Antigen-Antibody interactions (Precipitation)

Immunoassays

have been developed to detect either antigen or antibody, and they vary from easily performed manual tests to highly complex automated assays

precipitation or agglutination

Many assays are based on the principles of?

• Precipitation

• Agglutination

• Complement activation

• Types of interactions

• are immune system processes that are involved in defending the body against pathogens.

Precipitation

• It happens when soluble antigens, like toxins, bind to antibodies, forming a visible insoluble complex.

• This makes it easier for the immune system to remove those harmful substances

Agglutination

• Involves the clumping of cells, like bacteria, when antibodies bind to antigens on their surface

• helps phagocytes to efficiently engulf and destroy multiple pathogens at once

Complement activation

• It refers to the activation of the complement system.

• It can lead to the destruction of pathogens by promoting inflammation

Monovalent antigen

• Refers to an antigen that has a single epitope or a single antigenic determinant capable of binding.

• An antigen has only one binding site for antibodies, making it capable of forming only one antigen-antibody complex at a time.

Univalent antigen

• could be used to describe an antigen with a single reactive group

• Interaction with such antigens typically does not result in precipitation or agglutination.

Multivalent antigen

have multiple antigenic determinants (epitopes)

• Fab fragments

• F(ab)2 fragments

are antibody fragments obtained by the enzymatic digestion of immunoglobulins

Fab fragments

• is a monovalent fragment consisting of a single light chain homodimer.

• It is obtained by papain digestion of IgG, followed by reduction of the light chain disulfide bond. 

• It is a single Ag-binding site.

• It cannot cross-link Ags and cannot precipitate or agglutinate.

• Generated by digestion with papain, which cuts the IgG molecule above the hinge region.

F(ab)2 fragments

• fragment of IgG that is prepared by pepsin digestion of IgG.

• It lacks the heavy chains.

• Contains two antigen-binding sites.

• Divalent

• Produced by pepsin digestion, which cleaves below the hinge region.

IgG

is the most abundant antibody and can be enzymatically cleaved by papain and pepsin to produce different fragments.

Immune complexes

• help in destroying, neutralizing, and eliminating pathogens.

• They activate the classical complement pathway, leading to pathogen lysis.

Opsonization

Complement proteins coat the surface of pathogens, making them easier for immune cells to recognize and engulf through phagocytosis.

Cell lysis

Leads to the formation of the membrane attack complex (MAC), which creates pores or holes in the pathogen's membrane, causing cell lysis and death.

Inflammation

Complement proteins promote this by recruiting immune cells to the site of infection.

C3b

binds to immune complexes and helps in their clearance by RBCs and phagocytosis.

Affinity

• The initial strength of attraction between a single Fab site on an antibody and a single epitope (determinant site) on an antigen.

• Represents the likelihood that the antibody will bind to the antigen based on the complementarity of their shapes, charges, and molecular interactions.

Avidity

• The overall binding strength of multiple antigen-antibody interactions (summation).

• Refers to the formation of a network-like structure of antigen-antibody complexes, which is important for precipitation.

TRUE

TRUE OR FALSE: High avidity can compensate for low affinity to achieve a stable antigen-antibody interaction.

law of mass action

• Governs the interaction between antigens and antibodies, stating that the rate at which these molecules bin

• describes the equilibrium between Ag and Ab binding.

FALSE

TRUE OR FALSE: In lattice formation, a higher concentration of antigen and antibody increases the likelihood of binding, leading to a greater formation of immune complexes.

TRUE

TRUE OR FALSE: In law of mass action, if one component is in excess, then the reaction shifts to favor dissociation, potentially reducing the effectiveness of precipitation.

Principle of lattice formation

• It refers to the formation of a network-like structure of antigen-antibody complexes, which is important for precipitation.

TRUE

TRUE OR FALSE: For a visible precipitate to form, multiple binding sites on antibodies must interact with multiple antigen molecules, creating a lattice-like structure.

TRUE

TRUE OR FALSE: Optimal proportions of antigen and antibody are necessary for lattice to form effectively.

Immunoprecipitation

The process of precipitation of antigen-antibody immune complexes.

1. At least 2 available binding sites must be available to each antibody.

2. Multivalency and solubility for the antigens are a must.

3. Presence of correct proportions for the antigens and the antibodies.

Conditions for immunoprecipitation:

Flocculation

• Natural clumping

• It is observed as a "fleecy mass" when the antigen-antibody suspension is agitated.

Noncovalent bonds

• are weak, reversible interactions that play a crucial role in stabilizing antigen-antibody complexes and other biological structures.

• Requires a very close fit between antigen and antibody for effective binding.

Ionic bonds

• also known as electrostatic bonds.

• Attraction between oppositely charged particles.

• They are relatively strong but weakened in the presence of water or high salt concentrations.

Hydrogen bonds

• Attraction between polar molecules with slight charge separation, involving hydrogen atoms.

• They are essential in stabilizing secondary structures like alpha helices and beta sheets in proteins.

Hydrophobic bonds

• Association between nonpolar molecules that exclude water.

• In their presence, proteins fold, and the binding of antigens to antibodies occurs in aqueous environments.

Van der Waals forces

• Interactions between electron clouds of oscillating dipoles.

• These forces are weak, short-range forces that arise from transient dipoles in atoms or molecules.

• specificity

• cross-reactivity

• optimal binding

Strength of attraction is dependent on?

Specificity

Is the degree to which the antibody binds to a particular antigen, which depends on the shape and fit of the epitope with the antibody’s binding site.

Cross-reactivity

An antibody can bind to antigens structurally similar to the original antigen, but the strength of this bond depends on how closely the cross-reacting antigen resembles the original antigen (structural homology of antigens).

