Antigenic determinants on rat metallothionein: fine epitope mapping for a murine monoclonal antibody and rabbit polyclonal antisera.
In order to determine the epitope of metallothionein (MT) to a murine monoclonal antibody (MT 189-14-7) which had been produced by immunization with rat MT 2 (Kikuchi et al. (1988) Mol. Immunol. 25, 1033-1036), various lengths of synthetic oligopeptides were tested for their inhibitory activities in competitive radioimmunoassay (RIA). The amino-terminal acetylated pentapeptide, AcMDPNC, exhibited an inhibitory activity comparable to that of native MTs, whereas the acetylated tetrapeptide, AcMDPN, and the deacetylated heptapeptide, MDPNCSC, were much less inhibitory. The results suggest that the major part of the epitope structure of MT to the MT 189-14-7 monoclonal antibody is located within the amino-terminal acetylated pentapeptide, AcMDPNC. The specificities of polyclonal rabbit anti-MT antisera raised against the same immunogen were also determined by using various animal MTs and synthetic peptides as inhibitors in the RIA. Among three antisera tested, two reacted with several amino-terminal oligopeptides similarly to the MT 189-14-7 antibody. The major epitope structures to these polyclonal antibodies were shown to be located within the acetylated tetrapeptide, AcMDPN. Another antiserum contained at least two different populations of antibodies: one consisted of antibodies reactive with the amino-terminal synthetic peptides, while the other was not reactive with them. These results suggest that, in the rabbit also, the amino-terminal region common to various animal MTs can be an epitope to antibodies raised against rat MT, as shown in the mouse. Moreover, the results indicate that the synthetic amino-terminal peptides are useful for determination of the specificity of polyclonal rabbit anti-MT antibodies, which have been widely used for the quantification of MTs.
Kikuchi
Y
Y
Department of Microbiology, Hoshi College of Pharmacy, Tokyo.
Irie
M
M
Ikebuchi
H
H
Sawada
J
J
Terao
T
T
Nakayama
S
S
Iguchi
S
S
Okada
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D055598:Chemical Phenomena; D002621:Chemistry; D000939:Epitopes / Q000276:immunology*; D008668:Metallothionein / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009842:Oligopeptides; D010449:Peptide Mapping; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains
D000911:Antibodies, Monoclonal; D000939:Epitopes; D009842:Oligopeptides; D008668:Metallothionein