Immunoglobulin Structure and Function IVUSE AND ABUSE OF ANTIBODIESLecture 13Monday August 10, 2003Mike Wolcott :Immunoglobulin Structure and Function IVUSE AND ABUSE OF ANTIBODIESLecture 13Monday August 10, 2003Mike Wolcott Reading for Lectures 11-13: Kuby 5e &4e: Chapters 3,4, and 6:


polyclonal antiserum vs. monoclonal antibodies :polyclonal antiserum vs. monoclonal antibodies


What are Monoclonal Antibodies :What are Monoclonal Antibodies Monoclonal antibodies (MAbs) are: antibodies of exceptional purity and specificity components of the immune system able to recognize and bind to a specific antigen In 1975, Kohler and Milstein first fused lymphocytes to produce a cell line which was both immortal and a producer of specific antibodies. The two scientists were awarded the Nobel Prize for Medicine in 1984 for the development of this "hybridoma." The value of hybridomas to the field was not truly appreciated until about 1987, when MAbs were regularly produced in rodents for diagnostics.


Some Uses of Monoclonal Antibodies :Some Uses of Monoclonal Antibodies Monoclonal antibodies are currently utilized in many diagnostic procedures, including: measuring protein and drug levels in serum typing tissue and blood identifying infectious agents identifying clusters of differentiation for the classification and follow-up therapy of leukemias and lymphomas identifying tumor antigens and auto-antibodies identifying the specific cells involved in the immune response identifying and quantifying hormones Magic bullets


Production of Mab :Production of Mab Technology: The first step in the production of MAbs is to immunize a mouse with an antigen. When the mouse begins to produce antibodies to the antigen, its spleen is removed. Antibody-producing cells from the spleen are then fused with a selected myeloma cell line, one which is not Ab-producing and has been selected to be deficient in an enzyme in the nucleotide salvage pathway (hypoxanthine-guanine phosphotransferase, HGPRT).. The new fused cell line, which does produce antibodies, can be grown in culture or re-injected into another mouse's peritoneum. Helpful web site - http://ntri.tamuk.edu/protocols/tc_background.html


Production of Monoclonal Abs :Production of Monoclonal Abs http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/M/Monoclonals.html


Magic Bullets :Magic Bullets


Monoclonal antibodies as magic bullets. :Monoclonal antibodies as magic bullets.


Treatment of B-cell Lymphoma :Treatment of B-cell Lymphoma


Humanized (Chimeric) Monoclonal Antibodies :Humanized (Chimeric) Monoclonal Antibodies Examples: Anti-CD20 for treatment of non-Hodgkin’s lymphoma and “Herceptin for treatment of breast cancer.


Production of the Xenomouse(mouse with human immunoglobulin loci) :Production of the Xenomouse(mouse with human immunoglobulin loci)


Principal of Antibody Titration :Principal of Antibody Titration


Quantitative Precipitin Test :Quantitative Precipitin Test


Quantitative Precipitin Test :Quantitative Precipitin Test


Hemagglutination and Passive Hemagglutination :Hemagglutination and Passive Hemagglutination


Agglutination Inhibition :Agglutination Inhibition


Properties and Uses of ELISA :Properties and Uses of ELISA ELISA is a very sensitive assay The assay is easily automated Can be used to quantitate antigen or antibody Typically used for immuno-diagnosis of many bacterial, viral and parasitic infections. The standard screening test for HIV is the ELISA. However, there are many false positives, thus, a positve ELISA must be confirmed by a more specific assay. The most commonly used confirmatory test is the western blot.


Enzyme-Linked Immunosorbent AssayELISA :Enzyme-Linked Immunosorbent AssayELISA


ELSPOT Assay :ELSPOT Assay


Western Blotting :Western Blotting


Cellular proteins reacting with an antibody can be characterized by immunoprecipitation of labeled cell lysates :Cellular proteins reacting with an antibody can be characterized by immunoprecipitation of labeled cell lysates


Affinity Chromatography :Affinity Chromatography Successful separation by affinity chromatography requires that a biospecific ligand is available and that it can be covalently attached to a chromatographic bed material called a matrix. It is important that the biospecific ligand (antibody, enzyme, or receptor protein) retains its specific binding affinity for the substance of interest (antigen, substrate, or hormone). Methods must also exist for removing the bound material in active form with low pH, high pH, or high salt. Any component can be used as a ligand for purifying its respective binding substance. How to manual for your future reference. All you ever want to know about affinity chromatrography – and MORE/much MORE http://www.fm.uit.no/info/imb/amb/courses/bio360s/pdf/affinity.pdf


Affinity Chromatography :Affinity Chromatography


Immunological Cell Separation :Immunological Cell Separation Rare cell isolation: Stem cells (CD34+, AC133) Natural killer cells (CD56+) Cancer cells circulating in the blood 1 in 106 or less? Fetal cells in maternal blood Harmful Bacteria (O-157 etc) Undesired cell depletion: Removal of cancerous cells from cell population Removal of red blood cells from the leukocyte for cancer therapy


Cell Separation Technologies :Cell Separation Technologies Centrifugation : Density Filtration : Size Flow Cytometry (FACS) Size Granularity Fluorescence Batch affinity systems : antibody, avidin-biotin Batch magnetic systems


How can cells be separated magnetically? :How can cells be separated magnetically? Cells are incubated with an antibody specific for a particular cell surface epitope The antibody is linked to a magnetic bead (one step labelling), or a secondary reagent linked a magnetic bead is added that reacts with the first antibody (2 step labelling). These labelled cells are then flowed over a column consisting of a paramagnetic matrix and exposed to a strong magnetic field. Unbound cells are eluted, then the magnet is turned off and the specific cells are eluted.


Immunomagnetic Labeling of Cells :Two-Step Labeling One-Step Labeling Immunomagnetic Labeling of Cells


Magnetic-Bead Cell separation system :Magnetic-Bead Cell separation system Magnet Plunger Remove the magnet Unlabeled cells Immunomagnetically labeled cells Separation column


Subpopulations of cells can by physically separated using antibodies coupled to magnetic beads :Subpopulations of cells can by physically separated using antibodies coupled to magnetic beads