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Blood Bank

ELISA

While we often associate blood bank with agglutination tests (mixing cells and serum), ELISA, which stands for Enzyme-Linked Immunosorbent Assay, is a powerhouse technique used extensively behind the scenes, especially for ensuring the safety of the blood supply

Think of ELISA as a highly sensitive detection method, often performed in microtiter plates (those plastic plates with lots of little wells), that uses the specificity of antibody-antigen interactions coupled with an enzyme reaction to generate a measurable signal (usually a color change)

The Core Purpose: Detecting Specific Molecules

In the context relevant to blood banking and transfusion medicine, ELISA is primarily used for:

  • Infectious Disease Marker Screening: This is its BIGGEST role by far. Donor blood units are mandatorily screened for various transfusion-transmissible infections (TTIs) using highly sensitive methods, and ELISA is a workhorse here. We look for either:
    • Antigens: produced by the infectious agent (e.g., Hepatitis B surface antigen - HBsAg; HIV p24 antigen)
    • Antibodies: produced by the donor’s immune system in response to infection (e.g., antibodies to HIV-1/2, Hepatitis C virus - HCV, Hepatitis B core antigen - HBc, HTLV-I/II, Treponema pallidum - Syphilis, Trypanosoma cruzi - Chagas disease)
  • Detecting Certain Antigens or Antibodies (Less Common for Routine RBC Serology): While not the primary method for identifying red cell alloantibodies (we use agglutination for that), ELISA principles can be adapted for detecting specific protein antigens or certain types of antibodies (e.g., platelet antibodies, though flow cytometry is often preferred there)

The Basic Principle (Illustrated by Sandwich ELISA for Antigen Detection)

There are several variations, but the “Sandwich” ELISA for detecting an antigen (like HBsAg) is a great example:

  1. Coating The wells of the microtiter plate are coated with a specific “capture” antibody that recognizes the target antigen (HBsAg)
  2. Blocking Any remaining unbound sites on the plastic well surface are blocked using an irrelevant protein (like Bovine Serum Albumin - BSA or casein). This prevents non-specific binding later on, which could cause false positives
  3. Sample Addition The donor’s serum or plasma sample is added to the well. If the target antigen (HBsAg) is present, it binds to the capture antibody stuck to the well
  4. Washing The well is washed thoroughly to remove unbound components from the sample (everything except the captured HBsAg)
  5. Detection Antibody A second antibody, which also recognizes the target antigen (HBsAg) but at a different site, is added. This detection antibody is linked (conjugated) to an enzyme (e.g., Horseradish Peroxidase - HRP, or Alkaline Phosphatase - AP)
  6. Washing The well is washed again to remove any unbound enzyme-linked detection antibody
  7. Substrate Addition A chemical substrate specific for the enzyme is added. The enzyme acts on the substrate, converting it into a detectable product – usually causing a color change
  8. Reading The intensity of the color is measured using a specialized spectrophotometer called a microplate reader. The amount of color produced is directly proportional to the amount of target antigen (HBsAg) present in the original sample

Other Common ELISA Formats

  • Indirect ELISA: Used to detect antibodies in a sample (like anti-HIV). Here, the well is coated with the target antigen. Patient serum is added (patient antibodies bind). After washing, an enzyme-linked anti-human immunoglobulin antibody is added, which binds to the patient antibodies. Washing and substrate addition follow as above
  • Competitive ELISA: The target molecule in the sample competes with a labeled version of the target for binding sites on a limited amount of capture antibody. More target in the sample = less labeled target binds = weaker signal

Why ELISA is So Important in Blood Safety

  • High Sensitivity: ELISAs can detect very low concentrations of antigens or antibodies, which is crucial for identifying potentially infectious units early in the infection process
  • High Specificity: When designed correctly, they are very specific for the target molecule, minimizing (but not eliminating) reactions with unrelated substances
  • High Throughput & Automation: The microtiter plate format lends itself well to automation, allowing donor centers to screen thousands of samples efficiently using robotic systems
  • Quantitative/Semi-Quantitative Potential: While often used qualitatively (positive/negative) for screening, ELISA results (signal intensity) can correlate with the amount of target present

Limitations and Considerations

  • Window Period: Even highly sensitive ELISAs can miss very early infections before antigens or antibodies reach detectable levels (this is why Nucleic Acid Testing - NAT - is also used for viruses like HIV and HCV)
  • False Positives: Non-specific binding or cross-reactivity can occur, leading to initially reactive results that need further investigation with more specific confirmatory tests (e.g., Western Blot, Immunoblot, NAT)
  • False Negatives: Possible due to very low levels of target, specific viral strains not detected by the assay, or technical error
  • Requires Specific Equipment: Microplate readers, washers, precision pipettes are necessary
  • Turnaround Time: While automatable, the multiple incubation and wash steps mean results typically take a few hours

Quality Control is Key

Every ELISA run includes multiple controls:

  • Negative Controls: Ensure no significant background signal
  • Positive Controls: Ensure the assay system is working and can detect the target
  • Calibrators (Optional): Used to establish a cutoff value for determining positivity or for quantification
  • Kit Controls: Monitor specific steps or reagent integrity

Key Terms

  • ELISA (Enzyme-Linked Immunosorbent Assay): An immunoassay technique using an enzyme linked to an antibody or antigen to detect a specific substance, usually resulting in a color change measured in a microtiter plate
  • Immunoassay: A test that uses antibody-antigen interactions to detect a substance
  • Antigen: A molecule that can be bound by an antibody
  • Antibody: A protein produced by the immune system that binds specifically to an antigen
  • Microtiter Plate: A flat plastic plate with multiple small wells (commonly 96) used for running assays
  • Enzyme Conjugate: An antibody or antigen that has been chemically linked to an enzyme (like HRP or AP)
  • Substrate: A molecule acted upon by an enzyme to produce a detectable signal
  • Capture Antibody: An antibody immobilized on the solid phase (well surface) to bind the target antigen from the sample (in Sandwich ELISA)
  • Detection Antibody: An enzyme-linked antibody used to detect the captured antigen (in Sandwich ELISA) or the primary antibody (in Indirect ELISA)
  • Sensitivity: The ability of a test to correctly identify individuals who have the condition or marker being tested (low rate of false negatives)
  • Specificity: The ability of a test to correctly identify individuals who do not have the condition or marker being tested (low rate of false positives)
  • Window Period: The time between infection and when a test can reliably detect markers of that infection
  • Confirmatory Test: A second, usually more specific, test used to verify an initially reactive (positive) screening test result