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Applications of Ab Molecules
Chapter 4 Monoclonal Ab (p.104)
Chapter 5 Ab genes and Ab Engineering (p.139)
Monoclonal Antibodies
Clonal Selection of B Lymphocytes
Hybridoma
Köhler and Milsten (1975) - continuous culture
of specific antibody-forming cells
Hybrid = lymphoblast x myeloma cells
-oma = tumor
Formation and Selection
of Hybridoma Cells
Myeloma Cell Lines Commonly
Used to Make Hybridomas
_______________________________________
cell line
Ig produced
_______________________________________
P3-X63Ag8 (Ag8)
g1, k
NS1/1-Ag4.1 (NS1)
k (not secreted)
Sp2/0-Ag14 (Sp2/0)
none
X63-Ag8.653 (Ag8.653)
none
Y3-Ag1.2.3 (Y3) - rat
k
________________________________________
Principle of Selection
HGPRT - hypoxanthine guanine phosphoribosyl transferase
HAT - hypoxanthine, aminopterin, thymidine
Lymphocyte - HGPRT (+), can grow in HAT medium
not immortalized
myeloma cell – HGPRT (-), cannot grow in HAT medium
immortalized
hybridoma - HGPRT (+), can grow in HAT medium
immortalized
Metabolic pathways relevant to hybrid selection in
medium containing hypoxanthine, aminopterin and
thymidine (HAT medium).
When the main synthetic pathways are blocked with
the folic acid analogue aminopterin (*), the cell must
depend on the “salvage” enzymes HGPRT and TK
(thymidine kinase). HGPRT (-) cells cannot grow in
HAT medium unless they are fused with HGPRT (+)
cells.
5-Amino Imidazole4-Carboxy Ribonucleotide
*
5-Formido-Imidazole4-Carboxamine Ribonucleotide
PRPP
PP
Hypoxanthine
Inosine Monophosphate
Hypoxanthine Guanine
Phosphoribosyl Transferase
(HGPRT)
Guanine
Guanosine Monophosphate
(GMP)
PRPP
PP
Thymidine
Thymidine kinase
UDP
RNA
dTMP
dTDP
* Thymidylate
Synthetase
dUTP
dUMP
GDP
dGDP
GTP
dGTP
d TTP
DNA
dCTP
dATP
Production of mAb
Procedures
1. Immunization of BALB/c mice
2. Fusion of spleen cells and myeloma cells
with polyethylene glycol (PEG)
3. Selection of hybrid cells in HAT medium
4. Screening of antibody-producing cells
5. Cloning
6. Large-scale production of antibodies
Characterization
1. Determination of Ab class
2. Determination of Ab specificity
3.Analysis of antigens recognized by Ab
Applications
1. Study of antigens, e.g., microbial antigens,
histocompatibility antigens, tumor antigens,
differentiation antigens, etc.
2. Immunoglobulin structure and function
3. Immunodiagnosis
4. Immunotherapy
5. Affinity purification
Advantages of mAb
1. A monoclonal antibody reacts with a single
antigenic determinant.
2. Cross reactions are consistent.
3. Monoclonal antibodies are available in “unlimited”
supply.
4. We can produce antibodies to single molecules in
complex mixtures.
5. Monoclonal antibodies may detect components in a
mixture that are present in small quantities not
detectable by conventional antisera.
6. Antibodies can be “biologically” modified.
Disadvantages of mAb
1. Monoclonal antibodies cross-react due to
structural relatedness among antigens.
2. Biological function may be limited by heavy
chain class.
3. Most monoclonal antibodies will not precipitate
in immunodiffusion due to failure of cross-linking.
4. Single affinity and specificity may be more
influenced by pH, temperature, etc.
5. Sometimes, a monoclonal antibody may be too
specific.
Clinical Uses for mAb
Diagnosis, imaging, and therapeutic reagents
Immunotoxins: mAb conjugated to toxins,
such as ricin, Shigella toxin,
and diphtheria toxin
: inhibitory toxin chain
: binding component of the toxin
toxin
receptor
Catalytic mAb (Abzymes)
- A mAb that has catalytic activity.
- Similarities of the binding of an Ab to its Ag
and an enzyme to its substrate:
noncovalent interactions, high specificity, high affinity
- Ab does not alter the Ag, whereas the enzyme
catalyzes a chemical change in its substrate.
A central goal of catalytic Ab research
is the derivation of a battery of abzymes
that cut peptide bonds at specific amino
acid residues, much as restriction enzymes
cut DNA at specific sites.
Ab Genes and Ab Engineering
Problems of mouse mAb for clinical uses:
1. Human anti-mouse Ab
2. Formation of immune complexes
Human mAb
1. Human hybridoma
Human B cells x human myeloma cells
2. Human B cells transformed by Epstein-Barr
virus (EBV)
3. Humanized mAb
4. Human Ab constructed from Ig-gene libraries
Production of chimeric mouse-human mAb
or “transfectoma”
1. Less immunogenic
2. Fc retains the biological effector
functions of human Ab.
Chimeric and hybrid mAb engineered
by recombinant DNA technology
or “bispecific” Ab
mAb Constructed from
Ig-gene Libraries
Therapy for Non-Hodgkin’s Lymphoma
by a Genetically Engineered Ab
SCID-human Mouse
Mice with Human Ig Loci
The End