P7: Immunofluorescence Assay (IFA) (Immunology)

What is the immunofluorescence assay based on?

antibodies chemically conjugated with a fluorescent dye

What are the terms used to describe fluorescent dyes used in IFA?

fluorochrome, fluorophore

What are common dyes used in IFA?

fluorescein isothiocyanate, phycoerythrin, rhodamine, tetramethylrhodamine, Texas red

What colour is the light emitted when fluorescein isothiocyanate is used?

green

What colour is the light emitted when tetramethylrhodamine is used?

red-orange

What colour is the light emitted when R-phycoerythrin is used?

red-orange

What colour is the light emitted when rhodamine is used?

red

What colour is the light emitted when Texan red is used?

red

What does IFA allow for?

detection of antigen or antibody;
visualisation of specific proteins or antigens in cells or tissue sections

How does IFA work?

samples are illuminated with light of a specific wavelength, which is absorbed by fluorophores, causing them to emit light of a longer wavelength

How is fluorescence visualised?

fluorescence microscope

How is fluorescence quantified?

flow cytometer, spectrophotometer

What are the advantages of IFA?

identification of antigenic structures;
possible to identify different antigens in one sample

What are the disadvantages of IFA?

expensive equipment;
trained personnel

What are the two major types of IFA techniques?

direct immunofluorescence staining, indirect immunofluorescence staining

What is direct immunofluorescence staining?

primary antibody is labelled with fluorochrome

What is direct immunofluorescence staining used for?

tumour typing, detection and localisation of different sub-cellular antigens

What is indirect immunofluorescence staining?

two antibodies;
primary antibodies specifically bind to target antigens;
secondary reagent is conjugated to fluorochrome and recognises and binds to primary antibody

What are the secondary reagents in indirect immunofluorescence staining?

antiglobulin, protein A/G (labelled with fluorochrome)

How is one-step labelling of antigens carried out?

two or more antibodies conjugated with different fluorochromes;
can be direct or indirect;
antibodies derived from different animals can be mixed and incubated as a cocktail

How is immunofluorescence widely applied in diagnostic tests?

detection of infectious agents;
testing of autoimmune disorders;
analyse cell suspensions;
quantify amount of antibodies or antigens in test samples

Which infectious agent is IFA the choice method of detecting?

viruses (in biological samples)

What is the method for testing of autoimmune disorders using IFA?

tissue sections or cultured cells are applied to microscope slides;
sample is treated with patient's serum;
slide is incubated and washed;
secondary anti-IgG antibodies labelled with fluorescein are added;
incubation and washing;
fluorescence is detected using a fluorescence microscope

What is used to analyse cell suspensions using IFA?

flow cytometer

What is an example of the purpose of analysing cell suspensions using IFA?

identify and enumerate leukocyte subpopulations in blood samples

What is used to quantify the amount of antibodies or antigens in a test sample?

spectrophotometer

What is the method of quantifying the amount of antibodies in a test sample?

antigen is bound to a solid phase and exposed to a serum sample containing specific antibody;
unbound immunoglobulins are washed off and a fluorescein-conjugated anti-immunoglobulin is added to reveal antibody that reacted specifically with immobilised antigen;
spectrophotometer is used to measure fluorescence

How are the results of IFA using a spectrophotometer interpreted?

intensity of fluorescence is directly proportional to the concentration of antibody/antigen present

What is the method of quantifying the amount of antigens in a test sample?

antibody is bound to a solid phase and exposed to a test sample containing target antigen;
unbound antigens are washed off and a fluorescein-conjugated antibody is added to reveal antibody that reacted specifically with immobilised antigen;
spectrophotometer is used to measure fluorescence

What is the main advantage of immunoflourescence?

allow the identification of antigenic structures

What is the limitations of immunofluorescence?

all techniques require expensive equipment and reagents; assays based on microscopy require trained personnel and have a factor of subjectivity resulting in errors

What are the steps in immunofluorescence assays?

fixation, staining, flow cytometry

What fixation protocols can be used in immunofluorescence assays?

methanol fixation, formaldehyde fixation, paraformaldehyde/glutaraldehyde fixation

What is methanol fixation used for immunofluorescence?

cytoskeletal components

What is formaldehyde fixation used for in immunofluorescence?

membrane associated components

What is paraformaldehyde/glutaraldehyde fixation used for in immunofluorescence?

double-labelling of membrane-bound and cytoskeletal antigens

What is the advantage to methanol fixation in immunofluorescence?

easy method

What is the disadvantage to methanol fixation in immunofluorescence?

frequently solubilised and removes membrane-bound antigens and provides low structural preservation

What is the method for methanol fixation in immunofluorescence?

rinse cover glass with PBS;
fix cells by incubating the cells in pre-cooled 100% methanol at -20 C for 10 minutes;
wash cells with PBS

