Immunological Assays Notes

Immunological Assays

Background

  • In 1896, Max von Gruber and Herbert Durham discovered agglutination: blood from animals inoculated with pathogenic bacteria would clump when mixed with a culture of the same bacteria.
  • Von Gruber coined the term "agglutination" to describe this clumping.
  • Later that year, Georges-Fernand Widal's team found that mixing Salmonella typhi with blood serum caused agglutination in typhoid fever patients and healthy individuals.
  • Healthy individuals were identified as asymptomatic carriers: people colonized with bacteria but show no disease signs.
  • The Widal Test (mixing S. typhi with human blood serum) is one of the first agglutination immunoassays.
  • Immunological assays rely on the precise recognition of an antibody to its specific antigen to identify disease-causing microorganisms.
  • The Widal Test is an indirect immunoassay because it looks for human antibody to S. typhi bacterial antigens.
  • Direct immunoassays look for antigens in a patient sample.
  • All immunoassays are characterized by:
    • Sensitivity: The ability to correctly identify a negative result (true negative).
    • Specificity: The ability to correctly identify a positive result (true positive).
  • A more sensitive test means a negative result is more likely a true negative.
  • A more specific test means a positive result is more likely a true positive.
  • Immunoassays are inherently specific because of the precise antibody-antigen recognition.
  • Sensitivity can vary because agglutination must be visible.
  • Positive results are observed as clumping.
  • The power of any immunoassay relies on the ability of the diagnostician to observe clumping.
  • If clumping is not visible, the result will be incorrectly read as negative, decreasing the sensitivity.
  • Advancements in the last 100 years have sought to increase the visibility of positive results.
  • Three advancements are:
    • Latex Agglutination Immunoassays
    • Lateral Flow Immunoassays (LFAs)
    • Enzyme-Linked Immunosorbent Assays (ELISAs)

Latex Agglutination Immunoassay

  • Antigens (indirect assays) or antibodies (direct assays) are attached to large, sometimes colored, latex beads to make agglutination easily visible.
  • Latex beads with monoclonal antibodies are used for direct assays.
  • Latex beads with antigens are used for indirect assays.
  • Direct Latex Agglutination Immunoassay:
    • Antibodies are attached to latex beads in solution.
    • A patient sample (with possible antigens) is added and mixed.
    • If antigens are present, they bind to the antibody-bound beads and agglutinate (visible clumps).
    • If no antigens are present, clumping will not occur, and the solution remains a diffuse color.

Lateral Flow Immunoassays (LFAs)

  • The sensitivity of the agglutination reaction is increased using a pigmented conjugate.
  • Direct LFAs use monoclonal antibodies specific to patient antigens labeled with conjugate.
  • Indirect LFAs use conjugate-labeled antigens to recognize and bind patient antibody.
  • All necessary reagents are adsorbed to paper test strips, making them ideal for point-of-care testing.
  • Direct LFA procedure:
    • Patient samples are suspended in a reagent and drawn up via capillary action through the test strip.
    • The sample moves into the conjugate release pad, where conjugate-labeled antibody binds the patient antigens.
    • The conjugate-labeled antibody:antigen complexes move to the test line, where immobile antibodies bind to patient antigens, preventing further movement of the bound conjugate.
    • The concentrated bound conjugates of a positive sample create a visibly colored band in the test line.
    • Unbound conjugates move to the control line, interacting with immobile control antibodies and coloring the control line.
    • Unbound conjugate moves to the adsorbent pad to prevent false positives due to backflow.
  • The incorporated control line makes external controls unnecessary.
  • Test results are conclusive only if the control works properly.

ELISAs (Enzyme-Linked Immunosorbent Assays)

  • ELISAs are highly sensitive because an enzyme linked to a secondary antibody converts multiple substrates to colored products.
  • If antibodies and antigens are present, they form an antibody:antigen complex (positive result).
  • The results are made visible during an indicator stage where an enzyme-linked-secondary antibody (EL-Ab) binds to the antibody:antigen complex forming an antigen:antibody:EL-antibody complex.
  • When a chromogenic substrate is added, the enzyme converts it to a colored product.
  • Even a slight color change indicates a positive result.
  • Indirect ELISA: Antigens are bound to the well of a microtiter plate.
  • Direct ELISA: Antibodies are bound to the well.
  • Each step is completed by hand, and excess reagents are washed out to prevent false positives.
  • Positive results are indicated by a color change in the microtiter well.

ELISA Procedure (Indirect)

  1. Antigen is adsorbed to the well.
  2. Patient sample (with antibody) is added to the well.
  3. EL-anti-Ab is added to the well.
  4. Chromogenic substrate is added to the well for colorimetric detection.

Introduction to Lab Exercise

  • Identify Staphylococcus aureus from unknown bacteria using StaphTEX™ Blue (direct Latex Agglutination Immunoassay specific for coagulase and Protein A).
  • Identify Streptococcus pyogenes from unknown bacteria using BD Chek™ Group A Strep Test (direct Lateral Flow Immunoassay specific for the Strep A carbohydrate antigen).
  • Examine the results of an indirect Microtiter ELISA to determine the presence of anti-Human Immunodeficiency Virus (HIV) antibodies from patient sera.
  • Determine seroconversion of patients suspected of HIV infection.

Objectives

  1. Define immunoassay, agglutination, sensitivity, specificity, indirect, direct, and seroconversion.
  2. Understand the use and applications of the immunoassays discussed in this lab.
  3. Understand the mechanics of immunoassays (Latex Agglutination tests, Lateral Flow Immunoassays, and Microtiter ELISAs).
  4. Understand what makes all immunoassays specific.
  5. Understand what makes Latex Agglutination, Lateral Flow, and ELISA immunoassays sensitive.
  6. Identify the Staphylococcus aureus and Streptococcus pyogenes samples from your team isolates.
  7. Determine the seroconversion of each of the patients suspected of HIV infection.