Competitive vs non competitive analysis Flashcards
where are antibodies used in everyday life?
e.g.
* conventional pregnancy tests
* rapid tests in the doctors office
Antibody-based applications
– detection of antigen
A qualitative assay approach
Antibody-based applications for the detection of antigens often employ qualitative assay approaches to determine the presence or absence of specific antigens in a sample. These assays are designed to provide clear, yes-or-no answers regarding antigen detection.
Immunological assay set-ups
enables detection of
low levels of the target antigen
in the presence of
numerous irrelevant analytes
(some of which may be similar to the analyte of interest)
Immunological assay set-ups are…
very flexible and can be modified depending on the assay need at hand!
When designing an assay, it is critical to understand
problematic ”side-reactions” and adjust e.g. antibody speciticity so that such issues are eliminated / minimized!
By monitoring the binding of a labelled substance, you can…
i) detect the presence of the target analyte in the sample
and / or
ii) estimate the concentration of the target analyte
(when using a standard of known concentration)
Antibody-based applications
In other words i) yes or no answer
ii) amounts
Assay design principle
-Binding of antibody and antigen, where one
component is labelled for detection (directly/indirectly)
-Separation of bound components vs unbound components
-Detection of bound component
name some Immunological methods for quantitative assays
(based on detection principle)
ELISA –enzyme linked immunosorbent assay
FIA – fluorescence immuno assay
RIA – radio immuno assay
DELFIA – dissociation enhanced lathanide fluoro immuno assay
IRMA- immuno radiometric assay
PIA- polarization immun assay
SPRIA- scintillation proximity radio immuno assay
SPIA- scintillation proximity immuno assay
Common detection principles
Radioactive isotopes
Enzymes
Fluorescence
Chemiluminescence
Luminescence
Polarity of light
Surface plasmon resonance
Labelling of component problems for radioactive isotopes
Radioactiva isotopes
3H, 14C chemical synthesis
problem: low activity → poor assay sensitivity
125I oxidation process couples isotope to tyrosine
problem: chemical modification
General problems
- Handling of radioactive material – risk!
- Instability of the molecules.
what is used for labelling of enzymes?
Horseradish peroxidase
Alkaline phosphatase
what is used for labelling of Fluorochromes?
Covalent coupling. (e.g. N-hydroxysuccinimid chemistry)
High specific activity – every molecule can be measured at several occasions .
Large dynamic range
Low background (often)
→ Analysis with high sensitivity that enables the detection of
low-abundant target analytes.
e.g.:
FITC, Cy3, Cy5, ALEXA
Non-competitive analysis
Principle: antigen is captured by a large number of binding-sites before it is detected
Components: specific antibody (high concentration), sample,
labelled specific antibody (high conc.)
(i.e. secondary antibody)
Use of two antibodies may
enhance specificity
Typical quantitative assay of an antigen using ELISA
(non-competitive methodology)
- Coat wells with antibody specific for the analyte (≈0.1-10 µg/ml in PBS or Nacarbonate pH≈9)
- Incubate 2 h 37°C or over night at room temp or at +4°C
- Wash plate (e.g. PBS + 0.05 % Tween 20)
- Add sample diluted in PBS + 1%BSA or 1% milk powder and/or 0.05 % Tween 20
- Incubate 1 h 37°C
- Wash plate with PBS + 0.05 % Tween 20
- Add enzyme (HRP or AP)-labelled antibody specific for the analyte (≈1-10 µg/ml)
diluted in PBS + 1%BSA or 1% milk powder and/or 0.05 % Tween 20 - Incubate 1h 37°C
- Wash plate with PBS + 0.05 % Tween 20
- Incubate with substrate
- Read plate in ELISA spectrophotometer
- Compare sample signal with that of a standard curve
NB! There are many degrees of freedom in e.g. choice of buffer,
concentration of the reagents, enzyme, incubation times etc. All of
these paramaters can be optimized to give the best assay for the setup at hand.
Multiplexed assays
Multiplexed assays, for instance in bead-based formats, allows multiple analytes to be measured in a single sample conserving
precious biosamples.
Colour of bead defines the analyte
Intensity of fluorochrome (PE) defines amount of analyte
why are Small molecules a problem in non-competetive assays?
- Limited Epitopes
Few Binding Sites: Small molecules typically have fewer epitopes (antigenic sites) compared to larger molecules like proteins. This limitation makes it difficult to bind multiple antibodies simultaneously, which is a requirement for non-competitive assays such as sandwich ELISA.
Single Antibody Binding: In non-competitive assays, two distinct antibodies are used: a capture antibody and a detection antibody. Small molecules often cannot be simultaneously bound by both due to their limited surface area, making it impossible to form the required antigen-antibody-antibody complex. - Steric Hindrance
Space Constraints: The physical space on a small molecule can be too restricted to accommodate the binding of two antibodies at different sites. The proximity of binding sites can lead to steric hindrance, where the binding of one antibody interferes with the binding of the second.
Accessibility Issues: Even if multiple epitopes are present, they may not be accessible simultaneously to two different antibodies due to spatial constraints
eg. Testosterone (hapten) bound to one antibody
