Bloque 2: Antibodies

Question 1

Antibody definition

Answer 1

An antibody is a protein with the ability to bind and recognise an antigen. Antibodies can be either present in membrane receptors of B cells or secreted by plasma cells, which are differentiated B cells.

Question 2

General features of antibodies

Answer 2

An antibody is a protein with the ability to bind and recognise an antigen.

Antibodies can be either present in membrane receptors of B cells or secreted by plasma cells, which are differentiated B cells.

All antibodies share the same structural features, bind to antigens and participate in a limited number of effector functions.

Antibodies have the broadest antigen recognition spectrum, meaning that almost any kind of molecule can be recognised by antibodies.

Antibodies also have discriminatory specificity and the highest affinity.

Question 3

Basic structure of antibodies

Answer 3

Basic structure: Two identical light chains (kappa or lambda), made by a variable domain and a constant domain, and two identical heavy chains* with three or four constant domains.

*5 different isotypes according to the heavy chain: alpha, delta, epsilon, mu and gamma

Constant regions participate in effector functions of antibodies.

All antibodies share the same basic structure. However, they all have a different terminal region, the antigen recognition area, specific for each antigen.

Question 4

Immunoglobulins and main function

Answer 4

IgA: Mucosal immunity
IgD: Antigen receptor of young B lymphocytes
IgE: Defense against helminthic parasites and immediate hypersensitivity
IgG: Opsonisation, complement activation, antibody-dependent cell, neonatal immunity and feedback inhibition of B cells
IgM: B cell antigen receptor (monomeric form) and complement activation. It is part of the primary response

Question 5

IgA function

Answer 5

Mucosal immnity

Question 6

IgD function

Answer 6

Antigen receptor of young B lymphocytes

Question 7

IgE function

Answer 7

Defense against helminthic parasites and immediate hypersensitivity

Question 8

IgG function

Answer 8

Opsonisation
Complement activation
Antibody-dependent cell-mediated cytotoxicity
Neonatal immunity
Feedback: Inhibition of B cells

Question 9

IgM function

Answer 9

B cell antigen receptor (monomeric form)
Complement activation
Primary immune response

Question 10

IgG structure according to enzymatic digestion

Answer 10

Digestion with papain:

• 2 fragment antigen binding regions (Fab), which meant that IgG ability to bind to antigens was bivalent
• 1 fragment of crystallization (FC)

Digestion with pepsin:

• Only 1 F(ab’) region
• No FC

Question 11

Idiotype definition

Answer 11

It’s a set of antigen binding sites characteristic of an antibody and produced by a particular clone of antibody-producing cells. In conclusion, it is the specific structure of an antibody.

Question 12

Hinge region definition

Answer 12

The hinge region is a flexible amino acid stretch in the central part of the heavy chains of IgG and IgA, which link their two chains by disulphide bonds.
The hinge region is rich in Cysteine (favors heavy chain dimerisation through interchain disulphide bonds) and proline (provides flexibility) amino acids and has no resemblance to any other immunoglobulin region.
It is located between CH1 and CH2.

Question 13

Antiserum definition

Answer 13

Blood serum containing antibodies against specific antigens

Question 14

Regulatory network definition

Answer 14

Collection of regulatory molecules that interact with each other and with other substances in the cell to control the expression of mRNA and proteins

Question 15

Structure of different secreted immunoglobulins

Answer 15

Monomers:

• IgG: Gamma heavy chain (4 different subtypes)
• IgD: Delta heavy chain
• IgE: Epsilon heavy chain

Dimer:
- IgA: Alpha heavy chain (2 different subtypes)

Pentamer:
- IgM: Mu heavy chain

Question 16

Major functions of antibodies and immunoglobulins involved in each one

Answer 16

Opsonisation (IgG): Promotion of phagocytosis of antigens by macrophages. Fc receptors present on the surface of macrophages and neutrophils, binds to the constant region of most subclases of IgG

Activation of the complement (IgG and IgM): Serum of glycoproteins used in order to deal with pathogens that are inside cells.

Antibody-dependent cell-mediated cytotoxicity (IgG): Used in order to kill intracellular pathogens by the linking of an antibody bound to the infected cell and a natural killer cell.

