Lecture 1 & 2 Investigations into Immune System Function Flashcards
What do B cells have on their surface ?
B cell receptors
What are the 2 types of T lymphocytes?
- Cytotoxic T lymphocytes
- Helper T lymphocytes
What molecules do cytotoxic T cells have on their surface ? (2)
- T cell receptor
- CD8
What molecules do T helper cells have on their surface ? (2)
- T cell receptors
2 CD4
What does a B cell receptor consist of? (3)
- 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)
What do T cell receptors consist of ? (3)
- 1 alpha chain
- 1 beta chain
- linked by a disulphide bridge
(Also have variable and constant regions)
What are 2 ways in which antibodies eliminate pathogens?
- Pathogen is opsonised leading to faster phagocytosis
- Antibodies recruit complement proteins
Cause formation of Membrane Attack Complex
Causes holes to form in pathogen’s membrane so pathogen is destroyed
Why is T helper cell crucial in acquired immune response ?
T helper cell releases cytokines which activate cytotoxic T cells and B cells
What does the HIV virion infect?
T helper cells
Name the key parts of the HIV virion? (8)
- GP120 (docking glycoprotein)
- GP41 (transmembrane protein)
- Capsid (p24)
- Two identical RNA strands
- Matrix
- Viral envelope
- Reverse transcriptase
- Integrase
How does HIV virion infect T helper cells ? (2)
- GP120 binds to CD4
- GP41 binds to CXCR4 on surface of T helper cell
What does the infection of T helper cells by HIV virions lead to? (2)
HIV destroys T helper cell
B and Cytotoxic T cell cell-mediated immunity lost
What does AIDS stand for?
Acquired Immune Deficiency Syndrome
What causes AIDS?
Weakened immune system leads to susceptibility to opportunistic infections
Give 5 examples of infections people are more susceptible to with AIDS
- Toxoplasmosis
- Tuberculosis
-
Pneumonia
4 . Other fungal infections - Infection-related cancers e.g. Kaposi’s Sarcoma
What 3 things might be detected in the methods for detecting a HIV infection?
- Antibodies for HIV (seroconversion)
- Viral RNA
- Fall in T Helper cell numbers
When do antibodies against HIV develop?
Develop 2-8 weeks after infection (prior to this=‘window period’)
What do the antibodies bind to on a HIV virion? (2)
Often bind to p24 or gp41
What are 3 methods that detect the antibodies to HIV (seroconversion) ?
- ELISA
- Immunochromatography
- Latex particle agglutination
What does ELISA stand for?
Enzyme-Linked Immunosorbent Assay
What are the 4 main steps in an ELISA test?
- HIV antigen e.g. p24, gp41 bound to well . Then wash
- Patient serum added. Anti-HIV antibodies bind to antigen. Then wash
- Anti-human antibodies added and binds to anti-HIV antibodies. Enzyme linked to anti-human antibodies. Then wash
- Substrate added, converted to coloured product by enzyme (if anti-HIV antibodies present)
Give an example of and enzyme linked to an antibody and a substrate used in ELISA
- Antibody conjugated (linked) to Alkaline Phosphatase enzyme
- Substrate may be BCIP
- BCIP oxidised by AP from colourless to purple
How do you detect chromogenic substrates?
Detective by light absorbance
You can use different enzymes conjugated to antibody. Give an example of an enzyme and the 3 different substrates used
Horseradish peroxidase
Substrates = DAB, TMB, ABTS
What happens in immunochromatography? (2)
- Detection of antibodies to HIV
- Any anti-HIV antibodies bind to HIV antigen in test
What is added to the absorbent pad/sample port in immunohromatography?
Serum, plasma or whole blood
What will you find at the HIV test line?
Antibody which binds to antigen/antibody complex
What happens at the control line?
Control line binds to the HIV antigen (that have travelled up the test strip)
What is good about immunochromatography? (3)
Quick, simple, cheap
What happens in latex particle agglutination? (3)
- Latex particle covered with HIV antigen e.g. gp41
- Add blood which may contain anti-HIV antibodies
- If anti-HIV antibodies present latex particle agglutination occurs and can see visual clumping
What is good about latex particle agglutination? (3)
Quick, simple and cheap
Why is the detection of HIV Viral RNA useful (compared to antibodies) ?
Useful in ‘window period’ before antibodies to HIV detectable in serum
What method is used to detect HIV viral RNA
RT-PCR (reverse transcriptase polymerase chain reaction) detection
Why can is detecting viral RNA useful for detecting HIV infection?
Genome of HIV is RNA
How many copies of HIV RNA can RT-PCR detect per ml of plasma?
Can detect 40 copies of HIV RNA per ml of plasma
What are the 4 steps in PCR ?
- Double stranded DNA is denatured (separated) when heated to 95-96 degrees Celsius
- Primers anneal (bind) to sites either side of the target sequence
- DNA polymerase catalyses the extension of the target sequence by joining DNA building blocks (base + sugar + phosphate)
- Cycle repeats (123)
What does reverse transcriptase do?
Makes cDNA (DNA copy) using viral RNA
What method can be used to identify T helper cells on the basis of CD4/ analyse the number of T helper cells?
Flow cytometry and Fluorescence-Activated Cell sorting
What happens in Flow cytometry and Fluorescence-Activated Cell Sorting (FACS) ? (3)
- Blood cells mixed with antibody specific for CD4, which is engineered to contain a fluorescent label
- Cells are streamed through a flow cell
- Photomultiplier tubes detect light emitted
What is Flow cytometry and FACS useful? (3)
- 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
Flow cytometry/FACS using more than one fluorochrome?
- 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
What does flow cytometry/FACS using more than one fluorochrome tell us?
Gives you number of Cytotoxic T cells and Helper T cells etc.
What is the normal T helper cell range in flow cytometry/FACS?
Normal T helper cells 30-50% of lymphocytes
Expect 500-1200 cells/ul
What is the T helper cell range/number for a person with AIDS?
<200 CD4+/ul
What is the abnormal range of T helper cells per ul ?
200-500 CD4+/ul
What are the symptoms/presenting features of Multiple Myeloma (MM)? (5)
- Anaemia
- Weakness
- Tachycardia
- Recurrent infection
- Bone pain/fractures
What is multiple myeloma ?
Cancer involving plasma cells
Why do patients with MM get bone pain/fractures ? (3)
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
What method can be used to detect multiple myeloma?
Cellulose Acetate Electrophoresis
What causes Multiple Myeloma?
- Cancerous proliferation of a clone of immunoglobulin-producing plasma cells in the bone marrow
- so large amounts of one immunoglobulin (Ib) produced (PARAPROTEIN)
