Immunoassays Flashcards
What are immunoassays?
Utilize antibodies or antigens as reagents to measure analytes
Based on antigen:antibody interactions
Ag + Ab ↔ Ag:Ab
These reactions are reversible
What is the advantage of immunoassays?
Very sensitive and reliable.
What can immunoassays be used for?
Can use immunoassays to measure proteins, hormones, drugs (therapeutic and abuse), metabolites, tumour markers, etc.
What are the five primary classes of immunoglobulins? and their chains?
Antibodies are produced in response to antigen exposure
Five primary classes:
IgA, IgD, IgE, IgG, and IgM
Two identical heavy chains (α,δ,ε,γ, and μ) and two identical light chains (κ and λ).
What is the type of bonds between the chains?
Disulfide bonds between chains.
What molecule that the immune system recognizes as non-self can be used as a reagent in an immunoassay?
Antigens
What is affinity?
Strength of binding between an antibody and epitope of an antigen
Antibody attaches via the antigen-binding site of the Fab region
Determines sensitivity of method
What is avidity?
Binding strength between a number of binding sites
Likelihood of separation is inversely related to the binding strength of the antibody and epitopes
What is specificity?
Specificity allows detection of one epitope in the complex matrix of the sample.
Specific antibodies will react with one epitope and not others.
When does cross-reactivity occur?
Occurs when antibodies bind with structurally similar epitopes.
Dependent on chemical composition, physical forces, and molecular structure.
What is a monoclonal antibody?
Arising from one cell line
Antibody made by cloning
Each antibody is identical
How are monoclonal antibodies produced?
Produced by fusing mice cells (sensitized lymphocytes from spleen) with myeloma cells (cancerous cells) to produce one clonal line of antibodies
What are monoclonal antibody reagents specific for?
Monoclonal antibody reagents will have one antibody specificity
What are polyclonal antibodies? How are they produced?
- Arising from many cell lines
- Immunizing animals to produce antibodies –> Many B cells producing multiple antibodies
- Polyclonal antibody reagents will have a mixture of antibody specificities
Define ligand.
Analyte being measured (antibody, hormone, drug, etc.)
What is a conjugate?
Labeled reagent.
Antigen or antibody covalently attached to label.
What is a substrate?
A substrate creates the reaction to be measured.
Given an example of a substrate.
Example of the substrate peroxidase:
1. Reagent antibody labeled with horseradish peroxidase
(Ab-HRP) + Peroxidase → O2
2. O2 + chromagen → coloured product read by spec
What are labeled immunoassays?
- Tagged with a component that allows detection or visualization of immune complexes
- Measure concentration or activity of label
- Not part of ag:ab reaction
- Ag or ab can be labeled
What are some examples of types of labels used?
Labels may be: radioactive, enzymes, fluorescent, or luminescent.
What are common enzymes used with labeled immunoassays?
Common enzymes: alkaline phosphatase, horseradish peroxidase, glucose-7-P-dehydrogenase
How do you measure unlabeled immunoassays?
You can measure Ag:Ab complexes using nephelometry (scattered light).
Note: No colour change, complexes become insoluble.
Exists as particles in a solution when bound, soluble on own.
What may be added to unlabeled immunoassays to enhance the reaction?
Latex beads may be added to enlarge complex and make the reaction more visible.
What is a competitive immunoassay? How do you relate the signal to concentration?
Patient antigen and labeled reagent antigen with the same specificity compete for the same binding site on the antibody
Inversely proportional
More signal = less analyte of interest
Getting signal from labeled reagent antigen
What is one competitive immunoassay that is not inversely proportional to the analyte?
EMIT is competitive but directly proportional.
What is a noncompetitive immunoassay?
Labeled antibody to detect antigen OR labeled antigen to detect antibody
Does NOT compete with reagent that has the same labeled analyte of interest
Directly proportional relationship
More signal = more analyte of interest
What is a homogeneous immunoassay?
- Bound and free antibody does not need to be separated before label is measured
- Incubate sample antigen with labeled antibody
- Antibody label concentration or activity measured is directly related to amount of patient antigen.
What is a heterogeneous immunoassay?
- Bound and free (unbound) antibody must be separated before label is measured
- Separation achieved through use of solid surface such as latex beads, magnetic beads, or inner surface of microtitre wells
- Wash step required
In a sandwich assay, what occurs if the “hook” effect happens?
“Hook” effect
Antibodies are unable to form sandwich because high concentrations of antigen in the sample bind with capture and free labeled antibody in solution (B)
What is the result of the hook effect and how do you fix the problem?
Result: False negative.
Fix: Dilute patient sample.
What are heterophilic antibodies?
Antibodies that react with antigens other than its specified epitope.
What is the result of heterophilic antibodies?
False positive and false negatives.
