Immunological Techniques Summary

Question 1

Define antigen

Answer 1

Anything recognised by the immune system as non-self

Question 2

Define antibody

Answer 2

Proteins made in response to an antigen. Will only bind to the antigen that induced its formation

Question 3

Define epitope

Answer 3

Specific part of antigen that binds to the antibody

Question 4

Define affinity

Answer 4

Measure of binding strength between an epitope and antibody binding site. Higher affinity = stronger the attraction

Question 5

What is a titre of an antibody?

Answer 5

Lowest dilution of the sample that retains a detectable activity

Question 6

How to detect influenza?

Answer 6

Haemagglutination inhibition test

1. Influenza has haemagglutinin molecules on its surface
2. Haemagglutinin binds RBC and virus
3. Virus particles and red blood cells mix = cause haemagglutination = forms aggregate
4. In presence of specific Ab, haemagglutinin to RBC is inhibited. RBC settle to bottom of tube

Question 7

Haemophilus influenza detection?

Answer 7

1. CSF sample taken from pt with meningitis
2. Sample mixed with latex beads coated in anti-H influenzae Ab
3. Interaction between Ag and Ab causes agglutination of beads = positive diagnosis of H.influenzae

Question 8

Detection of streptococcus bacteria?

Answer 8

Anti-streptolysin O test

1. Serum taken from pt and diluted into tubes with sheep RBCs and O toxin
2. If pt has O toxin Ab = it will neutralise/inhibit the O toxin = stop it from lysing RBCs = clear tube
3. At low Ab conc there is not enough to stop the lysis = red tube
4. = Gives the Ab titre

Question 9

What are the uses of enzyme linked immunosorbent assay (ELISA)?

Answer 9

Detecting amount of Ab, Ag and protein in a sample
Non-aggultination and non-ppt tests
Quantifies levels of test protein in a sample using a standard curve
Uses Ab or Ag bound to a solid phase

Question 10

Types of ELISA? What do they measure?

Answer 10

Sandwich ELISA = measures cytokines, bac and virus products in serum
Antigen ELISA = measures conc of human Ab in serum to bacteria

Question 11

What is sandwich ELISA?

Answer 11

1. Capture Ab bound to plastic surface that is specific for the desired Ag
2. Add pt serum
3. Ab will bind specifically to its Ag. All over Ag is washed away
4. Add detection Ab - usually has an enzyme conjugated to it
5. Wash off excess Ab
6. Add substrate for enzyme - will turn blue in presence of enzyme
7. More colour = more Ag
8. Measure absorbance at 450nm
9. Calculate conc of Ag in the sample

Question 12

What occurs in antigen ELISA?

Answer 12

1. Bind Ag/bac/cell to solid phase - wash off excess Ag
2. Add primary Ab specific to Ag. Wash excess primary Ab
3. Add 2ndry Ab specific to primary Ab and is conjugated with an enzyme
4. Wash excess 2ndry Ab
5. Add substrate = turns from colourless to blue
6. Measure absorbance on spectrophometer
7. Calculate Ag conc

Question 13

How does flow cytometry work in HIV?

Answer 13

Determines number of CD4 T cells compared to CD8 T cells
In HIV = low CD4 progresses to AIDS

In flow cytometry - more emission intensity = more receptors on cells