Immunoassays Flashcards
Give examples of immunoassays.
Define: antigen.
Any molecule that induces the formation of antibodies and can bind to these antibodies.
Define: antibodies.
Immunoglobulin (Ig) proteins produced by animal B cells in response to an antigen.
What is an immunoassay?
Analytical techniques based on the specific and high-affinity binding of antibodies with particular target antigens.
Describe different types of antigens.
- Proteins: large molecules that induce antibody formation
-
Small molecules: do not develop antibodies, unless:
- Hapten: small molecule that must be linked to a large carrier protein before it can be used as an immunogen to induce antibodies
- Conjugate antigen: carrier protein-linked hapten
What are the 5 major classes of antibodies?
IgA
IgE
IgG
IgM
IgD
Describe IgG structure.
- Heavy chains
- Light chains
- Fragment antigen binding: two fragments capable of binding with antigen
- Fragment cystallizable: third fragment with no antigen-binding capability
Define: epitope.
A specific region bound by a single antibody.
Describe antigen-antibody binding.
- Noncovalent interactions
- Hydrogen bonds
- Electrostatic interations
- Hydrophobic interactions
- van der Waals forces
The binding strength (affinity) between an antigen and its antibody is among the strongest noncovalent interactions known between molecules.
What is a linear epitope?
Continuous amino acid sequence
What is a conformational epitope?
- noncontiguous amino acid sequences
- folded into close proximity
What is a polyclonal antibody? [4]
- Produced by different B cell clones
- Mixed population of antibody
- Bind to different epitopes on the antigen
- Cheap to produce
What is a monoclonal antibody? [5]
- Come from a single B cell clone
- Single antibody species
- Only one binding site
- Used as standard reagents in immunoassays
- Expensive to produce
What is hybridoma technology?
A specific B cell is fused with a myeloma cell to create a hybrid cell line that can be cultured indefinitely to produce large quantities of the monoclonal antibody.
What is the basic immunoassay theory?
- Use antibody as the capture molecule to search the target antigen
- Use the antigen as the capture molecule to trap the antibody in a complex sample
What are the two requirements for immunoassays?
- Be able to separate or differentiate free antigen from bound antigen
- Immobilizing protein on a hydrophobic solid phase
- 96-well microplate
- Immobilizing protein on a hydrophobic solid phase
- Antibody-bound antigens must be quantifiable at low concentrations for maximum sensitivity (use of labels):
- Radioactive iodine labeling (early stage)
- Enzymes
- Fluorochromes
- Gold nanoparticles
What is an enzyme immunoassay?
When an enzyme label are used to reveal the primary antibody-antigen binding.
Describe enzymes used for detection in immunoassays.
Describe the ‘ideal’ enzyme. [3]
What are two commonly used enzymes?
Use of enzymes for detection:
* Convert a colorless substrate to a colored soluble product in the solution
* For example: Colorless substrate: TMB (3,3’,5,5’-tetramethylbenzidine) soluble substrates yield a blue colour when detecting HRP
Ideal enzyme:
* Stable
* Easily linked to antibodies or antigens
* Rapidly catalyzes a noticeable change with a simple substrate
Two mostly used enzymes:
* Horseradish peroxidase (HRP)
* Alkaline phosphatase (AP)
What are the general steps in ELISA? [5]
- Coating of antigen or antibody onto the wells of a microtiter plate (solid phase)
- Blocking the remaining uncoated surface
- Incubating with different immunoassay reagents
- Washing the coated surface to separate free, unbound molecules from bound molecules
- Detecting the colour developed from the assay
What is the difference between direct and indirect ELISA?
Direct Assay
* Enzyme linked to detecting molecule (e.g. primary antibody)
* Directly measure the amount of the antibody-antigen complex
* Primary antibody: An antibody that binds the antigen
Indirect Assay
* Enzyme linked to intermediate reagent
* Indirectly measure the amount of antibody-antigen complex formed
* Secondary antibody: An anti-species antibody that binds primary antibody
What are the advantages of direct ELISA? [2]
- Faster: fewer steps as compared to indirect ELISA
- Less prone to error: less reagents and fewer steps
What are the advantages of indirect ELISA? [2]
- High sensitivity: several labelled secondary antibody can bind the primary antibody.
