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Immunology TutorialImmunology Tutorial

Introduction & Course outlineIntroduction & Course outline

By: Moh’d J. Al KhatatnehBy: Moh’d J. Al Khatatneh

© 2004 Wadsworth – Thomson Learning

Immunological Reactions• The ability to visualize Ab-Ag reactions is a powerful

tool for detecting, identifying, and quantifying antibodies or antigens.

• One may detect or identify an unknown antibody using a known antigen, or vice versa, one may use an antibody of known specificity to detect or identify an unknown antigen.

• This is the underlying basis of immunological testing.

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Characteristics aimed for in immunological testing:

• Specificity is the ability of a test to measure what is intended. A test with good specificity gives few false positive reactions. For example, an antibody which is specific will not cross-react.

• Sensitivity is the ability of a test to detect very, very small amounts of what is intended. A test with good sensitivity gives few false negative reactions. For example, an antibody that is very sensitive will detect minute quantities of its corresponding antigen.

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Serology• Serology is a branch of immunology that deals

with the in vitro diagnostic testing of antibodies and/or antigens (most often serum is used).

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Serological tests

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SerologyAntibody Type Antigen Called Reaction Called Definition

Precipitin Precipitinogen Precipitation The formation of an insoluble complex composed of a soluble Ag and a soluble Ab.

Agglutinin Agglutinogen Agglutination The cross-linking of a particulate or insoluble Ag by the corresponding Ab.

Hemolysin Hemolysis A reaction where the antibody is directed towards a red cell antigen and complement is activated to the C9 stage and the red cells are lyzed.

Cytolysin Cytolysis A reaction where the antibody is directed towards a cell other than a red cell and complement is activated to the C9 stage and the cell is destroyed.

Antitoxin Toxin In this reaction the antibody is directed towards a toxin, usually bacterial, and it neutralizes the toxin making it harmless.

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Precipitation reactions• Visible soluble precipitate

– mix soluble antigen and antibody– excess antigen or antibody--no precipitate– zone of equivalence--precipitate forms

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Zone of equivalence

• change the amount of antigen

• constant amount of antibody

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Gel precipitation

• Agar dish– solid medium

• One well contains antibody• Other well contains antigen• Allow diffusion

• Form precipitate at zone of equivalence

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Double immunodiffusion

• Two antigens and one antibody• Place in separate wells• Allow diffusion• Lines of precipitation

– continuous• identical antigens

– crossing lines• completely different antigens

– continuous with spur• partial identity

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Single immunodiffusion

• Antibody mixed into gel• specimens in well

– screening for presence of antigen

• precipitate forms band around well– indicate presence of antigen– size of band relative to concentration of antigen

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Immunoelectrophoresis

• Separate antigens before testing• put antigen in well• expose to electrical field• antigens are separated by size and charge• add antibody and allow diffusion and precipitation• precipitation with specific antibody gives identity of antigen

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Agglutination reactions

• Visible reaction because antigen or antibody is on larger molecule– cell– latex bead

• Interaction of antigen and antibody– clumping of large particles

• Similar to precipitation reaction

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Agglutination reactions

• Direct--detect antibodies– using cells with antigen on them

• Indirect--detect antigen or antibody– coated spheres or cells– observe agglutination

• Hemagglutination– Red blood cells agglutinate– certain viruses (influenza)

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Qualitative agglutination

• Known antigen in fluid• Unknown specimen added• Agglutination

– positive reaction

• No agglutination– negative reaction

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Quantitative agglutination

• Similar to qualitative

• Diluted samples of antibody

• Measure amount of agglutination for each dilution

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Complement fixation

• Positive reaction:– Antibody present in

serum– Serum added to test

antigen– Bound antibody

“fixes” complement– No available

complement to lyse indicator cells

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Complement fixation

• Negative reaction– No antibody in

serum– Complement not

“fixed”– Free complement

lyses indicator cells

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Immunoassays

• Detect antigen or antibody– use a secondary antibody– tagged with marker

• radioactive• fluorescent• enzyme

• Multiple samples tested at once• Great sensitivity

– dependent on type of tag– much greater than other tests

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ELISA

• Example of immunoassay• Indirect ELISA

– antigen coated to plastic well– protein blocks remaining plastic surface

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ELISA

– Serum added• primary antibody• if antibodies

– bind antigen

• if no antibodies– antigen not bound

– Indicator antibody• enzyme-linked anti-Ig antibody binds primary antibody

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ELISA

– Substrate• specific for enzyme linked to secondary antibody• enzyme causes substrate to change color

– Reactions• color change

– antibody in serum

• no color change– no antibody in serum

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Immunofluorescence

• Antibody with fluorescent label– Bind to cell– Visualize under UV light

• Purpose– detect specific proteins in cells– detect viruses in cells– identify microbial cells– identify and sort cells

• fluorescent activated cell sorter