RESUMEN
Some optimized methods of purifying actin from animal, plant, and bacterial cells were developed. Variants of the preparation of antiactin antibodies are described which use both traditional methods of immunization and phase display technology and antigen adsorption on colloidal gold particles. The conjugates of colloidal gold with phalloidin and heavy meromyosin as well as with antibodies were proposed. It was shown that these markers make it possible to reliably identify actins of different origin by the methods of light, and electron microscopy and dot-analysis.
Asunto(s)
Actinas/química , Oro Coloide/química , Actinas/inmunología , Animales , Anticuerpos/metabolismo , Azospirillum brasilense/química , Azospirillum brasilense/ultraestructura , Biomarcadores/química , Pollos , Oro Coloide/inmunología , Solanum lycopersicum/química , Ratones , Ratones Endogámicos BALB C , Subfragmentos de Miosina/química , Subfragmentos de Miosina/inmunología , Faloidina/química , Faloidina/inmunología , Conejos , Triticum/químicaRESUMEN
We studied the capacity of colloidal gold for enhancing specific and nonspecific immune response in laboratory animals (rabbits, rats, and mice) immunized with antigens of various nature. The antibody titers obtained with colloidal gold as a carrier were higher as compared to the standard immunization techniques (free antigen or Freund's adjuvant). Application of colloidal gold increased nonspecific immune responses as well: lysozyme concentration in the blood, activity of the complement system proteins, as well as phagocytic and bactericidal activities. The obtained antibodies were tested by immunodot assay using gold markers. Immunization of the animals with colloidal gold conjugates with haptens as well as complete antigens was shown to induce formation of highly active antibodies without using other antigens such as complete Freund's adjuvant. In addition, antigen quantities for animal immunization with colloidal gold was by one order of magnitude lower as compared to the complete Freund's adjuvant immunization. This fact can point to direct adjuvant activity of colloidal gold.
Asunto(s)
Anticuerpos/inmunología , Antígenos/inmunología , Oro Coloide/inmunología , Inmunización/métodos , Tilosina/análogos & derivados , Actinas/inmunología , Animales , Bacteriorodopsinas/inmunología , Cloranfenicol/inmunología , Adyuvante de Freund/inmunología , Gentamicinas/inmunología , Haptenos/inmunología , Ivermectina/inmunología , Macrólidos/inmunología , Ratones , Ratones Endogámicos BALB C , Proteínas Proto-Oncogénicas c-myc/inmunología , Conejos , Ratas , Albúmina Sérica Bovina/inmunología , Tilosina/inmunologíaRESUMEN
The electrophysical properties of Escherichia coli XL-1 cells interacting with helper phage M13K07 were studied as a function of the phage-to-cell ratio and the contact time. The electro-optical signal of bacterial cells changed considerably as soon as 10 min after the onset of their incubation with phage particles, presumably due to phage adsorption on the cell surface. The maximum changes in the orientational spectra of cell suspensions were observed when the phage-to-cell ratio was 20. Selectivity studies showed that E. coli XL-1 cells interacting with the helper phage M13K07 in the presence of foreign microflora, such as E. coli K-12 or Azospirillum brasilense Sp7, can be identified by using their electrophysical properties. Changes in the orientational spectra of cell suspensions are interpreted with the stage of phage-bacterium interaction taken into account. The results obtained can probably be used to devise a new rapid method for identification of microorganisms and to study the particular stages of cell infection by bacteriophages.
Asunto(s)
Bacteriófago M13/fisiología , Escherichia coli/virología , Bacteriófago M13/patogenicidad , Electrofisiología , Escherichia coli/aislamiento & purificación , Escherichia coli/fisiología , Especificidad de la Especie , Análisis EspectralRESUMEN
The study of the effect of ampicillin on the electrophysical properties of Escherichia coli cells showed that this antibiotic influences the orientational spectra (OS) of the ampicillin-susceptible E. coli strains K-12 and XL-1 within the frequency range 10-1000 kHz of the orienting electric field and does not affect the OS of the ampicillin-resistant strains K-12(pUC-18) and XL-1(pHEN1). The change in the electrooptical signal of the ampicillin-susceptible cells was maximum at an ampicillin concentration of 50 microg/ml and did not depend on the exposure time. The conclusion is drawn that changes in the OS of cells can be used to evaluate their resistance to ampicillin.
Asunto(s)
Resistencia a la Ampicilina , Ampicilina/farmacología , Antibacterianos/farmacología , Escherichia coli/efectos de los fármacos , Escherichia coli/fisiología , Campos Electromagnéticos , Escherichia coli/citología , Análisis EspectralRESUMEN
Single-chain miniantibodies (scFv's) to actin were obtained by the phage display method. A naive combinatorial phage display library of murine scFv's (containing 2x10(8) independent recombinant clones) was used to select miniantibodies. After three rounds of selection two clones producing miniantibodies to chicken smooth muscle actin with affinity constants of 1.4x10(7) and 1.2x10(6) M(-1) were chosen. The isolated miniantibodies could specifically detect various plant and animal actins.
Asunto(s)
Actinas/inmunología , Anticuerpos Monoclonales/inmunología , Fragmentos de Inmunoglobulinas/inmunología , Biblioteca de Péptidos , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/aislamiento & purificación , Especificidad de Anticuerpos/inmunología , Pollos , Ensayo de Inmunoadsorción Enzimática , Fragmentos de Inmunoglobulinas/genética , Fragmentos de Inmunoglobulinas/aislamiento & purificación , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/aislamiento & purificación , Ratones , Proteínas de Plantas/inmunología , Conejos , Especificidad de la Especie , Vicia faba/metabolismoRESUMEN
We have devised a protocol for the isolation and identification of a proliferative antigen of the initial cells of wheat stem meristems (termed PAI). We have carried out a variety of immunochemical and immunocytochemical methods, using colloidal gold (CG) complexed with monospecific antibodies to PAI as the marker for the detection of PAI. We have been able to determine the effectiveness of immunoaffinity chromatography in isolating PAI from plant tissues and have shown the advantages of CG over enzyme labels for identification of the antigen. Finally, we have obtained a purified preparation of PAI and have determined its molecular mass (approximately 83 kDa).