ANTIBODIES

Chalee R. Sienes-Reyes, RMT, MSMT Professor, Med. Lab. Sci. Dept., San Pedro College, Davao City

Outline
Antibody Production Basic Structure of Immunoglobulin and Function Polyclonal and Monoclonal Antibody Response Events in the Germline Center Effector Mechanisms of Humoral Immune Response

Affinity
Strength of the reaction between a single antigenic determinant and a single Ab combining site High Affinity Ab Low Affinity Ab

Ag

Ag

Affinity = attractive and repulsive forces

Avidity
The overall strength of binding between an Ag with many determinants and multivalent Abs

Keq =

104 Affinity

106 Avidity

1010 Avidity

Affinity and avidity

Ab1

Ab1

Immunologic Memory
Virgin lymphocyte pool

PRIMARY RESPONSE

effector cells

memory cell pool SECONDARY RESPONSE

effector cells

memory cell pool

Immunologically Naive
No previous experience No memory Must be educated

The Primary Immune Response

antibody titer
lag log phase plateau decline

antigen challenge

time

Class Switching

antibdy titer

IgM

IgG

time

Four phases of the primary response

a lag phase where no antibody is detected a log phase in which the antibody titer rises logarithmically a plateau phase during which the antibody titer stabilizes a phase (decline) during which the antibody is cleared or catabilized

Dynamics of Antibody Production

Comparison of Primary and Secondary Responses

Time course Antibody titer Antibody class Antibody affinity

Terms to remember

Opsonization Neutralization Complement activation Primary response Secondary/ anamnestic/ booster response Lag phase Log phase Plateau period Memory cells Class switching Affinity maturation/ change in affinity with time

Cellular Events
Antigen is processed by T lymphocytes and macrophages. Possess special receptors on surface. Termed antigen presenter cell APC. Antigen presented to B cell

Basic Antibody Structure

Two identical heavy chains
Gamma Delta Alpha Mu Epsilon

Two identical light chains

Kappa Lambda

Basic Antibody Structure

Basic Structure of Immunoglobulins

Papain Cleavage
Breaks disulfide bonds at hinge region Results in 2 fragment antigen binding (Fab) fragments. Contains variable region of antibody molecule Variable region is part of antibody molecule which binds to antigen.

Papain Cleavage

Pepsin
Breaks antibody above disulfide bond. Two F(ab )2 molecules The rest fragments Has the ability to bind with antigen and cause agglutination or precipitation

Papain and Pepsin Cleavage

IgG
Most abundant Single structural unit Gamma heavy chains Found intravascularly AND extravascularly Coats organisms to enhance phagocytosis (opsonization) Crosses placenta provides baby with immunity for first few weeks of infant s life. Capable of binding complement which will result in cell lysis FOUR subclasses IgG1, IgG2, IgG3 and IgG4

IgA
Alpha heavy chains Found in secretions Produced by lymphoid tissue Important role in respiratory, urinary and bowel infections. 15-10% of Ig pool Does NOT cross the placenta. Does NOT bind complement. Present in LARGE quantities in breast milk which transfers across gut of infant.

Secretory IgA
Exists as TWO basic structural units, a DIMER Produced by cells lining the mucous membranes.

IgM
Mu heavy chains Largest of all Ig PENTAMER 10% of Ig pool Due to large size restricted to intravascular space. FIXES COMPLEMENT. Does NOT cross placenta. Of greatest importance in primary immune response.

IgE
Epsilon heavy chains Trace plasma protein Single structural unit Fc region binds strongly to mast cells. Mediates release of histamines and heparin>allergic reactions Increased in allergies and parasitic infections. Does NOT fix complement Does NOT cross the placenta

IgD
Delta heavy chains. Single structural unit. Accounts for less than 1% of Ig pool. Primarily a cell bound Ig found on the surface of B lymphocytes. Despite studies extending for more than 4 decades, a specific role for serum IgD has not been defined while for IgD bound to the membrane of many B lymphocytes, several functions have been proposed. Does NOT cross the placenta. Does NOT fix complement.

 Polyclonal antibody
Antigens possess multiple epitopes Serum antibodies are heterogeneous,
To increase immune protection in vivo To reduces the efficacy of antiserum for various in vitro uses

To response facilitates the localization, phagocytosis, and complement-mediated lysis of antigen To have clear advantages for the organism in vivo

 Monoclonal antibody
Derived from a single clone, specific for a single epitope For most research, diagnostic, and therapeutic purposes

1975, by Georges Khler and Cesar Milstein - Be awarded a Nobel Prize in1984

Formation and Selection of Hybrid Cells

Hybridoma: the B cell X myeloma cell
To be produce by using polyethylene glycol (PEG) to fuse cells The myeloma cells: immortal growth properties The B cells: to contribute the genetic information for synthesis of specific antibody Selected by using HAT medium (hypoxanthine, aminoprotein, and thymidine) Myeloma cells are unable to grow B cells are able to survive, but can not live for extended periods

Two different pathways to synthesis nucleotide in mammalian cells

(Folic acid analog)

