Define antigen, antibody, epitope, affinity
Ag = anything recognised as non-self by the immune system Ab = protein produced in response to ag Epitope = specific part of ag that bind to ab Affinity = measure of binding strength between epitope and ab
Mechanism for production of polyclonal ab?
Use rabbit/ goat
Based on principle of ab being produced against ag
- Inject ag into animal - wait 4 weeks for primary immune response
- Booster injection - 4 weeks second antibody response - larger due to memory
- Collect blood and centrifuge to isolate serum
- Check serum specificity -ELISA
Mechanism for production of monoclonal ab?
- Mouse immunised - ab produced against ag
- Spleen removed - harvest plasma cells
- Plasma cells fused w/ immortal B cells using polyethylene glycol = immortal hybridomas
- Placed in well plate w/ HAT - kills of non-hybirdomas
- Dilute wells so one hybridoma per well = singe mAb
- Hybridoma with high affinity selected using ELISA
Ways of labelling?
Unlabelled
Enzyme - horseradish, peroxidase
Fluoresce - FITC
Gold - electron microscopy
Difference between direct and indirect testing?
Direct - add ab to ag -> label Ab to see where tagged
Indirect - use amplified signal
What is serological diagnosis?
Use ab specificity to detect ag
Titre of ab = lowest dilution of sample that retains detectable activity
Use: diagnose infection, identify microorganism, quantify protein in serum, type of blood
RETROSPECTIVE
What is immunoprecipitation?
Ouchteriony diffusion test
- Ab and ag placed in wells in agar
- Diffuse through gel and form precipitate at equivalence
Was used to detect diphtheria
What is identity?
Precipitn band formed w/ single Ag
What is single radial immunodiffusion?
Ag placed in ab contained gel - precipitation ring indicate reaction
Area of ring proportional to conc Ag
What is immunoelectrophoresis?
Ag palmed in well and separated
Ab placed in trough - precipitation lines as diffuse towards each other
What is agglutination test?
Uses cell or bead - rely polyclonal serum to cross link w. AB
Describe influenza test?
Detect Ab against influenza in pt serum or BAL
Haemagglutin influenza test
- Influenza has haemagglutin on surface - allow virus to bind RBC when exposed blood via haemaglutination
- This form aggregation
- In presence anti-haemagglutinin ab - binding inhibited
- RBC settle bottom tube
How to detect haemophiliac influenza?
CSF taken and mixed suspension of latex beads coated anti- h influenza ab
Reaction = immediate agglutination
How would you detect strepotoccous bacteria?
Gram+Ve group oral and dermal pathogens - secrete streptolysin O toxin - lyse RBC
Anti-streptolysin test - ASO
- pt serum mixed standard sheep blood and O toxin
- if pt has Ab - o toxin neutralised and no lysis
- If low conc ab - lysis - release haemoglobin
Explain mechanism of sandwich ELISA?
- Capture ab on plastic surface - specific to ag
- Add pt serum
- Ab will bind to ag raised to - other Ag washed away
- Add detection ab - has enzyme conjugated
- Add substrate enzyme = colour change
- Increased ag - increase colour - measured using absorbance
- Calculate conc ag in sample
What is antigen ELISA?
Measure conc. human ab in serum to bacteria
- Bind ag to solid phase and add primary ab
- Wash off primary ab and add secondary ab - specific to primary ab - has conjugated enzyme
- Add substrate - colour change
- Measure absorbance - calc conc on standard curve
What is flow cytometry?
Used to analyse cells and cell receptors
Ab conjugated to fluorescent tag - laser used to exite - increased emission = increased receptors
Measure 18 colours therefore 18 receptors
How does fluoresce work?
Molecule existed by laser - electron move to excited state at next energy level
Energy released as photon as move back down
Use flow cytometry
HIV - measure no CD4 compared CD8 cells
Signify progression
Difference between immunohistochemistry and immunocytochemistry
1 Immunohistochemistry - staining section of tissue
2. Immunocytochemistry - staining of cells
Use immunohistochem?
Use K167 - label proliferating cells
Used autoimmune disease - auto ab against ag in hemidesmosomes - break at basement membrane
Use of therapeutic ab?
Monoclonal ab specifically bind ag raised to
Ab can block activity of protein by binding
Problem
- Ab raised in mice therefore not human ab (human will raise ab to therapeutic ab = inactive)
How to therapeutic ab work?
Ab taken into cell via endocytosis - enter lysosome - proteolytic enzyme release drug
Drug taken nucleus - affect cell division - kill cell
