Immunisation techniques. Antibody Immunisation techniques. Antibody (polyclonal and monoclonal) (polyclonal and monoclonal) production, purification, labelling. production, purification, labelling. Hybridoma technology, Hybridoma technology, fermentation fermentation Department of Immunology and Biotechnology University of Pécs
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Immunisation techniques. AntibodyImmunisation techniques. Antibody(polyclonal and monoclonal)(polyclonal and monoclonal)
Department of Immunology and BiotechnologyUniversity of Pécs
Isotype
Idiotype
Structure of an antibody
Antibody and antigenic determinant
Antibody and antigenic determinant (epitope)
Types of antigenic determinants
A complex antigen generates polyclonal immuneresponse
Monoclonal antibodies:antibodies with the same specificity,produced by a cell line derived from only one activatedB cell,able to recognise only one epitpe
Polyclonal antibodies:mixture of antibodies with different specificities,product of more B cell clones,recognise different epitopes of an antigen
Y Y
Y
Ag
Y YY
Ag
Immunization
Selection of the adequate animal:• Monoclonal antibodies – mouse or rat• Policlonal antibodies – rabbit, sheep, goat
Adjuvants: delay the absorption of the antigen and non-specifically stimulate the immune system (activation ofmacrophages) resulting in increased antibody secretion.
Immunosorbent methods:Antigen specific isolation on the surface of Sepharose,
cellulose or polyacrylamid gels
Purification of polyclonal antibodies
Antibody testing
Testing of titer and specificity should be donein the same system where the antibody will be applied
e.g.: ELISA flow cytometry immunohistochemistry
Hybridoma technique,production of monoclonal antibody
With the hybridoma technique we are able to selecta specifically activated (e.g. antibody production),but limited life span immune cell and keep it in vitro(proliferation).
The basis of the technique is that we fuse in vitro anactivated immune cell with a B-cell/plasma cell-derived tumor cell line.
2. Selection: Selection of the desired hybrides (spleen cell + myeloma)
• Sp-2 cells are deficient for HGPRT and TK enzymes (purineand pyrimidine synthesis), but spleen cells have them, so bythe use of a selective medium (HAT) we can select the spleencell – Sp2 hybrids
Pathways of DNA synthesis
Selection of the antibody producing hybrid cultures, testing(ELISA)
• Cloning:dilution of the antibody producing cells so that on a96 well plate statistically less than 1 cell is seeded in awell. Testing antibody production of monoclones.
Antibody produced by a single clone of cells:MONOCLONAL ANTIBODY
Hybridoma production
Monoclone
4. Mass antibody production:(1) production of hybridoma supernatant in vitro;(2) administration of hybridoma cells ip. in mice (ascites);(3) fermentor (artificial mouse).
Hybridoma production
Applications of monoclonal antibodies
•• Preparative methodsPreparative methods–– Protein purificationProtein purification
•• Analytical methods (Diagnostic purposes,Analytical methods (Diagnostic purposes,research)research)–– Identification and isolation of lymphocyte subgroupsIdentification and isolation of lymphocyte subgroups
and clonesand clones–– TTumor detection and imagingumor detection and imaging–– Routine laboratory techniques- serologyRoutine laboratory techniques- serology
Enzyme converts substrates thatmanifests in colour reaction.
HRPO,ALP
Immunohistochemical slides
Fluorescent dye conjugates
FITC,PE
Imunofluorescence
PE
FITC
FITC
Flow cytometry
Toxin conjugates
Ricin
A toxin conjugated to an antibody binding tothe surface antigen of the target cell will beinternalized by the target cell. Then the toxinis released into the cytoplasm and exerts toxiceffect (e.g. Ricin can block the proteinsynthesis of the cell).
Radioisotope conjugates
• γ ray emission, low dose Diagnostic purposes – radioactive imagining Diagnosis of tumors and micrometastases
• α, β ray emission, high doseTreatment of tumors
Bispecific antibodies
TUMOR
Effector cell• NK• Tc• Macrophage
A bispecific antibody is created by chemical crossbinding of the Fab fragments of two different Igs.
Effector cell specific Fab part:• CD3 Tc• CD64 (FcγR1) high affinity FcγR; Mφ • CD89 FcαR; Mφ , granulocytes
Tumor specific Fab part
Therapeutic monoclonal antibodies registered by FDAName Specificity Type Application Year