ANTIBODY STRUCTURE and FUNCTION |

 also called antibody, that is produced by plasma cells with specificity to antigens
 composed of 82-96% polypeptide and 2-14% carbohydrate
- each immunoglobulins are made up of a number of regions called domains which are
approximately made up of 110 amino acids each

 appears primarily in the gamma band when subjected to electrophoresis at pH 8.6

 play an essential role in antigen recognition and biological activities such as opsonization and
complement activation
- one of immunoglobulins is highly associated with RBC sensitization
- Immunoglobulin classes’ names and classification is based on their Heavy Chains

o IgG has a  H chain

o IgM has a  chain
o IgA has a  chain
o has 4 basic tetrapeptide chain
o IgD has a  chain
 Fab region – Antigen-Binding Fragment
o IgE has  chain
 Fc region – Crystallizable Fragment
 Complement Fixation
 Opsonization

o Variable Region – 2 variable regions

 specific to that immunoglobulin – depends on antigen-presented
 varies from one immunoglobulin to another
 mixture of one region of both heavy chain and light chain
 molecular rearrangements depending on the antigens and some molecular level

o Constant Region – differs in different immunoglobulin classes

 Constant region of the heavy chain is unique to each class and it will give the immunoclasses their
names
 the name of the immunoglobulin is dependent on the heavy chain

o noncovalent bonds and disulfide bonds hold both chains together

- the number of disulfide bonds will give the different subclasses for each of the immunoglobulins

Difference Between Light Chain and Heavy Chain

- differs in the amount of amino acids present
- Light Chain – only few amino acids
- Heavy Chain – has more amino acids

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Gerald Edelman – Use of Analytical Centrifuge
 He found that intact IgG molecules had a sedimentation coefficient of 7S – Svedberg Unit
 Used 7M urea to obtain a purified preparation of IgG to unfold the molecule
 Once unfolded, the exposed sulfhydryl bonds could be cleaved by a reducing agent such as
mercaptoethanol
- he focused on IgG

 After such treatment, the material was subjected again to ultracentrifugation, and two separate
fractions (3.5 S and 2.2 S) were obtained
- 3.5 S – 50,000 Da  Heavy Chain
- 2.2 S – 22,000 Da  Light Chain

- He was the one who proposed the structure of immunoglobulins of having the heavy chain and light
chain
- He proposed that there are 2 pieces of each fraction, hence each immunoglobulin has this formula

2H = 2L

Rodney Porter – Cleavage with Papain

 Papain was used to cleave IgG into 3 pieces of about equal size, each having a sedimentation coefficient
of 3.5 S

 Carboxymethyl cellulose ion exchange chromatography separated the material into two types of
fragments
1. Fc fragment (Fragment Crystallizable) – spontaneously crystallized at 4C and had no The study of Light chains is discovered because of and based of the discovery of the Benz-Jones
antigen-binding ability and is now known to represent to carboxy-terminal halves of 2 H Proteins which is associated with a condition called Multiple Myeloma. This is having a high amount
Chain of protein in the urine.
- important in the effector functions of immunoglobulin molecules that includes
opsonization and complement fixation Benz-Jones Protein characteristic:
o When heated @ 60C – precipitates
2. Fab fragment (Fragment Antigen-Binding) – have antigen binding
o When heated at a higher temperature of 80C – re-
capacity and consists of 1 L Chain and ½ of an H chain held together
dissolve
by disulfide bonding
o occurs mostly in the Light chain. Hence the study of
light chain
Alfred Nisonoff – Pepsin Digestion
 Pepsin cleave IgG at the carboxy-terminal side of the interchain disulfide bonds, yielding 1 single Two main types of L chains
fragment with a molecular weight of 100,000 Da and the 𝐹(𝑎𝑏)2  kappa () and lambda ()
- this supports Porter’s work o Each contained between 200 and 220 amino acids
- he designated the 𝐹(𝑎𝑏)2    200 amino acids
- Fc fragment – disintegrated into smaller pieces thus, the 4-chain unit of immunoglobulin    220 amino acids
was obtained
- The 4 Basic Tetrapeptide Chain is because of Nisonoff o Constant region: same amino acid sequence from
position number 111
 The Fc fragment disntigrated into smaller pieces thus the four-chain unit of immunoglobulin was o Variable Region: known as the amino-terminal end
obtained, which indicate that each L chain was bonded to an H chain by means of S-S bond, and
o All kappa and lambda light chains have an almost identical carboxy-terminal end
the H chains were joined to each other by one or more S-S bonds
o Both kappa and lambda light chains are found in all five classes of immunoglobulins, but only one
type is present in a given sample
- not both kappa and lambda is present in each type of the molecules, it’s either kappa
The combined work of the 3 scientists provided us the basic structure of Immunoglobulins. It is a 4-chained
units and made up of 2L chains and 2H chains. or lambda
- IgG has activity against IgM and IgA because of the kappa and lambda light chain

