Immunology In The Clinic And Research Lab Flashcards
What are the two regions of a antibody?
Fc reigon - control reigon
Fab region - variable regions
how is a polyclonal antibody produced?
Set of B cells
Activated B cells
Antibody producing plasma cells
Antigen specific antibodies
How is a monoclonal antibody made?
Inject mouse with antigen
B cells harvested and fused with myeloma in polyethylene glycol
Select hybrid cells using HAT medium
- myeloma cells die as cannot make nucleotides
- B cells die as they have short life span
Grow hybrid cells in mass culture - clone cells - antibody’s produced and extracted
What happens to hybridomas after antibodies are produced?
Hybridomas can be stored indefinitely and grown to produce monoclonal antibodies
Antibody genes can be cloned from the hybridomas
What are anti-isotopic antibodies?
Polyclonal or monoclonal antibodies that bind to Fc regions of particular antibody classes
What are immunoassays?
Uses antibody-antigen interaction to measure the amount of antibody/antigen
What are the different labels in immunoassays?
Originally radioactive
Enzymes are used more commonly - detected by coloured product
Other alternatives are luminescent
What are the three types of solid phase. Immunoassays (ELISA)?
Direct
Indirect
Sandwich
Describe what occurs in the direct ELIZA test
- Antigen immobilised on solid support
- Test antibody solution covalently linked to enzyme added
- Enzyme substrate added, coloured product produced which can be measured by absorbance
What are the uses o a direct ELISA test?
Screen hybridoma supernatants
Detect exposure to infectious agent
Describe what occurs in the indirect Eliza test
- Antigen immobilised on solid support
- Primary antibody which binds to antigen is added
- Secondary antibody covalently attached to enzyme is subsequently added
- Secondary antibody bids to Fc region of primary antibody
- Enzyme substrate added, colour measured by absorbance
Describe what cures in the sandwich ELISA
- Antigens may be present in low concentration. Because antibodies have high affinity for antigen this technique the antigen
- Need 2 antigens reacting with different epitopes on the antigen
- One antibody mobilised on solid support
- Test antigen solution added, incubate and non-bound remove by washing
- Bound antigen detected by incubation with the other antibody which has been labelled, and non-bound removed by washing
How is a Elispot Assay conducted to measure cytokines secretion?
- Cytokine specific antibodies are bound to the surface of a plastic well
- Activated T cell are added to the well - these T cells are a mixture of different functions
- Cytokine secreted y some activated T cells is captured gy the bound antibody
- The captured cytokines is revealed by a second cytokines specific antibody, which is coupled to a enzyme, giving rise rise to a spot insoluble coloured precipitate
What is the use of SDS PAGE or western blotting?
- detect antigens and antibodies
- measure size of the protein analysed
- used to calculate protein concentration (intensity of a band comparison with a standard of a known concentration)
- May show if a protein has been degraded
Describe what occurs during purification
- Heterogenous population of lymphocytes is mixed with antibodies coupled to paramagnetic particles or beads and poured over iron wool mesh
- When a magnetic felid is applied the coupled cells stick to the iron wool, LABELLED cells are washed out
- When a magnetic field is applied the coupled cells which to the iron wool, UNLABELLED cells are washed out
- The magnetic felid is removed releasing he coupled cells
Describe what occurs during flow cytometry and fluorescence-activated cell sorter (FACS) analysis
- Individual cells within a mixed population are tagged by treatment with monoclonal antibodies which bind to surface molecules and are labelled with fluorescence
- Iced cells are then forced though a nozzle to for stream of single cells
What typed of samples are analysed by biomedical scientists?
Blood serum
Blood cells
Urine
Synovial fluid
Saliva
Mucus
Cerebrospinal fluid
What types of diseases can be identified by a sample analysed by biomedical scientists?
Transplant compatibility
Immunodeficiency
Autoimmunity
Allergy
Malignancy
What is histocompatibility?
Genetic differences between individuals are detected by the immune system, leads to rejection of Non-self
How is rejection avoided in transplants?
Bets transplant results are when the donor and recipient MHC are as similar as possible
MHC are major players in transplant rejection
What does MHC 1 bind to?
Fragments of intracellular proteins
What does MHC 2 bind too?
Fragments of proteins which have been taken up by endocytosis
What cells is MHC 1 seen by?
T cell receptor on cytotoxic T cells
With assistance from CD8
What cells is MHC 2 seen by?
T cell receptor on helper T cells, with assistance from CD4
How is MHC typing conducted for transplant compatibility?
MHC alleles of donor and recipient are identified by PCR
How can XLA (X-linked disorder) be diagnosed using flow cytometry?
The diseases. Causes a inability to produce mature B cells
B cells have CD19 surface protein which is a co-receptor for the B cell receptor
How can flow cytomety be used to monitor HIV infection?
Lymphocyte subset estimations are performed using monoclonal antibodies for infection : CD3, CD4 and CD8
The percentages f the cells in each subset is determine using FACS machine
Results are reported as a percentage
What occurs during neutrophil deficiencies?
High ris of infection
Deficiency in hagocyte function
Patients can’t form reactive oxygen species, succumb to bacterial and fungal infections
How can neutrophil function be measured?
Assay is conducted based on the principle that non fluorescent DHR when phagocytosed by normal activated neutrophil, can be oxidised by reactive oxygen species (ROS), produced during the activated neutrophil Respiratory oxidative burst.
Green fluorescent compound detected by flow cytmetry
How can antibodies be quantified?
Electrophoresis
Nephelometry - release on light scattering properties of antigen-antibody complexes
Describe how a RAST test is conducted to diagnose allergies
Suspected allergen is bound to an insoluble material and the patients serum is dead
If the serum contains antibodies to the llergen, those antibodies will bind to allergen
Radio labeled anti-human IgE is added where is is radioactively proportional to serum IgE fr the allergen
What is immunofluorescence?
Serum added to human cell line
Probed wiht fluorochrome-labelled anti-immunoglobulin antibody
Visualised by florescence microscopy
