RESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales , Cromatografía Líquida de Alta Presión , Inmunoensayo , Hígado/química , Tirosina/análogos & derivados , Tirosina/análisis , Zimosan/farmacología , Animales , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Western Blotting , Técnica del Anticuerpo Fluorescente , Haptenos/química , Haptenos/inmunología , Inmunohistoquímica , Hígado/efectos de los fármacos , Masculino , Ratones , Ratas , Ratas Endogámicas F344 , Distribución Tisular , Tirosina/inmunologíaRESUMEN
A previously described monoclonal antibody, S2B1, was highly selective for coplanar (non-ortho-chlorinated) PCB congeners in enzyme immunoassays that measured binding at equilibrium. In the present study, kinetic exclusion fluoroimmunoassay (KinExA) was used to determine the dissociation constants (Kd) and on and off rates (k(on), k(off)) for binding of various PCB congeners to affinity-purified S2B1 IgG and Fab fragments in solution. This method revealed that mono- and di-ortho-chlorinated PCBs were bound by S2B1, but the on rates were slower, and the off rates faster by 6-60-fold, than with congeners that had no ortho chlorines. Although the sensitivity of immunoassays may be improved by using competing haptens that S2B1 binds more weakly than the parent PCB, the KinExA results demonstrate that congener specificity is an intrinsic property of S2B1 and does not require weaker binding haptens. KinExA also provided new information on the percentage of active binding sites, valence, and effects of buffer, solvent, and biotinylation on S2B1. The advantages and drawbacks of KinExA for measuring antibody-ligand binding are described.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Bifenilos Policlorados/inmunología , Especificidad de Anticuerpos , Reacciones Antígeno-Anticuerpo , Inmunoensayo/métodos , Inmunoensayo/normas , Cinética , Bifenilos Policlorados/análisis , Sensibilidad y Especificidad , Espectrometría de FluorescenciaRESUMEN
Proteins can use aromatic side-chains to stabilize bound cationic ligands through cation-pi interactions. Here, we report the first example of the reciprocal process, termed pi-cation, in which a cationic protein side-chain stabilizes a neutral aromatic ligand. Site-directed mutagenesis revealed that an arginine side-chain located in the deep binding pocket of a monoclonal antibody (4D5) is essential for binding the neutral polynuclear aromatic hydrocarbon benzo[a]pyrene. This Arg was very likely selected for in the primary response, further underscoring the importance of the pi-cation interaction for ligand binding, which should be considered in protein analysis and design when ligands include aromatic groups.
Asunto(s)
Benzo(a)pireno/química , Benzo(a)pireno/metabolismo , Sitios de Unión de Anticuerpos , Cationes/química , Cationes/metabolismo , Región Variable de Inmunoglobulina/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Complejo Antígeno-Anticuerpo/química , Complejo Antígeno-Anticuerpo/genética , Complejo Antígeno-Anticuerpo/inmunología , Arginina/genética , Arginina/metabolismo , Secuencia de Bases , Sitios de Unión de Anticuerpos/genética , Sitios de Unión de Anticuerpos/inmunología , Ensayo de Inmunoadsorción Enzimática , Haptenos/química , Haptenos/inmunología , Haptenos/metabolismo , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Ligandos , Lisina/genética , Lisina/metabolismo , Ratones , Modelos Moleculares , Mutagénesis Sitio-Dirigida/genética , Mutación/genética , Electricidad Estática , TermodinámicaRESUMEN
Recombinant Fab antibodies (rFabs) specific for coplanar polychlorinated biphenyls (PCBs) were derived from a hybridoma cell line (Chiu et al. Anal. Chem. 1995, 67, 3829-3839). Immunoglobulin V(H)-C(H1) and V(L)-C(L) sequences from S2B1 messenger RNA were amplified by PCR and cloned into the M13 phagemid vector pComb3H. Phage displaying rFab were enriched by panning on a PCB hapten conjugate and expressed as soluble rFabs in Escherichia coli XL-1 Blue. Two rFab clones competitively bound PCBs 77 and 126 with half-maximal inhibition (I(50)) of 10-13 ppb in indirect and direct enzyme immunoassays (EIAs), with selectivity nearly identical to that of whole S2B1 IgG and its Fab fragments prepared by papain digestion. These results, and comparison of N-terminal amino acid sequences of MAb S2B1 and the rFab, indicated that rFab S2B1 is a functional copy of the MAb. The rFab S2B1 sequences have 75-89% sequence identity with antibodies that bind nitrophenyl haptens and are being used to construct a three-dimensional computational model of the PCB binding site.
