1. And antibody based techniques
Antibodies
And antibody based techniques

2. What are antibodies?
Antibodies are immunoglobulins which are free in the blood
Immunoglobulins are large Y shaped proteins
The different classes of antibody have different configurations
They have variable binding sites which recognise a range of antigens
This property of antibodies makes them useful for many research and diagnostic methods

3. Antibody (Immunoglobulin) structure
Antigen
Antigen binding site
Fab region (Fragment antigen binding)
Fc region

4. Antibody
Antigen
Binding site
IgE
IgD
IgA
IgM

5. Antibody types
The five classes of antibodies are based on their heavy chain structure – isotype
Within each class of antibody are subclasses
IgG – 4 subclasses
IgA – 2 subclasses
Each class of antibody may have different alleles – allotypes
The α, γ and κ chains all have allotypes
Antibodies which have specificity to the same antigenic determinants are called idiotypes
Based on V region of both heavy and light chains

6. Antigen recognition
Antibodies do not recognise whole molecules but a small portion of proteins – epitope
Different antibodies may recognise the same pathogen at different epitopes
Antibodies themselves do not destroy pathogens but attach and coat them making them “delicious” to phagocytes – opsinisation
Blocks pathogen attachment and entry – neutralisation
IgM and IgG (except IgG4) binding to antigen can activate the complement system – bacterial lysis and phagocytosis
Allows interaction of the antigen-antibody complex with other cell types via the Fc region

7. Pathogen
Fc Receptor
Phagocyte
C3b
C4b

8. Antibody function http://www. wiley
IgG is the major antibody in the blood, but it is able to enter tissue spaces.
Coats antigens, speeding antigen uptake
IgA concentrates in body fluids to guard the entrances of the body
IgM tends to remain in the blood,
Lead to efficient killing of bacteria
Is the largest antibody
IgD remains membrane-bound and somehow regulates the cell’s activation
IgE is found in trace amounts in the blood
Can trigger allergic reactions

9. Antibodies in the laboratory
IgG most important antibody in laboratory techniques
75-80% of antibody in blood
Human IgG injected to a mouse will be recognised as a foreign molecule by anti-IgG
Human IgG is an antigen in the mouse
Polyclonal antibodies can be generated by inoculating antigens into an animal and allowing its B cells to produce antibody against it
Animal is then bled and antibody purified
Polyclonal antibodies are produced by several B cells all recognising the same antigen but at different epitopes
More specific antibodies can be generated from a single immortalised B cell (hybridoma) that recognises one epitope- monoclonal antibodies

10. Immortalised B cells
Plasma cells that produce a specific antibody

11. Antibody uses Blocking or neutralising experiments
Used to investigate the role of a protein or other biological molecule
Add antibody to coat antigen and thus block/remove it
Can be used therapeutically to prevent the action of a particular protein
Detection experiments
Antibody is used to detect a protein - primary (1) antibody
But this doesn’t quantify the protein therefore need to add a secondary (2) antibody
2 antibody coupled to a tag, that can be measured

12. Add antibody specific for the protein of interest
Add secondary antibody with tag
Add antibody specific for the protein of interest
You have a mixture of proteins
Wash off excess antibody
On addition of substrate, the antibody (which relates to amount of protein) becomes detectable
Wash excess antibody

13. Antibody labels Radioactive tags (radiolabels) Enzyme labels
Used to be used frequently but for safety no longer used
Enzyme labels
Require a substrate to act on
Cause a “chromogenic” reaction - a colour can be produced
Horseradish peroxidase(HRP) substrates are aromatic phenols such as diaminobenzidine (DAB) (immunohistochemistry or blotting) or o-Phenylenediamine dihydrochloride (OPD) (ELISA)
Alkaline phosphatase substrate is any phosphate-containing molecule with an alkaline pH 
Fab fragment labels
Fab portion can be cleaved from the whole antibody and a label attached
Fluorophore can be directly visualised for fluorescence without the need of a secondary antibody
Biotin requires the addition of avidin horseradish peroxidase (sometime just called avidin peroxidase) which binds to biotin with high affinity
This now behaves as an enzyme labelled antibody and therefore requires the addition of a substrate in order to create a colour.
The advantage of the fab labelled antibodies is that a 2 antibody is not required for detection.

