Immunological Methods

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Immunological Methods

• Dr Kathy Triantafilou
• University of Sussex
• School of Life Sciences

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What is an antigen?

• Any substance capable of producing a specific
  immune response

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What is an antibody?

• Antigen-specific products of B-cells
• Present on the B-cell surface
• Secreted by plasma cells
• Effectors of the humoral immune response,
  searching and neutralising/eliminate antigens
• Two functions
• to bind specifically to molecules from the
  pathogen
• to recruit other cells and molecules to destroy
  the pathogen once the antibody is bound to it

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Structure of the antibody molecule

• The antigen-binding region of the antibody
  molecule is called the variable region or V
  region
• The region of the antibody molecule that engages
  the effector functions of the immune system is
  known as the constant region or C region.
• They are joined by a polypeptide chain that is
  known as the hinge region

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Structure of the antibody molecule

• X-ray crystallography has revealed that the
  overall shape is roughly that of a Y
• Each arm of the Y is formed by the association of
  a light chain with a heavy chain
• The leg of the Y is formed by the pairing of the
  carboxyl-terminal halves of two heavy chains

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Antibody-antigen interactions

• The biological function of an antibody is to bind
  pathogens and their products and facilitate their
  removal from the body
• Regions of the molecule that are specifically
  recognised by antibodies are called antigenic
  determinants or epitopes

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Antigen-antibody interactions

• Antigen-antibody interaction depend on four
  noncovalent forces
• hydrogen bonds
• ionic bonds
• hydrophobic interactions
• van der Waals interactions (between the outer
  electron clouds)

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Antibody-antigen interactions

• The interactions can be disrupted by
• high salt concentrations
• extremes of pH
• detergents

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Monoclonal antibodies

• The resulting serum antibodies are heterogeneous
  (mixture of antibodies against different
  epitopes)
• Antibody heterogeneity increases immune
  protection in vivo but reduces the efficacy of
  the antiserum in vitro
• For research, diagnostic and therapeutic purposes
  monoclonal antibodies are preferable

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Monoclonal antibodies

• Monoclonal antibodies are derived from a single
  clone, and thus are specific for a specific
  epitope
• In 1975, George Kohler and Cesar Milstein devised
  a method of preparing monoclonal antibodies
• They fused normal activated B-cells with a
  myeloma cell
• They generated a hybrid cell called hybridoma

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Hybridomas

• Hybridomas possess
• the immortal growth properties of the myeloma
  cell
• the secreted antibody produced by the B-cell
• The resulting clones secrete large quantities of
  a particular monoclonal antibody
• Hybridomas can be cultured indefinitely

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Use of antibodies

• Antibodies can be tagged to fluorophores,
  radioisotopes and enzymes
• Antibodies can be used to
• detect antigen
• purify proteins
• locate antigen

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Precipitin Reactions

• The interaction between an antibody and antigen
  in aqueous solution forms a lattice that
  eventually develops into a visible precipitate
• The precipitate develops as neighbouring antibody
  molecules within the lattice form ionic bonds
  with each other causing the lattice to lose its
  charge and thus become insoluble

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Precipitin reactions in gels

• Immune precipitates can form not only in solution
  but also in an agar matrix
• When antigen and antibody diffuse toward one
  another in agar or when antibody is incorporated
  into the agar and antigen diffuses, a visible
  line of precipitation will form
• Radial immunodiffusion
• Double immunodiffusion

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Immunodiffusion

• Radial immunodiffusion
• relative concentration of antigen can be
  determined
• Double immunodiffusion
• antigen and antibody diffuse radially from the
  wells towards each other

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Agglutination reactions

• The interaction between antibody and a
  particulate antigen results in visible clumping
  called agglutination
• Hemagglutination
• Bacterial agglutinations

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Agglutination Inhibition

• A modification of the agglutination reaction
• One of the early types of home pregnancy tests
• Included latex particles coated with human
  chrorionic gonadotropin (HCG), and anti-HCG
  antibody
• Addition of urine, which contained HCG, inhibited
  agglutination
• Absence of agglutination indicated pregnancy

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Agglutination Inhibition

• Agglutination inhibition is used
• to determine whether an individual is using
  certain types of illegal drugs, such as cocaine
  of heroin
• in clinical laboratories to determine whether an
  individual has been exposed to a certain type of
  virus (i.e. rubella)

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Western Blotting

• Used to identify a specific protein in a complex
  mixture of proteins
• A protein mixture is electrophoretically
  separated on a gel
• The proteins are then transferred onto a
  nitrocellulose membrane
• Individual protein bands are identified by the
  addition of specific antibodies
• The antigen-antibody complexes can be visualised
  by enzyme-linked antibodies

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Affinity Chromatography

• Attachment of antibody to a solid support, such
  as a column matrix
• An extract produced by disruption of cells is
  passed through the column
• The protein of interest is captured by the
  antibody, whereas everything else runs through
  the column
• In order to recover the protein of interest, the
  antibody-protein interaction is disrupted by
  change of pH (elution buffer)

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Enzyme-linked Immunosorbent Assay

• ELISA depends on an enzyme label on an antibody
• An enzyme conjugated with an antibody reacts with
  a colourless substrate to generate a coloured
  reaction product
• A number of enzymes have been employed, such as
  alkaline phosphatase, horseradish peroxidase and
  b-galactosidase

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Variations of ELISA

• Indirect ELISA
• Antigen is coated onto a well
• Serum (containing antibody) is added
• Presence of bound antibody is detected by adding
  an enzyme-conjugated secondary antibody
• Substrate is added

• Sandwich ELISA
• An antibody is immobilised on a well
• A sample containing antigen is added
• A second enzyme-linked antibody specific for the
  antigen is added
• Substrate is added

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