Advanced detection principles

Question 1

Biomarkers

Answer 1

A biomarker is a characteristic that can be objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes or pharmacological responses to a therapeutic
intervention

Question 2

What information can biomarkers give us?

Answer 2

Monitor progression
Treatment Efficacy
Disease Diagnostics
Prognostic value
Response to Treatment
Patient stratification
(Trials, treatment etc.)

Question 3

Biomarker discovery challenges

Answer 3

Access to
(well stratified)
patient samples

Sample origin:
invasiveness/
available amount

Abundance of
biomarker

Sensitivity

Specificity

Question 4

What tools do we have to…
- d e t e c t a n d m a k e ‘visible ’
- e n h a n c e ‘ v i s i b i l i t y ’
- o v e r c o m e t h e s p e c i f i c i t y < - > s e n s i t i v i t y c o m p r o m i s e for biomarkers?

Answer 4

Examples:
* Western blot
* ELISA
* Antibody microarrays
* in situ methods
Dependent on the assay method, the detection of antigens, antibodies or both
is possible

Question 5

what are Conjugating antibodies and labels?

Answer 5

Conjugating antibodies and labels refers to the process of attaching a specific label or tag to an antibody, which can then be used for detection, quantification, or visualization in various applications. These labels can be enzymes, fluorophores, radioactive isotopes, or other molecules that provide a detectable signal.

eg, streptavidin/biotin labels

Question 6

Streptavidin biotin systems

Answer 6

Streptavidin/Avidin
- purified from Streptomyces
avidinii/ egg
- binds to biotin
- fast!
- with very high affinity
- over a large pH range
- over a large temperature
range

Biotin (B7/Vitamin H)
- small size -> doesn’t
change properties of
antibody
- different derivates allow
binding to different reactive
groups (usually amines)

Question 7

Commonly used (traditional) detection labels

Answer 7

Radioactive isotopes:
-125I (radio-immuno-assay (RIA); rarely used today)
- other (in vivo diagnostics)

Enzymes:
- Horseradish peroxidase (HRP)
- Alkaline phosphatase
detection: chromogenic or chemiluminescence via application of a substrate
commonly used in: Western blotting, ELISA

Fluorophores:
aka fluorochromes, fluorescent dyes
- FITC (fluorescein isothiocyanate)
- rhodamine
- cyanines
detection: re-emission of light at a defined wavelength following excitation
commonly used in: flow cytometry/cell sorting, tissue stains

Question 8

Proximity ligation assays (PLA)

Answer 8

Combine the specificity of antibody* techniques with sensitivity of PCR. Transform detection of proteins into detection of nucleotide sequence

2 antibodies binding
different epitopes on the
same or different proteins
are coupled to specific
nucleotide probes (so called
proximity probe)

if close enough, the
proximity probes are
bridged by a connector
oligonucleotide

a ssDNA or dsDNA
template is created, and
amplified for detection

Question 9

Multiplexing

Answer 9

the simultaneous detection and quantification of multiple analytes (such as proteins, nucleic acids, or cells) in a single assay.

Question 10

Micro-Arrays

Answer 10

the ‘godfather’ of multiplexing
DNA
RNA
Peptides
Antibodies
Antibody fragments

to study and analyze the expression of numerous genes simultaneously or to detect specific DNA or RNA sequences. They consist of a grid of microscopic spots attached to a solid surface, usually glass or silicon, where each spot contains probes (short sequences of DNA, RNA, or antibodies) designed to hybridize with specific target sequences or proteins

each spot consists
of one kind of
antibody (fragment)

detection and
quantification via
spot-intensity