Asunto(s)
Anticuerpos Monoclonales , Fragmentos Fab de Inmunoglobulinas , Bifenilos Policlorados/análisis , Bifenilos Policlorados/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Secuencia de Bases , Sitios de Unión de Anticuerpos , Clonación Molecular , Cartilla de ADN , Técnicas para Inmunoenzimas , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/genética , Inmunoglobulina G/química , Inmunoglobulina G/genética , Cadenas Pesadas de Inmunoglobulina/química , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/química , Cadenas Ligeras de Inmunoglobulina/genética , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteínas RecombinantesRESUMEN
As a first step toward developing sensitive enzyme-linked immunosorbent assays (ELISAs) for multianalyte detection of polycyclic aromatic hydrocarbons (PAHs), haptens with different lengths of carboxylic acid spacers at various positions were derived from naphthalene, fluorene, anthracene, phenanthrene, pyrene, fluoranthene, chrysene, and benzo[a]pyrene (BaP). These haptens were coupled with bovine serum albumin (BSA) to form competitor conjugates. All of these haptens were recognized to different extents by monoclonal antibodies (MAbs) 4D5 and 10C10 originally derived by Gomes and Santella (Chem. Res. Toxicol. 1990, 3, 307-310). The most sensitive indirect ELISAs were obtained by coating wells with the least competitive conjugates. Direct ELISAs using horseradish peroxidase conjugates of pyrene and BaP were less sensitive. The MAbs bound BaP with spacers at either C1 or C6. The cross-reactivity profiles of the eight PAHs were different with each PAH-BSA conjugate used as coating antigen. The ELISA results for BaP closely correlated with those by gas chromatography (GC), but the detection limit of the ELISA was approximately 150-fold more sensitive than that of GC, with 2-600 nM spike recoveries of 80-127% from human urine and canal and tap water.
Asunto(s)
Anticuerpos Monoclonales/análisis , Ensayo de Inmunoadsorción Enzimática/métodos , Haptenos/análisis , Hidrocarburos Policíclicos Aromáticos/análisis , Antígenos/metabolismo , Benzo(a)pireno/análisis , Cromatografía de Gases/métodos , Reacciones Cruzadas , Haptenos/química , Humanos , Compuestos Orgánicos , Sensibilidad y Especificidad , Albúmina Sérica Bovina/química , Solventes , Agua/químicaRESUMEN
Polychlorinated biphenyls (PCBs) are ubiquitous environmental pollutants with diverse toxic, teratogenic, reproductive, immunotoxic, and tumorigenic effects. Three of the least abundant of the 209 PCB isomers (congeners) are the most toxic and most difficult to quantify. These are 3,4,3',4'-tetrachlorobiphenyl, 3,4,3',4',5'-pentachlorobiphenyl, and 3,4,5,3',4',5'-hexachlorobiphenyl (IU-PAC No. 77, 126, and 169, respectively). An immunizing hapten was designed to retain the 3,4,3',4' chlorine-substitution pattern and coplanarity characteristic of these toxic congeners. The optimal competitors for immunoassay were weaker binding distinctive single-ring fragments of the PCBs. A monoclonal antibody designated S2B1 was derived and used in direct (antibody-capture) competitive enzyme immunoassays (EIAs). The EIAs are highly specific for non-ortho-substituted congeners and do not recognize the more prevalent but much less toxic noncoplanar PCB congeners or 2,3,7,8-tetrachlorodibenzo-p-dioxin, 2,3,7,8-tetrachlorodibenzofuran, or dichlorobenzenes. Hapten and competitor design for this assay suggests a basis for development of sensitive EIAs for other classes of PCB congeners.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Bifenilos Policlorados/análisis , Animales , Unión Competitiva , Células Cultivadas , Reacciones Cruzadas , Haptenos/química , Hibridomas , Técnicas para Inmunoenzimas , Ratones , Bifenilos Policlorados/metabolismoRESUMEN
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).
