Immunological Techniques

Question 1

Define antigen, antibody, epitope, affinity

Answer 1

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

Question 2

Mechanism for production of polyclonal ab?

Answer 2

Use rabbit/ goat
Based on principle of ab being produced against ag

1. Inject ag into animal - wait 4 weeks for primary immune response
2. Booster injection - 4 weeks second antibody response - larger due to memory
3. Collect blood and centrifuge to isolate serum
4. Check serum specificity -ELISA

Question 3

Mechanism for production of monoclonal ab?

Answer 3

1. Mouse immunised - ab produced against ag
2. Spleen removed - harvest plasma cells
3. Plasma cells fused w/ immortal B cells using polyethylene glycol = immortal hybridomas
4. Placed in well plate w/ HAT - kills of non-hybirdomas
5. Dilute wells so one hybridoma per well = singe mAb
6. Hybridoma with high affinity selected using ELISA

Question 4

Ways of labelling?

Answer 4

Unlabelled
Enzyme - horseradish, peroxidase
Fluoresce - FITC
Gold - electron microscopy

Question 5

Difference between direct and indirect testing?

Answer 5

Direct - add ab to ag -> label Ab to see where tagged

Indirect - use amplified signal

Question 6

What is serological diagnosis?

Answer 6

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

Question 7

What is immunoprecipitation?

Answer 7

Ouchteriony diffusion test

1. Ab and ag placed in wells in agar
2. Diffuse through gel and form precipitate at equivalence

Was used to detect diphtheria

Question 8

What is identity?

Answer 8

Precipitn band formed w/ single Ag

Question 9

What is single radial immunodiffusion?

Answer 9

Ag placed in ab contained gel - precipitation ring indicate reaction
Area of ring proportional to conc Ag

Question 10

What is immunoelectrophoresis?

Answer 10

Ag palmed in well and separated

Ab placed in trough - precipitation lines as diffuse towards each other

Question 11

What is agglutination test?

Answer 11

Uses cell or bead - rely polyclonal serum to cross link w. AB

Question 12

Describe influenza test?

Answer 12

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

Question 13

How to detect haemophiliac influenza?

Answer 13

CSF taken and mixed suspension of latex beads coated anti- h influenza ab
Reaction = immediate agglutination

Question 14

How would you detect strepotoccous bacteria?

Answer 14

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

Question 15

Explain mechanism of sandwich ELISA?

Answer 15

1. Capture ab on plastic surface - specific to ag
2. Add pt serum
3. Ab will bind to ag raised to - other Ag washed away
4. Add detection ab - has enzyme conjugated
5. Add substrate enzyme = colour change
6. Increased ag - increase colour - measured using absorbance
7. Calculate conc ag in sample

Question 16

What is antigen ELISA?

Answer 16

Measure conc. human ab in serum to bacteria

1. Bind ag to solid phase and add primary ab
2. Wash off primary ab and add secondary ab - specific to primary ab - has conjugated enzyme
3. Add substrate - colour change
4. Measure absorbance - calc conc on standard curve

Question 17

What is flow cytometry?

Answer 17

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

Question 18

How does fluoresce work?

Answer 18

Molecule existed by laser - electron move to excited state at next energy level
Energy released as photon as move back down

Question 19

Use flow cytometry

Answer 19

HIV - measure no CD4 compared CD8 cells

Signify progression

Question 20

Difference between immunohistochemistry and immunocytochemistry

Answer 20

1 Immunohistochemistry - staining section of tissue

2. Immunocytochemistry - staining of cells

Question 21

Use immunohistochem?

Answer 21

Use K167 - label proliferating cells

Used autoimmune disease - auto ab against ag in hemidesmosomes - break at basement membrane

Question 22

Use of therapeutic ab?

Answer 22

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)

Question 23

How to therapeutic ab work?

Answer 23

Ab taken into cell via endocytosis - enter lysosome - proteolytic enzyme release drug
Drug taken nucleus - affect cell division - kill cell