RESUMEN
There are three different test generations of enzyme-linked immunosorbent assays (ELISA) for the detection of human immunodeficiency virus (HIV) infection, depending on whether virus lysate, recombinant proteins or synthetic peptides are used as solid phase antigen. Four different assays, i.e., three sandwich ELISAs and one competitive test, were used to demonstrate differences between the three systems with regard to the content of different diagnostically relevant virus proteins. The sensitivities and specificities of these assays were compared by using 312 anti-HIV positive sera and 500 sera of healthy blood donors. The highest sensitivity and specificity were achieved by the competitive ELISA based on recombinant proteins, and by the sandwich ELISA based on synthetic peptides.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Infecciones por VIH/diagnóstico , Western Blotting , Epítopos/análisis , Anticuerpos Anti-VIH/análisis , Anticuerpos Anti-VIH/inmunología , Humanos , Sensibilidad y EspecificidadRESUMEN
A sensitive non-isotopic assay for specific detection of reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) is described using 5-bromo-2'-deoxyuridine triphosphate (BrdUTP) instead of tritiated thymidine triphosphate. After the RT reaction the template primer is degraded by alkaline hydrolysis. Single-stranded poly.(BrdU) is detected in an immunoenzymometric assay using monoclonal anti-BrdU antibodies. The specificity of the assay is demonstrated by the isolation of RT from virus lysate by an insolubilised monoclonal anti-HIV-1 RT antibody prior to the RT reaction. Immunological RT binding leads to a tenfold increase in analytical sensitivity since substances inhibiting the RT reaction can be removed. This non-isotopic assay is some 30 times more sensitive than the classical radioisotopic RT assay. In terms of RT determination, however, there is a good correlation between these tests (r = 0.96). Several filtrations are no longer necessary to remove non-incorporated nucleotides. The test can be adapted to microtitre plates and hence is easy to automate.
Asunto(s)
VIH-1/enzimología , ADN Polimerasa Dirigida por ARN/aislamiento & purificación , Anticuerpos Monoclonales/inmunología , Nucleótidos de Desoxiuracil , Hidrólisis , Técnicas para Inmunoenzimas , ADN Polimerasa Dirigida por ARN/inmunología , Sensibilidad y Especificidad , Moldes Genéticos , Timidina/metabolismoRESUMEN
A highly conserved region of the transmembrane protein gp41 of the HIV-1 was found in which the amino acid sequence 606 to 620 was characterized as an immunodominant region. The binding behaviours of a human monoclonal antibody and of polyclonal specific antibodies in sera of HIV-infected persons to this particular sequence have been studied. In two sandwich-type ELISAs with synthetic peptides as antigen representing this region, sera of 312 HIV-infected persons at all stages could be clearly discriminated against 500 anti-HIV antibody negative sera of healthy blood donors.
Asunto(s)
Anticuerpos Anti-VIH/inmunología , Proteína gp41 de Envoltorio del VIH/inmunología , VIH-1/inmunología , Secuencia de Aminoácidos , Anticuerpos Monoclonales/inmunología , Sitios de Unión , Donantes de Sangre , Epítopos/análisis , Humanos , Datos de Secuencia Molecular , Mapeo PeptídicoRESUMEN
Peptides of HIV sequences are significant for antibody screening systems, and because of the limited number of amino acids they have to represent immunodominant regions of the virus proteins in order to maintain sensitivity. We detected, in a region of the outside loop of the transmembrane protein gp41 of the human immunodeficiency virus HIV-1 (amino acid 586-620), two immunodominant sequences which are distinct from each other. Whereas in the first immunodominant region the sensitivity and specificity of ELISA were inadequate, a neighbouring region is well suited for use as antigen for an anti-HIV screening ELISA.