Asunto(s)
Anticuerpos Monoclonales/genética , ADN Complementario/genética , Diurona/inmunología , Herbicidas/inmunología , Fragmentos Fab de Inmunoglobulinas/genética , Compuestos de Fenilurea , Secuencia de Aminoácidos , Animales , Bacteriófagos/genética , Secuencia de Bases , Clonación Molecular , Genes de Inmunoglobulinas/genética , Cadenas gamma de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/genética , Ratones , Datos de Secuencia Molecular , Péptidos , Proteínas Recombinantes de Fusión , Análisis de Secuencia de ADNRESUMEN
DNA polymerase I (pol I) from Escherichia coli has three well-defined activities: DNA polymerase, 3'-5' exonuclease, and 5'-3' exonuclease. We have raised monoclonal antibodies to pol I which selectively neutralize each of these three activities, thus supporting the model of separate active sites for each activity, heretofore exclusively demonstrated with proteolytic fragments of pol I. Antibodies from each class could bind pol I in the presence of antibodies of another class, indicating the existence of significant spatial separation between each of the three sites. In addition, several of the neutralizing antibodies were able to distinguish particular activities of the 5'-3' exonuclease. One of them, for example, inhibited the RNase H activity but not the DNase activity. Two other antibodies could, in addition to inhibiting the polymerase and the 3'-5' exonuclease, either stimulate or inhibit the 5'-3' exonuclease depending upon the assay conditions, particularly the ionic strength.
Asunto(s)
Anticuerpos Monoclonales , ADN Helicasas/metabolismo , ADN Polimerasa I/metabolismo , Escherichia coli/enzimología , Exodesoxirribonucleasas/metabolismo , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Complejo Antígeno-Anticuerpo , Sitios de Unión , ADN Polimerasa I/inmunología , Ensayo de Inmunoadsorción Enzimática , Exodesoxirribonucleasa V , Exodesoxirribonucleasas/inmunología , Cinética , Ratones , Ratones Endogámicos/inmunología , Pruebas de Neutralización , Concentración Osmolar , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismoRESUMEN
Aldrin and many other cyclodiene and polychlorocycloalkane insecticides interact with both the [35S]-tert-butylbicyclophosphorothionate ([35S]TBPS) binding site of the mammalian brain gamma-aminobutyric acid (GABA) gated chloride channel and several cyclodiene monoclonal antibodies (MAbs) at concentrations ranging from 0.06 to 8.7 microM. A survey of other classes of GABAA receptor antagonists (including picrotoxinin and several trioxabicyclooctanes) for possible interactions with the cyclodiene MAbs revealed only one potent inhibitor, the heteroadamantane tetramethylenedisulfotetramine (TETS) [mouse intraperitoneal LD50 0.24 mg/kg; TBPS binding site IC50 0.5 microM as a competitive inhibitor (Scatchard analysis); cyclodiene MAb IC50 3 microM]. These findings prompted comparative studies on the structure-activity relationships of other sulfamides as they apply to both the ligand-nerve and ligand-MAb interactions. TETS is active on only one (MAb 8H11) of four cyclodiene MAbs. Several hetero(homo)adamantanes were synthesized and compared with TETS for neurotoxicity and recognition by the TETS-sensitive cyclodiene MAb. The toxicity to mice and/or houseflies decreases in the following order: TETS much