And antibody based techniques Antibodies And antibody based techniques

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

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

Antibody Antigen Binding site IgE IgD IgA IgM

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

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

Pathogen Fc Receptor Phagocyte C3b C4b

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

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

Immortalised B cells Plasma cells that produce a specific antibody

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

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

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.

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)

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

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.

Blocking an ELISA plate

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

Standard curve y = mx + b Absorbance Concentration

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

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

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

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

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

Agglutination antigen on cells – direct agglutination

Agglutination antigen/antibody on beads – indirect agglutination

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

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)

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