Avtor/Urednik		Čurin-Šerbec, V
Naslov		Imunokemijsko proučevanje strukture amoditoksina
Tip		monografija
Kraj izdaje		Ljubljana
Založnik		Fakulteta za naravoslovje in tehnologijo
Leto izdaje		1988
Obseg		str. 72
Jezik		slo
Abstrakt		Ten monoclonal antibodies to a native amnodytoxin A were generated by hybridization of myeloma X63Ag8.653 cells with spleen blast cells of BALB/c immunized mice by the method of W.C.Davis. Antibodies secreting hybridomas were selected using the indirect test with pure antigen adsorbed to the surface of polystyrene microtiter plates. Positive hybridomas were further cloned by the limiting dilution method and retested for antibody production. The finally chosen clones were grown to the maximal cell density in DMEM supplemented with 13% fetal calf serum. The supernatants were harvested and the epitope specificity was studied for antibodies in pairs with the ELIZA cotitration assay. All ten hybridomas produced IgG monoclonal antibodies capable of binding the native ammodytoxin A at one of the two closely related epitopes. The toxicity and the phospholipase activity of antigen-antibody complexes were determined using a monoclonal antibody and monospecific polyclonal antibodies raised in rabbits against four peptide segments (L1, L2, L3, L4) from the primary structure of ammodytoxin A covalently bound to the hemocyanine carrier. L1 included all three differences between ammodytoxin A and ammodytoxin B, whereas L2, L3 and L4 were fragments of clustered basic amino acids in the structure of ammodytoxins. All complexes lost the enzymatic activity although to a different extent. Some complexes retained the toxicity whereas some were not toxic. We have established that the toxic site in the molecule of ammodytoxins is located somewhere else as the active centre is and it probably comprises at least a part if not the whole segments of the polypeptide chain L1 and L2.
Deskriptorji		ANTIBODIES, MONOCLONAL VIPER VENOMS PHOSPHOLIPASES A MICE MICE, INBRED BALB C RABBITS HYBRIDOMAS ENZYME-LINKED IMMUNOSORBENT ASSAY IGG ANTIBODY SPECIFICITY IMMUNODIFFUSION