Isolation and N-terminal sequence determination of a novel γ/δ T cell surface antigen

• Published in: Molecular immunology 1997-06, Vol.34 (8), p.583-591
• Main Authors: Nayeem, Naushaba, Barker, Patrick J., Binns, Richard M., Kanan, J., Chain, Benjamin
• Format: Article
• Language: English
• Subjects: Animals; Antibodies, Monoclonal - chemistry; Antibodies, Monoclonal - immunology; Bromelains - pharmacology; Epitope Mapping; Female; Glycoside Hydrolases - metabolism; Humans; Male; Molecular Weight; pig immunology; Receptors, Antigen, T-Cell, gamma-delta - chemistry; Receptors, Antigen, T-Cell, gamma-delta - immunology; Receptors, Antigen, T-Cell, gamma-delta - isolation & purification; surface antigens; Swine; T-Lymphocytes - chemistry; γ/δ T cells
• ISSN: 0161-5890; 1872-9142
• DOI: 10.1016/S0161-5890(97)00077-1

An antigen complex unique for porcine γ/δ T cells has previously been identified using the monoclonal antibodies MAC319 and MAC320. Here we use digestion with the proteolytic enzyme bromelain to selectively release the MAC319 antigen as a soluble fragment, for further characterisation. A cytofluorometric inhibition assay was developed to follow the purification of this fragment, as the conformation sensitivity of the MAC319 epitope prevented the use of immunoblotting techniques. The antigen has been purified using a combination of anion-exchange and hydrophobic-interaction columns, followed by separation on a size-exclusion column. Fractions from the size-exclusion column containing the antigen consisted of one major band at M r 95 000. This species was shown to be specifically absorbed onto MAC319-coupled Sepharose, thereby identifying the MAC319 antigen. N-terminal amino acid sequencing of this band has revealed a previously unidentified sequence. This fragment was also shown to be glycosylated, most likely with a single sugar moiety. Enzymatic removal of the sugars showed that they did not appear to be necessary for binding of the polypeptide to the MAC319 antibody.