L9: Immunoassay Configuration Flashcards
Name IA Categories: Label
Radioactive Isotopes – RIA, IRMA Enzymes – EIA, ELISA, CEDIA Fluorescent Molecules – FIA, FPIA Particles – PETIA/PENIA, PETINIA
For IA Categories: What is labelled?
Ligand – Ligand-labeled (i.e.. EIA, RIA) Antibody – Immunometric (i.e.. IRMA, ELISA) Nothing – The Ab-Ag complex is detected (i.e. precipitin rxn, immunoelectrophoresis, IFE, hemagglutination) Analogue – An analogue of the ligand is labeled
Name IA Categories: Assay Principle
Competitive – Labeled and unlabeled ligand compete for limited binding Non-Competitive – Excess Ab is present to bind to all Ag
Name IA Categories: Separation
Heterogeneous
– Bound and unbound elements are physically
separated prior to detection
Homogeneous
– Bound and unbound elements are not separated prior to detection
Name IA Categories: Incubation
One Step
– Ligand and detection molecule are incubated together
Two Step
– Ligand is applied first and unbound ligand is washed away prior to addition of the detection molecule
Describe the principles of competitive immunoassay
- Competition of unlabelled Ag with labeled Ag* for a limited amount of binding sites (antibody)
- A limited amount of Ab needed
- Simultaneous or sequential addition of Ag and labeled Ag*
- Sequential improves the detection limit 2-4X
- LoD is limited by the Ka of the Ab
Inversely related to concentration of Ag (see more into on sequential in slides)
Describe the principles of a noncompetitive immunoassay.
- An excess amount of Ab* is used to detect/extract Ag; excess unbound Ab* is removed (by various methods)
- Provides highest level of assay sensitivity and specificity
- LoD is dependent on the activity of the label
- Maximum sensitivity achieved with:
- High specific activity of Ab*
- Low nonspecific binding of Ab*
- High affinity constant of the Ab & Ag reaction
- High concentration of Ab (capture max. amount of Ag)
- Small experimental error in measuring bound Ab*
Described sandwich and one or two-step noncompetitive assay.
Most common is a sandwich assay,
• Ab attached to solid support (capture Ab) which binds Ag
• Ab* is second labeled Ab (detection Ab) detects bound Ag
• Requires Ag with at least two separate epitopes that can be bound simultaneously
As with competitive, can have 1-step or 2-step assays • 1-step: 1) Ag + Ab* + Ab-solid ↔ Ab*-Ag-Ab-solid + Ab* + Ab-solid 2) Wash 3) Detect • 2-step: 1) Ag + Ab-solid ↔ solid-Ab-Ag 2) Wash 3) Ab* ↔ Ab*-Ag-Ab-solid 4) Wash 5) Detect
Increases as analyte concentration increases
Describe a homogenous assay.
- No physical separation of bound and free components
- Activity of labeled Ag* is directly modulated by Ab binding:
- Conformation change of enzyme
- Inhibition of enzyme
- Fluorescence energy transfer, protection or polarization
- Technically simpler and faster vs. heterogeneous
- Allows for full automation
- Works with small antigen molecules: Drugs, T3/T4, etc.
- Both competitive and non-competitive assays can be homogeneous
Describe a heterogeneous assay.
- Physical separation of bound from free Ag and/or Ab
- Implicitly assumes kon»_space; koff
- Both competitive and non-competitive assays can be heterogeneous
Describe types of separation.
Absorption
• Charcoal, Florisil, talc
• Free antigen absorbed, bound Ag left in solution
Precipitation
• Double-antibody: a second antibody from a different species of animal used to precipitate primary Ag-Ab complex; Protein A can also be used.
• Polyethylene glycol, ethanol, dioxane, ammonium
sulfate precipitate Ab
Solid Phase
• Ab attached to solid phase
• e.g. reaction tube, microtiter plate, plastic or magnetic beads, cellulose, Sephadex, etc
Column Separation
• Ion exchange, Gel filtration
Miscellaneous
• Electrophoresis, radial partition
Describe types of solid phase support.
