Immunological Techniques in Diagnostics & Research Flashcards
What is the significance of A, B and RhD on RBCs?
Act as antigens eliciting an immune response in donor mismatches / blood transfusions
What is determined by flow cytometry?
- Whether a cell expresses a target protein
- The amount of expression of target protein
- The cells identity and analysis by protein expression
What technique is used to determine blood types?
Blood typing done by agglutination reaction
Describe the key features of ABO blood types
- A and B are glycoproteins on RBCs
- There are 4 blood types - A, B, AB, O
- Rhesus factors (D protein or RhD) proteins on RBC
surface determine charge -ve/+ve
How is western blotting used in research ?
Cell signalling proteins
Mechanism of action for cancer drug
Outline the process of flow cytometry
- Fluorochrome-conjugated antibodies specific for target
protein added to cells - Cells passed through lasers exciting fluorochrome
(blue laser excites HTC emitting green light) - Emitted light detected and plotted on graph
How is IHC used to diagnose cancer?
IHC used to stain B-Raf cancer protein in tissue sections from cancer patients and direct eligible for treatment with B-Raf inhibitors
How is flow cytometry used in research?
Identification and analysis of immune cells in infections and cancer immunotherapy
Outline how raising antibodies is carried out in a lab
- Mice immunised with target protein
- B cells harvested & fused with tumour cells forming
hybridoma - Hybridoma produces antibodies against target protein
which is selected and cloned - Antibodies are secreted by cloned hybridoma which
are collected & harvested to be used in immunological
techniques
What are the diagnostic uses of western blotting?
Pathogen used, proteins are separated on a gel and transferred to a membrane which is mixed with patient serum to catch ab
Excess is washed off then secondary ab are added to visualise
e.g. HIV, Parasites
Outline the process of IHC
- Thin sections of tissue are cut
- Primary antibodies recognising target proteins are
added to tissue - Ab-Ag complex visualised using chromogenic detection
- Secondary ab specific to primary ab conjugated to HRP
added - HRP catalyses chromogen3,3-DAB into a brown
precipitate at the protein’s location - Visualised using light microscope
Describe the structure of Antibodies
Antibodies have a Y shaped structure
- 2 Heavy chains and 2 light chains
- Fab and Fc compartments
- Fab specific for antigens
- 5 different types of heavy chains producing 5 isotopes
- IgA, IgG, IgM, IgD, IgE
What are the diagnostic applications of Flow cytometry?
Diagnosis of haematological malignancies
CD4 T cell counts in HIV
What is the significance of antibodies?
Important part of our immune response causing neutralisation and opsonisation in phagocytosis
Explain the role of B-Raf in cancer
Cancer caused by mutations in tumour suppressor /promoter genes causing uncontrolled cell division
B-Raf encodes B-Raf protein promoting cell division
many cancers have B-Raf mutation resulting in overproduction of B-Raf leading to uncontrolled cell division
How does flow cytometry determine amount of target protein in cell?
The amount of light emitted = amount of ab bound to protein = amount of protein expressed in cell
What is ELISA?
Enzyme linked immunosorbant assays
What are the applications of confocal microscopy?
- mainly research
- Identification and analysis of cells within tissues
- Co localisation of different antigens
What is IHC?
Immunohistochemistry
- shows the distribution and localisation of antigens in
tissue sections using antibody-antigen interactions
What is the role of ELISA?
Quantifies amount of protein / antibody in liquid samples such as sera / tissue culture supernatants
What are antibodies?
Specialised proteins produced by B cells that act as B cell receptors when membrane bound and can also be secreted
What are the key differences in confocal microscopy compared to flow cytometry?
The cells analysed aren’t in suspension
Tissue sections / cells analysed are attached to microscope slides
Light emitted isn’t plotted, its examined under microscope
Can visualise protein location on cell
What are the 4 different types of ELISA?
Direct
Indirect
Sandwich
Competitive
What are the applications of ELISA?
- ab titres in HIV & HepB patients serum
- Bacterial toxin detection in food (E.Coli)
- Home pregnancy testing detection of chorionic
gonadotropin hormone (HCG) in urine - Research quantification of cytokines / chemokines /
growth factors in tissue culture supernatants
Outline the process of blood typing
- Blood sample mixed with antibody raised against A, B
or RhD antigens - Sample checked for agglutination by eye
- Agglutination indicates presence of antigen in blood
sample
What is flow cytometry?
Used to analyse proteins on cells, in suspension
Often involves use of antibodies (commercially produced) that are conjugated to fluorochromes
What are immunological techniques?
Include both experimental methods to study immune response and methods to generate and use immunological agents as experimental tools
Outline how sandwich ELISA is carried out?
- Add capture ab
- Add sample serum (Serum/Tc)
- Add detection ab (HRP)
- Add substrate
- Determine optical density using spectrophotometer
Density of blue solution = amount of protein in sample
What are the most common immunological technique?
The most common techniques relate antibody production to detect specific proteins in biological samples
Describe the process of western blotting
- Sample prepped - cells lysed, protein denatured
- Electrophoresis - lysates laoded on gel, proteins
separated based on size - Transfer to membrane - fractionated proteins
transferred to membrane - Stain for protein of interest
- membrane incubated with primary ab specific for
target protein - then incubated with HRP conjugated secondary ab
specific for primary ab - Chemiluminescent HRP substrate added to membrane
exposed to Xray - bleaches when exposed to light
- membrane incubated with primary ab specific for
What is western blotting?
A technique used to detect proteins
Southern blot detects DNA
Northern blot detects RNA
