Biomedical subjects
Publications and source records attributed to J Burckard.
The biotin-avidin detection system was used in direct and indirect ELISA for detecting a broad range of serologically related tobamoviruses. When compared to standard ELISA procedures that use antibodies labelled with alkaline phosphatase, the biotin-avidin system increased the assay sensitivity and allowed a wider range of related viral serotypes to be detected.
Yolk immunoglobulins obtained from hens immunized with human rotavirus and two plant virus antigens were used in quantitative microcomplement fixation tests. Difficulties inherent in the use of avian complement were overcome by utilizing a mixture of the C1 component of chicken complement and guinea-pig complement devoid of its own C1. The method is suitable for detecting small quantities of viral antigen and antibody and for detecting antigenic activity present on peptide fragments of viral proteins.
The detection of human rotaviruses by routine electron microscopy examination of stool specimens has been compared with the sensitivity of detection obtainable by three different immunoassays. These assays are: 1) immunosorbent electron microscopy (ISEM), which consists of the serological trapping of viruses on electron microscopy grids coated with protein A and specific viral antiserum; 2) an enzyme-linked immunosorbent assay (ELISA), in which the primary antibody is rabbit anti-rotavirus immunoglobulin, the secondary antibody is chicken anti-rotavirus immunoglobulin extracted from egg yolk of immunized hens, and the indicator antibody is alkaline phosphatase-conjugated rabbit anti-chicken immunoglobulin; 3) counterimmunoelectrophoresis (CIE). A total of 63 stool specimens from infants with gastroenteritis were examined. Of these, 23 and 24 specimens were found to contain rotavirus by electron microscopy and CIE, respectively. When scored by ELISA and ISEM, 37 and 39 were found to be positive, respectively. Confirmatory inhibition assays were necessary to eliminate some false positive reactions in ELISA. Detection of human rotaviruses in stools by ISEM is as sensitive as by ELISA, but in weakly positive specimens, ISEM offers the additional advantage of a direct visual demonstration of the presence of the aetiological agent.
The suitability of two ELISA procedures for detecting serologically closely related as well as distantly related cucumoviruses has been compared. When antibodies to a single strain were used, closely related strains of cucumber mosaic virus could be detected by both direct and indirect ELISA. However, only the indirect ELISA procedure was capable of detecting distantly related viruses such as tomato aspermy, peanut stunt and cucumber mosaic viruses.
Those non-histone chromosomal proteins which are easily extractible from chick erythrocytes differ substantially from proteins similarly extracted from other tissues of various species. Although a protein P1 was isolated along with histone H1 by extraction of calf thymus chromatin with HC1O4, the same procedure did not extract this protein from chick erythrocyte chromatin of either normal or regenerating blood. Likewise , non-histone proteins extracted with 0.35 M NaCl from calf thymus differed from those of normal chick erythrocytes, which were qualitatively identical but quantitatively inferior to those of regenerating blood. The major protein of about 25 000 molecular weight, totally extracted with 0.35 M NaCl from calf thymus, was not found in chick erythrocyte chromatin, but rather another major protein of about 35 000 molecular weight was partially extracted from erythrocytes.
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Three peptides corresponding to residues 28-40, 138-154 and 380-387 of the coat protein of tomato bushy stunt virus (TBSV) were synthesized by the solid phase method and used to raise specific antibodies. These antibodies were used to follow the conformational changes that occur when TBSV particles swell under slightly alkaline conditions. Peptides 28-40 and 380-387 were found to correspond to continuous epitopes in the dissociated viral protein as well as in both compact and swollen virions. The region 138-154, which is also a continuous epitope of the monomeric protein, became accessible to antibody binding in the virion only when the particles were in the swollen state.
Eight peptides encompassing neutralization antigenic site 1 of poliovirus type 1 (residues 93-103 of VP1) were synthesized in linear or cyclized form and used to immunize rabbits. The resulting anti-peptide antibodies were tested for their ability to react with linear peptide 95-104, with infectious virus D-particles and heated C-particles and for their capacity to neutralize poliovirus infectivity. A good correlation was observed between the ability of different peptide antisera to immunoprecipitate D-particles and neutralize virus infectivity. The peptides that induced a neutralizing antibody response in the highest number of immunized animals contained flanking residues 104-115 in addition to the 93-103 residues of the epitope. However, a high neutralizing antibody titre was also obtained in two of ten animals immunized with peptide 93-104 cyclized via an amide bond between Asp93 and Lys103. It seems, therefore, that, at least in rabbits, the T-cell epitope recently identified in residues 103-115 of VP1 need not be present in the peptide immunogen in order to obtain poliovirus-specific neutralizing antibodies.