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 ANTIBODY STRUCTURE and FUNCTION |
 only IgG, IgA, and IgD has this; this is where the cleaving of immunoglobulins
happen
 The segment of H chain located the 𝑪𝑯 𝟏 and 𝑪𝑯 𝟐
 Has a high content of proline, which allows for flexibility and hydrophobic
residues
- flexibility of the hinge region allows both Fab regions to operate independently. Both can attach
to antigens

 The flexibility assists in effector functions such as initiation of the complement cascade
 ,  and  chains all have a hinge region, but  and  chains do not
-  and   no hinge region but 𝑪𝑯 𝟐 are paired in a way that they confer to flexibility

 All immunoglobulin contain a carbohydrate portion, which is localized between 𝐶𝐻 2 domains of the
two chains

Functions of carbohydrate include:

1. Increasing the solubility of immunoglobulin
2. Providing protection against degradation
 The first approximately 110 amino acids at the amino-terminal end constitute the variable domain.
- carbohydrates do not easily degrade especially in bacterias
Constant region is designated as 𝑪𝑯 𝟏, 𝑪𝑯 𝟐 and 𝑪𝑯 𝟑
3. Enhancing functional activity of the Fc domains
- most important because the glycosylation is critical in the Fc receptors found on the
phagocytic cells
 Isotypes
o unique amino acid sequence common to all immunoglobulin classes but is unique for that
immunoglobulin molecule in a given class and in a given human specie
- all our IgG has common amino acids for us human and there are different but IgG
common IgG for animals as well  known as the secondary response antibody
 Comprises approximately 75-80% of the total serum immunoglobulins
 IgG has a  H chain - this is why researches is based on IgG
 IgM has a  H chain
 IgA has a  H chain  Half-life: 23-45 days
 IgD has a  H chain  Subclasses: 𝐼𝑔𝐺1 (67%), 𝐼𝑔𝐺2 (22%), 𝐼𝑔𝐺3 (7%), and
 IgE has  H chain 𝐼𝑔𝐺4 (4%)
- these subclasses mainly differ in the number
and position of the disulfide bonds in the
 Allotypes gamma chains
o which contain minor variations of these sequences, may be present in some Individuals
o occurs in 4 of the IgG molecules, 2 in IgA molecules  𝐼𝑔𝐺3 has the largest hinge region largest number of interchain disulfide bonds followed by 𝐼𝑔𝐺1
o occurs also in the  light chain - 𝐼𝑔𝐺3 has larger hinge region – most efficient at binding complements
o These genetic markers are found in the constant region - That is why anti-IgG is mostly against 𝐼𝑔𝐺1 and 𝐼𝑔𝐺3 because these are more efficient in binding
- Examples of allotypes are variations of the  known as 𝑮𝟏 𝑴𝟑 and 𝑮𝟏 𝑴𝟏𝟕 complements compared to the other sublasses

Major functions:
 The variable portions of each chain are unique to the specific antibody molecule, and they constitute 1. Providing immunity for the newborn
the idiotype of the molecule 2. Fixing complement - 𝐼𝑔𝐺1 & 𝐼𝑔𝐺3
- idiotype of the molecule of amino-terminal ends of both Light chain and Heavy Chain 3. Coating antigen for enhanced phagocytosis - opsonization
constitutes the antigen-binding site; essential for the formation of FAB 4. Neutralizing toxins and viruses
- together, the whole structure forms the antigen-recognition unit 5. Participating in agglutination and precipitation reactions

