Monoclonal antibody analysis of keratin expression in the central nervous system.

A monoclonal antibody directed against a 65-kDa brain protein demonstrates an epitope found in keratin from human epidermis. By indirect immunofluorescence, the antibody decorates intracytoplasmic filaments in a subclass of astrocytes and Purkinje cells of adult hamster brain. Double-label immunofluorescence study using antibody to glial fibrillary acidic protein and this antibody reveals the 65-kDa protein to be closely associated with glial filaments in astrocytes of fetal mouse brain cultures. Immunoblot analysis of purified human epidermal keratin and hamster brain homogenate confirms the reactivity of this antibody to epidermal keratin polypeptides. All the major epidermal keratins were recognized by this antibody. It did not bind to the remaining major intermediate filament proteins. These findings suggest that monoclonal antibody 34C9 recognizes a cytoskeletal structure connected with intermediate filaments. In addition, the monoclonal antibody demonstrates that epidermal keratins share an epitope not only among themselves but also with a "neural keratin."

Loop arrays in mouse brain demonstrated with antisera to cytokeratins and monoclonal antibodies to several classes of intermediate filaments: strain differences and developmental expression.

Some monoclonal antibodies raised against mouse brain antigens display a novel loop array apparently localized within the cytoplasm of neurons in fresh frozen sections of adult mouse brain. By indirect immunofluorescence, these loops are detectable in the cerebral cortex, thalamus, brainstem, and are particularly striking in association with pyramidal neurons of the hippocampus. The loops are also seen with polyclonal antibodies to the cytokeratin class of intermediate filaments. The antibodies which react with these loops also react with ependymal cells. Western blot analysis of crude insoluble cytoskeletal components of mouse brain with antibodies of cytokeratins confirm the presence of reactive bands in the range of 40-60 kdalton, appropriate in molecular weight for this class of cytoskeletal filaments. This evidence suggests that the loops share antigenic determinants with non-neural cytokeratins. During development, immunoreactive structures are first seen as small punctate or curvilinear profiles, which change into a loop array at approximately 14 days postnatal age in several mouse strains. However, in 8 of 15 different mouse strains, these immature punctate profiles remain without morphological alteration to loops throughout adult age. The F1 crosses between strains with and without the loops develop loops, but on average they are of smaller size than in the positive parent.

The sequential development of spongiform change and gliosis of scrapie in the golden Syrian hamster.

The lesion profiles of spongiform change and gliosis in the hamster occurring after intracerebral (IC) inoculation of scrapie virus, are calculated and compared to the lesion profile of spongiform change of scrapie in mice and of scrapie and Creutzfeldt-Jakob disease (CJD) in the squirrel monkey. The profile of scrapie in hamsters differs considerably from that of a closely related strain of scrapie in mice, and both differ from scrapie and CJD in the squirrel monkey. These differences emphasize the effect of the host on the distribution of pathological changes in these unconventional virus infections. The sequential development of the lesions in the hamster shows that the earliest changes are detectable before the onset of clinical disease 49-57 days after inoculation, as assessed by light microscopy. Gliosis is detectable by indirect immunofluorescence 35-39 days after inoculation by use of a monoclonal antibody directed against astrocytes.

Incorporation of P0 protein into liposomes: demonstration of a two-domain structure by immunochemical and PAGE analysis.

