Immunology Flashcards
What is the structure of an antibody?
Immunoglobulin. Antigen binding region and region of biological activity. Fc is the constant region of antibodies, so can attach to other groups without affecting binding to antigens. Fab is the variable region of the antibody and makes antibodies specific to antibodies.
What can bind to the Fc region of antibodies? (x4)
• ENZYMES e.g. peroxidase. • FLUORESCENT PROBES e.g. dyes. • MAGNETIC BEADS to purify and pull out different cell/antibody types. • DRUGS attached to antibodies – antibodies direct drugs to targets e.g. cancer drugs.
What is indirect labelling using anti-antibodies?
• It is indirect labelling of an antigen, using an anti-antibody. • The first antibody that binds the antigen we are trying to detect is called the PRIMARY ANTIBODY. • The SECONDARY ANTIBODY binds to the constant region of the primary antibody – this is the anti-antibody. • Secondary antibody is conjugated to a REPORTER (the thing we use to report/identify the antibody’s presence e.g. dye/bead) – this is your labelling.
What three types of manufactured antibodies?
• ANTISERA FROM IMMUNISED ANIMALS (POLYCLONAL): This is the original method for producing antibodies. It involved immunising an animal against the antibody you wished to extract. The extracted antisera (serum containing antibodies against specific antigens) would contain a mixture of different antibodies i.e. many different specificities, so patients would get a POLYCLONAL response. • MONOCLONAL ANTIBODIES: this is how we now produce most of our antibodies. Monoclonal means producing antibodies of a single specificity. • GENETICALLY ENGINEERED ANTIBODIES: use the DNA segments that encode antibody specificities – genetically engineer antibodies using recombinant DNA techniques.
How are monoclonal antibodies generated?
- Mouse challenged with an antigen.
- Splenic B cells are harvested as a source of antibody producing cell.
- These splenic cells are fused with immortal myeloma cells (can divide indefinitely).
- Immortal hybridomas are formed and cultured to replicate.
- Hybridomas that produce the antibody of interest is selected and cloned.
- Monoclonal antibodies are harvested.
How are genetically engineered antibodies produced?
- Population of genes encoding antibody variable regions are isolated.
- Variable region gene is fused with bacteriophage coat protein gene. The fused variable region is subsequently displayed on the bacteriophage surface. Bacteriophages are viruses that infect bacteria.
- Bacteriophages are cloned, creating a random population of variable regions, and therefore a mixture of bacteriophages.
- Antigens are immobilised onto a plate and the mixture of bacteriophages applied. Bacteriophages that do not bind the antigen are washed away, leaving your desired variable region.
What are the THERAPEUTIC uses of manufactured antibodies? (x4) Examples for each. (x2, x1, x1, x1)
• Prophylactic protection against microbial infection e.g. IVIG (polyclonal antibodies for those that cannot produce antibodies), or Synagis (monoclonal antibodies to protect babies from RSV). • Anti-cancer therapy using monoclonal antibodies e.g. anti-HER2 (HER2 found on many breast tumours). • Removal of T-cells from bone marrow grafts, so graft will not attack host e.g. anti-CD3. • Block cytokine activity e.g. anti-TNFa stop inflammation in arthritis and Crohn’s disease.
What is the big problem with use of antibodies therapeutically?
Expensive.
What are the DIAGNOSTIC uses of manufactured antibodies? (x3)
• Blood group serology – found out patient blood group. • Quantitative immunoassays – quantify a specific substance in blood using antibodies. Hormones, antigens and antibodies may be quantified. • Immunodiagnosis – infection diseases, autoimmunity, allergy (IgE) and malignancy.
How do we use antibodies in quantitative tests?
• ELISA – Enzyme Linked ImmuoSorbent Assay. • Rapid testing
How does ELISA work in quantitative testing?
- Plastic plate contains wells; each well contains an antibody.
- You apply a test sample to each well.
- In one of the wells, the antibody may be complementary to a substance in the sample e.g. an antigen. The target antigen therefore binds to the antibody and excess is washed away.
- A detection antibody conjugated with the HRP enzyme at the Fc region is added.
- Detection antibody binds to the target antigen and excess is washed away.
- Clear solution is added, and the enzyme on Fc turns the solution coloured – used to quantify amount of antigen present.
How does rapid testing work in quantitative testing?
- Test sample is applied to a strip e.g. this testing is used in pregnancy testing.
- Test sample runs up the strip due to capillary action, and first encounters antibodies conjugated to gold nanoparticles.
- If the test sample contains the correct antigen, an antigen-antibody complex will form and continue running up the strip.
- TEST LINE: if antigen-antibody complexes are present, they will bind to antibodies on the test line.
- CONTROL LINE: antibody, in all cases will bind to antibodies in this line (anti-antibodies). This ensures test validity.
How is immunodeficiency measured in immunology? (x3) Including test used.
• Serum immunoglobulin levels using ELECTROPHORESIS/ELISA. • Test presence of specific antibodies that a patient should have been immunised to produce using ELISA. • Measure lymphocyte subsets e.g. CD3/4/8/19 using FLOW CYTOMETRY.
How is electrophoresis used to measure immunoglobulin levels? What would it look like when someone is fighting off a disease? Abnormal electrophoresis?
Middle bar is a healthy patient, with circled region showing a faint smear – indicative of presence of small amounts of antibodies. Left bar shows patient with an active immune response – so this ‘smear’ is deeper in colour as expected. Right bar shows patient has faint smear and a bold line – the bold line indicates monoclonal expansion e.g. in this case, there is monoclonal expansion of B cells which could mean B cell malignancy.
How does flow cytometry work to measure lymphocyte subsets?
Lymphocytes have different proteins on their surface e.g. CD3, 4…. In flow cytometry, colour labelled antibodies are added to serum, binding to different immune cells. The labelled serum is passed through a column, and a laser detects the fluorescence of passing labels to distinguish and quantify cell type.
