RESUMEN
The analytical performance of a membrane-based immunoassay for the simultaneous detection and differentiation of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia (including Prevotella nigrescens) was investigated. Positive reactions were observed for 71 of 71 reference strains and recent oral isolates of A. actinomycetemcomitans, P. gingivalis, and P. intermedia. No cross-reactivity was observed with 39 other common oral and environmental species. The specificity of the test was unaffected by the presence of potential oral interferents including whole blood, white blood cells, mucin, saliva, toothpastes, and oral rinses. A proficiency test by dental professionals using a standardized set of unknown simulated samples yielded a sensitivity of 97% (116/120) and a 100% specificity (240/ 240). An additional group including dental professionals and high school students was shown to be 99% proficient (1385/1397) in distinguishing proper from improper test function when processing control samples with normal test devices and devices with simulated error conditions. Comparisons to a culture standard for 104 subgingival plaque samples collected from 26 adult periodontitis patients yielded > 98% specificity for each of the test bacteria. In addition, the detection threshold for the test was determined to be equivalent to 10(4) cultivable test bacteria when compared to the culture standard. The data indicate that this membrane immunoassay is a valid and easy-to-use method for the detection of A. actinomycetemcomitans, P. gingivalis, and P. intermedia in subgingival plaque, at levels above the detection threshold of the test.
Asunto(s)
Aggregatibacter actinomycetemcomitans/aislamiento & purificación , Placa Dental/microbiología , Ensayo de Inmunoadsorción Enzimática/métodos , Porphyromonas gingivalis/aislamiento & purificación , Prevotella intermedia/aislamiento & purificación , Adulto , Proteínas Bacterianas/análisis , Pruebas Enzimáticas Clínicas , Recuento de Colonia Microbiana , Reacciones Cruzadas , Humanos , Periodontitis/diagnóstico , Periodontitis/microbiología , Control de Calidad , Juego de Reactivos para Diagnóstico , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Especificidad de la EspecieRESUMEN
We recently identified, by means of cotransformation of LTK- cells, a region of the Epstein-Barr virus (EBV) genome (the BamHI K fragment) that encodes or induces an EBV nuclear neoantigen (EBNA) serologically related to the EBNA found in lymphoid cells carrying the entire EBV genome. We now find that a second EBV DNA fragment, BamHI M, is also able to give rise to cotransformed LTK- cells with stable expression of a nuclear antigen. The BamHI K and M fragments have no apparent DNA homology. Many human sera that are reactive to EBNA in Raji cells detect both antigens; however, certain anti-EBNA-positive human sera are discordant and react only with the BamHI M or only with the BamHI K nuclear antigen. Every Raji cell appears to express both "M" and "K" antigens; D98 Raji cells, a somatic cell hybrid, express only "K" antigen. The K antigen is found on metaphase chromosomes of LTK cells and Raji cells. The M-induced antigen is not located on chromosomes when the cells are in metaphase but is present as granules within the nucleus.
Asunto(s)
Antígenos Virales/genética , Núcleo Celular/inmunología , Transformación Celular Neoplásica , Herpesvirus Humano 4/inmunología , Animales , Linfoma de Burkitt , Línea Celular , Cromosomas/inmunología , Enzimas de Restricción del ADN , Técnica del Anticuerpo Fluorescente , Herpesvirus Humano 4/genética , Humanos , Células L/fisiología , Ratones , Hibridación de Ácido NucleicoRESUMEN
All cells that harbor the Epstein-Barr virus (EBV) genome contain a neoantigen in the nucleus (EBNA). By transfection we located a segment of the genome that encodes or induces an antigen serologically related to EBNA. The responsible genes are found in the 3.4-megaldalton BamHI fragment K of EBV DNA, specifically in the left 1.9 megadaltons represented by HindIII fragment I1. Mouse LTK- cells were cotransformed with recombinant plasmids, containing the herpes simplex virus thymidine kinase gene and either EcoRI fragment B or BamHI fragment of K of EBV DNA. The TK+ cells surviving in selective medium were cloned. About 50% of the clones expressed the neoantigen in every nucleus. These mouse cells were used as antigens in immunofluorescence tests. Antibody to the nuclear antigen was found in 30 human sera known to contain antibody to EBNA; it was not detected in 18 sera that did not have antibody to EBNA. Mouse cells expressing EBNA as the result of acquisition of cloned EBV DNA fragments should prove useful in the characterization of the structure of this antigen and as reagents for the diagnosis of EBV infections.
Asunto(s)
Antígenos Virales/genética , Clonación Molecular , ADN Viral/genética , Herpesvirus Humano 4/genética , Animales , Núcleo Celular/metabolismo , Células Clonales , Enzimas de Restricción del ADN , Células L/metabolismo , Ratones , Peso Molecular , Plásmidos , Timidina Quinasa/deficienciaRESUMEN
The preparation and properties of cell cultures derived from human placental tissue are described and their usefulness for the propagation of animal viruses and as "feeder layers" is indicated.