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PATH 415 > Ag-Ab Reaction > Flashcards

Ag-Ab Reaction Flashcards

1
Q

What is affinity?

A

Affinity refers to the strength of a single Ab-Ag interaction. Each IgG Ag binding site typically has high affinity for its target.

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2
Q

What is avidity?

A

Strength of all interactions combined. IgM typically has low affinity Ag binding sites, but there are 10 of them so avidity is high.

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3
Q

What is cross reactivity?

A

Cross reactivity describes when an Ab binds not to the Ag that elicited its synthesis and secretion but to a different Ag

For example, Ab elicited with toxoids react with native toxins, allowing clinical application for vaccination with nonpathogenic Ag such as tetanus toxoid and diphtheria toxoid.

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4
Q

Describe 3 Ab mediated mechanisms that limit the infectivity of extracellular pathogens.

A
  1. Neutralization - Ab prevent virus or toxic protein from binding their target
  2. Opsonization - A pathogen tagged by Ab is consumed by macrophage/neutrophil
  3. Complement activation - Ab attached to the surface of a pathogen cell activate the complement system.
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5
Q

How does antibody neutralization work?

A
  1. Ab coat extracellular pathogens and neutralize them by blocking key sites on the pathogen that enhance their infectivity
  2. Neutralized Ab-coated pathogens can then be filtered by the spleen and eliminated in urine or feces
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6
Q

What does Ab neutralization prevent?

A

It can prevent pathogens from entering and infecting host cells

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7
Q

How does opsonization work?

A

Ab mark pathogens for destruction by phagocytic cells such as macrophages or neutrophils, because phagocytic cells are highly attracted to macromolecules complexed with Ab

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8
Q

How does complement fixation work?

A
  1. IgM and IgG in serum bind to Ag and provide docking sites onto which sequential complement proteins can bind
  2. Combination of Ab and complement enhances opsonization even further and promotes rapid clearing of pathogens
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9
Q

What is an antigen? What are the major classes of antigens?

A

Any substance that can be recognized by the immune system

  1. Carbohydrates
  2. Polysaccharides
  3. Proteins
  4. Glycoproteins
  5. Nucleic acids
  6. Lipids
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10
Q

What are immunogens?

A

Any substance that can evoke an immune response.

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11
Q

Are all Ag immunogens?

A

Not all antigens are immunogenic.
- Haptens are LMW compounds that are nonimmunogenic by themselves by are antigenic
- Haptens become immunogenic after conjugation to HMW immunogenic carriers

The rule to remember is that all immunogens are Ag, but not all Ag are immunogens. This concept is important when considering rational design of vaccines.

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12
Q

What are the two types of epitopes?

A
  1. Sequential epitopes - short stretches of amino acids (4-7 inches length) that can be recognized by Ab when the short peptide exists free in solution or when it is chemically coupled to another protein molecule
  2. Conformational epitopes require the native 3D configuration of the molecules to be intact, and antigenic determinants need not be contiguous; denaturation of the molecules destroys these kinds of epitopes
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13
Q

What are the requirements for immunogenicity?

A
  1. Physiochemical complexity
  2. Molecular weight > 6 kDa
  3. Foreignness (non-self)
  4. Degradability
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14
Q

List the number of available binding sites of each Ab.

A

IgG, IgD and IgE have two F(ab) binding sites
IgA has four binding sites
IgM has ten binding sites

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15
Q

Why do multivalent interactions increase functional affinity of Ab-Ag binding?

A

Interactions of repeating Ag with more than one functional Ag-binding region allow greater stabilization of binding and increase functional affinity.

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16
Q

What does the strength of binding to epitopes on the antigen (interaction affinity) depend on?

A

Multiple forces present within the binding site
1. Electrostatic interactions
2. Hydrophobic bonding
3. Hydrogen bonding
4. Van der Waal forces

Binding of Ag-Ab is a non-covalent interaction and are reversible in nature

17
Q

The specific binding between the epitope (Ag) and paratope (Ab) involves very small portions of the molecules, usually comprising only a few amino acids. These sites are critical in Ag-Ab reactions as specific binding has to overcome repulsion between the two molecules. How do they overcome the repulsion?

A
  1. When the epitope come in contact with the paratope, they are first attracted to each other by ionic and hydrophobic forces.
  2. These forces help them overcome their hydration energies and allow for the expulsion of water molecules as epitope and paratope approach each other
  3. Attraction becomes even stronger when VDW forces are employed later on to bring epitope and paratope even closer.
18
Q

What are the factors affecting Ag-Ab reactions?

A
  1. Temperature
  2. pH
  3. Ionic strength
  4. Concentrations of Ag and Ab
  5. Number of Ag-binding sites
  6. Structural arrangement
19
Q

How does temperature affect Ag-Ab reactions?

A
  • Optimum temperature for Ag-Ab reaction will depend on the chemical nature of the epitope, paratope and type of bonds involved in their interaction.
  • ex. H-bond tends to be exothermic and more stable at lower temperature and may be more important when dealing with carbohydrate Ag
20
Q

How does pH affect Ag-Ab reactions?

