5. Antibodies as diagnostic tools Flashcards
(19 cards)
What can antibodies be raised against?
- Almost any antigen
- Often, but not always proteins
- Including immunoglobins from other species
- Against antibodies: anti-antibody
What is rheumatoid factor?
- Anti-antibody
* IgM antibody against IgG
How can you generate monoclonal antibodies?
• Fuse 2 different cell types:
- B cells (spleen cells): positive for particular enzyme, produce antibodies
- myeloma cells (B cell tumour cells): grow indefinitely
• Hybridoma formed - immortal and produces antibodies
• Only select the cells where the enzyme can grow, using HAT medium
How do you produce antibodies using recombinant DNA technology?
- Make a library of all possible V segments
- Construct a fusion protein - V region with bacteriophage/coat protein
- Each bacteriophage displays different specificity V segments
- Poured onto plate with immobilised antigens
- Antibodies that bind will stick, others can be washed away
- End up with single bacteriophage of optimum specificity
- DNA segments that encode the variable part of the antibody then cloned
What are the therapeutic uses of manufactured antibodies?
- Prophylactic protection against microbial infection e.g anti-RSV (synagis)
- Anti-cancer therapy
- Removal of T-cells from bone marrow grafts
- Block cytokine activity e.g. anti-TNFα
What do the following monoclonal antibody suffixes mean:
- omab
- imab
- umab
- -omab: mouse monoclonal e.g. transplant immunosuppresion
- -imab: chimeric or partly humanised e.g. anti-TNFα, rituximab
- -umab: anti-RSV
Why can there still be problems, even when giving a patient a completely human antibody?
- Can still amount an immune response, which has negative effects
- Happens because they didn’t make the antibody themselves
What are the diagnostic uses of manufactured antibodies?
- Tissue typing
- Blood group serology
- Immunoassays (hormones, antibodies, antigens)
- Immunodiagnosis (infectious diseases, autoimmunity, allergy, malignancy)
How does ELISA (enzyme-linked immunosorbent assay) work?
- Wells coated with sample containing antigen
- Antibody, with a reported enzyme, is added
- If the antigen for the antibody is present in the well, the antibody will stick
- Then, add a colourless substrate
- If the antibody with the enzyme has stuck, the enzyme will break down the substrate into a coloured product
- Widely used to measure the levels of particular molecules
What is rapid testing?
- Dipstick tests, strip tests
- Use antibodies to develop coloured lines
- Pad absorbs liquid sample
- Antibodies against the sample, conjugates to small nanoparticles
- Capillary flow moves the particles
- Strip of antibodies against the thing you are trying to measure that will create complex - positive test
- Another strip - control line to show it’s a valid test
How do large and small immune complexes activate immune responses differently?
Depending on the ratio of antibody to antigen, you can get large or small complexes
• Large - good at directly activating complement, neutrophils, and platelets to form thrombi
• Small - don’t activate cell efficiently on their own, but efficient at activating immune components once immobilised on cell membranes
What tests can you do if you suspect a patient is immunodeficient?
• Serum immunoglobin levels - IgG, IgM, IgA, IgG1-4 - serum electrophoresis/ELISA/Nephelometry • Specific antibodies - protein antigens: tetanus + haemophilius - polysaccharide antigen: pneumococcus • Lymphocyte subsets (flow cytometry) - CD3/CD4/CD8/CD19/NK cells
How can serum electrophoresis show immunodeficiency?
- Normally, there should be a broad smear, with increased staining density in the gamma region
- Immunodeficiency - very sharp band indicating expansion of a single clone of B cells (possible malignancy)
What is lymphocyte subsets (flow cytometry) used for?
- Quantifying different lymphocyte subsets
* Different cells have different proteins on their surface, which can be used as markers
Which lymphocytes are the following markers used for: • CD3+ • CD4+ • CD8+ • CD19+ • CD56+
- CD3+ - pan T cell marker
- CD4+ - T helper cells
- CD8+ - cytotoxic T cells
- CD19+ - B cells
- CD56+ - NK cells
How does a flow cytometer work?
- You have antibodies against a specific marker with different fluorescent dyes
- Mix the antibodies with the lymphocytes and pass through the flow cytometer one at a time
- Cytometer detects the fluorescence emitted
- Also detect the forward and side scatter (info about their size and granularity)
How does the CD4+ count and HIV RNA count change over time in a patient without anti-RV treatment?
• Surge of viraemia and CD4+ count goes down in primary infection
• Immune system then recovers for a while where CD4+ increases and HIV goes down
• Long latent period where viral load is controlled but CD4+ depletes
• Eventually AIDS stage is reached after 7 years
- HIV increases
- CD4+ continues to deplete
- opportunistic infections appear
At what T cell count are patient given AVR therapy (and anti-reverse transcriptase drugs) for HIV?
Below 500/microlitre
What infections are ART naïve HIV-1 patients susceptible to at the following CD4+ counts: • 500 • 400 • 300 • 200 • 100
- 500 - bacterial/fungal skin infections, herpes simplex
- 400 - Kaposi’s sarcoma
- 300 - TB, hairy leukoplakia
- 200 - pneumocystis pneumonia (PCP), cryptococcis, toxoplasmosis
- 100 - CMV, lymphoma
