Lecture 1 & 2 Investigations into Immune System Function

Question 1

What do B cells have on their surface ?

Answer 1

B cell receptors

Question 2

What are the 2 types of T lymphocytes?

Answer 2

1. Cytotoxic T lymphocytes
2. Helper T lymphocytes

Question 3

What molecules do cytotoxic T cells have on their surface ? (2)

Answer 3

1. T cell receptor
2. CD8

Question 4

What molecules do T helper cells have on their surface ? (2)

Answer 4

1. T cell receptors
2. CD4

Question 5

What does a B cell receptor consist of? (3)

Answer 5

• 2 identical heavy chains
• 2 identical light chains
• linked by several disulphide bridges

(Also has constant and variable regions- variable regions form antigen-binding sites)

Question 6

What do T cell receptors consist of ? (3)

Answer 6

• 1 alpha chain
• 1 beta chain
• linked by a disulphide bridge

(Also have variable and constant regions)

Question 7

What are 2 ways in which antibodies eliminate pathogens?

Answer 7

1. Pathogen is opsonised leading to faster phagocytosis
2. Antibodies recruit complement proteins

Cause formation of Membrane Attack Complex

Causes holes to form in pathogen’s membrane so pathogen is destroyed

Question 8

Why is T helper cell crucial in acquired immune response ?

Answer 8

T helper cell releases cytokines which activate cytotoxic T cells and B cells

Question 9

What does the HIV virion infect?

Answer 9

T helper cells

Question 10

Name the key parts of the HIV virion? (8)

Answer 10

1. GP120 (docking glycoprotein)
2. GP41 (transmembrane protein)
3. Capsid (p24)
4. Two identical RNA strands
5. Matrix
6. Viral envelope
7. Reverse transcriptase
8. Integrase

Question 11

How does HIV virion infect T helper cells ? (2)

Answer 11

1. GP120 binds to CD4
2. GP41 binds to CXCR4 on surface of T helper cell

Question 12

What does the infection of T helper cells by HIV virions lead to? (2)

Answer 12

HIV destroys T helper cell

B and Cytotoxic T cell cell-mediated immunity lost

Question 13

What does AIDS stand for?

Answer 13

Acquired Immune Deficiency Syndrome

Question 14

What causes AIDS?

Answer 14

Weakened immune system leads to susceptibility to opportunistic infections

Question 15

Give 5 examples of infections people are more susceptible to with AIDS

Answer 15

1. Toxoplasmosis
2. Tuberculosis
3. Pneumonia
   4 . Other fungal infections
4. Infection-related cancers e.g. Kaposi’s Sarcoma

Question 16

What 3 things might be detected in the methods for detecting a HIV infection?

Answer 16

1. Antibodies for HIV (seroconversion)
2. Viral RNA
3. Fall in T Helper cell numbers

Question 17

When do antibodies against HIV develop?

Answer 17

Develop 2-8 weeks after infection (prior to this=‘window period’)

Question 18

What do the antibodies bind to on a HIV virion? (2)

Answer 18

Often bind to p24 or gp41

Question 19

What are 3 methods that detect the antibodies to HIV (seroconversion) ?

Answer 19

1. ELISA
2. Immunochromatography
3. Latex particle agglutination

Question 20

What does ELISA stand for?

Answer 20

Enzyme-Linked Immunosorbent Assay

Question 21

What are the 4 main steps in an ELISA test?

Answer 21

1. HIV antigen e.g. p24, gp41 bound to well . Then wash
2. Patient serum added. Anti-HIV antibodies bind to antigen. Then wash
3. Anti-human antibodies added and binds to anti-HIV antibodies. Enzyme linked to anti-human antibodies. Then wash
4. Substrate added, converted to coloured product by enzyme (if anti-HIV antibodies present)

Question 22

Give an example of and enzyme linked to an antibody and a substrate used in ELISA

Answer 22

• Antibody conjugated (linked) to Alkaline Phosphatase enzyme
• Substrate may be BCIP
• BCIP oxidised by AP from colourless to purple

Question 23

How do you detect chromogenic substrates?

Answer 23

Detective by light absorbance

Question 24

You can use different enzymes conjugated to antibody. Give an example of an enzyme and the 3 different substrates used

Answer 24

Horseradish peroxidase
Substrates = DAB, TMB, ABTS

Question 25

What happens in immunochromatography? (2)

Answer 25

- Detection of antibodies to HIV - Any anti-HIV antibodies bind to HIV antigen in test

Question 26

What is added to the absorbent pad/sample port in immunohromatography?

