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Flashcards in Immunological Methods Deck (20)
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Why can antibodies by used as a tool?

They can be used in research, diagnostics and clinical medicine
They are able to be used due to:
Antibodies are highly specific and can discriminate between very similar molecules
It is straight forward to immunise an animal with an antigen and generate an antigen specific antibody
You can generate monoclonal antibodies, which are specific for a single epitope
These can be made in large quantities


What are the applications of using antibodies as a tool?

Enzyme-Linked Immunosorbent Assay (ELISA) and Radioimmunoassay (RIA)
Immunofluorescence Microscopy


What is agglutination?

Antibodies can cause the particles to agglutinate (clump together)
This can be seen with the naked eye and does not require any sophisticated equipment to perform


What is the example linked to agglutination?

ABO blood group typing
There are 4 common blood groups: A, B, AB and O

Red blood cells (RBC) from an A individual express the A antigen on their cell surface
RBC from a B individual express B antigen
RBC from an AB individual express both A and B antigens
RBC from an O individual lack the A and B antigens


What is important with blood groups?

It is important for blood transfusions as both the donor and recipient must match
If there is a mismatch the recipient may reject the RBC, where they are destroyed, leading to fever and vomiting
Antibodies of the recipient need to recognised the donors RBC


What are the matches in blood tranfusions between recipient and donors?

Recipient A = Donor A or O
Recipient B = Donor B or O
Recipient AB = Donor A, B, AB, or O
Recipient O = DonorO

O = universal donor
AB = universal recipient


How does antibody determine the blood group?

Antibodies for A and B RBC antigens are tested on the donor and recipient RBC
If agglutination occurs this demonstrates that the antigen recognised by the antibody is present on the RBCs
This information and can be used to determine the subject’s blood group


What are some general facts about ELISA and RIA?

ELISA - Enzyme linked immunosorbent assay
RIA - Radioimmunoassay

They are simple to perform, sensitive and relatively inexpensive
Used widely to measure concentration of either antigen complexes or molecules in a sample
Used to measure the presence of specific antibodies


Describe the ELISA test?

Antigen is bound to the well
Antigen specific antibody is added to the well
A secondary reagent (eg an antibody that binds to the antigen specific antibody) that is coupled to an enzyme
An enzyme substrate is added and in the presence of the enzyme this is converted into a coloured/fluorescent product that can be quantified


Describe the RIA test?

Antigen is bound to the well
Antigen specific antibody is added to the well.
A secondary reagent (eg an antibody that binds to the antigen specific antibody) that is radiolabelled is added
Binding of the secondary reagent is quantified by counting the radioactivity


From the ELISA/RIA test how do we calculate antigen concentration?

Standards of known concentration are used to generate a standard curve (Abs readings v concentration of standard)
From this the antigen concentration in the sample can be calculated (interpolate on graph)


What are some applications of the ELISA/RIA tests?

Measurement of the levels of molecules in the blood: thyroid hormones
Measurement of antibodies against specific antigens in the blood: HIV test (positive = purple)
Measurement of levels of IgE antibodies (involved in type I hypersensitivity: allergic reactions)


Describe the importance of monitoring thyroid hormones?

Thyroid hormones - thyroxine and triiodothyronine (produced in the thyroid gland)
Too little = hypothyroidism, leads to weight gain, tiredness and feeling cold
Too much = hyperthyroidism, leads to increased heart rate, weight loss, muscle weakness, tremors, swollen eyes
The levels of these hormones can be measured by ELISA/RIA


What is immunoblotting used for?

This can determine whether an antigen is present in a complex sample, but it can also determine its size (molecular weight)
However, it can only be used to detect proteins


Describe the method of immunoblotting?

1. Separation of proteins in the sample by electrophoresis (SDS-PAGE)
2. Transfer of proteins onto a membrane (Western blotting)
3. Probe membrane with an antigen specific antibody
4. Detect antibody binding with a secondary reagent eg Horseradish peroxidase (HRP)-conjugated antibody
5. Add HRP substrate and detect light emitted using film or a sensitive camera

An image of the blot will be captured on film, showing the protein recognised by the antigen specific antibody


What are some applications of immunoblotting?

It can detect mutations in a protein e.g. FHL-4, where the syntaxin 11 gene is affected, resulting in a defective NK cell
Can detect HIV antibodies


What is immunofluorescence microscopy?

Microscopy - visualises cells at a high magnification, in order to see intra/cellular structures
Immunofluorescence microscopy enables the presence specific molecules in cells and tissues to be determined
It can also provide information on the position of where in the cell the molecule is localised


Describe the method of immunofluorescence microscopy?

1. Mount cells or sections of tissues onto glass slides
2. Stain with an antigen specific antibody
3. Stain with a fluorescently labelled secondary reagent (antibody)
4. Visualise fluorescent staining with a microscope


What else can you do with immunofluorescence microscopy?

It is possible to stain cells with 2 or more antibodies at once, by using different specific antibodies with different fluorescence
Secondary antibodies labelled with fluorescent dyes emit light at different wavelengths


What is an example of immunofluorescence microscopy?

Rabbit anti-US6 = Red
Mouse anti-Calreticulin = Green
Merged = Yellow (therefore regions containing both)