RESUMO
Antibodies raised against deoxyadenylate and deoxycytidylate were found to react with double stranded DNA as assessed by highly sensitive avidin-biotin microELISA. The binding was specific as it was completely inhibited by the homologous hapten. The antibodies did not react with tRNA and rRNA. These antibodies were also shown to react with supercoiled and relaxed forms of pBR322 DNA as demonstrated by gel retardation assay.
Assuntos
Anticorpos , DNA , Nucleotídeos de Desoxiadenina/imunologia , Desoxicitidina Monofosfato/imunologia , Nucleotídeos de Desoxicitosina/imunologia , Complexo Antígeno-Anticorpo , DNA/imunologia , Ensaio de Imunoadsorção Enzimática , Epitopos/análise , Cinética , MicroquímicaRESUMO
A radioimmunoassay (RIA) capable of quantitating dCTP in femtomolar amounts in cell extracts has been developed, and applied to human fibroblast cell lines and L5178Y mouse lymphoma lines. Cross reactivity of the antibody with CTP, though low (2.7%) has necessitated pre-RIA removal of CTP by either boronate affinity gel chromatography or sodium periodate oxidation. Fractions from the boronate gel column or aliquots of NaIO4-treated cell extract are quantitated directly by the RIA. Recovery of extracted dCTP standard taken through the entire procedure is quantitative and results are reproducible. Due to the high sensitivity of the quantitation step, dCTP can be accurately measured in relatively small numbers of cells--about 10(4) cells.
Assuntos
Nucleotídeos de Desoxicitosina/análise , Radioimunoensaio/métodos , Animais , Cromatografia de Afinidade , Reações Cruzadas , Citidina Trifosfato/imunologia , Citidina Trifosfato/isolamento & purificação , Nucleotídeos de Desoxicitosina/imunologia , Fibroblastos/análise , Humanos , Leucemia L5178/metabolismo , Camundongos , Microquímica , Ácido PeriódicoRESUMO
Antibodies raised in rabbits against deoxyguanylate and deoxycytidylate bind to 3H-lambda double stranded DNA and the binding is base specific. The concentrations of antibody populations that bind to double stranded DNA are much less than those binding to denatured DNA. Due to their low concentrations, these antibodies were not detected in earlier studies. These antibodies are expected to be useful to probe the conformational flexibilities of double stranded DNAs.
Assuntos
Anticorpos , DNA Viral , Desoxicitidina Monofosfato/imunologia , Nucleotídeos de Desoxicitosina/imunologia , Nucleotídeos de Desoxiguanina/imunologia , Complexo Antígeno-Anticorpo , Bacteriófago lambda , DNA de Cadeia Simples , HaptenosRESUMO
Antibodies to deoxycytidylate (dpC) were elicited in rabbits using a thyroglobulin (Tg) conjugate of dpC. The specificity of the antibodies was determined by measuring the inhibition of the binding of [3H]dpC to the antibodies by various non-radioactive nucleotides or derivatives. The antibodies were found to distinguish dpC from pC and DNA from RNA, probably due to their specificity for the conformation of the deoxycytidylate.
