Post on 12-Jan-2016
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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) 1, NS1/1-Ag4.1 (NS1) (not secreted) Sp2/0-Ag14 (Sp2/0) none X63-Ag8.653 (Ag8.653) none Y3-Ag1.2.3 (Y3) - rat
Principle of Selection HGPRT - hypoxanthine guanine phosphoribosyl transferaseHAT - hypoxanthine, aminopterin, thymidine
Lymphocyte - HGPRT (+), can grow in HAT medium not immortalizedmyeloma cell – HGPRT (, cannot grow in HAT medium immortalizedhybridoma - 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 Imidazole- 4-Carboxy Ribonucleotide * 5-Formido-Imidazole- 4-Carboxamine Ribo- nucleotide PRPP PP
Hypoxanthine Inosine Monophosphate Hypoxanthine Guanine Phosphoribosyl Transferase (HGPRT) Guanine Guanosine Monophosphate (GMP) PRPP PP Thymidine GDP dGDP Thymidine kinase RNA GTP dGTP
dTMP dTDP d TTP DNA * Thymidylate Synthetase UDP dUTP dUMP dCTP dATP
Production of mAb
Procedures 1. Immunization of BALB/c mice2. Fusion of spleen cells and myeloma cells w
ith polyethylene glycol (PEG) 3. Selection of hybrid cells in HAT medium4. Screening of antibody-producing cells5. Cloning6. Large-scale production of antibodies
Characterization 1. Determination of Ab class2. Determination of Ab specificity3.Analysis of antigens recognized by Ab
Applications
1. Study of antigens, e.g., microbial antigens, histocompatibility antigens, tumor antigens, diff
erentiation antigens, etc.2. Immunoglobulin structure and function3. Immunodiagnosis4. Immunotherapy5. 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 c
omplex 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 Ab2. 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 immunogenic2. 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