14. Antibody based laboratory techniques
Laboratory tests exploit a range of different responses to the binding of antigen by the antibody:
precipitation of the complex of antibody and antigen (precipitation)
agglutination of red blood cells either directly by antibody or after coating the cells with antigen (haemagglutination)
activation of complement (complement fixation test, CFT)
binding of antibody to antigens on the surface of microorganisms or within tissue sections (fluorescent antibody techniques)
binding of antibody to antigens adsorbed to plastic (ELISA)

15. Enzyme Linked ImmunSorbant Assay
Direct ELISA - primary antibody is labeled
Indirect ELISA - secondary antibody with label is added T
More sensitive than direct
Can cause nonspecific binding
Sandwich ELISA used to overcome nonspecific binding
Plate is coated with a primary antibody specific to your protein of interest.
Sample is then added
Unbound sample is washed away ensuring that only the protein of interest in left behind
Detected using another antibody specific to a different epitope which is coupled to a label

17. Blocking the plate A very important step in ELISA is blocking
Any protein will bind to the high binding plates surface
Need to block any surfaces of the plate where your antigen or primary antibody hasn’t bound
Usually done by the addition of feotal calf serum (FCS) or bovine serum albumin (BSA)
The blocking step will occur after the binding of the antigen/antibody to the plate.

18. Blocking an ELISA plate

19. Measuring amount of antigen
Measuring the colour that each sample produced tells if one contained more antigen than another.
But does not give the actual amounts of antigen
Need a standard curve.
A known amount of purified antigen serially diluted and added to your plate
Run alongside your samples.
Absorbance of colour that is produced at each concentration is plotted on a graph to create a standard curve
Curve used to calculate the amount of antigen in your samples by measuring where its absorbance lies on the graph

20. Standard curve
y = mx + b
Absorbance
Concentration

21. In an ELISA the antibody that first recognises the antigen is the
Primary antibody
Secondary antibody
Substrate
tag

22. In a direct ELISA, what binds to the plate?
Primary antibody
Secondary antibody
Sample
Substrate

23. Multiplex ELISA
Enables the measurements of several proteins from one sample
Two main commercial systems
Meso Scale Discovery (MSD)
Luminex

24. Precipitation
The binding of antibody to a soluble antigen often results in the formation of a precipitate
precipitin reaction – in a solution
Immunodiffusion - in a gel
Most biological antigens have more than one antibody binding site so complexes of many molecules can be generated.
Increasing amounts of soluble antigen are added to a fixed amount of soluble antibody and the amount of antibody–antigen complex precipitated is measured
ELISA has largely replaced precipitin reactions

25. Agglutination assays
These are antibody assays that look at the interaction between a pathogen and the host
When the assay involves red blood cell antigens and their antibodies it is called hemagglutination
Used in blood typing

26. Agglutination antigen on cells – direct agglutination

27. Agglutination antigen/antibody on beads – indirect agglutination

28. Flow cytometry
A laser-based technology to identify and quantitate different cell populations
The cells are first incubated with mAb specific for the surface molecules of interest
The antibody itself is covalently coupled to a fluorophore
Cells are suspended in a stream of fluid and passed by an electronic detection apparatus
Flow cytometry is routinely used in the diagnosis of health disorders, especially blood cancers, but has many other applications in basic research, clinical practice and clinical trials

29. Summary - 1 There are five classes of antibodies
They have different functions within the immune system
Mainly they recognise antigens and tag them for destruction by the immune system
They can be used in a variety of laboratory techniques
The most common one of these is the Enzyme Linked Immunosorbant Assay (ELISA)

30. Summary - 2
The specificity of antibodies allows them to be used in a range of laboratory methods
Antibodies can be used to either block, detect or tag an antigen of interest
ELISA, agglutination and Flow cytometry are three well used antibody based techniques
Immunoblotting (i.e. Western blot) is also an antibody based technique