Producción y caracterización de un anticuerpo monoclonal que marca selectivamente la población astrocitaria en el sistema nervioso central

Abstract

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The development of a monoclonal antibody that selectively labels the astrocitic population of the central nervous system in the three species tested (hurnan, monkey and rat) is described. The tissue used as immunogen conslsted in a sector of the human hippocampus (CAI) from a brain removed shortly after death. The neuropathological examination in this case revealed an congophilic angiopathy. The tissue was frozen until homogeneization and intraperitoneal injection in 8 weeks old BALB/C mice, after which lymphocites from the spleen were fused with NS-1 myeloma cells devoid of the enzyme Hypoxantine Guanine Phosphoribosyl Transferase (HPQT (-) ) following standard procedures. The resulting hybridomas were tested by ELISA assays, immunofluorescence and immunohistochemical (PAP and ABC) techniques. The monoclonal antibody obtained was identified as an IgM and the Western blot analysis indicated that it recognizes a protein of 43,000 dalton. Immunohistochemistry was performed in 30 or 50 um sections of human temporal lobe perfused through the carotid arteries with a mixture of 4% paraformaldehyde and 0.02% picric acid. Monkey tissue (sectioned at 50 um) was obtained from a brain with the same fixation. Rat tissue followed the same protocol, although better results were seen after shifting to 1% paraformaldehyde and 1.25% glutaraldehyde (sections were 30 um thick). Paraffin embedded human tissue (fixed in either 10% buffered formalin or in 4% paraformaldehyde) was also tested. All immunoreactions were intensified in a solution of osmium tetroxide and thiocarbohydrazide. The monoclonal antibody gave best results at a dilution of 1: 50-100, where it gave a sharp labeling of both fibrous and protoplasmic astocytes up to the most delicate processes, both in white and the grey matter. Rat tissue fixed in only 4% paraformaldehyde resulted in less optimal labeling, mostly restricted to limiting glia and Bergmann glia. The other fixation prolocol (1% paraformaldehyde and 1.25% glutaraldehyde) gave a labeling comparable to monkey and human tissue. Paraffin embedded tissue was positive and similar to that obtained in frozen tissue, only when used the 4% paraformaldehyde fixation, but not when it was fixed in 10% formalin. Therefore, this monoclonal antibody has high specificity for the ashocitic population in the nervous system of mammals and it can be useful in a variety of studies of the normal and pathological situations of the nervous system.