IMMUNOLOGY AND SEROLOGY – LECTURE  V is variable

 Light chain – Fab region

ANTIBODY
 Heavy Chain –
Immunoglobulins  Fab portion – fragment antibody region
 Fc portion – fragment crystallizable region; where
 Glycoproteins found in the serum portion of the coordination with opsonization and complement
blood secretion
 Appear primarily in the gamma band  Bond – disulfide bond
 Humoral Immunity  Hinge region – middle
 Biological activities such as Opsonin and  12 domain
complement activation
 Not all antibodies have gamma electrophoretic FOR MEMBRANE IgM
mobility
 Membrane bound IgM– bound in plasma membrane
of B cell
 Characteristic: Has additional Domain
Functions of Antibodies
 Antigen-binding site
 Mucosal immunity (IgA isotype II in tears, saliva,  Variable region – one for variable light and variable
secretions in serous fluid) heavy; has changes or varieties in receptor to bind to
 B cell antigen receptor (IgD found in the surface of B epitope
cell – indicator of maturing B cell)  Constant region – not changing
 Defense against helminthic parasites, immediate
hypersensitivity (IgE)
 Opsonization, complement activation, antibody
dependent cell mediated cytotoxicity, neonatal
immunity, feedback inhibition of B cells (IgG)  12 domains
 Naïve B cell antigen receptor (monomeric form),  Heavy and Light Chain
complement activation (IgM)

IgM – always first because surface antigen receptor just like

IgD

o H2L2 – 2 heavy chain and 2 light chain

 Carbohydrate
o At CH2, for protection from degredation,
increase solubility
 Residue of amino acid in the domain
 Disulfide bond in Hinge Region
o Proline – allows the flexibility and
hydrophilic residue; tend to open up this
region and thus making it accessible to
proteolytic cleavage
Antibody structure  2 antigenic binding site operate inbetween these two
o Gamma, Delta, and Alpha all have the
IgM – membrane-bound; primary response Hinge Region
 Gamma – prominent to solubility
Secreted IgG – in circulation
 Light Chain – 2 subtype: kappa (65) and lambda (35)

*kappa and lambda cannot be in the same antibody”

 IgM – no hinge region

Digestion with Papain and Pepsin

 Can yield a product by cleaving the structure of the

antibody;

Papain

 Digestion yields two F(ab) fragments and and Fc

portion (3 products)
FOR SECRETED IgG  Cleave antibody at the upper portion of hinge region
 N terminal – in the antigen-binding site
 C terminal
 Heterogenecity in the antigen-binding portion
 C is constant-binding region
Pepsin

 Digestion yields an F(ab) 2 fragment and Fc1

fragment (2 products)
 Cleaves antibody below the hinge region

F(ab) area – antigen-binding area


Fc area – function of opsonization and complement
component

Bivalent antigen binding fragment

Structure of a human IgG molecule as revealed by x-ray

crystallography. In this ribbon, diagram of a secreted
Tetrapeptide structure of Immunoglobulin IgG molecule, the identical heavy chains are colored
 Immunoglobulin molecules blue and red so that they can be easily visualized,
o Basic four-chain polypeptide unit although they are identical, and the light chains are
colored green; carbohydrate are shown in gray
 Heavy or H chains
 Light or L chains Three-dimensional structure of Antibodies
 1950s and 1960s, Gerald Edelman and Rodney
Porter  Immunoglobulin molecules does not actually exist as
o Experiment with immunoglobulin a straight shape, but in fact it is folded into compact
o Incorporated chemicals: globular subunits
 Beta-pleated sheet
7M urea and Mercaptoethanol   H chains line up with those of the L chain to produce
Ultracentrifugation  3.5S = H chain and 2.2S a cylindrical structure called an Immunoglobulin
= L chain Fold or Barrel
 Antigen binds in the middle of the CDRs
o 7M Urea is for unfolding or cleaving of the
(complementary determining region), with at least
molecule
four of the CDRs involved in the binding; CDR1,
o Mercaptoethanol used as Reducing agent
CDR2, and CDR3. More amino acid residue, it is
o Formula for IgG had to be H2L2 more variable and can bind to many structures of
o Sedimentation – bigat ng portion ng epitopes
antibody

