Alexander E. Karu

Characterization of the deoxyribonuclease determined by lambda reverse as exonuclease VIII of Escherichia coli.

Abstract Gottesman et al. (1974) detected a new DNAase in Escherichia coli infected with λ reverse, a recombination-proficient substitution mutant of phage λ which is deleted for the λ recombination genes. We have purified this enzyme, using the procedure developed for the purification of exonuclease VIII (Kushner et al. , 1974), a DNAase produced by E. coli K-12 strains carrying sbcA − mutations. The λ reverse exonuclease (Exoλ rev ) is identical to exonuclease VIII by several criteria. The two enzymes elute at similar salt concentrations from DEAE-cellulose and DNA-cellulose; sediment at the same velocity in glycerol gradients, corresponding to a molecular weight of about 1.4 × 10 5 ; migrate at the same R F in sodium dodecyl sulfate/polyacrylamide gels, indicating a polypeptide molecular weight of 1.4 × 10 5 ; exhibit maximum activity at 20 m m -Mg 2+ and pH 8 to 9; and are much more active on double-stranded DNA than on heat-denatured DNA. Both enzymes are rendered sedimentable by antiserum against Exoλ rev . This evidence supports the hypothesis that the non-λ DNA substitution in λ reverse includes recE , the structural gene for exonuclease VIII.

Immunochemical Detection of Polycyclic Aromatic Hydrocarbons and 1-Hydroxypyrene in Water and Sediment Samples

Abstract Several approaches were investigated to fully use an enzyme linked immunosorbent assay (ELISA) for accurate measurement of polycyclic aromatic hydrocarbons (PAHs) and their degradation products in surface water and sediment collected from the Pearl Harbor and James Campbell National Wildlife Refuges (NWRs) in Hawaii. Water samples were extracted by solid phase extraction (SPE) while the sediments were extracted by supercritical CO2. Parent PAHs and 1-hydroxypyene, selected as a marker, were quantitatively recovered with supercritical CO2 from sediments that were mixed with 5% Na4EDTA. Use of Na4EDTA or Na2SiO3 significantly improved recovery of 1-hydroxypyrene in sediments. Na4EDTA was more effective than Na2SiO3. The concentrations of PAHs as benzo[a]pyrene equivalents determined by ELISA were slightly higher than those determined by gas chromatography-mass spectrometry (GC-MS). ‘Overestimation’ by ELISA over GC-MS was attributed to, at least in part, the presence of PAH metabolites including 1-hydroxypyrene which were detected by ELISA, but not quantified by GC-MS.

Immunodetection of 3-nitrotyrosine in the liver of zymosan-treated rats with a new monoclonal antibody: Comparison to analysis by HPLC

Zymosan-induced peritonitis is associated with an increased production of reactive nitrogen oxides that may contribute to the often-observed failure of multiple organ systems in this model of acute inflammation. Quantitative biochemical evidence is provided for a marked 13-fold increase in protein-bound 3-nitrotyrosine (NTyr), a biomarker of reactive nitrogen oxides, in liver tissue of zymosan-treated rats. In order to investigate the localization of NTyr in this affected tissue, a monoclonal antibody, designated 39B6, was raised against 3-(4-hydroxy-3-nitrophenylacetamido) propionic acid-bovine serum albumin conjugate and its performance characterized. 39B6 was judged by competition ELISA to be approximately 2 orders of magnitude more sensitive than a commercial anti-NTyr monoclonal antibody. Binding characteristics of 39B6 were similar, but not identical, to that of a commercial affinity-purified polyclonal antibody in ELISA and immunohistochemical analyses. Western blot experiments revealed high specificity of 39B6 against NTyr and increased immunoreactivity of specific proteins from liver tissue homogenates of zymosan-treated rats. Immunohistochemical analysis of liver sections indicated a marked zymosan-induced increase in immunofluorescent staining, which was particularly intense in or adjacent to nonparenchymal cells, but not in the parenchymal cells of this tissue. Quantitative analysis of fractions enriched in these cell populations corroborated the immunofluorescent data, although the relative amounts detected in response to zymosan treatment was greatly reduced compared to whole liver tissue. These results demonstrate the high specificity of the newly developed antibody and its usefulness in Western blot and immunohistochemical analysis for NTyr, confirm the presence of NTyr by complementary methods, and suggest the possible involvement of reactive nitrogen oxides in hepatic vascular dysfunction.

