibody presence.

  • Western blot test

    • Identifies antimicrobial antibodies by their interaction with target antigens.

Rapid Test Card

  • Features: A quick, sensitive, and specific method for antibody detection.

  • Advantages: Provides fast results without needing expensive equipment.

Laboratory Procedures

Blood Collection for Brucellosis Test

  1. Collect whole blood (0.5-1mL) and centrifuge at 3000 rev/min for 3-5 minutes, or let stand at 4ºC overnight.

  2. Prepare test device on a flat, dry surface.

  3. Add serum or whole blood and dilution buffer to the sample well, and wait 5-15 minutes for results interpretation.

Interpretation of Test Results

  • Negative: No color on Test Line; Control Line shows color.

  • Positive: Color on both Test and Control Lines.

  • Invalid: No color on Control Line.

Brucellosis Testing: Rose Bengal Test

  • Purpose: Diagnose animal brucellosis via agglutination assay.

  • Procedure: Prepare antigen, mix with serum samples on a slide, and use a mechanical rotator for observation.

Judging Reaction Intensity

  • _-: No agglutination, evenly pink.

  • +: Slight agglomeration, liquid between slightly red.

  • ++: Clear curl, slightly clear liquid.

  • +++: Stronger agglutination reaction.

  • ++++: Cluster-like agglutination with clear liquid between.

ELISA: Enzyme-Linked Immunosorbent Assay

  • Purpose: Measure antibodies, antigens, and proteins in biological samples. Examples include FMDV diagnosis, pregnancy tests, and cytokine measurement.

Types of ELISA

  1. Direct ELISA

    • Uses enzyme-labeled primary antibody to bind directly to target antigen.

  2. Indirect ELISA

    • Utilizes a non-labeled primary antibody and an enzyme-labeled secondary antibody.

  3. Sandwich ELISA

    • Antigen binds to immobilized capture antibody, then detection antibodies reveal the signal.

  4. Competitive ELISA

    • Involves antigen competition for binding to antibody; useful for sensitivity.

Principles of ELISA

  • Detection based on color change from enzyme activity on a substrate.

Tools and Techniques in ELISA

  • Essential tools include micropipettes, microtiter plates, incubators, washers, ELISA readers, and various reagents.

Importance of Proper Techniques

  • Washing: Removes unbound reagents.

  • Incubation: Ensures adequate binding time for reactions.

  • Stop solution: Prevents excessive reaction, yielding inaccurate absorbance readings.