Transcytosis (IgM, IgG and IgA): Breastfeeding, crossing of the placenta…

Activation of mast cells, eosinophils and basophils (IgE)

Question 17

IgG functions according to their subtype

Answer 17

IgG1 and IgG2 neonatal immunity (are able to cross the placenta)

Capacity to activate the complement: 3>1>2>4

ADCC: IgG1 and IgG3

Neutralisation of toxins, bacteria and virus (all of them)

Opsonisation: IgG1 and IgG3

Question 18

Applications of antibodies

Answer 18

Natural constituents of immunity

Diagnostic tools

Laboratory reagents

Therapy: Neutralisation, depletion, agonism or antagonism and vehicles for drugs (specificity allows them to target some cells)

Question 19

Structure of Ig genes

Answer 19

B cells use parts of genes to create different possible antibodies by recombination.
The different genes segments or regions are:
- Variable (V)
- Diversity (D)
- Joining (J)
- Constant (C)
The recombination is controlled by a tightly regulated machinery

Question 20

Multigene organisation of light chain and genetic building of this chain

Answer 20

There are 2 possible light chains: lambda (40%) or kappa (60%)

Light chain is formed by a constant and a variable domain put together through joining regions. The first recombinational process is performed between V segment and J segment. As there are 35 V genes and 5 J genes, there are 175 different recombinations. In the case of Kappa chain, there is only one constant region so, after V-J recombination, Ck joins it, DNA is transcribed to mRNA and introns are eliminated. Lambda chain can be built from 7 different C regions, each of which is specific for a V-J domain and only 4 of these recombinations are suitable in human being.

Question 21

Multigene organisation of heavy chain and genetic building of this chain

Answer 21

In the case of the heavy chain, there are 4 different groups of genes that play a role in the building of this chain: Variable region (V), diversity region (D), joining region (J) and constant region (C).

Variable chains can be either CDR1, CDR2 or CDR3. CDR3 is the loop with the highest variability.

It is important to know that each subtype of immunoglobulin has its own constant regions.

In order to obtain the full sequence of the heavy chain, the V region must be built. These region will be formed first by the union of D and J genes, and finally, by putting together V genes with D-J. There are 45 different variable genes avaliable, 23 diversity ones and 6 possible joining genes, so in total there are 6000 possible V regions in the heavy chain.

After the Variable region is constituted, it must join one of the different nine constant regions. This event doesn’t take place until DNA transcription. The constant region chosen is specific for the immunoglobulin type and subtype that is needed.

Question 22

Differences between the building of the heavy and the light chains

Answer 22

Heavy chain variable region is formed by two recombinational events, one between diversity (D) and joining (J) genes and the other between D-J and variable genes (V), while the variable region of the light chain is constituted only by the recombination of V and J.

The constant region of the heavy chain can be: alpha (1 or two), gamma (1, 2, 3 or 4), delta, epsilon or mu, while the constant region of the light chain can be: kappa or lambda (in humans, there are 7 different combinations between lambda subtypes and V-J, from which only 4 are viable).

Question 23

V(D)J recombination

Answer 23

This recombination process is highly regulated. Many of the enzymes acting on this process, are also involved in DNA repairing functions.
This process is directed by recombination signal sequences.
Each region has a conserved nonamer and an heptamer sequence. Between a nonamer and an heptamer, lies either a 12 or 23 base pair spacer sequence. Recombination always takes place between the sequence with 12 pair bases and the one with 23. This ensures that, for example, if V sequence has 23 pair bases, J region also 23 and D region has 12, the recombination will always be D-J and then V-D-J. This is known by the 12-23 rule.

The most common thing is that the process ends here. However, there are some possible additional events:

• An exonuclease may eliminate some nucleotides located in the area, which may create a new Aa.
• P nucleotide are added after an asymmetric cut is produced.
• N nucleotides are added after a symmetric cut is produced.

Question 24

Mechanism to generate antibody diversity in naïve B cells

Answer 24

There are several mechanisms to provide antibody diversity to naïve B cells:

• COMBINATIONAL DIVERSITY, which is related to the combination of different heavy chains with different light chains.
• MULTIPLE GENE SEGMENT, which refers to the gene segments that are put together within a single chain
• P NUCLEOTIDE ADDITION, which is the addition of a nucleotide after an asymmetric cut is produced in the chain
• N NUCLEOTIDE ADDITION, which is the addition of a nucleotide after a symmetric cut is produced in the chain
• EXONUCLEASE TRIMMING, that occurs sometimes at junctions, where nucleotides are lost and the reading frame changes.

Question 25

Allelic exclusion in antibody production

Answer 25

Before talking about allelic exclusion, it’s important to know that antibody production is performed by both copies of a chromosome independently.

Allelic exclusion is the process that ensures that each B cell synthesises only one heavy chain and one light chain.

It is important to highlight that nonproductive arrangements lead to apoptosis during development.
Allelic exclusion must be seen in a binary way in order to be easily understood, in a “yes or no” way. If the recombination is correct, antibody production will go on. If the cell is not able to make a productive antibody, the cell will try to produce it again from the second chromosome. If the second chromosome is not able to generate a productive antibody, the cell will undergo apoptosis.

The first chain that will be produced is Kappa. If the first allele can’t produce Kappa chain, the second chromosome will try to make this chain.
If neither of them are able to produce Kappa chain, the first chromosome will try to create the Lambda light chain. Again, if this doesn’t work, the second chromosome will try to produce Lambda light chain. If this step can’t be fulfilled, the cell will die.