The amphiphilic nature of P0, the major glycoprotein of peripheral nerve myelin, has been suggested previously. In the present study, purified P0 from human peripheral nerve myelin was incorporated into an artificial lipid bilayer consisting of dimyristoyl lecithin and cholesterol. The liposomes were fractionated on a sucrose gradient. The continued expression of P0 antigenicity by the liposomes was shown by specific complement consumption with a multivalent antiserum against P0 or with an IgM monoclonal antibody. Both antibodies recognized P0 expressed on the surface of peripheral nerve myelin and the P0 liposomes. P0 liposomes and peripheral nerve myelin treated with trypsin lost the surface determinant that reacted with the monoclonal antibody. Analysis of the trypsin-treated liposomes and peripheral nerve myelin by polyacrylamide gel electrophoresis revealed molecular weights for this protein of 19,500 and 20,500, respectively. Similar treatment of the P0 in the fluid phase resulted in many smaller fragments. These results indicate that P0 consists of two domains, a hydrophilic domain accessible to trypsin digestion and a hydrophobic domain, which is potentially trypsin-sensitive, but shielded by the lipid bilayer. Binding studies with an anti-P0 monoclonal antibody and polyacrylamide gel analysis of the lipid-shielded P0 fragment in liposomes and peripheral nerve myelin suggest that the orientation of the protein in the liposome is similar to that in peripheral nerve myelin.

Monoclonal antibodies specific for Hantaan virus.

Six hybridoma cell line producing monoclonal antibodies to Hantaan virus were established by fusion of NS-1 mouse myeloma cells with spleen cells of mice immunized with Hantaan virus strain 76-118. The specificity of these monoclonal antibodies was established by immunoblotting analysis and immunofluorescence. Five of the clones reacted with antigens on the cell surface and in the cytoplasm, and one clone reacted with a determinant expressed only in the cytoplasm of the infected cells. Two of the clones produced antibodies that reacted with a Mr 50,000 polypeptide in virus-infected cellular extracts and purified virus preparations. The monoclonal antibodies were used to examine the antigenic relationship among Hantaan virus strains and between Hantaan virus and Prospect Hill virus and the virus of nephropathia epidemica. Three antibodies were capable of distinguishing between the Lee strain and the 760-118 strain of Hantaan virus and three additional antibodies reacted with determinants shared by both virus strains. None of the six reacted with Prospect Hill virus or the virus of nephropathia epidemica.

Monoclonal antibody specific for myelin glycoprotein P0: derivation and characterization.

A monoclonal antibody was produced against the major structural glycoprotein (P0) of human peripheral nervous system myelin. The hybridomas were generated by fusion of mouse myeloma line NS-1 with spleen cells of C3H mice immunized with purified human peripheral nervous system myelin. Hybridomas were screened by a two-step solid-phase radioimmunoassay, with P0 adsorbed on microtiter plates and with addition of 125I-labeled rabbit anti-mouse IgG as the second step. One derived clone, designated 41G10, bound P0 in the radioimmunoassay 4-fold over the background value obtained by using bovine serum albumin as the negative control antigen. Clone 41G10 was shown by immunofluorescence to bind to frozen sections of human intercostal nerve. Diffuse fluorescent staining occurred uniformly over the entire myelin sheath. The cylindrical axons were unstained. The same pattern of immunofluorescence was noted on rat, hamster, mouse, and rabbit sciatic nerve. Immunofluorescence was abolished when 41G10 was absorbed by P0. The monoclonal antibody 41G10 was absorbed by P0. The monoclonal antibody 41G10 is of the IgM class and activated complement in the presence of myelin vesicles or P0 liposomes but not in their absence.

Monoclonal antibodies to central nervous system antigens.

Thirty monoclonal antibodies produced by mouse hybrid myelomas which react with antigens in hamster or mouse nervous system tissues were derived. Using these antibodies as probes with indirect immunofluorescence and immunoperoxidase techniques, we can selectively identify by morphological criteria many of the structural components of the brain seen at a light-microscopic level, including the neutrophil, neuronal cytoplasm, nuclei, axons, astrocytes and ependyma. Some of the antibodies display cytoskeletal and filamentous structures, including intermediate filaments, microfilaments, neurofilaments, glial and ependymal filaments. The specificity to neural tissue components of these hybridoma antibodies was assessed by their reactivity to mouse and hamster non-neural tissues and selected mouse, hamster, rat and human cultured cell lines. Of the 30 clones analyzed, specificity ranged from 3 clones reacting only with grey matter of mouse and hamster brain, one clone reacting only with axons in animal and human brain, to 19 clones reactive with both neural and non-neural tissue components.