Antibodies/Immunoglobulins

antibodies/immunoglobulins

• Specialized proteins circulating through the blood or through a fluid called the lymph.

• They circulate through the blood and carry with them binding sites that attach to harmful invaders collectively known as antigens.

B cells’ surface

Where are antibodies located?

Differentiation

Antigen-induced B lymphocyte or B cell stimulation will result in a?

86% to 98%

Antibodies are glycoproteins that are composed of how many % polypeptide?

2% to 14%

Antibodies are glycoproteins that are composed of how many % carbohydrate?

• Antigen recognition

• Opsonization

• Complement fixation

Functions of Antibody:

Opsonization

function to hasten or enhance phagocytosis

20%

Antibody constitutes approximately how many % of plasma protein?

Heavy Chain Sequencing

• larger chain of the molecule 

• MW: 50,000 – 70,000 D

• Extends the full length

• Constant regions of the H chain are unique to each class and give each immunoglobulin type its name

The Nature of Light Chains

• Smaller chain of the antibody molecule

• MW: 22,000 Daltons

• Found on all types of immunoglobulin molecules but do not extend the full length.

• Only has 1 variable (VL) & 1 constant (CL) region.

Variable Region

• located in the amino terminal end of your antibody molecule.

• defines the specificity of an antibody

• first 110 amino acids

Constant Region

• found on the carboxy terminal end of the antibody.

• Starts from the 111th amino acid onwards for both H & L chains.

Gerald Edelman & Rodney Porter

made use of the IgG antibody for their research because it is the most prevalent in the body.

Gerald Edelman

• Separate out immunoglobulins on the basis of molecular weight using the analytic ultracentrifuge

• Found out that an intact IgG molecule had a sedimentation coefficient of 7s.

Sedimentation coefficient

is used to characterize a behavior of a molecule in sedimentation process, notably, centrifugation

Papain Cleavage

cleaves the IgG or the antibody molecule below the set of disulfide bonds that holds the 2 heavy chains together that would result in the formation of 3 fragments.

Alfred Nisonoff

Used pepsin to cleave IgG at the carboxyterminal side of the interchain disulfide bonds, yielding one single fragment with a molecular weight of 100,000 d and all the antigen-binding ability, known as F(ab′)2 fragment

FC Fragment

• spontaneously crystallizes @ 4°C

• represent the carboxy-terminal (tail) halves of two H chains which are all constant regions 

• No antigen binding capacity

• Responsible for the biological activity of the molecule

Fab Fragment

• Function is antigen binding

• (1) light chain & half of the heavy chain held together by disulfide bonds

• 2 identical fragments each representing one antigen binding site

Hinge region

• The segment of heavy chain located between the CH1 and CH2 regions

• Rich in hydrophobic residues and has high proline content

• Confers flexibility

• Susceptible to proteolysis because of the presence of disulfide bonds

IgG

• Major immunoglobulin in normal human serum comprising approximately 70-75% of the total serum immunoglobulins o Adults: 800 to 1,600 mg/dL

• MW: 150,000 D

• Sedimentation coefficient: 7S

• Has a high diffusion coefficient

• it appears late but persists longer because of its half-life of 23 days

• Provides immunity for the newborn

• Activates the classical pathway of complement / Fixing complement

• Coats antigen for enhanced phagocytosis (opsonization)

• Neutralizes toxins and viruses

• Participates in agglutination and precipitation reactions

• Targets virally infected cells for destruction by Antibody Dependent Cell Mediated Cytotoxicity (ADCC)

Functions of IgG in host immunity:

IgM

• Referred to as macroglobulin

• it is the largest of all immunoglobulin classes 

• MW: 900,000 D

• Sedimentation rate: 19 S

• 3rd most abundant immunoglobulin which accounts for 5-10% of the immunoglobulin pool o 120-150 mg/dl

• Considered as primary response antibody

• Is usually being detected during acute or current infections

IgA

• The predominant immunoglobulin in secretions and functions to protect your mucosal surfaces

• Is usually found in tears, saliva, milk, colostrum & intestinal fluids

• 2nd most abundant immunoglobulin accounting 10-15% of the circulating immunoglobulin pool o 70 to 350 mg/dl