Myeloma cells used in hybridoma technology are double mutants, they lack the HGPRTase and lose the ability to produce Ig

(Most common screening techniques are ELISA and RIA)

Low concentration (1-20 ug/ml)

High concentration (1-10 mg/ml)

Human Monoclonal Antibodies

Production of human monoclonal antibody There are numbers of technical difficulties
The lack of human myeloma cells to exhibit immortal growth, be susceptible to HAT selection, to not secrete antibody, and support antibody production in the hybridoma made with them Human B cell sometimes have immortality That is the difficulty of readily obtaining antigenactivated B cells

Human Monoclonal Antibodies

To culture human B cells in vitro to produce human monoclonal antibody

Transplant human cells with immune response into SCID mice (lack a functional immune system)

Clinical Uses for Monoclonal Antibodies

Very useful as diagnostic, imaging, and therapeutic reagents in clinical medicine
Monoclonal antibodies were used primarily as in vitro diagnostic reagents Radiolabeled monoclonal antibodies can also be used in vivo detecting or locating

Immunotoxins
To compose of tumor-specific monoclonal antibodies coupled to lethal toxin Valuable therapeutic reagent

Affinity Maturation and the Generation of Memory B Cells

Germline Organization Of Human Immunoglobulin gene

Germline Organization Of Human Immunoglobulin gene

Genomic organisation of Ig genes
LH1-123 VH 1-123 (No.s include pseudogenes etc.) DH1-27 JH 1-9 CQ

LO1-132 VO1-132

JO 1-5

CO

LP1-105 VP1-105

CP1 JP1

CP2 JP2

CP3 JP3

CP4 JP4

Ig light chain gene rearrangement by somatic recombination

VO Germline JO CO

Rearranged 1 transcript

SplicedmRNA

Ig light chain rearrangement: Rescue pathway

There is only a 1:3 chance of the join between the V and J region being in frame VO JO CO

Non-productive rearrangement

Light chain has a second chance to make a productive join using new V and J elements Spliced mRNA transcript

Ig heavy chain gene rearrangement

VH 1-123

DH1-27

JH 1-9

CQ

Somatic recombination occurs at the level of DNA which can now be transcribed

The constant region has additional, optional exons

CQ Primary transcript RNA AAAAA

Each H chain domain (& the hinge) encoded by separate exons

Secretion coding sequence CQ3

Polyadenylation site (secreted) pAs

Polyadenylation site (membrane) pAm

CQ1

CQ2

CQ4

Membrane coding sequence

Membrane IgM constant region

DNA Transcription
pAm

CQ1

CQ2

CQ3

CQ4

1 transcript

CQ1

CQ2

CQ3

CQ4

AAAAA

Cleavage & polyadenylation at pAm and RNA splicing

CQ1 h CQ2 CQ3 CQ4

mRNA

AAAAA

Protein

Membrane coding sequence encodes transmembrane region that retains IgM in the cell membrane Fc

Secreted IgM constant region

DNA Transcription
pAs

CQ1

CQ2

CQ3

CQ4

1 transcript

CQ1

CQ2

CQ3

CQ4

AAAAA

Cleavage polyadenylation at pAs and RNA splicing mRNA

CQ1 h CQ2 CQ3 CQ4

AAAAA

Protein

Secretion coding sequence encodes the C terminus of soluble, secreted IgM

Fc

The constant region has additional, optional exons

CQ Primary transcript RNA AAAAA

Each H chain domain (& the hinge) encoded by separate exons

Secretion coding sequence CQ3

Polyadenylation site (secreted) pAs

Polyadenylation site (membrane) pAm

CQ1

CQ2

CQ4

Membrane coding sequence

RNA processing
Primary transcript RNA
pAs

D J8

J9

CQ1

CQ2

CQ3

CQ4

AAAAA

mRNA

D J8 CQ1 h CQ2 CQ3 CQ4

AAAAA

The Heavy chain mRNA is completed by splicing the VDJ region to the C region

The H and L chain mRNA are now ready for translation VL VH JL DH JH CL h AAAAA CH AAAAA

Somatic hypermutation

Nature Reviews Immunology 2, 688-698 (2002)

Class Switching
DNA rearrangement
Antigen dependent Switch site Same VDJ TH cytokines

During B cell development, many B cells switch from making one class of antibody to making another, this process is called class switching.

Antibody Class Switching

Class switching in immunoglobulin heavy chain genes

Class Switching Recombination

Class-switch recombination (CSR) of immunoglobulin heavy chains is the genetic process by which a B cell switches from the production of IgM to the production of IgG, IgE or IgA.

Cellular interactions important for IgE class-switch recombination

Effector Mechanisms of Humoral Immune Response

Neutralization
Toxin receptors

Host cell

Bacterial toxins Neutralization by antibody

Forming phagosome Fc receptor

Phagocytosis of antibody-antigen complex by macrophage

Opsonization
Extracellular bacteria Macrophage Opsonization

Ingestion by macrophage

Digestion in lysosome

Complement Activation
Bacteria in plasma

Lysis and ingestion

C1 C2 C4 C1 C2 C4

Complement activation

Digestion in lysosome

IgE