1.

Are Complementarity Determining Regions and Hypervariable Regions on

antibodies the same thing? Define these two terms in your answer.
Complementarity determining regions (CDRs) are regions within antibodies (also known
as immunoglobulins) or T cell receptors where these proteins complement an antigen's
shape. Thus, CDRs determine the protein's avidity (roughly, bonding strength) and
specificity for specific antigens. The CDRs are the most variable part of the molecule, and
contribute to the diversity of these molecules, allowing the antibody and the T cell receptor to
recognize a vast repertoire of antigens.

In antibodies, hypervariable regions form the antigen-binding site and are found on both light
and heavy chains.[2] They also contribute to the specificity of each antibody.[2] In a variable
region, the 3 HV segments of each heavy or light chain fold together at the N-terminus to
form an antigen binding pocket.

Sketch of an antibody with the variable domains shown in blue, and the CDRs (which are
part of the variable domains) in light blue

Outline a procedure to produce Fab fragments from an antibody by a method that

will result in free sulphydryl groups on the Fab fragments.
Advantages of Antibody Fragments
Because of their smaller size as functional components of the whole molecule, antibody
fragments offer several advantages over intact antibodies:
1. Reduced nonspecific binding from Fc interactions
2. Ability to control Fc-binding to Protein A or Protein G in experiments involving
immunoprecipitation and Western blotting
3. More efficient penetration of tissue sections, resulting in improved staining in
immunohistochemistry (IHC)
4. Potentially higher sensitivity in antigen detection in solid phase applications as a
result of reduced steric hindrance from large protein epitopes
5. Elimination of Fc-associated effector functions (e.g,. complement fixation) in antigen-
antibody binding studies
6. Simpler system for studying the structural basis for immune recognition using X-ray
crystallography or NMR
7. Lower immunogenicity than intact antibody for experiments in vivo.

Types of Antibody Fragments

F(ab')2, Fab, Fab' and Fv are antigen-binding fragments that can be generated from the
variable region of IgG and IgM. These antigen-binding fragments vary in size (MW), valency
and Fc content. Fc fragments are generated entirely from the heavy chain constant region of
an immunoglobulin.

F(ab')2 Fragments
F(ab')2 (110,000 daltons) fragments contain two antigen-binding regions joined at the hinge
through disulfides. This fragment is void of most, but not all, of the Fc region.

Fab' Fragments
Fab' (55,000 daltons) fragments can be formed by the reduction of F(ab')2 fragments. The
Fab' fragment contains a free sulfhydryl group that may be alkylated or utilized in
conjugation with an enzyme, toxin or other protein of interest. Fab' is derived from F(ab')2;
therefore, it may contain a small portion of Fc.

Fab Fragments
Fab (50,000 daltons) is a monovalent fragment that is produced from IgG and IgM, consisting
of the VH, CH1 and VL, CL regions, linked by an intramolecular disulfide bond.

Fv Fragments
Fv (25,000 daltons) is the smallest fragment produced from IgG and IgM that contains a
complete antigen-binding site. Fv fragments have the same binding properties and similar
three-dimensional binding characteristics as Fab.

Fc Fragments
Fc (50,000 daltons) fragments contain the CH2 and CH3 region and part of the hinge region
held together by one or more disulfides and noncovalent interactions.

IgG - Preparing Fab, F(ab')2 and Fc Fragments

The hinge region of an immunoglobulin monomer (IgG) is readily accessible to proteolytic
attack by enzymes. Cleavage at this point produces F(ab')2 or Fab fragments and the Fc
fragment. The Fc fragment may remain intact or become further degraded, depending upon
the enzyme and conditions used.

Papain Digestion: Fab from IgG

Papain is a nonspecific, thiol-endopeptidase that has a sulfhydryl group in the active site,
which must be in the reduced form for activity. When IgG molecules are incubated with
papain in the presence of a reducing agent, one or more peptide bonds in the hinge region are
split, producing three fragments of similar size: two Fab fragment and one Fc fragment (1).
When Fc fragments are of interest, papain is the enzyme of choice because it yields an intact
50,000-dalton Fc fragment.

Pepsin Digestion: F(ab')2 from IgG

Pepsin is a nonspecific endopeptidase that is active only at acid pH. It is irreversibly
denatured at neutral or alkaline pH. Digestion by the enzyme pepsin normally produces one
F(ab')2 fragment and numerous small peptides of the Fc portion. The resulting F(ab')2
fragment is composed of two disulfide-connected Fab units.

Antibody F(ab')2 preparation by pepsin digestion and fragmentation.

F(ab')2 can be separated by mild reduction into two sulfhydryl-containing, univalent Fab'
fragments. The advantage of Fab' fragments is that they can be conjugated to detectable
labels directly through their sulfhydryl groups, ensuring that the active binding site remains
unhindered and active. Use 2-Mercaptoethylamine•HCl (2-MEA) for mild reduction of
F(ab')2 fragments. The free sulfhydryls of each Fab' can be targeted for conjugation, or they
can be blocked with an alkylating reagent, such as N-Ethylmaleimide (NEM) to prevent re-
formation of the F(ab')2.

The generated Fab fragments contains the hinge thiols available for conjugation through
standard maleimide chemistry.

Use FabRICATOR or FragIT to cut the IgG just below the hinge region. This procedure takes
approximately 30 minutes. You will end up with a homogenous pool of F(ab')2 fragments
and Fc fragments.
1. Generate F(ab')2 fragments using FabRICATOR or FragIT. Next, add 2-MEA (2-
mercaptoethanol amine) to a final concentration of 50 mM and incubate at 37ºC for 90
minutes.)