Small Particles (<20 μm)
• Latex, microcrystalline cellulose, magnetic particles
• Pros: Can be dispensed as liquids, high surface area
• Cons: Centrifugation or filtration required for separation long magnetic precipitation
Medium Particles (<1 mm)
• Sepharose, sephadex, magnetic particle
• Pros: No centrifugation required, short magnetic
precipitation, moderate surface area
• Cons: Agitation required, slower reaction time
Large Particle (>1 mm)
• Polystyrene, nylon
• Pros: No Centrifugation or agitation required
• Cons: Low surface area, difficult to dispense, poor
reaction kinetics
Solid Surfaces
• Coated tubes, dipsticks, microtitre plates, membranes
• Pros: No centrifugation or agitation required, no dispensing of reagent, simplest to use
• Cons: Lowest surface area and slowest reaction kinetics
Describe and provide examples of unlabelled immunoassay.
a.k.a. Precipitin Reaction
• Light is scattered by particles in suspension
• Optimal light scatter is dependent on the size of particles and wavelength of light
- Antibody is ~15nm in length
- Chylomicrons ~250nm
- RBCs ~1000nm
- Ab-Ag aggregates 250-1500nm
- Can quantitatively detect Ab-Ag complexes based on light scatter (turbidimitry or nephelometry)
- Optimal precipitation occurs at zone of equivalence
- Can couple Ab (or hapten) to a particle to enhance complex formation and light scatter
(See graph on slide 28)
Provide examples of competitive heterogenous assays.
Includes EIA, RIA, LIA (see slide 29)
Provide examples of competitive homogenous assays.
EMIT = Enzyme-Multiplied Immunoassay Technique
• Competitive; direct correlation between signal and [analyte]
• Not applicable for large antigens
(see slide 30)
CEDIA = Cloned Enzyme Donor Immunoassay • Competitive; direct correlation between signal and [analyte] • Not applicable for large antigens • Example: b-galactosidase (see slide 31)
FPIA = Fluorescence Polarization Immunoassay
(see slide 32)
PETINIA = Particle-Enhanced Turbidimetric Inhibition IA (slide 38)
Provide examples of noncompetitive heterogenous assays.
Includes IRMA, ELISA, ICMA (slide 34)
MEIA = Microparticle Enzyme Immunoassay (slide 35)
Provide examples of Non/competitive Homogeneous assays.
PETIA/PENIA = Particle-Enhanced Immunoassay
• Ab coupled to particles
• “Middle-range” analytical method in terms of sensitivity
• Simple and cost effective for measuring analytes that do not require great sensitivity e.g. albumin in CSF or urine; yet, an improvement of 1000-fold over conventional methods can easily be achieved.
• Increasingly popular as no specialized equipment is
required; often on routine clinical auto-analyzers as
turbidimetric measurements.
Describe 1-step analog immunoassay for free hormones.
1-step labeled hormone (Never really worked)
• Use a labeled hormone analog that does not bind to endogenous hormone binding proteins
• Ab affinity for the analog and free hormone are equal
• Incubate small amounts of Ab and analog together
with serum for a short period of time (do not shift the
free hormone equilibrium)
Hormone competes with the analog for Ab binding,
analog does not compete for binding proteins
1-step labeled antibody
• Use a hormone analog, bound to a solid phase, that
does not bind to endogenous hormone binding proteins
• Ab affinity for the analog and free hormone are equal
• Incubate small amounts of Ab and analog together with serum for a short period of time (do not shift the free hormone equilibrium)
Described direct free hormone assays and 2-step back titration.
- Separate the free hormone from the bound hormone
- Measure the free hormone
• Equilibrium dialysis
• Ultrafiltration
• RIA/EIA
2-step labeled hormone
• Incubate a limited amount of anti-hormone antibodies
with serum for a short period of time (avoid shifting the
free hormone equilibrium)
•Wash and then add labeled hormone to measure
unoccupied paratopes
• As long as the equilibrium is not dramatically disturbed, these assays can accurately estimate the level of free hormone
Immunoassay interference: describe factors limiting LoD.
- Background noise of detector
- Blank signal of detection reagent
- Non-specific binding of labeled reagent
- Assay factors:
- Measurement errors (i.e. pipetting)
- Analytical sensitivity (slope of cal curve)
- Assay design and optimization
Immunoassay interference: list clues to their presence.
• Result incompatible with other clinical and/or biochemical
findings
• Outlier in method evaluation/comparison studies –
suspicious but not proof!
• Non-linearity upon dilution
• Poor recovery in spiking experiments with standard or reference material
• Atypical blank reading from some automated
immunoassay analyzers
• Icteric, lipemic, and or hemolyzed samples
List immunoassay limitations.
