Immunoassay Types and Techniques

• Labeled immunoassays can measure small antigens and antibodies, even in low concentrations

Analyte

• The substance being measured in an immunoassay is called the analyte

Labeling Techniques

• Labeling techniques for immunoassays include radioactivity, enzymes, fluorescence, chemiluminescence, and chromatography

Immunoassay Formats

• Immunoassays are either heterogeneous or homogeneous
• Heterogeneous immunoassays require physical separation of bound and free components
• Homogeneous immunoassays do not require physical separation

Competitive & Noncompetitive Immunoassays

• Immunoassay method designs are subclassified as competitive or noncompetitive
• Competitive immunoassays involve adding all reactants simultaneously
• Labeled antigen competes with patient antigen for a limited number of antibody-binding sites
• In competitive immunoassays, the amount of bound label is inversely proportional to the concentration of the labeled antigen; more labeled antigen detected indicates less antigen is present in the patient sample

Noncompetitive Immunoassays

• Reagent antibody is passively absorbed onto a solid-phase material
• Excess capture antibody is present to ensure patient antigen will be bound
• A labeled antibody, directed against a different epitope of the antigen, is added
• The amount of label detected is directly proportional to the amount of patient antigen present

Antibodies

• Antibodies used in immunoassays must be specific and have a high affinity for the antigen
• High specificity reduces cross-reactivity, while affinity determines the stability of binding between antigen and antibody
• Specificity and affinity determine the sensitivity of immunoassays

Radioimmunoassays (RIAs)

• RIAs are based on a competitive principle and use a radioisotope label
• RIAs have high analytical sensitivity but inherent disadvantages due to the use of radioactive substances

Enzyme Immunoassays (EIAs)

• EIAs use enzymes to catalyze biochemical reactions
• They convert reagent substrates to produce chemically modified products that can be detected

Competitive Enzyme-Linked Immunosorbent Assays (ELISAs)

• Antigen in the sample competes with enzyme-conjugated antigen for a limited number of binding sites on antibody molecules attached to a solid phase
• Enzyme activity catalyzes the conversion of the substrate to a detectable product

Indirect ELISAs

• These are a type of noncompetitive immunoassay
• The enzyme-labeled reagent does not participate in the initial antigen-antibody reaction
• The associated antigen is bound to the solid phase
• Patient serum with an unknown antibody concentration is added
• A second antibody reacts with any patient antibody that is bound to the solid phase

Capture Immunoassays

• Capture immunoassays detect antigen
• The antibody is bound to a solid phase, and patient antigen is allowed to bind or be captured
• A second enzyme-labeled antibody is added
• The final complex forms a "sandwich" with the sample antigen between the two reagent antibodies

Homogeneous Enzyme Immunoassays

• These require no separation step
• Enzyme activity changes as specific antigen-antibody binding occurs
• Steric hindrance results in a loss of enzyme activity when antibody binds to enzyme-labeled antigen

Analytical Sensitivity

• Increased analytical sensitivity can be achieved by complexing biotin to the capture antibody and streptavidin to the solid-phase material
• Biotin-SAv labeling can be used in both indirect ELISAs and capture immunoassays

High-Dose Hook Effect

• The most commonly observed limitation of capture immunoassays
• Excess patient antigen causes falsely decreased detection, leading to an analyte concentration that appears low or normal

Antibody Interference

• Antibodies produced in vivo may interfere with immunoassays if similar to a test reagent
• Heterophile interference occurs when the individual produces antibodies similar to those used in the test reagent kit
• Test kits using a biotin-SAv complex can cause unpredictable interferences

Cross-Reactivity

• Cross-reactivity describes the detection of a substance other than the analyte of interest, typically a false-positive result

Homogeneous Immunoassays

• Used for detecting low-molecular-weight analytes such as hormones, therapeutic drugs, and drugs of abuse
• The cloned-enzyme donor immunoassay (CEDIA) is an example

Heterogeneous Enzyme Immunoassays

• One-step, easy-to-interpret formats which are Rapid immunochromatographic
• Test devices can capture antigen or antibody in a specific location on a membrane

Immunometric Assays

• Most are based on chemiluminescent label detection
• Chemiluminescence is produced by certain compounds when oxidized, serving as markers similar to RIA and EIAs

Fluorochromes

• Fluorescent compounds that absorb energy from an incident light source and convert that energy to light of a longer wavelength