greater than the heterotetracyclic compound hexamethylenetrisulfohexamine (HEXS) and two TETS analogues in which one sulfamide group is replaced with o-phenylenediamine or 1,1-dimethyl-1,2-diaminoethane much greater than seven other hetero(homo)adamantanes. The TETS-sensitive cyclodiene MAb recognizes HEXS (IC50 0.4 microM) and, to a lesser extent, two related sulfamides. However, the cross-reactivity noted for the cyclodiene insecticides and TETS relative to the GABA-gated chloride channel (inhibition of TBPS binding) and the cyclodiene MAb does not extend to several TETS analogues including HEXS.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Amidas/metabolismo , Anticuerpos Monoclonales/metabolismo , Hidrocarburos Aromáticos con Puentes/metabolismo , Cicloparafinas/inmunología , Antagonistas del GABA , Activación del Canal Iónico/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Amidas/farmacología , Animales , Unión Competitiva , Hidrocarburos Aromáticos con Puentes/farmacología , Canales de Cloruro , Cicloparafinas/farmacología , Moscas Domésticas , Técnicas para Inmunoenzimas , Insecticidas/farmacología , Cinética , Masculino , Proteínas de la Membrana/efectos de los fármacos , Ratones , Tejido Nervioso/efectos de los fármacos , Tejido Nervioso/fisiología , Relación Estructura-Actividad , Ácido gamma-Aminobutírico/fisiologíaAsunto(s)
Apolipoproteínas B/metabolismo , Epítopos/metabolismo , Lipoproteínas LDL/metabolismo , Anticuerpos Monoclonales/inmunología , Apolipoproteínas B/inmunología , Western Blotting , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Glicósido Hidrolasas , Humanos , Hidrólisis , Oxidación-Reducción , Especificidad por SustratoRESUMEN
Since Jerne proposed a "network" theory of immune regulation, the properties of anti-idiotypic antibodies (anti-IdAb) have been investigated widely. Anti-IdAb raised against antibodies to a variety of ligands have been shown to bind the ligands' receptors. Thus, the combining site of an anti-IdAb may contain information regarding the three-dimensional structure of an antigen. However, this remarkable property of "internal imagery" has not been exploited for structural investigation at the molecular level. In the present report, a monoclonal "auto"-anti-IdAb was raised against ganglioside GM1 (a cell-surface glycolipid that binds cholera toxin) and was shown to crossreact with the B subunit of cholera toxin. This antibody was presumed to recognize amino acid residues located within the GM1 binding domain. To identify these residues, the antibody was screened against homologous toxins purified from enterotoxigenic strains of Escherichia coli and chimeric peptides produced by recombinant methods. Amino acid variation at position 4 from the N terminus of these proteins was found to disrupt antibody binding. Since the toxins and chimera are all closely related in structure and function, the residue at position 4 (an asparagine in cholera toxin B subunit) appears to be in the epitope of the antibody and, by implication, in the GM1 binding site. Of particular significance, this structural detail could not be deduced with GM1 alone. It would seem that ligand and anti-ligand anti-IdAb encode similar stereochemical information but do so with different "chemical alphabets," giving rise to distinct binding specificities.