Answer 26

Serum, plasma or whole blood

Question 27

What will you find at the HIV test line?

Answer 27

Antibody which binds to antigen/antibody complex

Question 28

What happens at the control line?

Answer 28

Control line binds to the HIV antigen (that have travelled up the test strip)

Question 29

What is good about immunochromatography? (3)

Answer 29

Quick, simple, cheap

Question 30

What happens in latex particle agglutination? (3)

Answer 30

1. Latex particle covered with HIV antigen e.g. gp41 2. Add blood which may contain anti-HIV antibodies 3. If anti-HIV antibodies present latex particle agglutination occurs and can see visual clumping

Question 31

What is good about latex particle agglutination? (3)

Answer 31

Quick, simple and cheap

Question 32

Why is the detection of HIV Viral RNA useful (compared to antibodies) ?

Answer 32

Useful in ‘window period’ before antibodies to HIV detectable in serum

Question 33

What method is used to detect HIV viral RNA

Answer 33

RT-PCR (reverse transcriptase polymerase chain reaction) detection

Question 34

Why can is detecting viral RNA useful for detecting HIV infection?

Answer 34

Genome of HIV is RNA

Question 35

How many copies of HIV RNA can RT-PCR detect per ml of plasma?

Answer 35

Can detect 40 copies of HIV RNA per ml of plasma

Question 36

What are the 4 steps in PCR ?

Answer 36

1. Double stranded DNA is denatured (separated) when heated to 95-96 degrees Celsius 2. Primers anneal (bind) to sites either side of the target sequence 3. DNA polymerase catalyses the extension of the target sequence by joining DNA building blocks (base + sugar + phosphate) 4. Cycle repeats (123)

Question 37

What does reverse transcriptase do?

Answer 37

Makes cDNA (DNA copy) using viral RNA

Question 38

What method can be used to identify T helper cells on the basis of CD4/ analyse the number of T helper cells?

Answer 38

Flow cytometry and Fluorescence-Activated Cell sorting

Question 39

What happens in Flow cytometry and Fluorescence-Activated Cell Sorting (FACS) ? (3)

Answer 39

1. Blood cells mixed with antibody specific for CD4, which is engineered to contain a fluorescent label 2. Cells are streamed through a flow cell 3. Photomultiplier tubes detect light emitted

Question 40

What is Flow cytometry and FACS useful? (3)

Answer 40

- used to distinguish T helper cells from other blood cells by virtue of the CD4 on T helper cell surface - identify number of T helper cells in patients blood - so useful in monitoring HIV infection

Question 41

Flow cytometry/FACS using more than one fluorochrome?

Answer 41

- Mix lymphocytes with a range of antibodies (e.g to antigens CD4, CD8) each with a different fluorochrome (fluorescent label) - Detection of emitted wavelengths from both fluorochromes

Question 42

What does flow cytometry/FACS using more than one fluorochrome tell us?

Answer 42

Gives you number of Cytotoxic T cells and Helper T cells etc.

Question 43

What is the normal T helper cell range in flow cytometry/FACS?

Answer 43

Normal T helper cells 30-50% of lymphocytes Expect 500-1200 cells/ul

Question 44

What is the T helper cell range/number for a person with AIDS?

Answer 44

<200 CD4+/ul

Question 45

What is the abnormal range of T helper cells per ul ?

Answer 45

200-500 CD4+/ul

Question 46

What are the symptoms/presenting features of Multiple Myeloma (MM)? (5)

Answer 46

1. Anaemia 2. Weakness 3. Tachycardia 4. Recurrent infection 5. Bone pain/fractures

Question 47

What is multiple myeloma ?

Answer 47

Cancer involving plasma cells

Question 48

Why do patients with MM get bone pain/fractures ? (3)

Answer 48

Malignant plasma cells stay in bone marrow and activate osteoclasts Osteoclasts dissolve bone Their activity is up-regulated in MM So patients get Fractures of long bones, ribs and vertebrae

Question 49

What method can be used to detect multiple myeloma?

Answer 49

Cellulose Acetate Electrophoresis

Question 50

What causes Multiple Myeloma?

Answer 50

- Cancerous proliferation of a clone of immunoglobulin-producing plasma cells in the bone marrow - so large amounts of one immunoglobulin (Ib) produced (PARAPROTEIN)