Nász I

Concentrated, digestible DNA after hydroxylapatite chromatography with cetylpyridinium bromide precipitation

A method for the direct extraction of the DNA from the unfavorable phosphate eluant of hydroxylapatite chromatography is described. The DNA--reversibly precipitated with the cationic detergent cetylpyridinium bromide--can be subjected to further enzymatic manipulations within minutes. This method is applied to the rapid separation of pBR322 plasmid from the chromosomal DNA.

Molecular genetics of PKU in Eastern Europe: A nonsense mutation associated with haplotype 4 of the phenylalanine hydroxylase gene

Phenylketonuria (PKU) is a genetic disorder secondary to a deficiency of hepatic phenyalanine hydroxylase (PAH). Several mutations in thePAH gene have recently been reported, and linkage disequilibrium was observed between RFLP haplotypes and specific mutations. A new molecular lesion has been identified in exon 7 of thePAH gene in a Hungarian PKU patient by direct sequencing of PCR-amplified DNA. The C-to-T transition causes the substitution of Arg243 to a termination codon, and the mutant allele is associated with haplotype 4 of thePAH gene. The mutation is present in two of nine mutant haplotype 4 alleles among Eastern Europeans and is not present among Western Europeans and Asians. The rarity of this mutant allele and its restricted geographic distribution suggest that the mutational event occurred recently on a normal haplotype 4 background in Eastern Europe.

Immunofluorescent studies on circulating lymphocytes in oral mucosal diseases.

SummaryCirculating lymphocytes of patients with recurrent herpetic, aphthous and other oral mucosal diseases, as well as of persons with intact oral mucosa were examined with direct immunofluorescence method using HVH and adenovirus hyperimmune conjugated sera. Some of the circulating lymphocytes showed a specific fluorescence both in patients and in persons with intact oral mucosa. Increased herpes-positivity was observed only in erythema exsudativum multiforme and increased adeno-positivity in patients with recurrent aphthous stomatitis, especially in cases with constantly recurring aphthae.The possible pathologic role of the circulating virus-antigen-carrier lymphocytes is presumed.ZusammenfassungEs wurden Untersuchungen mit der direkten Immunfluorescenzmethode bei Kranken mit rezidivierenden herpetischen, aphthösen und anderen Mundschleimhautkrankheiten mit Antiherpes- und Antiadenoviruskonjugaten durchgeführt. Sowohl bei den Patienten wie bei den Personen mit intakter Mundschleimhaut wurde eine spezifische Fluorescenz beobachtet. Erhöhte Adenopositivität wurde bei Patienten mit rezidivierenden, besonders mit ständig rezidivierenden Aphthen, erhöhte Herpespositivität bei Kranken mit Erythema multiforme beobachtet.Die mögliche pathologische Rolle der virusantigen-haltenden Lymphocyten wurde angenommen.

Grouping of monoclonal antibodies to adenovirus hexons by their cross-reactivity

SummaryThirty two mouse ascitic fluids containing monoclonal antibodies (MAbs) directed against the hexon of human adenovirus type 1 were examined. The type and degree of cross-reactivity (CR) of the MAbs were determined by ELISA and hemagglutination methods with 10 heterologous hexon types. The similarity and the dissimilarity of the MAbs was also characterized by the correlation coefficient calculated from their reactivity values. On the basis of these, the 32 MAbs could be divided into five groups and fourteen individual MAbs, which altogether recognized 19 distinct epitopes. One of the recognized epitopes is the genus specific epitope of adenovirus hexons; the others are interspecies specific ones which can be found on the surface of the different hexon types in characteristic, mosaic-like combinations. The type and degree of CR of the MAbs lead to the conclusion that there exists a close antigenic relationship among the members of subgenus C of human adenoviruses and there is also a definite antigenic relationship between subgenera C and D. Hexons belonging to the oncogenic subgenera A and B display a much looser antigenic relationship with subgenus C.

Electron microscopic studies on the crystallization of adenovirus hexon capsomers.

Highly purified soluble adenovirus type 1 hexons were crystallized by dialyzing against 0.5 M acetate buffer, pH 4.5, for 3 days. Crystals of tetrahedral shape were formed after storage at 4 degrees within 1--10 days and ranged in size from 0.01 to 0.1 mm. In early phase of crystallization, diffusely dispersed hexons were observed by electron microscopy. Later, continuous monolayers of two-dimensional hexon paracrystalline arrays developed. Direct analysis of the electron micrographs and their optical diffraction patterns showed that the structure of the dense two-dimensional crystalline arrays of hexon capsomers was characterized by slightly skew and irregular hexagonal packing. Multilayered microcrystals with regular linear internal structure were also found. In addition, unique forms of aggregated hexons, such as holey hexon lattice and filamentous forms, were also found.