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 ANTIBODY STRUCTURE and FUNCTION |
 all IgG subclasses is capable of crossing the placenta except for 𝑰𝒈𝑮𝟐
 IgG is most efficient in precipitation compared to agglutination  The high valency of IgM antibodies contravenes the fact that they tend to have low affinity for antigen
- precipitation involves smaller substances than agglutination that is why in agglutination, IgM is  IgM is found mainly in the intravascular pool and not in other body fluids or tissues
most efficient because IgM can bring RBCs closer - in intravascular pool - because it is bigger
- AHG Testing  if RBCs are sensitized by IgG, it will only precipitate but no agglutination will
happen, thus it is not visible to us  IgM is known as the primary response antibody
- with the use of AHG reagent, there will be hemagglutination, we can now observe. Then, we can - during primary response and secondary response of
tell if there’s RBC sensitization or not immunity, there is a higher production of antibodies in the
secondary response.
 Macrophages, monocytes, and neutrophils have receptors on their surfaces that are specific for the Fc - Hence, it is IgM that is mostly produced during first exposure
region of IgG - IgM  past infection
- this enhances the contact between the antigen and phagocytic cells and generally increases the - IgG  current infection
efficiency of phagocytosis - if both are present in test kits, reinfection
- 𝐼𝑔𝐺1 and 𝐼𝑔𝐺3 are good at initiating phagocytosis since it has a larger hinge region, it is more
flexible. They also bind strongly at the Fc receptor

Why is IgM in primary response?

 IgG has high diffusion coefficient that allows it to enter extravascular spaces more readily than
other immunoglobulin types
- The  chain is made first in the Pro-B Cell stage. It is the
- thus the amount of IgG intravascularly and extravascularly are almost the same
same in cytoplasmic immunoglobulins and  chain is
always available.
- this is why it more efficient at neutralizing the toxins and viruses

 IgG is better at precipitation reactions than agglutination

 It is synthesized only as long as antigen remains present, because there are no memory cells for IgM
- without antigen-stimulation  IgM is absent because there are no memory cells

IgM
 Known as macroglobulin, because it has a sedimentation rate of 19 S, which represents a molecular
weight approximately 970,000
 Half-life: 10 days and accounts for between 5-10% of all serum immunoglobulin
 If treated with mercaptoethanol, it dissociates into five 7S units, each having a molecular weight of
190,000 and a 4-chain structure that resembles IgG
 The pentamer form is found in secretions, while the monomer form occurs on the surface of B
cells
- IgM appears in two structure: pentamer and monomer form
- monomer – surface of B cell
 starts in the pre-B cell stage
 the rearrangements of the  chain happens in the pro-B
cell then it appears in the pre-B cell stage up to the
mature B cell stage
 J or Joining Chain – holds together the five monomeric units
o glycoprotein with several cysteine residues
o serves as linkage points for disulfide bonds between two adjacent
monomers
o Linkage occurs at the carboxy-terminal end of two of the  chains
o plays a role in the initiation of polymerization by stabilizing the Fc-sulfhydryl groups so that the
cross-linking can occur
Functions include:
1. Complement fixation
- most efficient among Immunoglobulins at triggering the classical
complement pathway because a single molecule of IgM can
initiate the reaction and has multiple binding sites
2. Agglutination
- has larger binding sites, thus making it more efficient
3. Opsonization
4. Toxin neutralization
 Because IgM has a J chain, it can occasionally acquire a secretory component like IgA does, which
allows it to traverse epithelial cells and patrol mucous membranes
- facilitates secretion at the mucosal surfaces, that is why the pentamer structure is in the
secretions because of its J chain
 Also serves as antigen receptors for antigen
 The presence of membrane IgM classifies lymphocytes as mature B cells and also IgD

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 ANTIBODY STRUCTURE and FUNCTION |
- it will have an easier transport once the IgA has the secretory component
IgA o Makes the dimer more resistant to enzymatic digestion by masking sites that would be
 synthesized more compared to IgG but its location is more in the secretions but IgG is found susceptible to protease cleavage
intravascularly - we would know what subclass of IgA has a secretory component = 𝑰𝒈𝑨𝟐
 Half-life: 4-7 days
 Represents 10-15% of all circulating immunoglobulin, and appears as a monomer with a molecular Function of IgA
weight of approximately 160,000 1. Patrol mucosal surfaces and act as a first line of defense
 Has a sedimentation coefficient of 7S and migrates between the  and  regions on electrophoresis 2. Plays an important role in neutralizing toxins produced by microorganisms
 Subclasses 𝐼𝑔𝐴1 and 𝐼𝑔𝐴2 3. Prevent bacterial adherence to mucosal surfaces
o differ in content by 22 amino acids, 13 of which are located in the
hinge region and are deleted in 𝐼𝑔𝐴2  Not capable of fixing complement by the classical pathway
 Neutrophils, monocytes and macrophages possess specific receptors for IgA
 𝑰𝒈𝑨𝟏
o has 13 amino acids at the hinge region The actions of IgA are the basis for Oral Vaccination. Through the activity of the IgA it makes oral
o found in the serum vaccines more reactive since it is found in secretions.