2. Mild reduction of hinge thiols

Now your solution conatins Fab fragments, Fc fragments, 2-MEA and possibly
FabRICATOR enzyme.
3. Final step

Conjugation: (replace protein for Fab-NH2)

Q. List in point form the different mechanisms that the immune system uses to generate
diversity in antibody structures. Present your list in order of the occurrence of events.
There are two classes of light chain: Kappa -  and Lambda - 

Gene rearrangements to form light chains in germ line DNA

Deletion of DNA during B-cell development
Random deletion/addition of nucleotides at junctions (P-nucleptide addition and N-nucleotide
addition)

Gene rearrangements to form heavy chains in germ line DNA

Deletion of DNA during B-cell development
Somatic hypermutation of VDJ and VJ regions
 Q. Explain the biochemical mechanisms that are used by a B-lymphocyte to switch from
producing IgM as a cell surface protein (IgMm) to secreting IgM (IgMs).

Membrane-bound versus secreted immunoglobulins. A virgin B-cell bears IgM (and possibly
IgD) in its membrane; following stimulation it begins to secrete IgM into its surrounding
environment. These two forms of IgM are structurally different. The mu heavy chain of
membrane-bound IgM has an amino acid sequence at its carboxy- terminal end which
anchors the molecule into the membrane; the secreted form of IgM has a different C-terminal
sequence which lacks a membrane anchoring region. The membrane-bound form of IgM is
also incapable of associating with J-chain and forming its normal pentameric structure;
membrane IgM, therefore, exists exclusively in monomeric form (H2L2), also known as
IgMs or "sub-unit" IgM.
A virgin B-cell produces only the membrane-bound form of IgM. As it develops into an
antibody-secreting plasma cell it may transiently produce both membrane and secreted IgM,
but it eventually produces only the secreted form. Likewise, a memory B-cell which
synthesizes only membrane-bound IgG will shift to producing exclusively the secreted form
of IgG as it develops into a plasma cell following secondary antigen stimulation.

1. Give a description of how the precipitin reaction can be used as the basis for a
quantitative assay for an antigen such as a blood protein in pathology testing.

The maximum amount of precipitate forms when antigen and antibody are present in similar
molar amounts (the equivalence zone). At a low antigen:antibody ratio, few antibody
molecules will bind more than one antigen molecule, hence the multiple intermolecular links
 necessary for a lattice again do not form (antibody excess zone). At a high antigen:antibody
ratio, all antibody combining sites will be saturated with antigen, and few antigen molecules
will be shared between two antibodies (antigen excess zone). The zones are defined
empirically as those sets of reactions whose supernatants give additional precipitate upon
adding a small increment of antibody (in the antigen excess zone) or antigen (in the antibody
excess zone). In the equivalence zone, little antibody or antigen remains in the supernatant,
and no additional precipitate can be induced. With some antigens and antisera, complete
antibody precipitation occurs over an extended range of antigen additions, while excess
antigen is still observed in the supernatant. In such cases, the term "inhibition zone" refers to
the region of the antigen excess zone in which the absolute recovery of precipitate declines.

Q. Surface Plasmon Resonance is a phenomenon that can be used to detect small

changes in the refractive index of an aqueous medium close to a surface. Explain how
this can be used to analyse the interaction of an antibody with its antigen.

The surface plasmon resonance (SPR) phenomenon occurs when polarized light, under
conditions of total internal reflection, strikes an electrically conducting gold layer at the
interface between media of different refractive index: the glass of a sensor surface (high
refractive index) and a buffer (low refractive index).

Surface plasmon resonance (SPR) detection requires no labeling of antigen or antibodies and
allows quantification of two or more interacting molecular species. A first molecule is
immobilized to the dextran modified surface of the sensor chip. By sequential introduction,
the stepwise formation of multimolecular complexes can then be monitored.

Specificity: The extent to which different molecules interact with a single partner
immobilized on a sensor surface reveals the specificity of an interaction.
Kinetics: rates of reaction: The kinetics of an interaction, i.e. the rates of complex
formation (ka) and dissociation (kd), can be determined from the information in a sensorgram.
If binding occurs as sample passes over a prepared sensor surface, the response in the
sensorgram increases. If equilibrium is reached a constant signal will be seen. Replacing
sample with buffer causes the bound molecules to dissociate and the response decreases.

The traditional approach to a kinetics assay is to inject a series of analyte concentrations over
the sensor surface, one at a time, with regeneration of the surface between each analysis
cycle.

Affinity: the strength of binding--The affinity of an interaction is determined from the level
of binding at equilibrium (seen as a constant signal) as a function of sample concentration.
Affinity can also be determined from kinetic measurements. For a simple 1:1 interaction, the
equilibrium constant KD is the ratio of the kinetic rate constants, kd/ka.

Concentration analysis
Concentration is determined by monitoring the interaction of a molecule with a prepared
sensor surface in the presence of a target molecule in solution (solution inhibition) or excess
analyte (surface competition). concentrations are calculated either by interpolation of the
binding responses on a calibration curve
 Concentration can be determined for purified molecules or for molecules in complex
mixtures such as serum or food samples.
Concentrations in the nanomolar range can be measured
Q. Describe with the aid of diagrams an experimental procedure that could be used to
determine the affinity of an antibody for a small antigen molecule. Write an equation to
calculate the affinity constant (Ka) and show how the terms would be estimated from the

experimental set up. Show the units that Ka would have.

Describe the reagents and reaction that could be used to link the polysaccharide agarose (an
1,6 linked polymer of glucose) to a carrier protein in order to raise antibodies to this
carbohydrate.