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Applications of Ab Molecules
Chapter 4 Monoclonal Ab (p.104)
Chapter 5 Ab genes and Ab Engineering (p.139)
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Monoclonal Antibodies
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Clonal Selection of B Lymphocytes
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Hybridoma Köhler and Milsten (1975) - continuous culture of specific antibody-forming cells Hybrid = lymphoblast x myeloma cells -oma = tumor
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Formation and Selection of Hybridoma Cells
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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
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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
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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.
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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
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Production of mAb
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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
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Characterization 1. Determination of Ab class2. Determination of Ab specificity3.Analysis of antigens recognized by Ab
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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
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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.
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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.
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Clinical Uses for mAb
Diagnosis, imaging, and therapeutic reagents Immunotoxins: mAb conjugated to toxins, such as ricin, Shigella toxin, and diphtheria toxin
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: inhibitory toxin chain
: binding component of the toxin
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toxin receptor
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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.
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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.
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Ab Genes and Ab Engineering
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Problems of mouse mAb for clinical uses:
1. Human anti-mouse Ab2. Formation of immune complexes
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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
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Production of chimeric mouse-human mAb
or “transfectoma”
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1. Less immunogenic2. Fc retains the biological effector functions of human Ab.
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Chimeric and hybrid mAb engineered by recombinant DNA technology
or “bispecific” Ab
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mAb Constructed from Ig-gene Libraries
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Therapy for Non-Hodgkin’s Lymphoma by a Genetically Engineered Ab
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SCID-human Mouse
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Mice with Human Ig Loci
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The End