Hinge Region

 Located between the CH1 and CH2 regions and

sulfide bond
 Gamma, Delta, and Alpha chains
 Mu and Epsilon has NO hinge region
 Increase proline and hydrophobic residues;
o the high proline content allows for flexibility
 Assists in effector functions such as initiation of the
complement cascade
 Four polypeptide chains = contain a carbohydrate
portion, in CH2 domain of the two H chains

Carbohydrate: CDR – Complimentarity Determining Region

CDR1, CDR2, and CDR3 – flexible, long, that projects toward

 Increase solubility of the immunoglobulin
from the amino terminal end of the variable Light domain.
 Provide protection against degradation
“Antigen is captured with the barrel”; bind with a small
 Enhance the functional activity of your Fc domain
number of an antibodies.

Structure of an Immunoglobulin Domain

Variable light and Variable Heavy o Of amino-terminal variable (V) regions that
participate in antigen recognition and
 Composed of two unparallel arrays of Beta strand carboxy-terminal constant (C)\ regions
o The regions of the heavy chins help mediate
some of the
o protective or effector functions of
antibodies
o Effector functions such as opsonization and
complement component binding
 Heavy Chain C regions interact with other molecules
and cells of the immune system and therefore help
mediate most of the biologic functions of antibodies

Heavy exist in two forms that at their carboxyl-terminal end

 One form is heavy chain anchors to the membrane of

the antibody in the plasma membrane
Complementarity Determining Region  The other form is in the secreted antibody
 The higher the number of the residue, the more it is
variable; high variability
Immunoglobulin Variants
Variability – access to many epitopes
 Is the specific chemical determinant group or
 Orange is Heavy molecular configuration which the immune response
 Red is Light is directed
 Have many variants because there are many
differences of epitopes that infect us
Model of Globular Protein Antigen

Antigen – BLUE
Isotype
Light Chain – Yellow
 Present in all the member of the same SPECIE (e.g.
Heavy Chain – Red Human – present; have the same heavy chain for each
class)
 Same heavy chain for each class
Fc and Fab Region
Allotype
 Antibody heavy chains and light chains both consist
 Present in the member of the same FAMILY
 Variation in constant regions
o Also used for paternity testing  The antibody is catabolized

Idiotype

 Different for each and every individual

 Has differentiation or variation in variable region
 Produce many variation to become ready to bind to
many epitopes

Antibody Synthesis

 The production of antibody is induced when the host

lymphocyte come into contact with foreign antigen IgM is first if PRIMARY RESPONSE
substance and binds to the receptor
 After 3-5 days, clone to be produced and to IgM is a receptor in the cell (membrane-bound)
differentiate into antibody-producing cell (Plasma IgG is next to increase
cell)
o This allows time for most pathogens to
damage the host cell
 When the host B lymphocyte is activated by
microbes, it will produce different daughter cell,
plasma cell, and memory cell for amnestic response

Clonal selection

 Activation and proliferation of B lymphocytes and

Plasma Cell

Amnestic response (memory cell)

 Memory response due to subsequent exposure to the

same antigen

Naïve B lymphocyte – virgin lymphocyte, has not experience

Antibodies are synthesized only by cells of the B lymphocyte antigen; activated with contact with antigen; once activated
lineage and exist in two forms: undergo activation and differentiation

1. Membrane-bound antibodies (IgD and IgM) on the Naïve T lymphocyte – presented with APC to be activated
surface of B lymphocytes functions as antigen 1. Clonal Expansion
receptors 2. Differentiation
2. Secreted antibodies (IgA – dimer part, IgG, and IgE) 3. Increased Antibody
function to protect against microbes
Effector T lymphocyte – does the work
Memory Cell – “Perfect Receptor”
4. Degradation and Decrease

