Immunology & Serology - Lecture - 7 - Antigen-Antibody Reactions - Part 2 - Complete

Immunodiffusion

Is the term used to describe the diffusion of antibodies and/or antigens

Agarose gel or agar

Immunodiffusion is typically done using what media?

0.3-1.5%

What concentration of agarose gel is used if used in immunodiffusion?

Agar

This support medium can be used in immunodiffusion and it provides a stable matrix against temperature and mechanical induced currents, and can still permit an unimpeded flow of proteins

Makes precipitin bands visible, Stabilizes the diffusion process

Advantages of using agarose or agar for immunodiffusion

Agar

This immunodiffusion medium is derived from red algae & seaweed

Gracilaria, Gelidium

What genus of red algae and seaweed are used for agar in immunodiffusion?

Agarose, agaropectin

Agar components in immunodiffusion

Agarose

This immunodiffusion medium is a purified form of agar and the predominant component of agar

Linear polysaccharide

Agarose composition

Agar

Agar or Agarose

Which one is cheaper?

Agarose

Agar or Agarose

Which one is more expensive?

Agar

Agar or Agarose

Production is less time-consuming

Agarose

Agar or Agarose

Production is more time-consuming

Microbiology studies, substitute for gelatin, used to make jelly

Uses of agar

Electrophoresis, immune diffusion, bacterial culture

Uses of agarose

To detect antigen-antibody interaction using precipitation as a measure of the reaction

Main goal of immunodiffusion systems

Single, Double immunodiffusion techniques

What are the different types of immunodiffusion techniques?

Single immunodiffusion techniques

In this type of immunodiffusion technique, one of the reactants (either antigen or antibody incorporated into the agar) remains gel fixed, and the other reactant is allowed to move and interact with the immobilized reagent.

Liquid

In Single immunodiffusion techniques, to make the reactant be fixed to the gel, it is a prerequisite that it has to be added to the gel medium while it is in what form?

Radial or linear

In Single immunodiffusion techniques, the free reactant (antigen) diffuses either a ________ pattern to react with the immobilized reagent (antibody).

Single linear immunodiffusion

In this type of single immunodiffusion technique, one reagent (either antigen or antibody) is gel-fixed, while the other reagent moves only in one direction.

Antibody

In Single linear immunodiffusion, what reagent is usually immobilized in a gel within a glass tube?

Antigen

In Single linear immunodiffusion, what is evenly layered into the surface, forming a thin layer, then it will diffuse downward into the gel forming a precipitin band once it can interact with the antibody in correct proportion?

Formation of precipitin band

It is the endpoint in Single linear immunodiffusion

Single radial immunodiffusion

In this type of single immunodiffusion technique, it is a method used to determine antigen concentration in a sample.

Antigen

In Single radial immunodiffusion, what is the mobile reagent?

Antibody

In Single radial immunodiffusion, what is fixed in the gel?

Radial pattern

In Single radial immunodiffusion, what pattern does the formation of the precipitin band manifest?

Formation of precipitin ring

Endpoint of Single radial immunodiffusion

Standard curve

Single radial immunodiffusion can be quantitative if this was constructed from which the antigen concentration can be extrapolated

Apoprotein determination

Clinical use for Single radial immunodiffusion

Mancini, Fahey and McKelvey method

Two techniques used for the measurement of Single radial immunodiffusion

Mancini method

In this method of measuring single radial immunodiffusion, the antigen is allowed to diffuse to completion, and when equivalence is reached, there is no further change in the ring diameter

Endpoint method

Other name for Mancini method

Fahey and McKelvey method

In this method of measuring single radial immunodiffusion, the measurements are taken before the point of equivalence is reached.

Kinetic method

Other name for Fahey and McKelvey method

Rocket immunoelectrophoresis

In this type of single immunodiffusion technique, the reagents move through the gel medium under the influence of an applied field/voltage

Voltage facilitated single immunodiffusion

Other name for Rocket immunoelectrophoresis

One

In Rocket immunoelectrophoresis, how many directions does the antigen move because it is pushed through the gel by the applied field?

Antibody

In Rocket immunoelectrophoresis, what reagent is in the gel?

Antigens

In Rocket immunoelectrophoresis, what are moved into the gel by the electrical field?

Rocket-shaped precipitation pattern

What is the endpoint for Rocket immunoelectrophoresis?

Electroimmunoassay, Electroimmunodiffusion, Luarell technique, Voltage facilitated single immunodiffusion

Other names for Rocket immunoelectrophoresis

Yes

Yes or No

Can Rocket immunoelectrophoresis be used to determine the concentration of Carbon, Hydrogen, Oxygen, Nitrogen (Protein) in a mixture?

Yes

Yes or No

Can Rocket immunoelectrophoresis be used to detect abnormal monoclonal antibodies (mAbs) related to lymphocytic diseases (i.e., multiple myeloma)?

Yes

Yes or No

Can Rocket immunoelectrophoresis be used to detect allergens in food?

Charge-based separation

Electrophoresis principle

Electrophoresis

This type of single immunodiffusion technique involves the movement of charged particles like proteins, DNA, or RNA through a gel or medium under the influence of an electric field.

Size, Shape

In Electrophoresis, charge is based on what 2 factors?

Positively-charged molecules

In Electrophoresis, these molecules move toward the cathode

Negatively-charged molecules

In Electrophoresis, these molecules move toward the anode

Cathode

It is the negative electrode

Anode

It is the positively-charged electrode

Gel

The medium used in electrophoresis is often this material, such as agarose or polyacrylamide, which provides resistance that causes small molecules to move faster than larger ones.

