A two-step rapid procedure for the isolation of pure human IgM para-proteins at high yield from plasma samples is described. It involves gel filtration on Ultrogel ACA34 followed by anion exchange chromatography on DEAE-Sepharose CL6B. Covalently bonded human 7S IgM (IgMs) and non-covalently bonded human 7S IgM (IgMr) were prepared from myeloma 19S IgM by reduction with cysteine-HCl and dithiothreitol (DTT), respectively. The effect of varying the concentration of cysteine-HCl and the reduction time on the yield of was investigated. Both types of 7S IgM were found to be antigenically identical to 19S IgM when reacted with rabbit anti-human Fabmu, and antigenically deficient when reacted with a sheep anti-human mu-chain and two rabbit anti-human (Fc)5 mu antisera. Human myeloma 19S IgM, IgMs and IgMr were digested with the enzymes trypsin, pronase, proteinase K, elastase, mu-chymotrypsin and clostripain. The degradation products were isolated by gel filtration and characterised immunologically and by SDS-PAGE. The order of increasing susceptibility to degradation was IgMr >> IgMs > IgM. The major products were Fabmu and (Fab)2mu fragments. The latter fragment consisted of two (Fab)mu pieces and two Cmu2 domains disulphide bonded. The relative amount of each fragment produced varied with the enzyme and the IgM species used (19S IgM, IgMs or IgMr). The Fcmu fragment was mostly degraded into small peptides. (Fc)5mu digestion by some of the enzymes was also performed. Antibodies, specific for Cmul, Cmu2 and the Fcmu regions of human IgM mu-chain were prepared from sheep and rabbit antisera by immunosorption on insolubilised proteolytic fragments of IgM. The domain specificity of these antibodies was confirmed by passive haemagglutination, double antibody radioimmunoassay and immunodiffusion, using the appropriate proteolytic fragments as antigens. Rabbit and sheep anti-human IgM domain antisera were used in the rosette and indirect immunofluorescence tests for the detection of mIgM on the surface of two human lymphoid cell lines, CLA-4 and Daudi. For each type of cell lines, the Cmul, Cmu2 and light chain determinants were equally expressed on the same number of cells as determined by the rosette test, while the Fcmu determinants were expressed on a significantly lower number of cells. When the indirect immunofluorescence test was used, the Cmul, Cmu2 and Fcmu domains were expressed on a similar number of cells (for each cell line) with equal intensity. The proteolysis of mIgM on the surface of CLA-4 and Daudi cells with the enzymes, trypsin, mu-chymotrypsin, elastase, pronase, proteinase K and papain was monitored with the rosette and indirect immunofluorescence tests. None of the enzymes completely removed mIgM from the cell surface, even when used at 1mg/ml for 1hr. Papain caused reduction in the percentage of rosettes and staining intensity with anti-Cmul and Cmu2 antisera but not with anti-Fcmu, while elastase, proteinase K and pronase slightly reduced both with the three anti-domain antisera. Immuno- precipitation of fragments released and those remaining, on the cell surface seem to suggest the release of Fabmu fragments by papain, pronase, elastase and proteinase K and (Fab)2mu fragments by elastase and pronase. The Fey fragment left on the cell surface being degraded in all the cases except with papain.
|Date of Award||1982|