Locating epitope(s) in protein reactive with monoclonal antibodies - by testing immuno-reactivity of truncated polypeptide(s) translated from fragments of C-DNA

摘要

Process for locating the interaction site between a specific protein (I) and at least one monoclonal antibody (MAb) which recognises the entire protein comprises: (1) either isolating a DNA fragment which includes a cDNA encoding (I), under control of a transcriptional promoter, plus the elements necessary for in vitro translation of corresp. RNA, or prepg. a mixt. of fragments of such DNA contg. differently-sized portions of the cDNA under control of the same promoter; (2) transcribing the cDNA, then in vitro translation of the resulting RNA into polypeptides (Ia) in a cell-free medium; (3) testing (Ia) for reaction with specific MAb; (4) sepg. (Ia) according to size (this step may precede reaction with MAb); (5) identifying the largest and smallest (Ia) recognised by MAb; and (6) identifying the essential element of t e-epitope specific for MAb in the smallest (Ia); it consists of the C-terminal sequence of one or more amino acids still present in the smallest (Ia) recognised by (Ia) but missing from the largest (Ia) not recognised. Also new is a support (pref. a gel) contg. labelled, truncated proteins, pref. in order of size, sepd. from each other and fixed to the support in such a way that they can still undergo immunological reaction with specific MAb which recognise the intact protein (from which all (II) are derived) or its homologues. USE/ADVANTAGE - The method is used to define immunogenic and functional domains of proteins; for in vitro mutagenesis testing, and for mapping biological molecules (e.g. receptors or antigens). Compared with known methods, this method is simpler and requires fewer steps.