Comparative studies of adenovirus hexon antigens

Purified soluble hexon antigens of type 5, 7, and 8 adenoviruses, belonging to three different subgroups have been studied. The electrophoretic mobility of the three hexons was found to be different in immunoelectrophoresis, and the calculation of absolute electrophoretic mobility gave different values too. Significant dissimilarities were observed in the elution patterns of the hexons in anionexchange chromatography. However, no essential differences between the molecular weights of the hexons were demonstrable, they appeared to be with all three types about 300,000. Determination of the smallest hexon quantities demonstrable by immunodiffusion, immuno-osmophoresis and complement fixation (CF) reaction, showed the CF reaction to be the most sensitive. 0.02 μg of type 5 and type 8 hexons and 0.08 μg of type 7 hexon were detectable using homologous anti-hexon sera. The anti-hexon sera neutralized the cytopathic effect of the homologous adenovirus types only. In the immunodiffusion experiments any two antigens of the three proved to be identical, examined with the third (heterologous) serum. Using homologous serum they revealed only a partial identity. Absorption experiments were successful only when homologous antigen was used, even excess amount of heterologous hexons failed to remove the antibodies from the anti-hexon sera. These results demonstrate the presence of closely connected group-, subgroup or type-specific components in the hexons of adenoviruses, and definite differences in the physico-chemical properties of hexons belonging to different serotypes.

Delineation of antigenic determinants of adenovirus hexons by means of monoclonal antibodies.

60 hexon-specific mouse hybridoma clones were selected by using crystallized hexon capsomers of human adenovirus type 1 as immunizing and selecting antigen. The reactivities of the monoclonal antibodies were tested with purified hexon preparations from 10 human adenovirus types by enzyme-linked immunosorbent assay and passive hemagglutination techniques. The reactivity patterns delineated a total of 41 adenovirus-type-1-related determinants present on a number of heterologous hexons in different interspecies combinations. The pattern of interspecies specificities did not coincide with the current grouping of adenoviruses into subgenera.

Antigenic homogeneity among the adenovirus hexon types of subgenus C

SummaryAntigenic relationships of hexons of human adenovirus (Ad h) types 1, 2, 5 and 6 of subgenus C were studied with 61 monoclonal antibodies (MAbs) raised against human Ad h1, Ad h35 and bovine adenovirus 2. The reactivity pattern (RP) and the titers of the MAbs were determined in indirect ELISA. In previous experiments with hexons of different subgenera 49 MAbs displayed numerous different intertype specificities besides genus specific and type specific ones. With the four hexon types of subgenus C all MAbs gave identical RPs except the type specific ones. Data reveal the existence of a remarkable homogeneity in the antigenic structure among the hexon types of subgenus C defined by the presence of identical or closely related intertype specific epitopes on the surface of the hexons. The possible significance of the results in the experimental gene therapy is discussed.

Determination of different antigenic sites on the adenovirus hexon using monoclonal antibodies

SummaryEighteen mouse ascitic fluids containing monoclonal antibodies (MAbs) directed against crystallized hexon of adenovirus (AV) type I were used to map the antigenic structure of the capsomer in reciprocal competitive binding ELISA. With the help of peroxidase-labelled MAbs at least nine epitopes (epitope clusters) located on three distinct antigenic sites were identified on the hexon. Epitope on antigenic site I recognized by two MAbs could be the genus specific antigenic determinant based on the broad reactivity patterns of the MAbs. Epitopes on the antigenic site II recognized by fifteen MAbs could be divided into seven epitope clusters according to the competition patterns. Antigenic site III recognized by one MAb completely differs from the antigenic site I and on the basis of one-way blocking with all the MAbs specific for antigenic site II, should be also different from the latter one. The data suggest that the seven epitope clusters of antigenic site II contain partially overlapping epitopes and may be a part of a large single immunodominant antigenic region on AV1 hexon as well as on hexons of heterologous types.

Differentiation of adenovirus hexon epitopes with monoclonal antibodies by gel diffusion assays

Nine monoclonal antibodies (MAbs) directed against crystallized human adenovirus type 1 (Ad h 1) hexon were tested with purified homologous and heterologous hexon preparations by gel diffusion. Six MAbs formed a single line with the homologous hexon in a 2-well pattern, and 3 MAbs formed lines only in biclonal combinations with an appropriate MAb. All of the 6 precipitating MAbs formed a continuous line of complete identity when tested simultaneously against homologous and different heterologous hexons. With Ad h 1 hexon a line of double partial identity (double spur) was formed when some pair combinations of 2 MAbs were placed in 2 juxtaposed wells. Other MAbs in the adjacent wells formed a line of identity. The MAbs could be divided into 2 antibody groups (groups A and B) based on this phenomenon. Members of antibody groups A and B apparently identified 2 sterically distinct epitopes: one of them is presumably the genus-specific epitope of the hexons (group A) and the other(s) should be intertype-specific epitope(s). Thus, the gel diffusion method can be used for selecting pairs of MAbs for their specificity to sterically independent epitopes. Mixtures of 2 MAbs belonging to the different antibody groups formed double lines with Ad h 1 hexons. Members of group A showed some helper effect to the members of group B for their precipitin line formation.