Cell-mediated – T lymphocyte

Humoral-mediated – B lymphocyte

Apoptosis – programmed cell death

Memory Cell – long lifespan specific for antigens that

triggered B lymphocyte

Primary Antibody Response

Four phases after a foreign antigen challenge

1. Lag Phase
 No antibody is detectable

2. Log Phase
 The antibody titer increases logarithmically

3. Plateau Phase
 The antibody titer stabilizes

4. Decline Phase
 Heated to 600C = precipitate from
urine (become cloudy)
Secondary (Anamnestic) Response o Heating to 800C = they redissolve (become
 Why Anamnestic? There has been memory produced clear)
 Two main types of L Chains
Differs from a primary response as follows: o Designated Kappa and Lambda
1. Time. A secondary response has a shorter Lag phase, Both the Light Chain (Kappa and Lambda) are found in the 5
longer Plateau phase, and more gradual Decline class of Immunoglobulins, but only ONE type are present in a
2. Type of Antibody. IgM-type antibodies are the given molecule
principal class formed in the primary response.
Although some IgM antibody is formed in a
secondary response, the IgG class is the
Isotypes
predominant type formed

Acute infection – IgM is predominant

Chronic infection – IgG is predominant

3. Antibody titer. In a second response, antibody levels

attain a higher titer. The plateau levels in a secondary
response are typically 10-fold or greater than the
plateau levels in the primary response

When we have produced antibody, a negative effect:

1. IgG
 75 to 80% of total serum in immunoglobulins
 Major immunoglobulin in normal serum
o IgG1 = 67%
o IgG2 = 22%
o IgG3 = 7%
o IgG4 = 4%
 IgG3 is the most efficient at binding complement,
followed by IgG1, IgG2, and IgG4 have shorter
hinge segments
 All subclasses of IgG appear to be able to cross the
placenta, although IgG2 is the least efficient
[Link] titer  IgG1 and IgG3 are particularly good at initiating
phagocytosis
 Hemolytic disease of the newborn – develop anti-Rh
antibody immunoglobulin, that can attack to the Subclass differs mainly in number and position of the
second pregnancy; for IgG Disulfide bridges between the chains
 Hepa B vaccine booster – administered 5 years after
second or third dose, given to boost immune system IgG1 and IgG3 are good at initiating phagocytosis because
for Hepa B virus; increase in antibody titer and they bind most strongly to the Fc portion
increase in clone of memory cell

2. IgM (Macroglobulin = 19S)

The Nature of Light Chains  Highest sedimentation rate of 19
 No hinge region but with J chain
 Multiple Myeloma or Waldenstrom’s o J chain connect monomers of IgM
Macroglobulinemia (pentamer)
o Bence-Jones proteins in urine of patients  Effective in agglutionation and cytolytic reaction
o Maglignant plasma cells = increase L  Produced early in an immune response and is largely
chain production confined to the blood
o Determined with:  Efficient in multivalent antigens
 IgA2 Secretory component mass or
protect the site that could be
susceptible to the protease cleavage
 This is a good opsonin to mucosal
surface

Additional domain in CH4

Because of its large size, it is confined in the BLOOD (than in

placenta or CSF)

In primary response, a long Log phase reaching it can

occasionally be acquired as a secretory component like IgA,
allow to transverse epithelial cell and can patrol mucus
membrane

Presence of membrane-bound IgM classifies lymphocytes as

mature B cell

4. IgE
 Paired of Kappa and Alpha light chain and Two
Epsilon heavy chain
 For parasite and allergic reaction
 It easily bind in histamine and heparin that cause type
I Hypersensitivity
 Monomer
 Lung and in the skin
 Fc region binds strongly to a receptor on mast cells
and basophils and, together with antigen, mediates
the release of histamines and heparin from these cells
3. IgA
 Recruits neutrophils and eosinophils to the area
 IgA1 – mainly found in serum
 IgA2 – mainly found in the mucosa
 Most Heat-labile of all immunoglobulins (heating at
 Has J chain and has no Hinge region
the 560C for 30 mins and 3 hours result in the
o Mainly Dimer
conformational change and loss of ability to bind to
o Differ in content of 22 amino acids, 13 of target cell)
which are located in the Hinge region, and
are deleted in IgA2 (has deleted Hinge
region)
o IgA2 is more resistant to some bacterial
proteinases that are able to cleave IgA1
o IgA2 is the predominant form in the
secretion of the mucosal surface
o IgA1 is mainly found in the serum

