Advanced Detection Principles and Microarrays using Antibodies

Question 1

what is a biomarker

Answer 1

is a characteristic that be objectively evaluated and measured as an indicator of normal biological process

Question 2

how can a protein, DNA, RNA be monitored or evaluated

Answer 2

by using antibodies

Question 3

what information can biomarker give us

Answer 3

• patient treatment or trials
• response to treatment
• monitor progression
• treatment efficacy
• diseases diagnostic

Question 4

biomarker challenges

Answer 4

• access to patient sample
• specificity and sensitivity
• sample origin and sample amount

Question 5

what do we use biomarker tools

Answer 5

• to detect and make visibility
• enhance visibility
• overcome sensitivity and specificity

Question 6

which methods do we use to evaluate antigen-antibody binding

Answer 6

ELISA, western blot, in situ methods

they can be used to detect antigen or antibody it depends on the method

Question 7

how many methods is used to detect antigen variation using ELISA

Answer 7

• direct: where primary antibody directly bind to antigen, here no secondary antibody.
  *here it can switch where fluorescence can bind to antigen/ antigen can be labelled instead of antibody
• indirect: where primary antibody bind to antigen and enzyme conjugated secondary antibody will be used to detect antigen.
• sandwich: where primary and enzyme conjugated secondary antibody will bind to 2 epitope of the antigen, here secondary antibody directly bind to antigen

Question 8

how Conjugating antibodies and labels is done

Answer 8

by using streptavidin/avidin and biotin system.
because the affinity between streptavidin/avidin and biotin is high, biotinylatedantigen/antibody will bind to avidin making it possible to be detected.

Question 9

does antibody contain biotin

Answer 9

yes and it doesn’t change its property because is in small size

Question 10

what are commonly used detection label

Answer 10

• enzyme: application of substrate
  ex: horseradish peroxide (HRP)
• HRP react to give substrate which give detectable product
• fluorophores: emission of light
  ex: FITC

Question 11

what is Tyramide signal amplification (TSA

Answer 11

is when enzyme and fluorophore detection are combined to increase signal for low abundant protein or to reduce use of expensive antibodies

Question 12

in Tyramide signal amplification (TSA) which signal dominate

Answer 12

fluorophore signal

Question 13

Proximity ligation assays (PLA)

Answer 13

is a transform of protein detection to detection of nucleotide sequence by combining specificity of two antibodies and create ssDNA and dsDNA template.

Question 14

what does a rule of thumb give us about multiplexing of combining strength

Answer 14

specificity is inversely proportional to sensitivity

Question 15

what is Micro-Arrays

Answer 15

godfather of multiplexing

Question 16

where is Micro-Arrays used

Answer 16

to achieve high flexibility when we want to detect different number and combination of biomarkers

Question 17

answer this questions

Answer 17

-Antibodies in biomarker detection
-Protein biomarkers: large number, areas of use, challenges
-Recap on antibody-antigen binding and the variations commonly used Different labels for detection and how to conjugate them
- Signal amplification incl. increasing sensitivity using nucleotide tags in PLA
-Multiplexing to exploit information from multiple biomarkers simultaneously (Microarrays as example)

Question 18

hey