Monoclonal antibodies were obtained against the membrane‐bound lysosomal enzyme β‐glucocerebrosidase (acid β‐glucosidase), which is deficient in Gaucher's disease.
BALB/c mice were immunized with homogeneous enzyme protein extracted from a sodium dodecyl sulphate/polyacrylamide gel. The mice were subsequently hyperimmunized with partially purified enzyme prior to fusion of spleen cells with myeloma cells. After fusion, 32 primary hybrid cell populations were obtained which continued to produce antibodies against β‐glucocerebrosidase after prolonged time of culture. All antibodies reacted with both native and denatured enzyme.
Four primary cell populations were subcloned and the antibodies produced were characterized. The antibodies were all four of the IgG1 subclass. Three of these antibodies bind to protein A whereas one does not. The results of binding assays indicated that three of the antibodies react with the same antigenic domain (epitope 1), but the fourth with a different one (epitope 2). Probably two antigenic determinants are present in epitope 1 since one of the antibodies with specificity for epitope 1 is inactivated after iodination by the chloramine‐T procedure whereas a second one is not.
The acid hydrolases alpha-glucosidase, beta-galactosidase, N-acetyl-beta-D-hexosaminidase, beta-glucocerebrosidase and cathepsin D were studied immunocytochemically in normal and mutant human cells using monoclonal and affinity-purified polyclonal antibodies. For light microscopy, Rhodamine or Fluorescein-labelled conjugates were used, and for electron microscopy protein A-gold conjugates were employed. With the double labelling procedure, it was found that in normal fibroblasts every lysosome contained all the enzymes studied. The method described also enabled us to demonstrate the presence or absence of mutant enzyme protein in fibroblasts derived from patients with a genetic lysosomal enzyme deficiency. Immunoreactive acid hydrolases or their precursor forms were found in the rough endoplasmic reticulum, the cisternae of the Golgi complex, Golgi associated vesicles and lysosomes. This is in agreement with the present concept that the Golgi complex plays an essential role in the processing and targeting of lysosomal enzymes.
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