• MW: approximately 160,000 D

• Sedimentation coefficient: 7S

IgA1 / Monomeric IgA

• Found mainly in the serum with a MW of 160,000 D

• Exists as a single immunoglobulin molecule

• Responsible for antigenic clearance & immune regulation

• Acts as an anti-inflammatory agent

• DOWNREGULATE phagocytosis, chemotaxis, bactericidal activity and cytokine release

IgA2 / Dimeric IgA / Secretory IgA

• Predominant form in secretions (found in mucosal surfaces)

• 2 monomers joined together by the joining chain/J Chain in the middle

• Also appears in breast milk, colostrum, saliva, tears and sweat

• Has a secretory component (70,000 D) in the middle which makes the dimer more resistant in proteolytic digestion (It is more resistant to bacterial proteinases that are able to cleave IgA1)

• Synthesized by plasma cells at a much greater rate than IgG, found mainly in mucosal-associated lymphoid tissue

• Patrol mucosal surface and act as first line of defense

• Neutralizes toxins

• Prevents bacterial and viral adherence to mucosal surfaces

• Aggregation of immune complexes caused by antigen antibody reactions of IgA may trigger the ALTERNATE complement pathway.

Function of IgA

IgD

• First discovered in 1965, in a patient with multiple myeloma

• Constitutes less than 0.001% of the total immunoglobulins (immunoglobulin pool)

• Half-life: 1 to 3 days

• MW: 180,000 D

IgE

• Least abundant in the immunoglobulin pool – accounts for only 0.0005% of total serum immunoglobulins

• Has a MW of 190,000 D

• Sedimentation coefficient: 8S

• Most heat labile/sensitive to heat of all antibodies

Isotypes

• Unique amino acid sequences that are common to all immunoglobulin molecules of a given class or subclass.

• Constant regions of the heavy chains that are unique to each immunoglobulin class and give each type its name.

Allotypes

• Minor variations of amino acids sequences that are present in some individuals of the same species but not others

• Referring to the differences in the constant regions of both the heavy and light chains

• occur in the four IgG subclasses, in one IgA subclass and in the ʎ light chain

Idiotypes

• Variations in the variable region that give each immunoglobulin molecule its specificity 

• Example: hypervariable region of each immunoglobulin class

• Contained in the amino-terminal ends of both L and H chains

Primary antibody response

• Occurs when an antigen comes in contact with the immune system for the first time. The body does not recognize the antigen yet

• Antibody is IgM

• Lag phase

• Log phase

• Plateau phase

• Decline phase

After an antigenic stimulation (a foreign antigen challenge), an antibody response proceeds in four phases:

Lag Phase

No antibody is detectable yet because the body is still learning how to recognize the antigen

Log Phase

• There is an increase in your antibody titer

• Antibody has a logarithmic increase

Plateau Phase

The antibody titer is stable

Decline Phase

The antibody is catabolized, and the titer starts to decrease

Antibody titer

• A measurement of how much antibody an organism has produced that is able to recognize a particular epitope

• In the laboratory, it is expressed as the inverse of the greatest dilution that shows a positive result

Secondary antibody (Anamnestic) response

• the immune system can eliminate the antigen which has been encountered by the individual during primary response more rapidly and efficiently

• This phase is already the subsequent exposure to the same antigenic stimulus

• Antibody is IgG

• Clones of memory cells are stimulated to proliferate

Affinity

• The measure of the binding strength of a single interaction. antibody-antigen

Avidity

The measure of the total binding strength / the strength of all interactions combined.

Hydrophobic Bonds

When antigen and antibody molecules come together, these sidechains interact and exclude water molecules from the area of interaction.

Hydrogen Bonds

Formation of Hydrogen bridges between two electronegative atoms

Electrostatic Forces

Results from the attraction of oppositely charged amino acids located on the side chains of two amino acid residues

Van der Waals Forces

Nonspecific, attractive forces generated by the interaction between electron clouds and hydrophobic bonds

Monoclonal antibody

Defined as identical antibodies that are produced from a single clone of plasma cells

Hybridomas

Referring to the fusion of two different types of cells:

• Myeloma cells

• Activated B-cell