 Bifunctional; one region of the molecule involves

the binding to antigen, and a different region
mediated binding of the immunoglobulin to host
tissues There is additional domain = Constant Epsilon 4
 Transcytosis = secretory component (SC)
o Does act to facilitate transport of IgA
o Dimer (IgA2) is more resistant to enzymatic
digestion than IgA1
Antigen-Antibody Interaction: Specificity and Cross-
reactivity

Specificity – ability of a particular antibody to combine with a

particular antigen (specific antigen and specific antibody)

Sensitivity – ability of an individual antibody to locate antigen

4. IgD even when it is greatly diluted
 Has a long Hinge region causes it to be more Cross-reactivity – when some of the determinants of an
susceptible to proteolysis among the other antigen are shared by similar antigenic determinants on the
immunoglobulins surface of apparently unrelated molecules, a proportion of the
 Less than 0.001 percent of total immunoglobulin antibodies directed against one type of antigen will also react
 Regulating B-cell maturation and differentiation with the other type of antigen
 Short half-life
e.g. Rheumatic Fever, we have antibody against group A
streptococcal cell, that antibody can cross-react with human
heart tissue (heterophile antibody- cross-react with other type
of antigen)

Antibody Affinity, Antibody Avidity, and Immune

Complexes

Summary Affinity – initial force of attraction that exists between a single

Fab site on an antibody molecule and a single epitope
determinant site on the corresponding antigen

Avidity – the functional combining strength of an antibody

with its antigen

Immune Complex – noncovalent combination of antigen with

its respective specific antibody

Molecular Basis of Antigen-Antibody Reactions

The four types of noncovalent bonds involved in antigen-

antibody reactions are:

1. Hydrophobic bonds
*Sedimentation Coefficient* 2. Hydrogen bonds
*H chain* - indicate its Isotype or what class of 3. Van der Waals forces
Immunoglobulin 4. Electrostratic forces

*Constant Region* - 5 subclass for IgG, 2 subclass for IgA,

extra region or domain in IgM and IgE (4) Genes coding for Immunoglobulins
*Percent Total Immunoglobulin* - IgD<1 because of longer Chromosome contain no intact immunoglobulin genes, only
Hinge region building blocks from which genes can be assembled
*Concentration* Three unlinked clusters:
*Half-life* - IgG high half-life, IgD more susceptible H Chain genes – are located on Chromosome 14, Kappa Chain
*Carbohydrate Content* - greater carbohydrate content in IgD genes – are on Chromosome 2, and Lambda Chain genes – are
on Chromosome 22
*Electrophoretic migration*

*Complement fixation* - IgM and IgG can fix a complement

Once this rearrangement does occur, it permanently changes
*Crosses placenta* - IgG can only cross placenta the DNA of the particular lymphocytes
Rearrangement of Heavy Chain genes Light Chain Rearrangement

 H chain genes are located on Chromosome 14  L chain rearrangement occurs only after mu chais
 H chain genes = VH, D, and J appear
 VJD region to be coupled with a different C region to  Lack a D region
produce antibody of a different class  Recombination of segments on chromosome 2-kappa
 Class Switching – daughter plasma cells can produce chromosome 22-lambda
antibody of another type  The process of VJ joining is accomplished by an
 Contact with T cells and with Cytokines provides the excision of intervening DNA
signal for switching to take place (in the Constant  Only if a nonfunctioning gene product arises from
region) rearrangement does lambda chain synthesis occur

o Lambda chain has lesser number than kappa

Coding of Immunoglobulin H Chain chain

Coding for Light Chain

Vh – variable heavy – 39 genes

D – diversity – 23 genes
Absence of D
J – joining – 6 genes (Diversity) region

Constant region – will identify what immunoglobulin or V/J joining – deletion and combination
isotype will be produced
Transcription
1st Rearrangement between D and J – close proximity –
RNA splicing – removal of Introns
causing them to combine first
= V/J3/Ck
 Deletion of a portion and then joining (combination)
D/J joining All are mixed or rearranged together to create a unique
binding site for different antigen because there are so many
2nd needs to combine to variable region, another
antigen in environment
rearrangement, delete and then adjoining = V/D/J joining -
V1V2D2J1J2J3J4 o Function or Essentials of the rearrangement
of the different regions or genes
Transcription – RNA Synthesis
(Recombination)
V2D2J3Cmu