Asunto(s)
Antígenos VIH/inmunología , VIH-1/inmunología , Proteínas del Envoltorio Viral/inmunología , Secuencia de Aminoácidos , Animales , Ensayo de Inmunoadsorción Enzimática , Epítopos/análisis , Epítopos/inmunología , Anticuerpos Anti-VIH/análisis , Anticuerpos Anti-VIH/inmunología , Antígenos VIH/análisis , Proteína gp41 de Envoltorio del VIH , VIH-1/análisis , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/inmunología , Proteínas del Envoltorio Viral/análisisRESUMEN
The ELISA is the established screening technique for the detection of antibodies directed against HIV. The first generation assays, mostly based on the sandwich principle, employed purified virus from cell culture and gave both false-positive and false-negative results. Sandwich-type assays preferentially detect IgG antibodies, require a high serum dilution and are two-step procedures. In order to detect an immune response as early as possible after infection anti-HIV antibodies of the IgM class should also be measured. To this end a competitive ELISA has been developed using a solid phase-adsorbed recombinant HIV envelope protein and an enzyme-labelled human monoclonal antibody. This detects both IgM and IgG antibodies, the results are available within 1 h and a serum predilution is not necessary.
Asunto(s)
Anticuerpos Monoclonales , Ensayo de Inmunoadsorción Enzimática/métodos , Anticuerpos Anti-VIH/análisis , Proteínas del Envoltorio Viral/inmunología , Unión Competitiva , Relación Dosis-Respuesta Inmunológica , Epítopos , Seropositividad para VIH/diagnóstico , Humanos , Proteínas Recombinantes/inmunologíaRESUMEN
Intermediate filament keratin is regarded as a good marker for epithelial and mesothelial tumors. In the intracranial and intraspinal spaces keratin has been demonstrated only in the endocrine cells of the adenohypophysis, squamous epithelial islands in the pars tuberalis of the hypophysis and in the choroid plexus epithelium. Since gliomas and meningiomas do not express keratin, this marker provides an additional help for differentiating between primary and secondary CNS tumors. Indirect immunofluorescence using an anti-keratin serum was used in a retrospective search for keratin in 80 tumors of the cranium and intraspinal space. Of the primary CNS tumors keratin positivity occurred in craniopharyngiomas, epidermoid tumors, pituitary adenomas, chordomas, a plexus papilloma as well as in the majority of germ cell tumors. Only 3 renal cell carcinoma metastases of 21 metastatic epithelial cell tumors (7 bronchial carcinomas, 6 breast cancers, 6 renal carcinomas, 1 rectum carcinoma, 1 cervix carcinoma) were keratin-negative. Similar findings were made in two melanoma metastases which we examined, whereas in a seminoma metastasis a few keratin expressing cells were found. Primary CNS tumors such as myxopapillary ependymomas, medulloepitheliomas, malignant meningiomas and paragangliomas which are often difficult to distinguish from these metastases proved to be keratin negative.
Asunto(s)
Neoplasias Encefálicas/patología , Queratinas/análisis , Neoplasias de la Médula Espinal/patología , Neoplasias Encefálicas/clasificación , Neoplasias Encefálicas/secundario , Diferenciación Celular , Células Epiteliales , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Masculino , Metástasis de la Neoplasia , Neoplasias de la Médula Espinal/clasificación , Neoplasias de la Médula Espinal/secundarioRESUMEN
Rabbits were immunized with 10 nm filaments of a mixture of cytokeratins which has been isolated from human heel callus material and reconstituted to filaments in vitro. The antisera to keratins (ASK) have been tested histologically at fixed and unfixed tissue samples by means of the indirect immunofluorescence and PAP technique. The ASK recognized specifically only the epithelial cells of skin, of the mucous membranes of mouth and digestive tract, of salivary glands, sweat gland and mammary gland, but did not react with hepatocytes or kidney cells. The following tumors, tested till now, reacted with the antikeratin antisera: epithelial and lymphoepithelial carcinomas of skin, mouth and digestive tract, carcinomas of salivary glands, mammary gland and thyroid gland, adamantinoma, basalioma of skin, and metastases from carcinomas.