RESUMEN
Telomerase is an important tumor marker but few antibodies to the enzyme have been described or used without difficulty in histochemical detection. Here we report specific detection of the enzyme in cell and tissue preparations using a new monoclonal antibody (mAb 476) and a new antigen-retrieval buffer (Enhancing buffer). When used to detect telomerase under normal immunostaining conditions in HL-60 cells or tissue sections of hepatocellular carcinoma or metastatic choriocarcinoma, unexpectedly, the antibody stained the cytoplasm rather than the nucleus. Nuclear staining, however, was revealed using the Enhancing buffer. Since other nuclear antigens in the HL-60 cell could be stained both ordinarily and in the Enhancing buffer, nuclear telomerase appears to be shrouded by the nuclear matrix or blocked by accessory proteins. The cytoplasmic activity seen in normal buffer but absent largely from the Enhancing buffer may be an artifact or the nascent, "naked" enzyme. With a known cytoplasmic antigen (proteinase-3) chosen arbitrarily for comparison, the antigenicity was found enhanced, instead, by the Enhancing buffer. The mode of action of the Enhancing buffer differs from that of microwave irradiation or the signal amplification (CSA) used by some investigators. The latter was found to enhance the cytoplasmic reactivity rather than the nuclear reactivity of mAb 476.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Biomarcadores de Tumor/inmunología , Núcleo Celular/enzimología , Telomerasa/inmunología , Animales , Especificidad de Anticuerpos , Tampones (Química) , Carcinoma Hepatocelular/enzimología , Carcinoma Hepatocelular/patología , Núcleo Celular/inmunología , Coriocarcinoma/enzimología , Coriocarcinoma/secundario , Citoplasma/enzimología , Citoplasma/inmunología , Epítopos , Células HL-60 , Humanos , Inmunohistoquímica , Neoplasias Intestinales/enzimología , Neoplasias Intestinales/secundario , Neoplasias Hepáticas/enzimología , Neoplasias Hepáticas/patología , Ratones , Ratones Endogámicos BALB C , ARN Mensajero , Telomerasa/genéticaRESUMEN
OBJECTIVE: To investigate why the serum of a pediatric patient with systemic lupus erythematosus was persistently (>30 months) and strongly positive for antibodies to double-stranded DNA (dsDNA) as revealed by enzyme-linked immunosorbent assay (ELISA), but yielded negative results on the antinuclear antibody test (HEp-2 immunofluorescence [IF]). METHODS: The patient's antibodies were isolated on dsDNA and single-stranded DNA (ssDNA) supports, which were then examined by dsDNA ELISA and HEp-2 IF. Tests included the use of various inhibitors to determine the fine specificity of the antibodies. Other tests performed included immunoblotting, immunoprecipitation, Crithidia luciliae IF, and neutrophil IF. RESULTS: The antibodies isolated from the dsDNA and ssDNA supports were similar, in that they were of the IgG type, bound well in the dsDNA ELISA, and recognized a normally hidden nucleolar RNA antigen in HEp-2 cells. With both the dsDNA ELISA and nucleolar antigens, inhibition studies revealed that the epitope recognized was guanosine 5'-triphosphate (GTP). Binding of the antibodies was better to GTP than to guanosine 5'-monophosphate or cytidylyl (3'-5') guanosine, and, in turn, was better than to guanosine, while N7-methylated GTP was unreactive. The antibodies did not bind to dsDNA present in solution or in HEp-2 or Crithidia cells, but bound transfer RNA well and recognized a cytoplasmic RNA antigen in neutrophils. CONCLUSION: A new problem in dsDNA ELISA is revealed in the occurrence of a hitherto-unknown and unusual buckling of the insolubilized DNA molecule, which, absent in dsDNA found in solution or in whole cells, presumably creates gaps of single-strandedness in the molecule. A new antibody specific for GTP is described in this patient, which may be clinically important.