- Ab/Ag deformation (steric hindrance at solid phase)
- > Reduces sensitivity
- Non-specific binding of labeled Ag or Ab to solid surface
- > Increases noise, reduces sensitivity
• Reduced label activity due to conjugation
• >Incubate longer (allow for more signal) or increase
labeling (can have other negative consequences)
Describe matrix effects.
- The matrix includes everything present within a sample excluding the analyte
- A matrix effect is an interference caused by a difference in reactivity of an analyte due to the differences in the environment of the sample
- Ab-Ag binding reactions can be sensitive to
- [Protein], [Lipid], pH, Ionic strength
- Differences between artificial matrices of standards, QC and PT samples and that of patient samples often cause marked biases between different assays
Describe high hook dose effect.
Antigen Excess
• Affects 1-step sandwich assays and Precipitin reactions
• Does not affects 2-step sandwich assays
List interferences for homogenous IA
• More susceptible to sample interferences (no washing)
• Fluorescence
• Enzyme activity
• Label-like substances
• Hemolysis/Icterus/Lipemia
• Can be partially compensated for by dilution or kinetic
measurements
• Are more precise than heterogeneous assays (no wash)
List possible interferences.
1. Exogenous substances • Anticoagulants can inhibit enzyme activities • e.g. EDTA and ALP • Biotin (in high doses) • Hemolysis, Icterus, Lipemia • Biologics / Therapeutic antibodies • Foodstuffs, drugs, herbals/supplements • Carryover contamination
Endogenous substances
• Cross reacting proteins, hormones or drug metabolites
• Hormone binding proteins
• Interfering antibodies can be present within individuals,
producing variable responses (i.e. pos or neg interference)
• Heterophilic antibodies
• HAMA/HAAA
• RF
• Analyte auto-antibodies
• Paraproteins
Heterophilic antibodies
• Poorly defined group of antibodies that react with a wide spectrum of antigens
• Originally defined as a group of IgM antibodies
associated with mononucleosis
• Seen in patients with chronic or acute diseases
• Bind to immunoglobulins from other species leading to false results
Human anti-mouse antibodies (HAMA)
• May develop as a result of both iatrogenic or
noniatrogenic causes
• Are distinguished from heterophilic Ab in that they are more specific and have a high affinity
• Can be found against any animal (HAAA)
Rheumatoid Factor (RF) • Are autoantibodies that bind to multiple antigenic determinants on the Fc portion of IgG • Like HAAA, are specific antibodies produced against a defined immunogen
Paraproteins
• Due to multiple myeloma at very high concentrations
• Various effects on photometric and immunoassays
Analyte auto-antibodies
• Antibodies that bind to the analyte of interest, often
termed ”macro-complexes”
• Usually remain in circulation but not biologically active
• Location of binding of auto-antibody to the analyte affects the immunoassay Ab to cause positive or negative interference
Biotin
• Ingestion of high doses found in supplements
• Nature of interference
• Sandwich assay: falsely lowered results
• Competitive assay: falsely elevated results
• Theoretical interference not always observed
• Some biotin-based assays are designed with biotin prebound to streptavidin before the sample is present
• For patient recall, wash-out period may take 1-3 days
Describe mechanisms of interference.
- Immunoglobulin Aggregation
• Two-site immunometric assays
• Can crosslink the capture and detection antibody in
the absence of the analyte
• Light scatter assays
• Can add to the size of the precipitating immune
complex - Blocking of binding site
• Two-site immunometric assays
• Can inhibit binding of analyte to capture (or
detection) Ab, leading to falsely low results
• Competitive Assays
• Can block the binding of labeled analyte to Ab,
leading to falsely elevated results
3. Polyspecific binding to an antigen • Can affect assays designed to measure endogenous antibodies, leading to false positive results • e.g. serological assays
Minimizing interferences through assay design.
• Including non-immune immunoglobulins from the species
used to raise the reagent antibodies may block the
interferences due to HAAA
• Does not always work, may need Ig from multiple species
• Idiotypic antibodies will not be eliminated
• Using Fab fragments as the capture antibodies (eliminate Fc portion of Ab)
• Use Capture and Detection Ab from different species
• Use Chicken antibodies (do not react with RF)
Investigating interferences.
• ‘Non-linearity’ is often a clue to the presence of some type of interference
• Test the sample using a different immunoassay method
“Blocking” resins/reagents are commercially available and used to treat samples suspected to have
interferences
• Incubate sample with Protein A sepharose to remove immunoglobulins. Also streptavidin (magnetic) beads to remove biotin.
• Compare urine results to serum results (e.g. hCG)
