Immunology Flashcards
Immunoassays
Conjugation in Immunoassays
Labels are covalently bonded to antibodies/antigens for stable attachment during assays.
Indirect Detection Principle
Test results are measured by detecting the label (e.g., enzyme, fluorescent tag), not the antibody-antigen complex directly.
Role of Enzyme Labels in Immunoassays
Enzymes (e.g., HRP/ALP) are covalently linked to Ab/Ag. When substrates are added, enzymes convert colorless substrates (chromogens) into colored products for detection.
Example of Enzyme-Substrate Reaction
Colorless → Colored Product (Enzyme Immunoassay)
Adding a substrate (e.g., TMB) to an enzyme-linked Ab-Ag complex triggers a color change, enabling visual or spectrophotometric detection.
What is a Chromogen?
A colorless substrate (e.g., TMB) that reacts with enzymes (e.g., HRP) to produce a colored product in immunoassays.
Think: “Enzymes act like signal boosters.”
Why are enzymes popular labels in immunoassays?
Enzymes (e.g., HRP, AP) amplify signals by catalyzing reactions (e.g., colorless → colored product), enabling sensitive detection.
Example: ELISA uses enzyme-substrate reactions for visual/quantitative
Radioimmunoassay (RIA) Key Feature
Largely replaced by safer methods ( ELISA) due to radioactivity hazards.
Uses radioactive isotopes (e.g., iodine-125) to tag antibodies/antigens; radiation levels indicate target concentration.
Indirect Detection Principle
Test results are measured by detecting the label (e.g., enzyme, fluorescent tag), not the antibody-antigen complex directly.
Immunoassay Result Types
Qualitative (presence/absence of target) and quantitative (concentration measurement).
Role of Enzyme Labels in Immunoassays
Enzymes (e.g., HRP/ALP) are covalently linked to Ab/Ag. When substrates are added, enzymes convert colorless substrates (chromogens) into colored products for detection.
Common Labels in Immunoassays
Enzymes: ELISA (e.g., HRP, Alkaline Phosphatase).
Radioactive isotopes: RIA (e.g., iodine-125).
Fluorogenic reporters: FIA (e.g., fluorescein, phycoerythrin).
Chemiluminescent tags: CLIA (e.g., acridinium ester).
DNA reporters: Combined with qPCR for quantification.
Labels convert undetectable Ag-Ab interactions into measurable signals (color, light, radioactivity).
Horseradish Peroxidase (HRP)
Role: Converts chromogens (e.g., TMB) into colored products.
Substrate Example: TMB (colorless → blue).
Detection: Spectrophotometry at 450 nm.
Preferred for cost-effectiveness and rapid reaction times.
Alkaline Phosphatase (AP)
Role: Hydrolyzes substrates (e.g., pNPP) to produce yellow color.
Detection: Spectrophotometry at 405 nm.
Used when prolonged reactions are needed (slower degradation than HRP).
Principle of Competitive Immunoassays
Used when matched antibody pairs are unavailable (e.g., small molecules like hormones).
Labeled (Ag-E) and unlabeled antigens compete for limited antibody binding sites.
Higher unlabeled Ag → less labeled Ag binds → lower signal.
Why use competitive immunoassays?
When no matched antibody pair exists for the analyte.
Prompt: Think: “No second antibody? Use competition!”
Drug testing (e.g., cortisol, THC).
Think: “No second antibody? Use competition!”
Adsorption Method (Dextran-Coated Charcoal)
Mechanism: Charcoal adsorbs small, unbound molecules (e.g., free antigens/drugs).
Separation: Centrifugation/filtration removes charcoal-bound unreacted analyte, leaving complexes in supernatant.
Footnote: Charcoal’s high surface area traps small molecules; dextran coating reduces non-specific binding.
Analogy: Like a sponge soaking up tiny particles, leaving larger complexes behind.
Precipitation Method (Ammonium Sulfate/Ethanol)
Ammonium sulfate “salts out” proteins by reducing solubility; ethanol dehydrates molecules.
Mechanism: Concentrated ammonium sulfate or ethanol precipitates large antigen-antibody complexes.
Separation: Centrifugation pellets precipitated complexes; unbound analyte remains dissolved.
Use Case: Common in antibody purification or older RIA protocols.
Outcome of Poor Separation
Unremoved unbound analyte causes background noise, skewing results (e.g., overestimation in competitive assays).
Example: In RIA, unlabeled antigen left in solution competes with labeled antigen, reducing accuracy.