Examples: rheumatic factor or human-anti-mouse antibodies (HAMA)
False positive – crosslinks between capture and detection antibody = signal (Bridging)
False negative – binds with detection or capture antibody so they cannot react with intended target = no signal (Blocking)
What is a Immunonephelometric Assay (INT)? How is it measured?
Immunonephelometric Assay (INT):
1. Ag:Ab form complex in solution which causes light to scatter
2. Measures light scatter other than at 180˚
No complex = no scatter as both components are soluble on their own
What is critical in an Immunonephelometric Assay (INT)? What causes false negatives?
Ratio of ag and ab is critical
Ab excess = prozone (false negative)
Ag:ab = zone of equivalence (reliable resutls)
Ag excess = postzone (false negative)
What is ELISA?
ELISA (Enzyme-Linked Immunosorbent Assay):
Heterogeneous immunoassay
Can be: competitive or noncompetitive with labeled antigen or labeled antibody
Use enzyme or fluorescence detection
Not as sensitive as chemiluminescence or radiodetection
Widely used and has been automated
What is the general procedure for ELISA?
ELISA General procedure:
1. Plate coated with antibodies or antigen
(The opposite of what we are trying to find in patient sample).
2. Sample is added and plate is incubated
(Formation of the ag:ab complex if specificity present).
3. Wash to remove unbound substance
4. Enzyme labeled antibody is added
Will bind if ag:ab complex has formed
5. Wash
6. Add substrate to cause enzymatic colour change
7. Measure product using an appropriate detector (ie. spectrophotometer or flurometer)
Why is the wash step important in ELISA?
Wash steps are important as left over unbound label can result in a false-positive or falsely elevated result.
Why is it important to incubate the sample?
Reactions in the body typically occur at 37C, body temperature.
What is EMIT?
EMIT (Enzyme-Multiplied Immunoassay Technique)
Competitive and homogeneous
Most tests use an enzyme-labeled ag (ie drug) + specific ab + substrate + test (or patient) ag
Unlabeled patient ag competes with labeled ag
Enzyme is active when labeled ag is free and not bound to the ab
Ag:ab interaction inhibits enzyme activity
What happens as the concentration of unlabelled Ag increase in the Enzyme-Multiplied Immunoassay Technique (EMIT)?
As the concentration of unlabeled ag increases, less enzyme labeled ag can bind to the ab
More labeled antigen is free = ↑ enzymatic activity = ↑ signal = ↑ patient antigen is bound to antibody
The exception to the “competitive” rule being inversely proportional as this assay is competitive by directly proportional
What is IRMA?
IRMA (Immunoradiometric Assay)
Noncompetitive assay with a radiolabeled ab to measure antigen in patient sample
Radioisotopes like I125 or I131
Gamma emission
Unstable nuclei release electromagnetic radiation energy in very short λ
Geiger counter used as a detector
Signal is directly proportional
What are the pros and cons of IRMA?
Pro: Extremely sensitive and precise
Con: Health concerns, specialized equipment, disposal of hazardous waste, and expensive
What is FPIA?
FPIA (Fluorescent Polarization Immunoassay)
Homogeneous assay
A fluorescent dye (attached to an antigen or an antibody fragment) can be excited by plane-polarized light at the appropriate wavelength
Small fluorophore-labeled antigen is a small quickly rotating molecule
Interrupts polarized light, therefore, unpolarized light emitted
Larger molecules (fluorophore-labeled antigen bound to antibody) rotate slower
Emits polarized light
What is CIA or CLIA?
CIA or CLIA (Chemiluminescence Immunoassay)
Competitive or noncompetitive and heterogeneous
Chemiluminescence refers to light energy produced by chemical reaction
Excited compounds emit light when reverting back to ground state
Chemicals such as luminol and acridinium esters are oxidized to produce signal
Signal measured by luminometer
Inversely proportional
Highly sensitive
CMIA = Chemiluminescent Microparticle immunoassay
Uses microparticles to assist in separation and washing steps
What is ECLIA?
Electrochemiluminescence Immunoassay (ECLIA)
What is lateral flow?
Lateral Flow
Aka immunochromatographic test
Sample containing the analyte passes through the membrane via capillary action
Will pick-up labeled antibody if specificity is present on its way through the strip
Capture antibody (antibody specific for the target analyte) is immobilized on a specific area of the strip
Formation of sandwich (labeled antibody – antigen – capture antibody) = colour change and positive result
Strips have control line present to ensure strip was not ripped and that sample was added and made it all the way to capture antibodies
Example: pregnancy or urine drug tests
What is the Geenius(TM) HIV 1/2 Confirmatory Assay?
The Geenius™ HIV 1/2 Confirmatory Assay is a immunochromatographic assay for the confirmation and differentiation of individual antibodies to Human Immunodeficiency Virus Types 1 and 2 (HIV-1 and HIV-2).
For HIV why would we test for the antibody and not the antigen?
Looking at unchanged parts as HIV tends to mutate quickly.