Translation
Clonal
Mu heavy chain (gamma immunoglobulin)

Constant region tells it is gamma (Cmu)

No switchingd done, IgM molecule

In a acute inflammation – increase concentration of IgM

because decrease signaling coming from cytokines and T cell

When RNA synthesis occur, one constant region is attached to

the V/D/J region, heavy chain are made first but the cell
retains it capacity to produce immunoglobulin of another class Selection

*Only one  Process of antibody formation

of these  Cell-mediated
constant  Lymphocytes are genetically preprogrammed to
region is produce one type of immunoglobulin and that a
selected at specific antigen finds or selects those particular cells
any one capable of responding to it, causing them to
time* proliferate
 Antigen select which lymphocytes will undergo Myeloma Cell – deficiency of the enzyme hypoxanthine
clonal expansion and produce more lymphocyte with guanine phosphoribosyl transferase (HGPRT) and thymidine
the same type of antigen receptor kinase synthesis = DIE
 Included is memory cell for anamnestic response
Aminopterin is a drug that prevents myeloma cells from
making their own purines = DIE

Normal spleen cells do not survive in culture, Normal B cells

cannot be maintained continuously in cell culture

= Hybridoma Cell will be left and grow

Hybridoma Production

That Hybridoma cell, are able to use the Hypoxanthine and

Thymidine in culture = SURVIVE
Monoclonal Antibody They produce antibody specific for antigen
Only attach to one antigenic site  Diluted out and placed in microtiter wells, where they
are allowed to grow
 B cells are genetically preprogrammed to synthesize
very specific antibody has been used in developing  Antibody-producing clones lose their ability to
antibodies for diagnostic testing synthesize or secrete antibody after being cultured for
several months
 Purified antibodies cloned from a single cell
 The cells may then be grown in mass culture or
Hybridoma (Cell Hybrid) injected intraperitoneally into mice. Because
hybridomas are tumor cells, they grow rapidly and
 the multiplying hybrid cell culture; can be clone induce the effusion of large quantites of fluid into the
 single cell involved in the specificity of the peritoneal cavity. The ascites fluid is rich in MAbs
monoclonal antibody (Monoclonal Antibodies) and can be easily harvested
Myeloma Cells (Plasma cells derived from malignant tumor Clinical Applications
strains)
 Identifying and quantifying hormones
 is not capable of producing antibody  Typing tissue and blood
Monoclonal Antibody  Identifying infectious agents
 Identifying clusters of differentiation for the
 a monoglobulinderived from a single clone classification of leukemias and lymphomas and
follow-up therapy (specificity can identify markers of
Monoclonal Antibody production
leukemia or lymphoma)
 Identifying tumor antigens and autoantibodies (attach
to autoantibody; needs to be conjugate with indicator
that can be measured)
 Delivering immunotherapy (HIB)

Primary response

 Lag phase – does not produce antibodies

 Lag phase – produce many antibodies
Mouse is injected with a certain antigen and let the mouse  Plateau – stabilizes
develop the primary and secondary response  Decline phase – interaction of antibody and antigen;
immune response has already the optimum
When there is development, there is extraction of spleen cells
amount/activity
in the mouse

Spleen cells combined with mouse myeloma cells

Anamestic Response
Fused in presence of PEG
Time
 PEG = only in in 200,000 spleen cells actually forms
a viable hybrid with a myeloma cell Antibody type
 After fusion, cells are placed in culture using a
Selective Medium contining hypoxanthine, - Shorter Log phase, longer plateau, more gradual
aminopterin, and thymidine (HAT) decline
- IgM primary responded, but IgM are the predominant
HAT will separate the hybridoma cell by allowing them to still
grow selectively
Antibody titer

B lymphocyte
Protein usually found in urine with a multiple myeloma and
wild macroglobinulemia

- Bence jones proteins

- Proteins should not be in kidney