 𝑰𝒈𝑨𝟐
o found as a dimer along the respiratory, urogenital and intestinal IgD
mucosa and it also appears in milk, saliva, tears and sweat
 Representing less than 0.001% of the total immunoglobulins
o no amino acid at the hinge region
 Half-life: 2-3 days
o this is an advantage because it will make 𝐼𝑔𝐴2 more resistant to the
 The  H chain has a molecular weight of 62,000
degradation of some bacterial proteinases which cleaves 𝐼𝑔𝐴1
 Appears to have an extended hinge region consisting of 58 amino acids
o this is why 𝐼𝑔𝐴2 is more predominant in the mucosal surfaces compared to 𝐼𝑔𝐴1
 Present on the surface of immunocompetent but unstimulated B
lymphocytes
Remember that:
 It has a high level of surface expression and its intrinsic flexibility make it
Mucosal surfaces are part of the first line of
an ideal early responder to an antigen
defense in the Innate Immune System. 𝐼𝑔𝐴2
- since it has a bigger hinge region, it is more flexible than the others
is present in the mucosal surfaces because it
 May play a role in regulating B-cell maturation and differentiation
is more predominant and resistant to the
 More susceptible to proteolysis than other immunoglobulins
cleaving by bacterial proteinases

IgE
Plasma cell that are dedicated to produce IgA have a higher tendency to be associated with and tend to
seek or homing the Subendothelial Cells since the secretory component is found here, like the epithelial
cells. Which is why the plasma cells producing IgA is near the epithelial cell so that they can easily acquire
the secretory component.

 Formation of secretory IgA: Secretory component

 Hypersensitivity reactions and Allergic Reactions
o A receptor that binds IgA and exits the cell along with it
 Accounting for only 0.0005% of total serum immunoglobulins
Endothelial cell has poly-Ig Receptor.
 Is the most heat-labile of all immunoglobulins; heating 56 C for between 30 min and 3 hrs results in
1. Through the process of endocytosis, this IgA will
conformational changes and loss of ability to bind to target cells
bind to the secretory component.
 Attaches to basophils and tissue mast cells by means of specific surface proteins,
2. Through the process of transcytosis, the secretory
component will attach to IgA that has exited the termed high-affinity Fc  R1 receptors. The molecule binds at the 𝐶𝐻3domain
plasma cell. on the Fc region
3. The IgA that has exited the endothelial cell will now  Plasma cells that produce IgE are located primarily in the lung and in the skin
acquire the secretory component. - since IgE is associated with hypersensitivity and allergic reactions, it can react by rashes in the
o Facilitate transport of IgA to mucosal surfaces skin and difficulty in breathing in the lungs
 Serve a protective role by triggering an Acute Inflammatory Reaction

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 ANTIBODY STRUCTURE and FUNCTION |
 Tonegawa discovered that chromosomes contain no contact immunoglobulin genes, only building
blocks from which genes can be assembled
- thus the immunoglobulin and its diversity

 Human immunoglobulin genes are found in 3 unlinked clusters

o H genes – chromosome 14
o  chain genes – chromosome 2
o  chain genes – chromosome 22
 A selection occurs
- the chromosome does not contain intact gene for the coding of immunoglobulins. Only
building blocks
- the DNA will be the one guiding the selection process

Rearrangement of Heavy Chain Genes

 All H chains are derived from a single region on chromosome 14

 The genes that code for the variable region are divided into 3 groups
 Neutralization – they will mask dangerous parts of bacterial exotoxins or viruses thus it cannot bind o 𝑽𝑯 (39)
o D (23)
When will H chain rearrangement occur?
to binding sites therefore they cannot exhibit clinical signs and symptoms and cannot infect
- Pro-B Cell
 Agglutination – IgM is most effective o J (6)
 Precipitation – IgG is most effective
 Complement Fixation – IgM and IgG  There is a set of genes (Constant region) that codes for the constant region which includes one gene
for each H chain isotype they are in this order:
1. C
2. C
Cloninal Selection 3. C3
 1950s by Niels Jerne and Macfarlane Burnet
4. C1
 Key premise: Individual lymphocytes are genetically preprogrammed to produce one type of
5. C1
immunoglobulin and that a specific antigen finds or selects those particular cells capable of
6. C2
responding to it, causing them to proliferate
7. C4
 Main drawback: consideration of the genetic basis for the diversity antibody molecules
8. C
 Dryer and Bennet proposed that the constant and variable portions of immunoglobulin chains are
9. C2
actually coded for by separate genes

All immunoglobulin classes are present here.

