Immunological methodsImmunization and the detection, measurement and characterization of antibodies

Ramona Busch

11.07.2018 – Immunology Lecture

https://www.mybiosource.com/learn/ELISA

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Contents

Immunization and the detection, measurement and characterization of antibodies

Immunization

• Some definitions

• factors that influences the immunogenicity of proteins

Detection, measurement and characterization of antibodies

• Affinity chromatography

• ELISA and RIA

• blood typing

• Measurement of antibody affinity and avidity

• Production of monoclonal antibodies or antibody V-region

• Immunofluorescence microscopy

• …

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Immunization – some definitions

Immunization and the detection, measurement and characterization of antibodies

• Immunization: deliberate induction of an immune response

• Antiserum: fluid phase of clotted blood taken from immunized

individual

• Immunogen: substance, that can elicit an immune response

• Antigen: substance, that can bind to a specific antibody

→ all antigens are immunogens but not vice versa

• Cross-reaction: binding of an antibody to an antigen other than the

immunogen

→ only proteins elicit fully developed adaptive immune response

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Influencing factors

Immunization and the detection, measurement and characterization of antibodies

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Affinity chromatography

Immunization and the detection, measurement and characterization of antibodies

• Separates molecules on the basis of their affinity for one another

• Here: specific binding between antibody and antigen

• antigen is bound to solid matrix

→ specific antibody can be isolated from an antiserum

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ELISA and RIA

Immunization and the detection, measurement and characterization of antibodies

ELISA: enzyme-linked immunosorbent assay

RIA: Radioimmunoassay

RIA

• Radioactive label: 125I

• Radioactivity is measured directly

Labeled anti-immunoglobin antibodies

• Detection of unlabeled antibody to

unlabeled antigen

• Second layer

ELISA

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Competitive

inhibition assay

Immunization and the detection, measurement and characterization of antibodies

• Fixed amount of

unlabeled antibody

• Standard reference

preparation of a labeled

antigen

• Add test samples or

unlabeled standards

• Displacement of labeled

antigen is measured

→ comparison the curves

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Hemagglutination and blood typing

Immunization and the detection, measurement and characterization of antibodies

• Antibody binding: ability to alter the physical state of the antigen

• Detection by agglutination: when antigen is displayed on the

surface of a large particle (e.g. a bacterium)

→ antibodies can cause the bacterium to clump or agglutinate

• Hemagglutination: antigens are on the surface of red blood cells

→ is used to determine the blood group

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Hemagglutination and blood typing

Immunization and the detection, measurement and characterization of antibodies

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Precipitin reaction

Immunization and the detection, measurement and characterization of antibodies

• Precipitate consisting of large aggregates of antigen:antibody complexes

• Depends on amount of antibody and antigen and their ratio

• Small amounts of antigen, antibody excess

→ each antigen is bound to antibodies and crosslinked to molecules

• Large amounts of antigen, antigen excess

→ small (often soluble) complexes are formed

• Zone of equivalence

→ all molecules are in the precipitate

→ very large lattices formed by cross-linking

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Precipitin reaction

Immunization and the detection, measurement and characterization of antibodies

Affected by:

• Number of binding sites of each AB

• Maximum number of AB that can be

bound by an AG

→ these quantities are defined as the

valence of the AB or AG

→ valence has to be 2 or greater

• valence of AB: structural class

• valence of AG: epitopes and steric

considerations

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Measurement of antibody affinity and avidity

Immunization and the detection, measurement and characterization of antibodies

• Affinity: strength of binding of a monovalent ligand to a single antigen-

binding site on the antibody

• Determination by equilibrium dialyses

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Measurement of antibody affinity and avidity

Immunization and the detection, measurement and characterization of antibodies

• Avidity: overall strength of binding of an antibody

molecule to an antigen or particle

• For IgG:

- bivalent binding can increases avidity

• For IgM:

- 10 identical antigen-binding sites

- affinity of each site is quite low

- avidity of binding of the whole antibody can

be very high

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Coombs test (Detection of Rhesus incompatibility)

Immunization and the detection, measurement and characterization of antibodies

Erythroblastosis fetalis

(hemolytic disease of newborn)

→ occurs, when mother makes IgG for Rhesus antigen

Coombs test

• Directly: taking fetal red blood cells, washing

adding anti-human antibody

• Indirectly: taking maternal serum

incubating with Rh-positive cells, washing

adding anti-human antibody •

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Production of monoclonal antibodies

Immunization and the detection, measurement and characterization of antibodies

Heterogeneous:

Antibodies/antisera from natural immune response or after immunization

Disadvantages: - all antisera are different

- only limited volume

- other antibodies

Aim:

Unlimited supply of antibodies of homogeneous structure and known

specificity

→ monoclonal antibodies from cultures of hybrid antibody-forming cells or by

genetic engineering

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Production of monoclonal antibodies

Immunization and the detection, measurement and characterization of antibodies

Fusion of:

• Spleen cells from an immunized mouse

• Myeloma cells from mouse

Hybrid cells/hybridomas:

• Ability to make specific antibodies

• Ability to grow in culture

→ production of antibody specific for the antigen used

to immunize the spleen donor cells

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Production of antibody V-region

Immunization and the detection, measurement and characterization of antibodies

Fusion of:

• Genes encoding V domains of antibodies

• Genes encoding the coat protein of a bacteriophage

Phage display library: collection of different recombinant phage

Select phages by specific binding to antigen

Production of monoclonal antigen-binding particle

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Immunofluorescence microscopy

Immunization and the detection, measurement and characterization of antibodies

• Antibody or anti-immunoglobulin antibody is labeled with a fluorescent dye

• Dye is excited by light of one λ and emits light of a different λ

• Fluorescein (emits green light), Texas Red (emits red light), Rhodamine

(emits orange/red light)

• Can be used to detect the distribution of any protein

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Immunoelectron microscopy

Immunization and the detection, measurement and characterization of antibodies

• High resolution

• Antibodies are labeled with gold particles

• ultrathin sections

• examination in the transmission electron

microscopy

Studying two proteins:

• Using gold particles of different diameters

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Immunoblotting (Western blotting)

Immunization and the detection, measurement and characterization of antibodies

• Identifying the presence of a protein in a cell lysate

• Unlabeled cells

• Detergent to solubilize all cell proteins

• SDS-PAGE to separate the proteins by size

• Transfer proteins to nitrocellulose membrane

• Treatment with antibodies against specific proteins

• Antibodies are revealed by anti-immunoglobulin

antibodies labeled with radioisotopes or an enzyme

→ Often used to test antisera for the presence of

specific antibodies

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Take home masseges/summary

Immunization and the detection, measurement and characterization of antibodies

Immunization

• Deliberate induction of an immune response

• Immunogen: substance, that can elicit an immune response

• Antigen: substance that can bind to a specific antibody

Immunological methods

• A lot of methods to detect, measure and characterize antibodies

• Depend on interaction between antibody and antigen

• Label: radioisotope, enzyme or fluorescence dye

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Thank you for your attention!

Immunization and the detection, measurement and characterization of antibodies

Questions?

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Measurement of antibody affinity and avidity

Immunization and the detection, measurement and characterization of antibodies

• Affinity: strength of binding of a monovalent ligand to a single antigen-

binding site on the antibody

• Determination by equilibrium dialyses