Data Analysis Workshop 1 Flashcards
What is target identification and target validation?
List the processes that are involved in target validation.
- Target identification is the process of identifying a biological process that plays a role in a disease.
- Target validation is the process of confirming the role of that biological process in the disease. This might involve:
1 - Studying effect of bioactive molecules on the target.
2 - Applying the target to cell-based models.
3 - Studying protein interactions.
4 - Signalling pathway analyses.
5 - Functional analysis of genes.
6 - In vitro genetic manipulation.
List in chronological order the stages of drug design.
1 - Early drug discovery.
2 - Preclinical research.
3 - Clinical trials.
4 - FDA review.
Give an overview of the process of early drug discovery.
1 - Hit discovery:
- A hit is a compound which has the desired activity in a compound screen.
- A hit discovery process is therefore the development of a screening assay for that compound / hit.
2 - Hit to lead:
- Hits that are likely to be pharmacologically useful are known as ‘lead compounds’, and the process of identifying lead compounds is known as ‘hit to lead’.
- Screening assays known as high throughput screening (HTS) can identify the lead compound from a group of hits.
3 - Lead optimisation:
- Lead compound optimisation is the process by which a drug is designed after a lead compound is identified.
Give an overview of the process of preclinical studies.
- Preclinical studies are proof-of-concept studies that involve testing the new drug on non-human subjects to identify:
1 - ADMET information.
2 - Potential mechanisms of action.
3 - Appropriate doses.
4 - Appropriate routes of administration.
5 - Possible side effects.
6 - Drug interactions.
7 - Effectiveness compared to similar drugs.
What is the difference between in vivo, in vitro, ex vivo and in silico preclinical studies?
- In vivo studies are carried out using tissues within the body.
- In vitro studies are carried out using extracted cells or tissues.
- Ex vivo studies are carried out using whole tissues outside the body with minimal alteration of the physiological conditions.
- In silico studies are assays performed on a computer.
List the phases of clinical trials.
What are the aims of each phase?
- Phase I of clinical trials determines the safety of the drug in healthy volunteers.
- Phase II of clinical trials determines the safety and efficaciousness of the drug in a small patient population.
- Phase III of clinical trials determines the safety and efficaciousness of the drug in a larger patient population.
List 5 protein quantification methods.
Briefly describe how each one works.
Spectrometry methods:
1 - High-performance liquid chromatography (HPLC).
- ‘High performance liquid chromatography is basically a highly improved form of column chromatography’.
- Instead of being dripped through a column like in column chromatography, pumps in HPLC pass a pressurized liquid solvent containing the sample mixture (mobile phase) through a column filled with a solid adsorbent material (stationary phase).
- The components of the mobile phase separate according to the interactions with the stationary phase.
- The retention time of each component can be used to identify the substance
2 - Liquid chromatography-mass spectrometry (LC/MS).
- As above but mass spectrometry also provides structural identity of the components left on the adsorbent material.
Antibody-dependent methods:
3 - Enzyme-linked immunosorbent assay (ELISA).
- Capture antibodies are bound to a surface on a plate.
- The sample being tested is added, and target antigen from the sample attaches to the capture antibodies to a degree which reflects the quantity of antigen present in the sample.
- Then, an enzyme-bound antibody is applied over the surface so it can bind the antigen (so now the antigen is sandwiched between two antibodies).
- Any unbound antibodies are removed.
- A substance containing the enzyme’s substrate is added and, if there was enzyme-bound antibody binding to the antigen, the reaction produces a detectable signal (most commonly a colour change).
4 - Immunoelectrophoresis.
- An antigen mixture is first separated into its component parts by electrophoresis and then tested by double immunodiffusion.
5 - Protein immunostaining.
- Any use of an antibody method to stain a substance in a sample, e.g. in immunohistochemistry and immunocytochemistry.
What is the difference between ELISA and immunohistochemistry?
- In ELISA, substrate is extracted, immobilised and labelled on a fixed surface such as a nitrocellulose membrane, with antibodies.
- In immunohistochemistry, labelling antibodies are applied directly onto the tissue sections.
List 4 pitfalls of the enzyme-linked immunosorbent assay (ELISA) protein quantification method.
Pitfalls of the enzyme-linked immunosorbent assay (ELISA) include:
1 - Specificity of the antibodies, which is liable to change under different experimental conditions.
2 - Sensitivity of the antibodies, as above.
3 - Interference from the sample antigen with antibody binding and with the enzyme substrate.
4 - ELISA requires calibration on every plate (because the substrate being tested will interfere with the calibration curve given in any commercially available ELISA test, which is only valid for the samples in which it was validated).
What is LLOD and LLOQ?
- LLOD and LLOQ are values that should be calculated as part of basic quality control.
- LLOD (lower limit of detection) is the lowest concentration at which 95% of positive samples are accurately detected in an analytical procedure.
- LLOQ (lower limit of quantitation) is the lowest concentration in the calibration curve which can be determined with the required precision and accuracy, which usually must be +/-20%.
What is the difference between immunohistochemistry and immunocytochemistry?
- Histochemistry applies to the level of the light microscope.
- E.g. can be used to visualise the distribution of protein expression on a whole tissue sample.
- Cytochemistry applies to the level of the electron microscope.
- E.g. can be used to visualise the distribution of protein localisation within a single cell.
What is an imputation?
Imputation the process of replacing missing data with substituted values by extrapolating from gathered data.
What is statistical power?
Statistical power is the probability of a hypothesis test of finding an effect (rejecting the null hypothesis) if there is an effect to be found.
What is coefficient variation?
- Coefficient of variation is a measure of relative variability.
- It is calculated as the ratio of the standard deviation to the mean.
