Immunology in the Clinic and Research Lab Flashcards
Describe the structure of an antibody molecule
2 heavy chains, 2 light chains - held together by disulphide bridges
- Fc and Fab region
What is the role of the Fc antibody region?
Mediates process:
- Antibody dependent cell-mediated cytotoxicity (ADCC)
- Antibody dependent cellular phagocytosis (ADCP)
- Complement dependent cytotoxicity (CDC)
- Pharmacokinetics/half-life
What is the role of the Fab region?
binds antigen to specific epitope via CDRs
What is antibody repertoire?
total possibility of antigen binding sites
What is antibody affinity?
strength of single interaction between antigen and epitope
What is antibody avidity?
different affinities e.g. multimeric
What type of antibody response is initially generated when exposed to pathogens?
Polyclonal antibody response mounted when infected with pathogen
How does a polyclonal antibody response occur?
Infected by an antigen with several epitopes
~1 week later an antibody response is generated against an antigen - polyclonal antibody produced
How is a specific antibody response generated against pathogens?
B cells have unique specificities for each antigen
Epitope binding to B cell induces proliferation and a monoclonal antibodies are produced
Outline how monoclonal antibodies are produced by hybridoma culture
- B cells
- Immortal, derived from B cell tumours do not produce antibodies. Lack hypoxanthine-guanine phosphoribosyl transferase (HGPRT) gene
- Hypoxanthine-aminopterin-thymidine (HAT) selection for fused cells
- Dilute to individual cells.
- Culture cells individually – they will proliferate to form a clone of cells identical to the original parent
- Antibody of one specificity will be produced
How are hybridomas selected for in hybridoma culturing for monoclonal antibodies?
In HAT medium:
Myeloma cells die - can’t make nucleotides due to lack of HGPRT gene
B cells die - have short life span
Only hybridomas grow and proliferate
Why are hybridomas a good culturing technique for monoclonal antibodies?
Hybridomas can be stored indefinitely and grown to produce monoclonal antibody when required
Why is hybridoma culturing useful for antibody production?
Antibody genes can be cloned from the hybridomas which allows antibodies to be engineered for different applications
What are anti-isotypic antibodies?
Polyclonal / monoclonal antibodies produced which bind to Fc regions of particular antibody classes e.g. to IgG’s, IgA’s etc.
What are immunoassays?
immuno
- uses antibody-antigen interaction (one of which is “labelled” or “tagged” to allow its detection)
assays
- measures (amount, concentration) of antibody or antigen
What antibody type do immunoassays use?
Immunoassays use polyclonal or monoclonal antibodies
What is the advantage of using immunoassays?
Very sensitive and specific hence, widely used in research and analytical labs
What is the original immunoassay label used?
originally radioactive radioimmunoassay (RIA)
What is the new commonly used label for immunoassays?
commonly now enzyme e.g. horseradish peroxidase (HRP) or alkaline phosphatase -usually detected by coloured product (colorimetric) e.g. ELISA
other alternatives are luminescent
What is ELISA?
enzyme-linked immunosorbent assay (ELISA)
What are the 2 type sof ELISA?
Direct/Indirect
- Often used to quantify an antibody
Sandwich (Capture)
- Often used to quantify an antigen
What is the purpose of ELISA testing?
Using these assays, analyte in sample can be calculated by comparison to analyte standards of known concentration
Outline how a direct ELISA test is done
- Antigen immobilised on solid support
- Test antibody solution covalently linked to enzyme (e.g. HRP or AP for colorimetric ELISA) added
- Enzyme substrate added
- Coloured product produced which can be measured by absorbance
What are the uses of direct ELISA test?
- screen hybridoma supernatants
- detect exposure to infectious agent
Outline how an indirect ELISA is carried out
- Antigen immobilised on solid support
- Primary antibody which binds to antigen is then added
- Secondary antibody covalently attached to enzymes is subsequently added
- Secondary antibody binds to Fc region of primary antibody
- Enzyme substrate added
- Colour measured by absorbance
Why can the second antibody added in Indirect ELISA bind to multiple eptiopes?
Secondary antibody is often polyclonal and so may bind to different epitopes on a primary antibody
How does the polyclonal nature of the secondary antibody amplify the indirect ELISA signal?
Allows multiple secondary antibodies to bind to the same primary antibody thereby amplifying the signal and increasing the sensitivity of the test
When is sandwich ELISA used?
When antigens may be present in low concentration
Why can sandwich ELISA be used on low [antigen]?
Because antibodies have high affinity for antigen this technique can concentrate the antigen
Outline how a sandwich ELISA is conducted
Need two antibodies reacting with different epitopes on the antigen
- One antibody immobilised on solid support
- Test antigen solution added
incubated - Non-bound removed by washing
- Bound antigen detected by incubation with other labelled antibody
- Non-bound removed by washing
What are elispot immunoassays used for?
Used to detect cytokine secretions
How are elispot immunoassays carried out?
- Cytokine specific antibodies bound to plastic well surface
- Activated T cells added (different effector functions)
- Cytokine secreted by some activated T cells captured by bound antibody
- Labelled cytokine-specific antibody captures cytokines and produces insoluble coloured precipitate
What genomic testing is used alongside ELISA?
SDS PAGE/Western blotting often used alongside ELISA
What is SDS PAGE?
Immunoassays: Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE) and Western Blotting
What are the uses of SDS PAGE and western blotting?
