Spleen ceils obtained from BALB/c mice immunized with methylamine-inactivated α2M were fused with non-producing Sp 2/0-Ag14 myeloma cells. Fifty anti-α2M-producing hybridoma clones were obtained. In a search for anti-α2M receptor recognition site antibodies, all supernatants were screened for inhibition of endocytosis of 125I-α2M-trypsin complexes by normal human fibroblast cell layers. One strongly inhibiting clone, B2, was detected. Antibodies produced by this clone and by a non-inhibiting clone, D9, were purified on affinity columns. Ouchterlony double immunodiffusion indicated both antibodies to be of IgG1 isotype. Antigen binding assays with purified B2 antibodies indicated the B2 determinant to be exclusively present as a neo-antigen on α2M-complexes prepared with proteases or methylamine. In contrast to this, D9 antibodies recognized antigenic sites on both native and complexed α2M. Supernatant of clone B2, B2 antibodies, and Fab fragments of B2 antibodies were equally potent in inhibiting endocytosis of 125I-α2M-trypsin by normal human fibroblasts, which suggests a close relation between the receptor recognition site on α2M complexes and the neo-antigen recognized by B2 antibodies. Incubation of α2M-complexes with an excess free trypsin at 4°C induced a specific loss of β-2-determinants on α2M-complexes without major degradation of α2M. Inhibition of endocytosis of 125I-α2M-trypsin complexes by cold α2M-trypsin was correlated with the presence of the B2 antigenic sites on α2M-trypsin. No inhibition was observed with α2M-trypsin lacking the B2 antigenic determinants, which suggests that proteolytic loss of the B2 antigenic sites also affected the receptor recognition sites on α2M.
No takes yet. Share an insight, caveat, or question.
Marynen et al. (1981) studied this question.