DIAGNOSTIC IMMUNOLOGY

* A brief history of antibodies

* 1891: The term “Antikörper” coined by Paul Ehrlich

* Substance in the blood that confers immunity

* "if two substances give rise to two different antikörper, then they themselves must be
different”

* “Lock-and-Key” theory

* 1920’s: Heidelberger and Avery identified antibodies as proteins

* 1940’s: Linus Pauling confirms lock-and-key theory

* 1948:Immunoprecipitation: use of antibodies for detection

* 1956: Glick and Chang-Bursa of Fabricius: Antibodies come from B cells

* 1976: Hozumi and Tonegawa, antibody gene rearrangement

DEFINITION OF TERMS

* Antigen

-foreign substance that, when introduced into the body, is capable of stimulating an immune reaction
e.g. foreign molecules in bacteria, viruses, protozoa, serum components, etc.

* Antibody

- also known as “immunoglobulin” =large protein produced by plasmocytes which identifies and
neutralizes antigens

* Immmune reaction

- reversible binding of antigen to homologous antibody (high specificity)

DIAGNOSTIC IMMUNOLOGY

Diagnostic immunology is a study that deals with diagnostic method that relies on antigen-antibody
reaction for detection of the disease.

Immunologic methods are used as tools in the treatment and prevention of the infectious
diseases and immune-mediated diseases. They have high specificity and sensitivity which all depends on
the value of antibody detection.

Diagnostic immunology involves using antibodies to acquire clinical data using procedures such as:

a) Precipitation Reactions
 - the formation of insoluble Ab:Ag complexes

b) Agglutination

- the formation of visible Ab: Ag aggregates

c) Fluorescent Antibody Staining

- reveal the presence of specific pathogens

d) ELISA

- automated technique revealing presence of Ab or Ag

Diagnostic immunology

* Tests based on the interactions of antibodies and antigens

* These tests determine the presence of antibodies or antigens in a patient.

Antigen -Antibody Reactions

* Antigen – antibody reactions are performed to determine the presence of either the
antigen or antibody. This test is called serological test.

* One of the two components has to be known.

* e.g. with a known antigen, such as influenza virus , a test can determine whether
antibody to the virus is present or not .

Immunologic-based diagnostic tests

Serological Tests

* Agglutination: Particulate antigens

* Hemagglutination: Agglutination of RBCs

* Precipitation: Soluble antigens

* Fluorescent-antibody technique: Antibodies linked to fluorescent dye

* Complement fixation: RBCs are indicator

* Neutralization: Inactivates toxin or virus

* ELISA: Peroxidase enzyme is the indicator

Tests Based on Ag/Ab Reactions

* All tests based on Ag/Ab reactions will have to depend on lattice formation or they will have to
utilize ways to detect small immune complexes

* All tests based on Ag/Ab reactions can be used to detect either Ag or Ab

 AFFINITY

* -a liking for or an attraction to something

AVIDITY

* -the overall strenght of binding between an antibody and antigen

Ag:Ab Ratio

* -ratio of the antibody and the the antigen

Antibody-based Diagnostic Tests

* problem that must be overcome in antibody-based diagnostic tests:

* antibodies cannot be seen directly

* Antibodies produced in an animal were mixed and limited quantity.

Antibody Structure

* Two identical light chains

* Two identical heavy chains

* Linked by S-S bonds

* Monoclonal Antibodies (Mabs)

* Pathogens contain many different antigens (and many more epitopes)  Polyclonal antibody
response

* Monoclonal antibody: single type of antibody directed against one specific epitope. Produced
by single B cell clone.

ANTIBODIES

* Monoclonal antibodies

* MAbs produced from a single clone of B cells

* Monoclonal antibodies all have identical antigen-binding sites. Thus they all bind to the same
epitope with the same affinity

* Mostly produced by fusing a B cell secreting the desired antibody with a myeloma cell capable of
growing indefinitely in tissue culture

Production of MAbs

* Monoclonal Antibodies (Mabs)

 * Serological identification (tissue and blood typing)

* As immunotoxins to treat cancer

* Measure serum protein and drug levels

* Identify infectious agents

* Identify and quantify hormones

* Treatment of specific diseases

* Development of New Generations of MAbs

* Chimeric MAbs (66% human): Genetically modified mice produce Ab with a human constant
region.

* Humanized MAbs (90% human): MAbs that are mostly human, except for mouse antigen-
binding.

* Fully human antibodies

* Bacterial, plant and animal systems under investigation to increase production volumes.

IMMUNOCHEMICAL TESTS:

Serological Tests

* Precipitation Reactions

* Interaction of soluble antigens with IgG or IgM antibodies to form large, interlocking molecular
aggregates called lattices.

* When optimal proportions of antigens and antibodies  Lattice formation.

* Excess of either component decreases lattice formation and subsequent precipitation.

* Precipitin ring test performed in small tube.

* Immunodiffusion procedures: precipitation reactions carried out in agar gel medium

* Precipitation

* Precipitation :

* Agglutination Reactions

* Antibody Titer

* Is the concentration of antibodies against a particulate

 Agglutination reactions are used

 in blood typing, the diagnosis of certain diseases, and the identification of viruses.
* Agglutination

* Neutralization Reactions

* Viral Hemagglutination-Inhibition Tests

* Fluorescent Antibody Techniques

* ELISA