Laboratory Methods Flashcards
Describe haemagglutinin inhibition assays
Used to measure Ab level in serum to particular flu viruses for antigenic characterisation
Add virus, serum, RBCs (chicken), if Ab binds virus then the RBCs won’t agglutinate (button at bottom of well)
A titre is reported to determine similarity of viruses
What are the aims of Phase 1-4 clinical trials?
Phase 1: Focuses on safety, dosage, and pharmacokinetics (20–100 healthy volunteers).
Phase 2: Assesses efficacy and side effects in patients with the condition (100–300 patients).
Phase 3: Confirms effectiveness, monitors side effects, and compares with current treatments (300–3,000+ patients).
Phase 4: Post-marketing surveillance to monitor long-term effects and rare side effects after the drug is approved and available for general use.
Examples of automated PCR analysis software
Quantstudio
Ugentec FastFinder
PCRAi
Buzz words for phylogenetic trees
Outgroup for rooting
Neighbour joining/maximum likelihood
Clustering
Nodes ,
Background data
Common genetic origin
Multiple genes improve analysis
What test can be used for immune monitoring in CMV?
IFNg ELISpot
Quantiferon-CMV
Can aid in risk stratification and clinical decision making
What is CAR-T?
Personalised immunotherapy - T cells taken from the patient and genetically modified to make chimeric antigen receptor (CAR) designed to recognise particular cancer antigen (ie CD19 pos cells)
These are then grown and expanded in the lab then reinfused into patient
Highly immunosuppressed, at risk of viral infection and reactivation
Ingredients of PCR reaction and program
Ingredients:
Primers/probe
Taq polymerase
dNTPs
Buffer with MgCl2
Water
Denaturation (95°C) – breaks hydrogen bonds between complementary DNA strands
Annealing (50-60°C) – primers bind, temp depends on primer length and GC content
Extension (72°C) – optimal temp for DNA polymerase
How many points are required for a standard curve?
What is an acceptable slope?
What is a 100% efficient slope?
What is the ideal R2 value?
5 points
Slope = -3.3 to -3.8
-3.322
1
What is SYBR green?
How is it analysed?
Fluorescent dye which non specifically binds to dsDNA
Melt curve analysis to check product is specific, each peak is specific product
How does PRNT work? And what is it used for?
Serum and virus mixed, incubated and plated onto cells
Agar added to avoid spreading and plaque units can be counted
Concentration of serum needed to reduce number of plaques by 50% is the PRTN50
Used to quantify titre of antibodies
How do IgG avidity tests work?
Measures the binding strength between IgG antibodies and virus.
The test works by binding antibody to antigen and then adding urea, if the antibody-antigen bond is high this will remain intact, if binding is weak (low avidity) urea will destroy the bond