Ehrlich’s Side-Chain Theory
Remember that: The heavy chain gives the name of the different immunoglobulin
 1900s by Paul Ehrlich
 certain cells had specific surface receptors for antigen that were present before contact with antigen
The constant region includes one gene of each of the type of H chain. Therefore, IgM is considered as
occurred
the primary response antibody since it is the first one that is rearranged
 Once antigen was introduced, it would select the cell with the proper receptors, combination would
take place, and then receptors would break off and enter the circulation as antibody molecules
Once antigen was introduced, it would select the cell with the proper receptors, combination
would take place, and then receptors would break off and enter the circulation as antibody
 For synthesis of the entire H chain, a choice is made from each of the selections so as to include
 Key premises:
o 1 𝑉𝐻 gene
1. Lock-and-key concept of the fit of antibody for antigen
o 1 D gene
2. Antigen selected cells with the built-in capacity to respond to it
o 1 Constant region
 this theory did not explain the kinetics of the immune response or the idea of immunologic memory,
it laid the foundation for further hypotheses

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 ANTIBODY STRUCTURE and FUNCTION |
H Chain Gene Rearrangement L Chain Gene Rearrangement

 Occurs only after  chains appear

 L chains exhibit a similar genetic rearrangement, except they lack a D region
- only V gene and J gene
 Recombination of segments on chromosome 2 coding for  chains, occurs prior that on chromosome
22, which codes for  chains
 The process of VJ joining is accomplished by an excision of intervening DNA resulting for the 𝑉𝐾 and 𝐽𝐾
segments to be permanently joined to another
- same process from H gene rearrangement but only V gene and J gene

 Joining of the segments (variable region) occurs in two steps:

1. At the DNA level, one D and J randomly chosen and are joined with deletion of the intervening
DNA
2. A V gene is joined to the DJ Complex, resulting in a rearranged V(D)J gene

The selection process is not specific, thus giving us more heterospecific immunoglobulins.
 A productive rearrangement of the genes with subsequent protein production keeps the other
chromosome 2 from rearranging, and it shuts down any recombination of the –chain locus on
chromosome 22
 Alleic Exclusion: if a successful rearrangement of DNA on one chromosome 14 occurs, then the
 L chains are then joined with  chains to form a complete IgM antibody
genes on the second chromosome are not rearrange
-  chain rearranged from the H gene rearrangement since it is always the Heavy chain that is
 The variable and constant regions are joined at the ribonucleic acid level, the conserving DNA of
rearranged first
the constant regions and allowing for class switching

That is why there are different variable regions produced by plasma cell. Because they will undergo
class switching. For example, in one of the set of the constant region, will be selected if the  chain will
be selected, then it will give us the  heavy chain = IgM

RAG-1 and RAG-2  associated in gene rearrangement in the Pro-B Cell Stage

At a DNA level, the same process will happen except in alleic exclusion. 1 D and 1 J will be chosen, then
the V gene will join. After a successful DNA rearrangement, the variable and constant region will join at
the ribonucleic acid level during the transcription phase.

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 ANTIBODY STRUCTURE and FUNCTION |
1. Two immunoglobulin classes that can fix the complement or that can facilitate complement
fixation
- IgM and IgG
2. The half-life of IgG
- 23-25 days
3. What structure type of IgM is found in the serum?
- Pentamer
4. It is known as the secondary response antibody
- IgG
5. Give two functions of the Fc region
- Opsonization and complement fixation
6. His work led to the discovery of Fc fragment and Fab fragment
- Rodney Porter
7. Which IgG subclass is incapable of giving protection to newborns?
- 𝑰𝒈𝑮𝟐
8. Which IgA subclass is resistant to bacterial proteases?
- 𝑰𝒈𝑨𝟐
9. Plasma cells that produce IgE are located primarily in what organs?
- Lungs and Skin
10. This immunoglobulin class plays a role in regulating B cell regulation and differentiation
- IgD
11. What are the three groups of genes that code for the variable region?
- 𝑽𝑯 Gene, D gene, J Gene

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