Immunoassays Flashcards
(41 cards)
1
Q
Give two examples of lateral flow immunoassays.
A
2
Q
Define: antigen.
A
Any molecule that induces the formation of antibodies and can bind to these antibodies.
3
Q
Define: antibodies.
A
Immunoglobulin (Ig) proteins produced by animal B cells in response to an antigen.
4
Q
What is an immunoassay?
A
Analytical techniques based on the specific and high-affinity binding of antibodies with particular target antigens.
5
Q
Describe different types of antigens.
A
- 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
6
Q
What are the 5 major classes of antibodies?
A
IgA
IgE
IgG
IgM
IgD
7
Q
Describe IgG structure.
A
- Heavy chains
- Light chains
- Fragment antigen binding: two fragments capable of binding with antigen
- Fragment cystallizable: third fragment with no antigen-binding capability
8
Q
Define: epitope.
A
A specific region bound by a single antibody.
9
Q
Describe antigen-antibody binding.
A
- 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.
10
Q
What is a linear epitope?
A
Continuous amino acid sequence
11
Q
What is a conformational epitope?
A
- noncontiguous amino acid sequences
- folded into close proximity
12
Q
What is a polyclonal antibody? [4]
A
- Produced by different B cell clones
- Mixed population of antibody
- Bind to different epitopes on the antigen
- Cheap to produce
13
Q
What is a monoclonal antibody? [5]
A
- Come from a single B cell clone
- Single antibody species
- Only one binding site
- Used as standard reagents in immunoassays
- Expensive to produce
14
Q
What is hybridoma technology?
A
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.
15
Q
What is the basic immunoassay theory?
A
- 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
16
Q
What are the two requirements for immunoassays?
A
- 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
17
Q
What is an enzyme immunoassay?
A
When an enzyme label are used to reveal the primary antibody-antigen binding.
18
Q
Describe enzymes used for detection in immunoassays.
Describe the ‘ideal’ enzyme. [3]
What are two commonly used enzymes?
A
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)
19
Q
What are the general steps in ELISA? [5]
A
- 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
20
Q
What is the difference between direct and indirect ELISA?
A
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
21
Q
What are the advantages of direct ELISA? [2]
A
- Faster: fewer steps as compared to indirect ELISA
- Less prone to error: less reagents and fewer steps
22
Q
What are the advantages of indirect ELISA? [2]
A
- High sensitivity: several labelled secondary antibody can bind the primary antibody.
- High flexibility: the same secondary antibody may be used for several primary antibodies
23
Q
What are the disadvantages of direct ELISA? [2]
A
- Less flexible: each target needs a specific conjugated primary antibody
- No signal amplification
24
Q
What are the disadvantages of indirect ELISA? [1]
A
- Longer protocol if compared to direct ELISA
25
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.
26
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
27
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
28
Competitive ELISA is mainly used for [...]
Small molecule analysis (only one epitope or even only part of one epitope)
## Footnote
e.g., pesticides are small molecules that are detected with this method
29
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.
## Footnote
Bound Hapten Format: A small version of the antigen (hapten) is coated on the plate and competes with sample antigen for binding to a limited amount of antibody.
Bound Antigen Format: Antibody is coated on the plate, and sample antigen competes with a labeled antigen for binding to the antibody.
30
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.
## Footnote
Bound Hapten Format: A small version of the antigen (hapten) is coated on the plate and competes with sample antigen for binding to a limited amount of antibody.
Bound Antigen Format: Antibody is coated on the plate, and sample antigen competes with a labeled antigen for binding to the antibody.
31
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
32
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.
33
What is a magnetic immunoassay?
* Magnetic particles are used to purify the target analyte using an external applied magnetic field.
34
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)
35
Describe an application of magnetic immunoassays.
36
What is a lateral flow assay?
## Footnote
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
37
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
38
Describe a competitive lateral flow assay.
* Detection of multiple pesticide residues
39
What are the benefits of lateral flow assays? [4]
* Portable
* Easy to use and run
* Results <15 minutes usually
* Cheap
40
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
41
What are applications of lateral flow assays? [7]
* Pesticides
* Drugs
* Toxins
* Microorganisms
* Viruses
* Proteins (e.g., allergens)
* Hormones (e.g., pregnancy)