Optimal binding

Maximum affinity occurs when there is a perfect “lock-and-key” fit between the epitope and the antibody binding site.

represents the sum of all attractive forces between the antigen and antibody, and measures the overall stability of the antigen-antibody complex.

Function of avidity

Equilibrium constant

represents the ratio of the concentrations of the products to the concentrations of the reactants when equilibrium is reached.

TRUE

TRUE OR FALSE: A higher Ka indicates a stronger binding affinity because the antigen and antibody tend to stay bound.

TRUE

TRUE OR FALSE: A lower Ka suggests weaker interactions because more antigens and antibodies remain in their free states.

Zone of antibody excess (Prozone)

• Early part of the curve

• Presence of free antibodies

• The antigen combines with only 1 or 2 antibody molecules.

• Large immune complexes can be formed due to antigen insufficiency.

Zone of equivalence

• Lies between the early and later parts of the curve.

• The maximal/optimum amount of precipitation occurs.

• The number of multivalent sites on the antigen and antibody is approximately equal.

Zone of antigen excess

• The latter part of the curve.

• Free antigens are present in the solution.

• Every available antibody site is bound to a single antigen, and no cross-links are formed.

immunodiffusion

• used to describe the diffusion of antibodies and or antigens.

• Typically done using agarose gels (0.3% - 1.5%) or agar (support medium).

to detect antigen-antibody interactions by using precipitation as a measure of the reaction.

Main goal of immunodiffusion system

Agar

• Derived from the red algae and seaweed (Gracilaria and Gelidium).

• Mixture of components: agarose and agaropectin

• Cheaper

• Usage: in microbial studies, a substitute for gelatin, used to make jelly.

Agarose

• It is a purified form of agar and the predominant component of agar.

• A linear polysaccharide.

• Used in electrophoresis, immune diffusion, and bacterial culture.

Single immunodiffusion techniques

One of the reactants (antibody or antigen) remains gel fixed, and the other reactant is allowed to move and interact with the immobilized reagent.

Antibody

In single immunodiffusion techniques, what is the immobilized reagent?

Antigen

In single immunodiffusion techniques, what is the one that is moving and diffusing?

Single linear immunodiffusion

• One reagent either the antibody or the antigen is gel-fixed, while the other reagent moves only in 1 direction.

• The antigen is evenly layered into the surface, forming a thin layer, then it will diffuse downward

Precipitin band

Endpoint of single linear immunodiffusion

Single radial immunodiffusion (RID)

• It is a method used to determine the antigen concentration in a sample.

• The formation of the precipitin band manifests a “radial pattern”.

Precipitin ring

Endpoint of single radial immunodiffusion

Apoprotein determinations

Clinical use of single radial immunodiffusion

1. Mancini

2. Fahey and McKelvey

Two techniques for the measurement of radial immunodiffusion:

Mancini

• Endpoint method

• The antigen is allowed to diffuse to completion, and when equivalence is reached, there is no further change in the ring diameter.

Fahey and McKelvey

• Kinetic method

• The measurements are taken before the point of equivalence is reached.

Rocket immunoelectrophoresis/Voltage facilitated single immunodiffusion

• The reagents move through the gel medium under the influence of an applied field/ voltage.

Rocket-shaped precipitation pattern

Endpoint of Rocket immunoelectrophoresis

1. Electroimmunoassay

2. Electroimmunodiffusion

3. Laurell technique

4. Voltage facilitated single immunodiffusion

Other names for Rocket Immunoelectrophoresis:

• Determining the concentration of a CHON in a mixture.

• Detecting abnormal mAbs related to lymphocytic diseases (i.e., multiple myeloma). 

• Detecting allergens in food

Clinical usage of rocket immunoelectrophoresis

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

Principle of electrophoresis

charged-based separation

How does electrophoresis work?

Cathode

In electrophoresis, positively charged molecules move toward the negative electrode, known as the?

Anode

In electrophoresis, negatively charged electrode, known as the?

Double immunodiffusion techniques

• Both the reactants (antibody or antigen) diffuse into the gel medium.

Radial boundary intersection

In double immunodiffusion techniques, where is the site where the ratio of the antibody and antigen is appropriate?

1. Very time-consuming and not very sensitive.

2. It might take 3 to 4 days to detect the reactants with low concentrations.

Disadvantage of double immunodiffusion techniques

1. Ouchterlony

2. Simple immunodiffusion

Classic double immunodiffusion methods:

Double simple immunodiffusion

Both the antibody and the antigen diffuse through the agarose gel or agar medium until they meet at an equivalence ratio.

Precipitation of immune complexes

endpoint of double simple immunodiffusion

Ouchterlony or double diffusion

• Also known as Double Immunodiffusion or Double Angular Diffusion.

• It is a method based on the diffusion of 3 reagents within an agarose or agar medium in a petri dish.

1. Pattern of identity / Full identity / Serological identity

2. Pattern of partial identity

3. Pattern of non-identity

The three basic patterns that can be observed:

Pattern of identity / Full identity / Serological identity

• The results when the unknown and known antigens are placed in the wells are identical and specific for the antibody present in the 3rd well.

• There is a fusion of the lines at their junction to form an arc that represents serological activity, or that a common epitope is present.

Pattern of partial identity

Fusion of 2 lines with a spur signifies?

Pattern of non-identity

A pattern of crossed lines means 2 separate reactions and indicates that no common epitopes are shared by the compared antigens.

Counter immunoelectrophoresis

• Also known as Counter-Current Electrophoresis or Voltage Facilitated Double immunodiffusion.

• In the agarose gel, the movement of the antigen-antibody is due to the applied voltage rather than diffusion.