Immunology Lab Notes

Immunoassays

  • Immunoassays are analytical methods using antibody-antigen interactions.
  • This exercise introduces three types of tests used in clinical microbiology and point-of-care diagnostics:
    • Lateral Flow Immunoassays (LFIAs)
    • Latex agglutination (direct)
    • Enzyme-linked Immunosorbent Assay (ELISA)

Lateral Flow Immunoassay (LFIA)

  • Unidirectional.
  • Detects a target in a sample.
  • Meant for point-of-care testing.
  • Pigment-conjugated antibodies (specific to patient antigen, e.g., Streptococcus pyogenes) are adsorbed on one end of a membrane/paper strip.
  • On the other end, antibodies against the same antigen (but not conjugated to pigment) are immobilized as a test line.
  • A control line with antibodies against conjugate antibodies is further down the test line.
  • Patient sample moves towards conjugate (labeled) antibodies via capillary action.
  • If specific antigens are present, they bind to the antibody and move towards the test line.
  • Antibodies on the test line also bind to the antigen, forming a sandwich.
  • Excess conjugate antibody moves further and binds to control line antibodies.
  • Sample is resuspended in reagents.

Latex Agglutination Reactions

  • Antibodies and antigens interact and form agglutination (clumps).
  • Anti-coagulase and anti-Staphylococcus protein A antibodies are attached to colored latex spheres to make clumps visible.
  • Antigens are on the bacterial cells/patient sample.
  • Forms visible clumps due to agglutination.

Enzyme-Linked Immunosorbent Assay (ELISA)

  • Antibodies conjugated with an enzyme are used in testing a sample.
  • The activity of the enzyme indicates the presence of the antigen or antibody being tested for.
  • Types:
    • Direct ELISA: tests for an antigen.
    • Indirect ELISA: tests for an antibody.
  • This exercise uses indirect ELISA to test for HIV antibody in a patient sample.

Indirect ELISA Procedure (for HIV antibody detection)

  • HIV envelope protein (antigen) is adsorbed onto the wells of a microtiter plate.
  • Any unbound area is blocked with non-specific protein (milk protein).
  • Patient sample is added. If the patient is HIV-positive, the sample will contain antibodies against HIV and will bind to the adsorbed antigen.
  • Wells are washed to remove unbound antibodies.
  • A secondary antibody (against human antibody) is added. This antibody has been conjugated with an enzyme.
  • The wells are washed after the binding.
  • When a substrate is added, a colored product is formed due to the enzyme’s activity.
  • A positive indirect ELISA to detect antibodies (refer to Figure 18.14b)
    • Antigen is adsorbed to well.
    • Patient serum is added; complementary antibody binds to antigen.
    • Enzyme-linked anti-HISG is added and binds to bound antibody.
    • Enzyme's substrate is added, and reaction produces a product that causes a visible color change.