Recombinant antibodies to small analytes and prospects for deriving them from synthetic combinatorial libraries

During the past 4 years, several laboratories have developed new methods of cloning antibody combining site genes from hybridomas or B‐lymphocytes, and functionally expressing them in bacteria, yeast, mammalian cells or plants. At least three research groups have also constructed ‘antibody display’ libraries with vastly diverse sequence permutations of combining site genes in bacteriophage. These semi‐synthetic combinatorial libraries present an antibody repertoire orders of magnitude greater than that accessed by conventional hybridoma technology. They may yield antibodies not obtainable through conventional immunization. We have cloned the immunoglobulin genes from a hybridoma specific for the phenylurea herbicide, diuron, into a phage display vector. The cloned antibody fragments (Fabs) were as sensitive as the parent monoclonal antibody for detecting free diuron in competition enzyme immunoassays. We also derived diuron hapten‐specific clones from synthetic combinatorial phage libraries, but only a fe...

Use of multivariate statistical methods to identify immunochemical cross‐reactants

Quantitative competition immunoassays with appropriate combinations of antibodies give consistent dose‐response patterns which may be used to identify and estimate amounts of cross‐reacting compounds. Previously reported methods of analyzing cross‐reaction patterns include multiple regression, principal components analysis and minimum estimates of variance (MEV). Four other techniques which are preferable in theory have been surveyed: discriminant analysis (DA), maximum likelihood estimates (MLE), classification and regression trees (CART), and computational neural networks (NN). MLE and simple back‐propagation neural networks can estimate the concentration, as well as the identity, of individual compounds. These four methods worked well with unfitted, unscaled data from monoclonal assays of triazines, phenylureas and avermectins. Immunoassays must be properly designed to provide adequate data for pattern recognition. Cross‐reactivity pattern analysis will make multi‐analyte, multi‐antibody immunoassays f...

Sequences of the cDNAs encoding the heavy- and light-chain Fab region of an antibody to the phenylurea herbicide diuron.

The cDNAs from a hybridoma (mAb 481.1) specific for diuron, a widely used phenylurea herbicide, were cloned into the phage display vector pComb8. Antigen-binding clones were selected by panning on diuron-hapten-BSA conjugates. The nucleotide and deduced amino-acid sequences encoding the Fab regions of the light (kappa) and heavy (gamma) chains were determined. The light chain was from mouse kappa chain subgroup III and the heavy chain was a member of the mouse H chain subgroup III(d).

INcreased immunoreactivity of apolipoprotein B epitopes during prolonged storage of low density lipoproteins

Human low density lipoproteins (LDL) serve as the major carriers of plasma cholesterol. LDL are heterogeneous in their lipid content, size and density [l] and certain LDL subspecies may increase risk of atherosclerosis [l], possibly due to differences in the conformation of apolipoprotein (APO) B in the particle. Attempts to determine the structural organization of Apo B in LDL have been largely frustrated due to the extreme insolubility and aggregation of delipidated apo B. More recent investigations have utilized monoclonal antibodies (MAbs) [2,3] to probe apo B structure in LDL. A number of these investigations have established that apo B is immunochemically heterogeneous [2,3] and that alterations in the lipid [2] or protein [3] components of LDL result in changes in immunoreactivity. In the present study we report our finding that the LDL isolated from some, but not all, individuals show a time related increase in enzyme-linked immunosorbent assay (ELISA) rates for three monoclonal antibodies.

Recognition of Coccidioides Immitis Antigens with Monoclonal Antibodies

This paper summarizes recent observations on the antigenic specificity and suitability for diagnostic use of seven IgM monoclonal antibodies (MAbs) prepared in 1984 with C. immitis Silveira spherules and endospore/spherule culture filtrate (ESCF) as immunogens.