Asunto(s)
Toxina del Cólera/metabolismo , Idiotipos de Inmunoglobulinas/análisis , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Complejo Antígeno-Anticuerpo/análisis , Toxina del Cólera/inmunología , Femenino , Cinética , Sustancias Macromoleculares , Ratones , Ratones Endogámicos BALB C , Modelos MolecularesRESUMEN
The immune response of humans and mice to temperature-specific, plasmid- or chromosome-encoded proteins of yersinia pestis and Yersinia enterocolitica was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Extracts from Y. pestis and Y. enterocolitica strains with and without the virulence plasmids pYV019 and pYV8081, respectively, were resolved by denaturing electrophoresis, and the major antigens were visualized with sera from convalescing plague patients, individuals immunized with plague vaccine, and mice and rabbits immunized with avirulent live yersiniae. The Y. pestis grown in vitro in this study did not express detectable amounts of plasmid-encoded antigens. The sera from plague patients recognized Y. pestis and Y. enterocolitica antigens ranging from 15 to 72 kilodaltons (kDa), whereas sera from immunized subjects recognized four antigenic components in Y. pestis ranging from 17 to 64 kDa and five antigens in Y. enterocolitica ranging from 16 to 68 kDa. Sera from mice reacted with 7 antigens in Y. pestis and 12 antigens in Y. enterocolitica ranging from 14 to 68 kDa, and sera from rabbits reacted with 7 and 10 antigens in Y. pestis and Y. enterocolitica, respectively. All of the plague patient sera, as well as the sera from immunized mice and rabbits, reacted with a 22-kDa Y. enterocolitica plasmid-associated polypeptide, and five of the patient sera also recognized a Y. enterocolitica plasmid-associated 31-kDa protein. The results indicate a common immune response to at least these two plasmid-specified Yersinia outer membrane proteins. Y. pestis apparently expresses these components only in vivo, and in vitro, Y. enterocolitica expresses a greater number of plasmid-associated antigens than does Y. pestis.
Asunto(s)
Antígenos Bacterianos/inmunología , Cromosomas Bacterianos , Plásmidos , Yersinia/inmunología , Adolescente , Adulto , Animales , Anticuerpos Antibacterianos/análisis , Proteínas Bacterianas/análisis , Niño , Preescolar , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Inmunización , Ratones , Persona de Mediana Edad , Péptidos/análisis , Conejos , TemperaturaRESUMEN
Murine B-cell hybridomas made with the P3X63-AG8.653 myeloma showed increases in cloning efficiency and efficiency of growth in hypoxanthine-aminopterin-thymidine (HAT) medium of 50-100-fold in the presence of medium conditioned by primary mouse peritoneal macrophages (MCM). Similar effects were elicited by MCM from 3 continuous macrophage lines. The J774A.1 line conditioned the medium as efficiently as primary macrophages without induction. Conditioning by the P388D1 line was several-fold less efficient, but could be increased by treating the cells with Escherichia coli lipopolysaccharide. By contrast, the BJ-1 macrophage line required treatment with the lipopolysaccharide to induce expression of the hybridoma growth factor(s). Four commercially available serum supplements could not substitute for MCM, but addition of MCM and the supplements together stimulated the growth rate of hybridomas in media with 4% or less fetal bovine serum. The rate of antibody synthesis paralleled the growth rate, and the amount of antibody synthesized per cell was approximately the same for hybridomas grown in medium supplemented with MCM or adapted to growth in the absence of MCM. The results indicate that MCM has advantages as an alternative to 'feeder cells' and serum supplements in hybridoma cultures, and suggest that MCM may be useful for hybridoma culture at reduced serum concentrations. The nature of the soluble factor(s) in MCM which promote these effects remains unknown.
Asunto(s)
Formación de Anticuerpos , Hibridomas/inmunología , Macrófagos/inmunología , Animales , Sangre , División Celular/efectos de los fármacos , Línea Celular , Células Cultivadas , Medios de Cultivo , Sustancias de Crecimiento/biosíntesis , Sustancias de Crecimiento/farmacología , Hibridomas/citología , Macrófagos/metabolismo , RatonesRESUMEN
A Dynatech Autoprep liquid sampling system has been modified to perform fully automated aseptic sampling, feeding, and expansion of hybridoma cultures in standard 96- and 24-well culture plates. The system is controlled by an Apple IIe computer, and uses a single teflon probe to transfer culture medium from randomly located wells to EIA plates and deliver fresh medium to the sampled wells. An 'expansion mode' allows suspension of cells for transfer to another plate. The sampling probe may be washed with sterile medium, buffer, or water between each transfer. Any combination of up to 6 assay plates, sterile growth plates, and expansion plates may be operated on at one time, and each transaction is recorded on a floppy disk file. Experiments with various hybridoma cultures indicated that transfers were reproducible, sterility was maintained, and the washing procedure reduced cross-contamination of cultures with other cells or antibodies to negligible levels. The APPLE BASIC computer programs which perform the functions and record the transactions are described in the paper and the Appendix, and are available upon request.