- High flexibility: the same secondary antibody may be used for several primary antibodies
What are the disadvantages of direct ELISA? [2]
- Less flexible: each target needs a specific conjugated primary antibody
- No signal amplification
What are the disadvantages of indirect ELISA? [1]
- Longer protocol if compared to direct ELISA
Why can a single secondary antibody be used with any primary antibody produced in that species?
- Secondary antibodies are generated to recognize and bind to a specific species’ constant region (Fc region) of IgG.
- Since the Fc region is conserved within species, a single secondary antibody can be used with any primary antibody produced in that species.
Compare non-competitive and competitive ELISA.
Non-competitive
* Large molecule analysis (e.g. proteins)
* Positive correlation between antigen amount and colour intensity
* Sandwich ELISA
Competitive
* Small molecule (< 5000 Da)
* Inverse relationship between the amount of colour developed and antigen
What is sandwich ELISA?
- One of the most popular non-competitive enzyme immunoassay
- Analyze protein in food (e.g., adulterant, allergen)
- Two Primary antibodies
- Capture antibody: a primary antibody immobilized onto a solid phase
- Detection antibody: another primary antibody that also binds the antigen forming an antibody-antigen-antibody complex
- More target, antigen gives stronger signals
Competitive ELISA is mainly used for […]
Small molecule analysis (only one epitope or even only part of one epitope)
e.g., pesticides are small molecules that are detected with this method
Describe direct competitive ELISA in bound hapten format.
- The competition is between the free as well as the hapten-carrier protein conjugate.
- More free antigens means less of the enzyme-conjugated primary antibody will interact with the hapten-carrier protein conjugate, and more of the enzyme will be washed away - therefore more antigen results in a lower signal.
Describe direct competitive ELISA in bound antibody format.
- The competition is between the free antigen and the enzyme labeled hapten-carrier protein conjugate.
- More free antigen means more enzyme will be washed away and the overall signal will be less.
Describe applications of ELISA in food analysis. [3]
- Detection of proteins in food and agricultural products
- Determination of allergens
- Meat species content, seafood species adulteration
- Chemical contaminants analysis
- Toxins, antibiotics and pesticides
- Identification of bacteria and viruses
Describe the use of ELISA for pork adulteration detection in beef meat.
- Direct ELISA is used to detect Pig IgG by using HRP-labeled anti pig IgG antibody.
- The addition of substrate, TMB, will trigger the enzymatic catalysis of HRP for the colour development.
What is a magnetic immunoassay?
- Magnetic particles are used to purify the target analyte using an external applied magnetic field.
What are the benefits of magnetic immunoassays?
- More surface area to capture Abs/Ags (sphere vs plane)
- Diameter depends on the sub-field of magnetic immunoassays
- Generally the core is magnetic
- Covered in a layer of non-reactive resin (polystyrene; polystyrene-divinylbenzene)
Describe an application of magnetic immunoassays.
What is a lateral flow assay?
Non-competitive
Same basic principles as ELISAs; Liquid sample added
Capillary action is the main driver
* First brings analytes across a strip containing labelled Abs
* Then brings Ag-Ab-label conjugates to capture antibodies
Nano gold particles are often used
* Appear red
* Depends on size and shape
What is lateral flow multiplexing?
Can make a device to analyze several target Ags at once
Example:
* Silver nanoparticles and two types of gold nanoparticles
* Abs for casein, ovalbumin (egg), and hazelnut proteins
* Each Ab is associated with a different end colour
Describe a competitive lateral flow assay.
- Detection of multiple pesticide residues
What are the benefits of lateral flow assays? [4]
- Portable
- Easy to use and run
- Results <15 minutes usually
- Cheap
What are the disadvantages of lateral flow assays? [3]
- Miniaturization of sample volume may present issues
- Accuracy of small volumes or analyte content
- Often semi- quantitative or qualitative instead of firmly quantitative
- May be difficult to present analyte in soluble form that is compatible with the test (materials or Abs)
- e.g., organic solvents may give better extraction, but damage test integrity
What are applications of lateral flow assays? [7]
- Pesticides
- Drugs
- Toxins
- Microorganisms
- Viruses
- Proteins (e.g., allergens)
- Hormones (e.g., pregnancy)
