Immunoassays & ELISA Flashcards
What is Indirect ELISA Immunoassay
- Antigen is coated on surface
- Specific primary antibody is bonded to the antigen
- Secondary enzyme labelled antibody that is specific to the primary antibody is used
Can also use a fluorescent tag instead of an enzyme
How to interpret results of a Direct ELISA Immunoassay
Presence of colour change means antigen has bonded to the antibody conjugate with an attached enzyme
- The enzyme is metabolizing the substrate creating a colour
No presence of colour means the antigen bonded with nothing and all the antibody conjugates with the enzyme were washed out
What stabilizes binding between epitopes and paratopes
Non-covalent Forces
- Ionic Bonds
- Hydrogen Bonds
- Van Der Waals Attraction
- Hydrophobic Effect
What is Avidity
The combined strength of multiple binding sites on the Antigen
Formula for association constant of Aby-Ag interactions
- How do we increase the amount of complexes?
[Aby-Ag] / [Aby] x [Ag]
Increasing antigens and antibodies simultaneously will lead to more formation of complexes
Non Competitive assays signal?
The more target antigens the more captured antibodies capture them. Thus, detection antibodies are able to bind to them. Producing a larger signal
Proportional Correlation
Competitive assays signal
The more labeled Ag - Aby complexes the brighter the signal
The more Ag of interest - Aby complexes the dimmer the signal
Inverse Correlation
What makes the ideal home test kit
- High specificity
- High sensitivity
- Easy to use, easy to interpret results
Explain the different sized bands in the detection method used to detect Aby-Ag precipitation
The thicker the band the larger the protein molecule (IgM) as it bumps into the gel more, making it take longer to diffuse
Thinner molecules (IgG) will make smaller bands
What is the other type of Radioimmunoassay?
Heterogenous Non-Competitive Assay
- Radiolabeled Antibody binds to Antigens captured by an Immobilized antibody
- Unbound antigens and Radiolabeled antibody get washed out
Bounded radiolabeled antibody will emit if target antigen is present
What is a Hapten?
Small molecules that have no antigenic properties themselves
- When they bind to larger carriers they can then produce an immune response
How would a positive and negative result look on a Lateral Flow Type Test
Positive: Two lines
- Antigen bonded with the signaling antibodies at the start
- Ag-Aby complex then binds with the immobilized antibody, sandwiches the antigen (First line forms here)
- Signaling Antibodies that did not pick up the antigen continue to move up where they bind with an immobilized immunoglobulin (Second line forms here)
Negative: One line
- Antigens do not bond with the signaling antibodies at the start
- Antigens do not bind to the immobilized antibody (First line does not form)
- Antigens bind to the immobilized immunoglobulin (Second line forms here)
What is Direct ELISA Immunoassay
- Antigen is coated on surface
- Antigen binds to Antibody
- Antibody with a corresponding enzyme
- Mixture is washed and swished
- If antigen present the antibodies will bind to it and not get washed out
- If antigen is not present the antibodies will bind to nothing and get washed out - As a substrate is added the enzyme attached to the antigen will start to convert it into a colour
Can also attach a fluorescent tag instead of an enzume
Can immunoassays be used to confirm diagnosis
Immunoassays are only qualitative or semi-quantitative at best
- Often need more sensitive blood/tissues to be analyzed at a lab to confirm diagnosis
Core traits central to immunoassay
Affinity, Avidity, Specificity, Sensitivity
Heterogenous Immunoassays
The immuno-complexes are separated from the unbounded forms
- All unbound antigens are washed out as the bound antigens are bounded to an immobilized antibody
What kind of distribution do Precipitates have?
- How?
Normal Distribution
- If low antibodies then only few crosslinked complexes are formed
- If high antibodies then antigens have no available epitopes for crosslinking, leading to no crosslinking of epitopes
Meaning of results from Radioimmunoassay?
High signal means more Labeled Ag formed complexes with the antibody
Low signal means more Sample Ag formed complexes with the antibody
What are Non-Competitive Assays?
- A captured antibody is immobilized to the surface of the test tube
- This antibody is specific for the target antigen
- Target antigen binds to the captured antibody
- A detection antibody specific to the antigen binds to the target antigen and can produce colour
What are Homogenous Immunoassays?
Do not require separation of immuno-complexes from the reaction mix
- Good for point of care testing
What is sensitivity
Detection limit of an antibody for an antigen
Explain Agglutination
Large latex particles are coated with the antibody are crosslinked with antigens within a sample, Agglutination is visible due to the large size of the latex particles
- If Aby crosslinks with Ag then the latex will create little granules
- If Aby do not crosslink with Ag then the latex will stay milky white
What do immunoassays measure
Measure the interactions between antibodies and antigens
Advantages of Direct ELISA Immunoassay
Quick: Only one antibody is needed
Cross reactivity of secondary Antibody is eliminated
What is affinity?