A

Effect of pH on the equilibrium constant of the Ag-Ab complex lies in the pH range of 6.5-8.4.
- pH < 6.5 and pH > 8.4, the Ab-Ag reaction is strongly inhibited
- At pH 5 or 9.5, the equilibrium constant is 100x lower than at pH 6.5-7
- Under extreme pH conditions, Ab may undergo conformational changes that can destroy the complementarity with the Ag

21
Q

How does ionic strength affect Ag-Ab reactions?

A
  • Effect of ionic strength on Ag-Ab reaction is particularly important in blood group serology
  • Reaction is significantly influenced by Na and Cl ions
  • Na and Cl ions cluster around the complex and partially neutralize charges, potentially interfering with Ab binding to Ag which can be problematic when low-affinity Ab are used
  • When exposed to very low ionic strengths, gamma-globulins aggregate and form reversible complexes with lipoproteins of RBCs, leading to their sedimentation
22
Q

How does concentrations of Ag and Ab affect Ag-Ab reactions?

A

Increase in the conc. of Ag and Ab enhances the rxn.

23
Q

How does number of Ag-binding sites affect Ag-Ab reactions?

A
  • An increase in # of Ag-binding sites on the Ab allows for an increased chance of interaction
  • For example, IgM is a pentamer and has 10 binding sites whereas IgG is a monomer and has only 2 binding sites so IgM will bind more efficiently with Ag
24
Q

How does structural arrangement affect Ag-Ab reactions?

A

If the structure of epitope and paratope is such that they could fit well as lock and key then it enhances the interaction between Ag and Ab

25
Q

What are the two types of Ag-Ab interaction?

A
  1. In vivo
  2. In vitro
26
Q

What is an in vivo Ag-Ab interaction?

A

General Ab-mediated immune response occurring in our body against any Ag

27
Q

What are 5 examples of in vivo Ag-Ab interaction?

A
  1. Agglutination - reaction between insoluble (large) Ag and soluble Ab leads to agglutination
    - Formation of visible clumps occurs
    - Occurs on the surface of the particle involved
    - Ab involved are called agglutinins
  2. Precipitation - rxn betwen soluble (small) Ag and soluble Ab forms an insoluble ppt. Proportion of Ag and Ab rxn must be equal for rxn to occur.
    - In the presence of electrolytes at specific pH and temp.
    - Ab involved are called precipitins
  3. Complement fixation - process of binding/fixing the serum complement proteins with the Ag-Ab complex which further initiates a series of immune responses against the Ag
    - Used in detection of any specific Ag or Ab in the patient’s serum
  4. Neutralization - biological effects of viruses & toxins are neutralized by homologous Ab called neutralizing Ab
28
Q

What is an in vitro Ag-Ab interaction?

A

Serological tests performed in labs to detect Ag or Ab in case of many diseases

29
Q

What are 5 examples of in vitro Ag-Ab interaction?

A
  1. ELISA - one of the sensitive techniques for the detection of the presence of Ag or Ab and quantification as well in case of clinical diagnosis of many diseases such as AIDS, EBOLA, etc.
    - Enzymes used for labelling: detects free Ag or Ab
  2. Radioimmunoassay - type of IA in which radioisotopes are used for labeling the Ag or Ab to detect the formation of the Ag-Ab complex
    - Can determine very small quantities of Ag and Ab in the serum
    - Used for the quantification of hormones, drugs, and, viral antigens
  3. Western blotting
    - Protein separation is done by electrophoresis
    - Used to detect proteins
    - Confirmatory test for HIV
  4. Immunofluorescence - type of IA in which fluorescent dyes are used for the visualization of Ag-Ab reactions
    - Detect surface Ag/Ab
    - Fluorescent dyes such are fluorescein isothiocyanate and lissamine rhodamine used
30
Q

What is isotype switching?

A

A process to change the biologic properties of the secreted Ab
- Deletion of intervening DNA sequences occurs, allowing Ab to switch from IgM and IgD to another isotype

31
Q

How does isotype switching happen?

A
  1. Isotype switching results when Ag-stimulated B cells receive a cytokine signal from T helper cells
  2. The V region does not change during isotype switching therefore, the same antigenic specificity is retained
  3. Switching involved the deletion of intervening DNA between specific recombination sites called switch regions
  4. Because intervening DNA is lose, the B cell cannot “switch back” to an isotype that has already been deleted
  5. The V region and C regions are transcribed together, and RNA splicing and translation results in expression of the new isotype
32
Q

What is affinity maturation?

A

A differentiation process to change the biologic properties of the secreted Ab in which germline DNA is subject to mutational change, allowing coding for Ab with increased affinity for binding to Ag

33
Q

How does affinity maturation happen?

A
  1. This process is a result of somatic hypermutation in which V regions of the Ab heavy and light chain genes undergo more than 10000x higher rate of mutation that “regular” DNA
  2. Some of these mutations increase the affinity of Ab for Ag, and those B cells expressing Ab with higher affinity will be selectively stimulated, increasing the proportion of high-affinity Ab in secondary responses
34
Q

What are the applications of Ag-Ab interactions?

A
  1. Blood typing
  2. Rapid diagnosis test kits (ex. pregnancy, malaria)
  3. Quantification of drugs, hormones
  4. Presence/absences of protein in serum
  5. Study characteristics of different immunodeficiency diseases
  6. Confirmatory tests (ex. Western blotting for HIV)
  7. Serological ascertainment of exposure to infections agents

PATH 415 (10 decks)

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