1) Can be used to detect antigens or antibodies
2) Used to measure size of the protein being analysed
3) Can be used to calculate protein concentration
4) May show if protein has been degraded
How is [protein] measured in western blotting?
By comparing intensity of band we are detecting to band from a protein standard of known concentration
Why is western blotting used to detect degraded proteins?
Some of degradation fragments may contribute to signal in ELISA if both coating and detecting antibody are able to bind to them
What is PICS?
Antibody-Antigen Interaction detection method
> Purification of Immune Cell Subsets antibody coated magnetic beads
Outline PICS method
- Antibodies coupled to paramagnetic beads
- Heterogenous mix of lymphocytes added
- Mixture poured over iron wool mesh
- Magnetic field applied
- Coupled cells stick to iron mesh, unlabelled cells washed out
- Magnetic field removed to release coupled cells
How is flow cytometry and FACS conducted?
- Individual cells within mixed population tagged with monoclonal antibodies
- Bind to surface molecules and are labelled with fluorescent dyes
- Mixed cells forced through a nozzle to form stream of single cells
- Individual cells pass through laser beam
- Laser scatters light, causing dye to fluoresce
- Cell sorter can separate specific subpopulations of cells
What samples are used in antibody analysis?
- Blood serum
- Blood cells
- Urine
- Synovial fluid
- Saliva
- Mucus
- Cerebrospinal fluid
What types of disease require antibody testing?
- Transplant compatibility
- Immunodeficiency
- Autoimmunity
- Allergy
- Malignancy
What is the use of antibody testing in transplant patients?
Avoiding Unwanted Immune Responses
Histocompatibility – genetic differences between individuals are detected by immune system, leads to rejection of non-self
Which molecules cause transplant rejection?
Major histocompatibility proteins (MHC) are major players in transplant rejection
What makes a good donor match?
Best transplant results when donor and recipient MHC are as similar as possible
What are MHC Molecules?
In humans known as HLA (Human Leukocyte Antigens)
Describe the genetic make up of HLA proteins
The MHC is located on chromosome 6 and contains 3 MHC class I proteins and 3 MHC class II proteins
Highly polymorphic - 100s of different variants
How are HLA alleles detected for donor matching?
MHC alleles of donor and recipient are identified by Polymerase Chain Reaction
What technique is used to quantify cell populations?
Flow cytometry: Antibodies to cell surface markers
What is X-linked agammaglobulinemia (XLA)?
X-linked disorder- inability to generate mature B cells
B cells have CD19 surface protein which is a co-receptor for the B cell receptor
When is flow cytometry used in clinic?
monitoring HIV
Lymphocyte subset estimations performed using monoclonal antibodies to: CD3, CD4 and CD8 on whole blood and analysed by flow cytometry
How are cells quantified in flow cytometry?
Percentages of cells in each subset determined using a FACS machine
Results reported as percentages and absolute counts
How are T cell responses tracked?
MHC-peptide complexes made that bind specifically to TCR
Fluorochrome (phycoerythrin) added
Visualised by flow cytometry
Where are large numbers of neutrophils expected to be found?
Neutrophils are found in acutely inflamed tissue
What is the role of neutrophils?
Ingest pathogens and kill using reactive oxygen species
Why does inflammation lead to pus?
Neutrophils rapidly die after phagocytosis which generates pus
What is neutropenia?
Deficiency in neutrophil numbers – neutropenia – high rate of infection
What is the consequence of phagocyte function deficiency?
Deficiency in phagocyte function; chronic granulomatous disease – patients cannot form reactive oxygen species, succumb to bacterial and fungal infections
How can we measure neutrophil function?
Neutrophil Oxidative burst assay
How does Neutrophil Oxidative burst assay measure neutrophil function?
- Neutrophils stimulated with PMA – phorbol myristate acetate
- Normal activated neutrophils phagocytose Nonfluorescent DHR
- DHR oxidized by reactive oxygen species (ROS), produced during activated neutrophil respiratory oxidative burst
- Rhodamine 123 produced; green fluorescent compound,
- Detected by flow cytometry
How are antibodies quantitated?
- Electrophoresis
- Nephelometry
What is nephelometry?
Automated and rapid method used to measure serum immunoglobulin levels
How does nephelometry work?
Measures Light Refraction
Relies on the light-scattering properties of antigen-antibody complexes
What is nephelometry used for?
To study amount of antibody from different classes present in serum e.g. IgG, IgA etc
Here, serum is mixed with anti-isotype antibodies
How is an allergic reaction detected?
IgE responses predominate
- skin prick test
- RAST RadioAllergoSorbent Test)
Name some common allergens
House dust mites; Cat, Dog, Trees, Grasses, Moulds, Egg, Milk, Cod, Soya, Peanut etc
How does a skin prick test identify an allergen?
- IgE binds to allergen
- Fc region of antibody binds on mast cell receptors
- Mast cells degranulate causing release of mediators (e.g histamine)
- Reddening + swelling of skin
Describe the characteristics of Autoimmunity
Autoimmune disease characterised by autoantibodies to nuclear antigens e.g. DNA, RNA
What is the significance of antibody detection
Detection of autoantibodies is useful for diagnosis and monitoring disease activity
Give an example of autoimmune disease
Systemic Lupus Erythematous (SLE)
Autoimmune disease characterised by production of autoantibodies causing a range of symptoms, often dermatological