Direct Immunofluorescence Assays

• Involve antigen detection through a specific antibody labeled with a fluorescent tag
• The presence of fluorescence is detected with a fluorescent microscope that utilizes UV light

Indirect Immunofluorescent Assays

• The original antibody is unlabeled
• Incubation with antigen is followed by addition of a second fluorescent-labeled anti-immunoglobulin that detects antigen-antibody complexes

Multiplex Immunoassay (MIA)

• Uses polystyrene beads as the solid phase
• Beads conjugated to different antigens detect antibodies in patient serum
• MIA technology allows simultaneous detection of multiple antibodies or antigens

Fluorescence Polarization Immunoassay (FPIA)

• Was a type of homogeneous fluorescent immunoassay based on the principle that polarization of light increases when an antigen is bound to antibody

Newer Immunoassays

• Single-use disposable immunoassays are rapid tests based on immunochromatography
• Designed to detect analytes such as hCG and drugs in urine

Homogeneous EIA

• No physical separation step of bound and labeled antigens required
• Patient antigen and labeled antigen react with reagent antibody in solution
• Enzyme label is inactivated when reagent antigen binds to antibody
• Patient antigen concentration is directly proportional to the signal

Heterogeneous Immunoassay

• Requires solid-phase material to allow for antigen or antibody binding
• Includes both competitive and noncompetitive immunoassay designs
• In a noncompetitive immunoassay, the concentration of patient antigen is directly proportional to the signal
• In a competitive immunoassay, patient antigen concentration is indirectly proportional to the signal

Competitive Immunoassay

• Patient antigen (Ag) competes with labeled antigen (Ag*) for limited antibody-binding (Ab) sites
• Final detected signal is AgAb (labeled antigen bound to antibody), as shown in the reaction: Ag, Ag, Ab→Ag*Ab + AgAb
• The more patient antigen is present, the less label is detected

Noncompetitive, Indirect ELISA for Antibody Detection

• Excess solid-phase antigen binds patient antibody, and a second labeled antibody (anti-human immunoglobulin) is added
• All patient antibody is allowed to bind
• Amount of label is directly proportional to the amount of patient antibody present

Capture or Sandwich Immunoassay

• Excess solid-phase antibody binds patient antigen, and a second labeled antibody (to the antigen) is added after a wash step
• All patient antigen is allowed to bind
• Amount of label is directly proportional to the amount of patient antigen present

Enzyme-Multiplied Immunoassay Technique (EMIT)

• Reagent antigen is bound to an enzyme tag
• Change in enzyme activity is observed as specific antigen-antibody interaction occurs in solution
• All patient antigen is allowed to bind
• Amount of signal generated is directly proportional to the amount of patient antigen present
• If reagent antibody binds to the enzyme-antigen pair, enzyme activity is blocked, otherwise, enzyme is active
• Active enzyme (commonly glucose-6-phosphate dehydrogenase) catalyzes the reduction of NAD+ to NADH, leading to increased absorbance in the UV-wavelength region

Direct Immunofluorescence

• Patient sample (e.g., tissue) is attached to a slide, and specific fluorescent-labeled antibody is added
• In cell-based immunoassays, patient cells are incubated with fluorescent-labeled antibody to a cell surface antigen
• If fluorescence is detected, patient sample is positive for the antigen
• Fluorescence on cell surfaces indicates that the cell is positive for the antigen

Indirect Immunofluorescence

• To detect patient antibody, reagent antigen is attached to a slide and incubated with the patient sample, then a second fluorescent-labeled antibody (anti-human immunoglobulin) is added
• To detect antigen, patient sample is fixed to a slide and incubated with antibody to the antigen (primary antibody), followed by labeled secondary antibody, directed against the primary antibody
• If fluorescence is detected, antibody or antigen is present in the sample, and the test is positive

Fluorescence Polarization

• Fluorescent-labeled antigen competes with patient antigen for a limited number of soluble antibody-binding sites
• When patient antigen binds to antibody, less reagent-labeled antigen binds, and less polarized light will be detected
• The relationship is an Inverse ratio between patient antigen and amount of polarization

Rapid Immunochromatographic

• Patient sample is added to a test strip and migrates through the strip
• Patient antigen complexes to antibody-labeled particles or competes with antigen- Presence of test line may indicate either positive or negative result, depending on manufacturer design