What is the method for formaldehyde fixation in immunofluorescence?

rinse cells with PBS at room temperature;
fix in 3-4% paraformaldehyde in PBS for 15 minutes at room temperature;
wash 3 times each with PBS containing 100 mM glycine;
permeabilise cells with 0.1% Triton X-100 in PBS for 1-4 minutes;
rinse with PBS

What is the method for paraformaldehyde/glutaraldehyde fixation in immunofluorescence?

rinse cells with PBS at room temperature;
fix in 3% paraformaldehyde, 0.02% glutaraldehyde in PBS for 15 minutes at room temperature;
permeabilise the cells by dipping cells for 10 seconds in 100% methanol at -20 C;
incubate cells 3 times for 10 minutes in 0.5 mg/ml NaBH4 in PBS, pH 8.0 to reduce aldehyde groups;
rinse with PBS

What methods of staining are there in immunofluorescence?

direct staining, indirect staining

What is the method of direct staining in immunofluorescence?

drain culture medium and rinse cover slips with PBS;
fixate cells;
wash with PBS 3 times for 5 minutes;
permeabilise cells with 0.01% Triton X-100 in PBS for 30 seconds (if needed);
wash in PBS 3 times for 5 minutes;
incubate in 1% BSA in PBS pH 7.5 for 30 minutes to block unspecific binding of antibodies;
incubate with fluorochrome-labelled antibodies in 1% BSA in PBS pH 7.5 for 60 minutes at room temperature in a humid chamber in the dark;
wash with PBS 3 times for 10 minutes

What is Triton X-100 used for?

permeabilisation of cells

What is the method for indirect staining in immunofluorescence?

drain culture medium and rinse cover slips with PBS;
fixate cells;
wash with PBS 3 times for 5 minutes;
permeabilise cells with 0.01% Triton X-100 in PBS for 30 seconds (if needed);
wash in PBS 3 times for 5 minutes;
incubate in 1% BSA in PBS pH 7.5 for 30 minutes to block unspecific binding of antibodies;
incubate with the primary antibody in 1% BSA in PBS pH 7.5 for 60 minutes at room temperature in a humid chamber in the dark;
wash with PBS 3 times for 5 minutes;
incubate with a fluorochrome-labelled secondary reagent in 1% BSA in PBS pH 7.5 for 60 minutes at room temperature in a humid chamber;
wash with PBS 3 times for 10 minutes

What are alternative blocking solutions to BSA used in staining of immunofluorescence?

1% gelatine, 1% bovine or horse serum

What are examples of the secondary reagent used in indirect staining in immunofluorescence?

antiglobulin, protein A/G

What is flow cytometry used for?

analysing the expression of cell surface and intracellular molecules, characterising and defining different cell types in a heterogeneous cell population, assessing the purity of an isolated subpopulation and analysing cell size and volume

What is flow cytometry used to measure?

fluorescence intensity, ligands that bind to specific cell-associated molecules

What is an example of a ligand binding to specific cell-associated molecules that can be measured using flow cytometry?

propidium iodide binding to DNA

What is another term for cytometer?

fluorescence-activated cell scanner

How can the cyclical process of fluorescence be summarised?

excitation of a fluorophore through the absorption of light energy;
a transient excited lifetime with some loss of energy;
return of the fluorophore to its ground state, accompanied by the emission of light. The light energy emitted is always of a longer wavelength than the light energy absorbed, due to the energy lost during the transient excited lifetime

What are the steps of flow cytometry?

cell suspension is run through a cytometer with sheath fluid;
tiny stream of fluid takes cells past laser light one cell at a time;
light scattered from the cells or particles is detected as they go through the laser beam;
detector in front of the laser measures forward scatter and several detectors to the side measure side scatter;
fluorescence detectors measure fluorescence emitted from stained cells or particles

What is the purpose of sheath fluid in flow cytometry?

used to hydrodynamically focus the cell suspension through a small nozzle

How are the results of flow cytometry interpreted?

forward scatter correlates with the size;
side scatter is proportional to the granularity of cells

What is flow cytometry used for as a clinical tool?

immunophenotyping, detection and classification of leukaemia

What is immunophenotyping?

based on the use of fluorochrome-labelled monoclonal antibodies which bind to their membrane molecules (CD antigens), which are only on cells of a certain lineage and in a particular stage of development

What CD marker is used for identification of neutrophils?

CD15

What CD marker is used for identification of monocytes?

CD14

What CD marker is used for identification of B-lymphocytes?

CD19

What CD marker is used for identification of T-lymphocytes?

CD3

What CD marker is used for identification of Th-lymphocytes?

CD4

What CD marker is used for identification of Tc-lymphocytes?

CD8

What CD marker is used for identification of NK cells?

CD16