Electrical current

In this electrical field, an ______ is applied which creates a potential difference that drives the migration of the charged molecules through the gel.

Rocket-shaped precipitates

The separation of these molecules allows for analysis as they are separated into bands based on their movement or in the case of rocket immunoelectrophoresis, we can observe ___________.

These bands can then be analyzed and visualized to determine their presence, size and concentration of their specific molecules

Double immunodiffusion techniques

This type of immunodiffusion technique is termed as "double" because both the reactants (antigen and antibody) are diffusing into the gel medium, forming a precipitate.

Antigen diffuses to antibody, antibody diffuses to antigen

These form the precipitate in double immunodiffusion techniques

Post-gel setting

When are the reagents in Double immunodiffusion techniques added?

Yes

Yes or No

In Double immunodiffusion techniques, both the antigen and antibody are mobile since they are both diffusing in the gel.

No

Yes or No

In Double immunodiffusion techniques, only the antigen is fixed. The antibody is not.

Yes

Yes or No

In Double immunodiffusion techniques, neither antigen or antibody is fixed.

Radial diffusion

In Double immunodiffusion techniques, each reagent is placed on a well and allowed to make a ________

Radial boundary intersection

In Double immunodiffusion techniques, it is the site when the ratio of the antibody and antigen is appropriate (zone of equivalence).

Ouchterlony, Simple immunodiffusion

These are the classic double immunodiffusion techniques

Yes

Yes or No

Are Double immunodiffusion techniques very time consuming?

No

Yes or No

Are Double immunodiffusion techniques very sensitive?

3-4 days

How long can Double immunodiffusion techniques take to detect the reactants with low concentrations?

Double simple immunodiffusion

This type of Double immunodiffusion technique has both the antigen and antibody diffuse through the agarose gel or agar medium until they meet at an equivalence ratio.

Double simple immunodiffusion

This type of Double immunodiffusion technique, the antigen and antibody are placed at some distance from one another in parallel wells that have been cut out of the agarose or agar that has solidified on a petri dish or glass slide.

Precipitation of immune complexes

Endpoint of Double simple immunodiffusion

Time-consuming

It is the disadvantage Double simple immunodiffusion

Ouchterlony Double Immunodiffusion

This type of Double immunodiffusion technique is based on the diffusion of 3 reagents within an agarose or agar medium in a petri dish

Double diffusion, Double Immunodiffusion, Double angular diffusion

Other names for Ouchterlony Double Immunodiffusion

Ouchterlony Double Immunodiffusion

This type of Double immunodiffusion technique is more complicated than double simple diffusion.

3

In the Ouchterlony Double Immunodiffusion, how many wells are cut out from the gel?

At the apexes of an imaginary equilateral triangle

How are the wells arranged in the Ouchterlony Double Immunodiffusion?

All directions

In what directions are the 3 reagents in Ouchterlony Double Immunodiffusion diffusing?

Pattern of identity, Pattern of partial identity, Pattern of non-identity

What are the 3 basic patterns that can be observed in the Ouchterlony Double Immunodiffusion?

Pattern of identity

This pattern in the Ouchterlony Double Immunodiffusion is a continuous line.

Pattern of identity

This pattern in the Ouchterlony Double Immunodiffusion results when the unknown and known antigens into the wells are identical and specific for the antibody present in the 3rd well

Pattern of identity

In this pattern in the Ouchterlony Double Immunodiffusion, there is fusion of the lines at their junction to form an arc, representing serological identity (a common epitope is present).

Full identity, Serological Identity

Other names for Pattern of identity

Pattern of partial identity

This pattern in the Ouchterlony Double Immunodiffusion is a continuous line with a spur at one end.

Pattern of partial identity

In this pattern in the Ouchterlony Double Immunodiffusion, there is fusion of 2 lines with a spur signifying partial identity

Pattern of non-identity

This pattern in the Ouchterlony Double Immunodiffusion has the 2 lines crossed completely.

Pattern of non-identity

This pattern in the Ouchterlony Double Immunodiffusion has a pattern of crossed lines meaning 2 separate reactions and indicate that no common epitopes are shared by the compared antigens

Pattern of identity

Identify Ouchterlony diffusion pattern

Pattern of partial identity

Identify Ouchterlony diffusion pattern

Pattern of non-identity

Identify Ouchterlony diffusion pattern

Antibody

In the Ouchterlony Double Immunodiffusion, it is a mixture of anti-1 and anti-2 and is placed in the central well.

Unknown antigens

In the Ouchterlony Double Immunodiffusion, these are placed in the outside wells.

Pattern of identity

This pattern in the Ouchterlony Double Immunodiffusion, the arc indicates that the 2 antigens are identical

Pattern of non-identity

This pattern in the Ouchterlony Double Immunodiffusion shows crossed lines representing 2 different precipitation reactions; Antigens share no identical determinants

Pattern of partial identity

In this pattern in the Ouchterlony Double Immunodiffusion, antigen 1a shares a determinant that is part of Antigen 1 but is not as complex. The spur formed always points to the simpler antigen.

Counter Immunoelectrophoresis

In this type of Double immunodiffusion technique, in the agarose gel, the movement of the antigen and antibody is due to the applied voltage rather than diffusion (application of an electric field).

Counter Immunoelectrophoresis

In this type of Double immunodiffusion technique, the antigen and antibody are placed in parallel wells and move toward one another under the influence of an electric field.

Counter current electrophoresis, Voltage facilitated double immunodiffusion

Other names for Counter Immunoelectrophoresis

Formation of precipitin band

In Counter Immunoelectrophoresis, what is the result once the antigen and antibody meet in the proper ration?