Degree of attraction between the epitope on an Antigen and the paratope on an Antibody
Explain the Ouchterlony Immunodiffusion used to detect Precipitin formation
Antigen placed in centre
Control Serums placed on top and bottom
Patient Serum placed on left and right
Antibodies in the serums will start to diffuse towards the antigens. If the concentrations are suitable then precipitates start to form as seen through white bands
How does Lateral Flow Type Tests work
- Tip is dipped into sample
- The antigens in the sample migrate up
- Antigens encounter signaling Antibodies
- Ag-Aby complex forms
- Complex migrates further, until it is captured by an immobilized antibody
- Antigen is sandwiched here where the signaling antibody causes a line to appear
- Lots of signaling antibodies that did not pick up the antigen continue to migrate further, until it is captured by an immobilized anti Immunoglobulin, causing a line to appear here showing the test does work
Disadvantages of Test Tube / Multi-Well Plates
- May need to rinse multiple times
- Have to consider enzyme kinetics and stop all the test reactions at the same time
- Have to be stored at 4°C, some formats can be frozen
- After the expiry date the antibody’s denature and the test can not be used
Advantages of Indirect ELISA Immunoassay
- Primary Aby immuno-reactivity is not compromised
- Allows for signal amplification, primary antibody contains multiple sites for the labelled secondary antibody.
–> Improves assay sensitivity
What is Immune Precipitation
Aby-Ag complexes form cross links with each other creating a precipitin line
- Occurs with antigens with multiple epitopes
What is an immunoassay
Analytical test that uses highly specific antibody-antigen complexes to produce a signal
What are competitive assays?
Un-labelled analyte (Ag of interest) is measured based on its ability to compete with a labeled Ag
- Competition between the two Ag in the system
The more labeled Ag - Aby complexes the brighter the signal
What is specificity
Property of antibodies that allow it to react with some antigens instead of others
Uses of Immunoassays
- Detection of Pathogen Antigens (Bacterial, Viral, Fungal)
- Detection of Drug substances (In patient plasma, can be used for PK)
- Detection of Hormones / Cytokine levels in test body fluid sample
- Detection of Cancer Antigens Theranostics (Antigens that can detect and treat cancer)
- Detection of Serological Antibody response in patients (Indication that the body is producing antibodies in response to a past infection / vaccination)
What is a Serological Test
Blood Antibody Test
- Deted the production of antibody produced in response to a previous infection / vaccination
Ordered by:
- Control
- IgG: Means production of specific antibody to pathogen
- IgM: Indicates recent exposure
Disadvantages of Direct ELISA Immunoassay
- Attaching the enzyme/fluorescent tag may compromise the immuno-reactivity and denature the antibody
- Labelling the antibody can be time consuming and expensive
- Limited flexibility in choice of antibody used
- Less scope for signal amplification (1:1 ratio, the one antigen will bind to one antibody conjugate)
What is the Test Tube / Multi-Well Plate
Non-competitive Heterogenous
1. Captured antibody is coated on surface
2. Sample antigen is added to tube, binds with antibody
3. Washed and rinsed out
4. Signaling antibody is added
5. Mixture is incubated
6. Enzyme assay solution is added
7. Wait for colour to develop
The intensity of colour (absorbance reading) determines the concentration of Antigen
Disadvantages of Indirect ELISA Immunoassay
Secondary antibody could bind to something besides the primary antibody (Cross Reactivity)
- Usually need to use albumin to absorb to the surface of the plate and reduce any non-specific adsorption of the secondary antibody
How do OTC Pregnancy Kits work
Uses ELISA to detect Human Chorionic Gonadotropin (hCG)
- A hormone produced by the placenta during pregnancy
hCG molecules
- 2 subunits: Alpha and Beta
- Alpha subunit: Common in hormones
- Beta subunit: Unique to hCG
Antibodies that detect Beta subunit are used in immunodiagnostic kits
What is Radioimmunoassay
Heterogenous Competitive Assay
- Uses a radio-labeled Ag as the tracer
- Antibody Coated Tube
- Sample (Un-labeled Ag) is added
- Tracer (Radio-labeled Ag) is added
- Incubated (Competition between the sample and tracer occurs)
- Decant and count
Advantages of Test Tube / Multi-Well Plates
- Can test lots of samples
- Semi quantitative read out of antigen amount using a colourimeter
- Kits come with a positive and negative control for validation
Immunoassays are considered as ____?
Semiquantitative
- Tells us if there is a lot of aby-ag interactions, but, not the exact amount
Qualitative
- Tells us if an analyteis there or if it is absent
What is the Coated Dip Stick
- Capture Antibody is coated on plastic at one end
- Stick is dipped into sample
- Rinsed
- Stick is then dipped into signaling antibody (enzyme-antibody conjugate)
- Extensive rinsing to remove any unbound signaling antibody
- Stick is dipped into solution for enzymatic assay
- Appearance of colour indicates a positive result
Limitations of Lateral Flow Test Kits
- Increase in antigen concentration from unrelated conditions can cause a fals positive
- Dead organisms can give false positives
- Infectious disease kits for specific bacterial/viral antigen may not be able to detect variants/mutants
- Complex biological samples can have inhibitors/activators of enzymes used in assay
- Improper storage can impact the stability of antibody/enzymes in the kit
- Human error
How is Hapten used
Free hapten are harvested using absorbent materials like charcoal and membranes
- It is then injected into the blood of an animal
- Where it then binds to albumin thus, activating it causing it to produce an immune response and produce antibodies
How to interpret results of an Indirect ELISA Immunoassay
Presence of colour change means antigen has bonded to the secondary antibody conjugate with an attached enzyme
- The enzyme is metabolizing the substrate creating a colour
No presence of colour means the antigen bonded with nothing and all the antibody conjugates with the enzyme were washed out
What isotype has 10 Antigen Binding Sites
- What is the effect of this?
lgM, even if affinity is low, it will have strong avidity (10 binding sites vs just 2)
What is cross reactivity
Antibodies can bind with low affinity to closely related epitopes
