A monoclonal antibody, synthesized by an interspecies lymphocyte-myeloma hybrid cell line, and which binds to antigenic determinant of acetylcholine receptors (AChR) remote from the binding site for cholinergic ligands, was conjugated to agarose to purify AChr solubilized in Triton X-100 from the electric organ of Torpedo californica. Protein was eluted from antibody-agarose in high yield (62 to 95 mg/kg of organ) at pH 10 (0.2 M glycine NaOH, with 0.5 M NaCl and 1% sodium cholate). When neutralized promptly, specific binding of the cholinergic ligand α-bungarotoxin (4.9 ± 0.35 nmol/mg) did not differ significantly from that of Torpedo AChR purified on α-neurotoxin-agarose (yield = 14 mg/kg; α-bungarotoxin binding = 5.9 ± 0.75 nmol/mg). Intradermal inoculation of 2.5 μg of the antibody-purified AChR with adjuvants induced experimental autoimmune myasthenia gravis in rats. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed four polypeptide subunits (Mr=44,000, 52,000, 58,000 and 66,000). These were indistinguishable from those of AChR prepared on α-neurotoxin-agarose. Subsequent elution of antibody-agarose at pH 2.5 (1.0 M propionic acid in 0.1% Tween 20) yielded additional protein (20 mg/kg of organ). Although this protein had only 1% of the 125I-α-bungarotoxin binding activity of protein eluted at pH 10, is noteworthy that it retained antigenic determinants common to mammalian muscle AChR. Immune-mediated loss of autologous muscle AChR occurred in rats inoculated with 6.6 μg of protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of protein eluted in propionic acid revealed multiple components of molecular weights ranging downward from 66,000 with a major accumulation below Mr=20,000. Monoclonal antibodies of appropriate specificity offer the potential for separating antigenically distinct subpopulations of muscle AChR (e.g. those of innervated and noninnervated muscle.).
|Original language||English (US)|
|Number of pages||4|
|Journal||Journal of Biological Chemistry|
|State||Published - 1980|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology