Enzymatic and immunological characterization of the Mycobacterium fortuitum complex.
(9/8142)
The arylsulfatase isozymes of Mycobacterium fortuitum, M. peregrinum, M. chelonei subsp. chelonei, and M. chelonei subsp. abscessus were examined to determine the isozymal and immunological relationship among the members of the M. fortuitum complex. Cell extracts were subjected to electrophoresis on agarose and polyacrylamide gel, and arylsulfatase activity was localized using beta-naphthyl sulfate as substrate. Unique zymograms were produced for M. fortuitum, M. peregrinum, and M. chelonei which were characteristic for each species. The immunological relationship among the sulfatases was assayed by using immunodiffusion and immunoelectrophoresis followed by sulfatase staining for the enzyme. One of the isozymes of M. fortuitum and M. peregrinum cross-reacted, showing immunological identity. Antisera to sulfatases of M. fortuitum and M. peregrinum did not react with sulfatases of M. chelonei. The characterization of sulfatase isozymes in extracts of organisms in the M. fortuitum complex suggests the division of the M. fortuitum complex into two species, M. fortuitum and M. chelonei, with subspecies designations. (+info)
Characterization of monoclonal antibodies for prostate-specific antigen and development of highly sensitive free prostate-specific antigen assays.
(10/8142)
BACKGROUND: The recent elucidation of the importance of serological free prostate-specific antigen (PSA) in the diagnosis of prostate cancer has created a demand for immunoassays specific for free PSA. METHODS: We developed and characterized 11 monoclonal antibodies with high affinities for PSA (Ka values from 1.1 x 10(8) to 1.8 x 10(10)L/mol), only 3 of which cross-react with human glandular kallikrein (hK2). Using these antibodies and PSA antibodies developed by others, in conjunction with time-resolved fluorometry, we developed ultrasensitive sandwich immunoassays specific for the free form of PSA. RESULTS: The analytical detection limit of these immunoassays is 0.001 microg/L. To our knowledge, this is the most sensitive free PSA assay reported to date. The free PSA immunoassays exhibit <1% cross-reactivity with PSA-alpha1-antichymotrypsin, show no cross-reactivity with hK2, and correlate well with established free PSA kits. The 11 antibodies developed by our group, in conjunction with 4 commercially available antibodies, were used to generate a putative epitope map of the PSA molecule. CONCLUSION: The highly sensitive free PSA immunoassays may be used for measuring PSA subfractions in female serum, an application currently impossible with other reported free PSA immunoassays. (+info)
Performance and specificity of monoclonal immunoassays for cyclosporine monitoring: how specific is specific?
(11/8142)
BACKGROUND: Immunoassays designed for the selective measurement of cyclosporin A (CsA) inadvertently show cross-reactivity for CsA metabolites. The extent and clinical significance of the resulting overestimation is controversial. A comprehensive assessment of old and new methods in clinical specimens is needed. METHODS: In a comprehensive evaluation, CsA was analyzed in 145 samples with the new CEDIA assay and compared with the Emit assay with the old and new pretreatments, the TDx monoclonal and polyclonal assays, the AxSYM, and HPLC. All samples were from patients with liver and/or kidney transplants. RESULTS: The CEDIA offered the easiest handling, followed by the AxSYM, which showed the longest calibration stability. The TDx monoclonal assay provided the lowest detection limit and the lowest CVs. The mean differences compared with HPLC were as follows: Emit, 9-12%; CEDIA, 18%; AxSYM, 29%; and TDx monoclonal, 57%. The CycloTrac RIA paralleled the Emit results. In contrast to the mean differences, substantial (>200%) and variable overestimations of the CsA concentration were observed in individual patient samples. Metabolic ratios, estimates of the overall concentrations of several cross-reacting metabolites (nonspecific TDx polyclonal/specific reference method), correlated with the apparent biases of the various monoclonal assays. Metabolic ratios varied up to 10-fold, which translated into biases for individual samples between -7% and +174%. The higher the cross-reactivity of an assay was, the higher was the range of biases observed. The interindividual differences markedly exceeded other factors of influence (organ transplanted, hepatic function). CONCLUSION: Because assay bias cannot be predicted in individual samples, substantially erratic CsA dosing can result. The specificity of CsA assays for parent CsA remains a major concern. (+info)
Genetic control of cytolytic T-lymphocyte responses. I. Ir gene control of the specificity of cytolytic T-lymphocyte responses to trinitrophenyl-modified syngeneic cells.
(12/8142)
The ability of cytotoxic T lymphocytes (CTL) induced in vitro to trinitrophenyl (TNP)-modified syngeneic cells to cross-reactively lyse a TNP allogeneic spleen target varies among inbred mouse strains. The cross-reactive CTL phenotype was found to be histocompatibility 2 (H-2) linked and to be dominant in F1 hybrid mice. All strains investigated demonstrated cross-reactivity except for some strains bearing portions of the H-2k haplotype. The gene(s) controlling this response maps to the K and/or I-A region of the H-2 complex. We have termed the immune response (Ir) gene responsible for controlling the specificity of CTL induced to TNP-modified syngeneic cells Ir-X-TNP. (+info)
Genetic control of cytolytic t-lymphocyte responses. II. The role of the host genotype in parental leads to F1 radiation chimeras in the control of the specificity of cytolytic T-lymphocyte responses to trinitrophenyl-modified syngeneic cells.
(13/8142)
Bone marrow cells from C3H (H-2k) mice, a strain that does not exhibit cross-reactive lysis of trinitrophenyl (TNP)-modified allogeneic targets, were allowed to mature in heavily irradiated (B6 times C3H)F1 (H-2b/k) recipients, an F1 hybrid that does demonstrate cross-reactive lysis. Spleen cells from these chimeric mice were removed after 3-4 mo and by H-2 typing shown to be of C3H origin. These cells were found to be tolerant to B6 alloantigens by mixed lymphocyte reaction and cell-mediated cytotoxicity and, when stimulated in vitro with TNP-modified syngeneic cells, now cross-reactively lysed TNP-modified allogeneic targets. These studies demonstrate that the host environment where T cells differentiate influences the specificity of the primary cytolytic T-lymphocyte (CTL) response to TNP-modified syngeneic antigens. (+info)
Cytotoxic T-cell responses in mice infected with influenza and vaccinia viruses vary in magnitude with H-2 genotype.
(14/8142)
Secondary effector T-cell populations generated by cross-priming with heterologous influenza A viruses operate only in H-2K or H-2D compatible situations, when assayed on SV40-transformed target cells infected with a range of influenza A viruses. The H2-Kb allele is associated with a total failure in the generation of influenza-immune cytotoxic T cells, though this is not seen for the primary response to vaccinia virus. In both influenza and vaccinia development of effector T cells operating at H-2Db is greatly depressed in B10.A(2R) (kkkddb) and B10.A(4R) (kkbbbb), but not in B10 (bbbbbb), mice. However, there is no defect in viral antigen expression at either H-2Kk or H-2Db in B10.A(2R) target cells. This apparently reflects some inadequacy in the stimulator environment, as (A/J X B6) F1 T cells can be induced to respond at H-2Db when exposed to vaccinia virus in an irradiated B6 but not in a B10.A(4R) recipient. The present report, together with the accompanying paper by Zinkernagel and colleagues, records the first rigorous demonstration of both a nonresponder situation and a probable Ir-gene effect for conventional infectious viruses. Possible implications for the evolution of H-2 polymorphism and mechanisms of Ir gene function are discussed. (+info)
Competition of Abeta amyloid peptide and apolipoprotein E for receptor-mediated endocytosis.
(15/8142)
The genetic polymorphism of apolipoprotein E (apoE) is associated with the age of onset and relative risk of Alzheimer's disease (AD). In contrast to apoE3, the wild type allele, apoE4 confers an increased risk of late-onset AD. We demonstrate that the beta-amyloid peptide isoforms Abeta (1-28), Abeta (1-40), and Abeta (1-43) compete for the cellular metabolism of apoE3 and apoE4 containing beta-very low density lipoproteins. An antibody raised against Abeta (1-28) cross-reacted with recombinant apoE. Epitope mapping revealed positive amino acid clusters as common epitopes of Abeta (13 through 17; HHQKL) and apoE (residues 144 through 148; LRKRL), both regions known to be heparin binding domains. Abeta in which amino acids 13 through 17 (HHQKL) were replaced by glycine (GGQGL) failed to compete with the cellular uptake of apoE enriched betaVLDL. These observations indicate that Abeta and apoE are taken up into cells by a common pathway involving heparan sulfate proteoglycans. (+info)
Prophenoloxidase-activating enzyme of the silkworm, Bombyx mori. Purification, characterization, and cDNA cloning.
(16/8142)
Prophenoloxidase-activating enzyme (PPAE) was purified to homogeneity as judged by SDS-polyacrylamide gel electrophoresis from larval cuticles of the silkworm, Bombyx mori. The purified PPAE preparation was shown to be a mixture of the isozymes of PPAE (PPAE-I and PPAE-II), which were eluted at different retention times in reversed-phase high performance liquid chromatography. PPAE-I and PPAE-II seemed to be post translationally modified isozymes and/or allelic variants. Both PPAE isozymes were proteins composed of two polypeptides (heavy and light chains) that are linked by disulfide linkage(s) and glycosylated serine proteases. The results of cDNA cloning, peptide mapping, and amino acid sequencing of PPAE revealed that PPAE is synthesized as prepro-PPAE with 441 amino acid residues and is activated from pro-PPAE by cleavage of a peptide bond between Lys152 and Ile153. The homology search showed 36.9% identity of PPAE to easter, which is a serine protease involved in dorso-ventral pattern formation in the Drosophila embryo, and indicated the presence of two consecutive clip-like domains in the light chain. A single copy of the PPAE gene was suggested to be present in the silkworm genome. In the fifth instar larvae, PPAE transcripts were detected in the integument, hemocytes, and salivary glands but not in the fat body or mid gut. A polypeptide cross-reactive to mono-specific anti-PPAE/IgG was transiently detected in the extract of eggs between 1 and 3 h after they were laid. (+info)