chapter 4: serology concepts. what is an antigen? an antigen is any substance that elicits an...

Forensic Biologyby Richard Li

Chapter 4: Serology Concepts

What is an antigen?

An antigen is any substance that elicits an immune response and is then capable of binding to the subsequently produced antibodies.

Antigens are generally proteins or polysaccharides, but other substances such as nucleic acids can also be antigens.

What is an antibody?

An immunoglobulin that is capable of combining with specificity to the antigen that elicited its production.

How, Why and Where are Antibodies Produced?

(Massively condensed version)

Produced in Vertebrate Cells in response to a ‘non-self’ substance termed an antigen.

The process begins with macrophages roaming the bloodstream. Macrophages engulf ‘non-self’ molecules and then display portions of the engulfed molecules on their outer plasma membranes.

After many recognition steps B-cells are stimulated to produce specific antibodies.

The antibodies then go off and bind to the foreign substance thereby marking the substance for destruction.

What is an Epitope

An epitope is the small site on the antigen which is recognized by the antibody.

Usually between one and six sugars or amino acids on the surface of the antigen.

Antibody-Antigen Interactions

Binding of antibody to antigen is dependent on hydrogen bonds, electrostatic attractions and Van der Waals attractions.

These bonds are weak compared to covalent bonds but the large number of weak bonds result in a stable complex.

Antibody-antigen binding is reversible. Binding site differences are due to amino

acid sequence differences. The variable region is made up of 110 –

130 amino acids.

Polyclonal Antibodies

Produced by immunizing an animal with the appropriate antigen.

The immunized animal’s serum is collected.

Antibodies can then be purified from the serum.

Since one antigen induces the production of many antibodies the result is a ‘polyclonal’ mixture of antibodies.

Polyclonal antibodies are much less expensive than monoclonal antibodies.

Monoclonal Antibodies

Much more complicated to produce than polyclonal antibodies.

Process begins by immunizing an animal (most commonly a mouse) with an antigen.

The animal’s spleen is removed. B-cells are fused with myeloma cells resulting

in hybridomas. Hybridomas are screened to find those

producing antibodies to the antigen with which they were immunized.

Each hybridoma cell is derived from one B-cell so the antibodies that a clonal population of hybridoma cells produce are monoclonal antibodies.

Monoclonal antibodies recognize one epitope only.

Producing Monoclonal Antibodies

In the creation of a monoclonal antibody, a normal B cell (a type of lymphocyte, or white blood cell) is united with a myeloma cell (a type of cancer). This union results in the formation of cloned hybridomas (hybrid cells) that have the cancer cell’s trait of dividing endlessly and the B cell’s ability to produce a specific type of antibody. Monoclonal antibodies are designed to target specific molecules in the body. They have a variety of uses from pregnancy testing to diagnosing and treating certain diseases.

In the creation of a monoclonal antibody, a normal B cell (a type of lymphocyte, or white blood cell) is united with a myeloma cell (a type of cancer). This union results in the formation of cloned hybridomas (hybrid cells) that have the cancer cell’s trait of dividing endlessly and the B cell’s abilityto produce a specific type of antibody. Monoclonal antibodies are designed to target specific molecules in the body. They have a variety of uses from pregnancy testing to diagnosing and treating certain diseases.

Producing Monoclonal Antibodies

Serological Reagents

Antibodies- capable of binding to an antigen Also known as Immunoglobulins

▪ Found in serum portion of blood▪ Five classes

▪ IgG▪ IgA▪ 1gM▪ IgD▪ IgE

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Serological Reagents

Polyclonal Antibody Produced by different B lymphocyte

clones in response to the different epitopes of the immunogen

Avian- B Type Rabbit- R Type Horse- H Type Polyclonal antiserum Serum

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Serological Reagents

Monoclonal Antibody Spleen cells are utilized Fused cells are called Hybridoma Cells Limitations for use in serology assays

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The Strength of Antigen-Antibody Binding

Mediated by the interaction between epitope of the antigen and the binding site of the antibody

Strongest binding occurs only if the shape of the epitope fits the binding site

Depends on:▪ Affinity▪ Avidity

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The Strength of Antigen-Antibody Binding

Affinity- the energy of the interaction between a single epitope on the antigen and a single binding site on the corresponding antibody Cross-reaction

Avidity- the overall strength of binding between an antibody and an antigen.

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Antigen-Antibody Binding Reactions

Three types: Primary

▪ Most sensitive techniques Secondary

▪ Less sensitive but easier to perform Tertiary

▪ Not commonly employed in a forensic setting

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Antigen-Antibody Binding Reactions

Secondary Reactions

Precipitation Agglutination

Complement Fixation

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Form the basis for many serologic assays in forensic labs

Used less frequently in forensic serology

Antigen-Antibody Binding Reactions

Precipitation

Precipitin curve▪ Prozone▪ Zone of Equivalence▪ Postzone

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Antigen-Antibody Binding Reactions

Agglutination If antigens are located on the surface of

cells or carriers, the interaction of antibodies and antigens will cause the cells or carriers to aggregate and form larger complexes▪ Initial Binding▪ Lattice Formation

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