Antibodies
Polyclonal : * A set of B-cells is stimulated with various Epitopes * 2 Injections within a few weeks + adjuvans (Freundsches, Al-hydroxide, bacteria, detergent Quil A,..) * Small molecules (haptene) only on immunogenic support or as macro-polymer * amount of serum: rabbit 80 ml, mouse 1.5 ml * working dilution about 1:10000 !! * Mostly more avid than monoclonal ABs * Increased specificity by saturation Monoclonal : * One cell line = 1 Epitop * Lymphocytes of immunized mice + myeloma (B-Zell)- tumor cells are fused with e.g. polyethylene glycol * Selection system: Myeloma cell line deficient in HGPRT (Hypoxanthinguanine-phosphoribosyltransferase) and TK Thymidinekinase) (no nucleotide synthesis via Salvage pathway) After fusion both cells types the necessary enzymes are delivery via the B-cell Plate them in microtiter plates (add irradiated feeder cells if required) grow and dilute them --> Cell clone * Humane monoclonal ABs from B-Cells + Eppstein-Barr-Virus Problem: unstable cell line New techniques: * SCID-Mice with humane immune system* Clone from IgG-Genes in phages (phage display)* Expression in milk producing glands in cows, as egg protein, in plants,... Antibody fragments: * Pepsin: F(ab’)2 - Fragment and Fc’ - Fragment* Papain: 2 x Fab - Fragment and Fc - Fragment