Asunto(s)
Hibridomas/análisis , Manejo de Especímenes , Animales , Autoanálisis/instrumentación , Programas Informáticos , Factores de TiempoRESUMEN
We have prepared hybridomas which secrete antibodies against E. coli recA protein, by fusing spleen lymphocytes from immunized mice with P3X63-AG8 myeloma cells. This paper describes a preliminary survey of properties of antibody from a hybridoma population designated 156, which inhibits the strand pairing, strand assimilation, repressor cleavage, and DNA-dependent ATPase activities of recA protein. The 156 antibody consists of one or two species which have been tentatively identified as IgG2b. 156 Globulin reacts 4.6-fold more efficiently with denatured recA protein, and it reacts partially with the native tif-1, lexB30, recA44, and recA629 proteins, as well as with peptide fragments which are not common to all four proteins.
Asunto(s)
Anticuerpos Monoclonales , Proteínas Bacterianas/genética , Epítopos/análisis , Escherichia coli/genética , Mutación , Alelos , Animales , Proteínas Bacterianas/inmunología , Línea Celular , Reacciones Cruzadas , Variación Genética , Hibridomas/inmunología , Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Plasmacitoma/inmunología , Rec A RecombinasasRESUMEN
An enzyme-linked immunosorbent assay (ELISA) has been adapted to measure E. coli recA protein in the 1 to 10 ng range in whole-cell sonicates, membrane extracts, and osmotic shock fluid from 2 x 10(8) cells. The specific activity of recA protein is maintained at a relatively constant "basal' level (800 to 1,200 molecules per cell for wild-type E. coli in L-broth, salt-depleted broth and minimal media) during early-log and mid-log phase growth, but it increases by two- to ten-fold as the culture approaches saturation density. Nalidixate-induced levels are 20- to 50-fold higher, and 100-fold higher in a constitutive tif- spr- mutant. Induction of recA protein synthesis by nalidixic acid, which normally requires functional recBC enzyme, also occurs in recB- and recC- cells by pathways activated by mutation in the sbcA and sbcB indirect suppressors. In recB- sbcA- mutants, exonuclease VIII, the recE gene product, is required for induction of recA protein. Abolition of exonuclease I activity by mutation in sbcB allows induction of recA protein by nalidixate in recB- and recC- cells. Mutation in recF does not affect induction by nalidixate in RecBC+ cells, but it enables induction to occur in RecBC- cells, suggesting that recF gene product is involved in regulation of recA protein.
Asunto(s)
Proteínas Bacterianas/biosíntesis , Proteínas de Escherichia coli , Recombinación Genética , Desoxirribonucleasas/metabolismo , Inducción Enzimática/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Exodesoxirribonucleasa V , Exonucleasas/metabolismo , Mutación , Ácido Nalidíxico/farmacología , Rec A RecombinasasRESUMEN
A computation and analysis program has been assembled to facilitate the use of competition ELISA and similar assays for studies of antigen regulation and turnover. The program fits sigmoidal standard curves using a 4-parameter logistic function, determines amounts of antigen from the equation which defines the standard curve, and calculates specific activities by linear regression of the levels of antigen in varying amounts of total protein. An optional weighting function is provided to adjust for systematic non-uniform variance. Outlying points are identified during the linear regression, and the user may delete them or redefine the acceptable working range of the standard curve. The program provides a complete print-out of the data and optional plots of the fitted standard curve and the regression analysis of the samples, as well as statistics which are useful for quality control. It also provides the option of storing the data points from the standard curve on magnetic diskettes. The package is written in BASIC for a Wang Model 2200 computer equipped with a magnetic diskette drive, line printer, and flat-bed X-Y plotter, but it is readily adaptable to other systems and input/output devices.