Intermittent microwave irradiation facilitates antigen-antibody reaction in Western blot analysis.

We established a shortened protocol for western blot analysis using intermittent microwave irradiation. With this method, the procedure is completed within 1 h after applying the primary antibody, and thus greatly saves time. This procedure appears to be applicable to any antibody based on our experience of several years.

Effects of 100 GHz radiation on alkaline phosphatase activity and antigen-antibody interaction.

Equipment that generates microwave radiation (MWR) spanning the frequency range of 300 MHz-100 GHz is becoming more common. While MWR lacks sufficient energy to break chemical bonds, the disagreement as to whether MWR exposure is detrimental to cellular dysfunction may be difficult to clarify using complex systems such as whole animals, cells, or cell extracts. Recently, the high frequency range of terahertz (THz) radiation has been explored and sources of radiation and its detectors have been developed. THz radiation is associated with the frequency interval from 100 GHz to 20 THz and constitutes the next frontier in imaging science and technology. In the present study, we investigated the effect of radiation in the low frequency THz range (100 GHz) on two defined molecular interactions. First, the interaction of soluble or immobilized calf alkaline phosphatase with the substrate p-nitrophenylphosphate and second, the interaction between an antibody (mouse monoclonal anti-DNP) and its antigen (DNP). Irradiation of enzyme either prior to addition of substrate or during the enzymatic reaction resulted in small but significant reductions in enzyme activity. These differences were not observed if the enzyme had previously been immobilized onto plastic microwells. Exposure of immobilized antigen to radiation did not influence the ability of the antigen to interact with antibody. However, irradiation appeared to decrease the stability of previously formed antigen-antibody complexes. Our data suggest that 100 GHz radiation can induce small but statistically significant alterations in the characteristics of these two types of biomolecular interactions.

Design and characterization of stimuli-responsive FLAG-tag analogues and the illumination-induced modulation of their interaction with antibody 4E11.

An azobenzene group containing beta-amino acid N-Fmoc-4-aminomethyl phenylazobenzoic acid was synthesized and with the exception of the C-terminal amino acid residue was substituted by solid-phase peptide synthesis into all positions of the FLAG sequence (DYKDDDDK), an octapeptide capable of specific interaction with the monoclonal antibody 4E11. The trans state of the beta-amino acid was thermodynamically more stable than the cis state. However, the molecule could be switched into the cis conformation by illumination at 340 nm. Peptides containing the artificial amino acid also became photoresponsive. In the absence of light, the spontaneous back-isomerization into the trans conformation of the photoresponsive was extremely slow (>8 h no significant increase in trans content). When illuminated with visible light (440 nm), the back-isomerization from the cis to the trans state was accelerated and occurred with a half-life of approximately 10 min. The cis form of the photopeptides was more hydrophilic than the trans form, as evidenced by differences in the retention time of the two isomeric forms in reversed-phase chromatography. Photopeptides that contained the intact sequences responsible for binding of the FLAG tag to the antibody, namely, the DYK motive at the N-terminus, showed binding to the antibody in both a dot blot immunoassay and in Biacore binding studies, albeit with lower affinity than the unmodified FLAG sequence. Peptides with a substitution in positions 4-6 showed differences in binding strength between the trans and the cis form in the Biacore studies, no such difference could be observed for the peptide with a substitution in position 7.

[The immunological and hormonal effects of combined exposure to a bitemporal ultrahigh-frequency electrical field and to decimeter waves at different sites]. / Immunologicheskie i gormonal'nye éffekty sochetannogo vozdeistviia élektricheskogo polia ul'travysokoi chastoty bitemporal'no i detsimetrovykh voln razlichnoi lokalizatsii.

Bitemporal UHF electric field is shown to enhance glucocorticoid adrenal function unlike inhibition of the thyroid function suppressing a primary immune response (PIR) in the productive phase. The combined exposure to bitemporal UHF electric field and decimeter waves of the adrenals doubles glucocorticoid synthesis abolishing the inhibitory action of the UHF therapy on thyroid function resultant in much more suppressed PIR. Both modalities inhibit thymic production. Decimeter waves alone are less effective. The exposure of the thyroid to decimeter waves initiated PIR by 2.5-fold activation of medullar lymphocytes and by a 80% increase in the thymic function. No response was achieved in combined action on the thyroid of the electric field and decimeter waves.

[The immunomodulating properties of erythrocytes induced by physical factors]. / Immunomoduliruiushchie svoistva éritrotsitov, indutsirovannye fizicheskimi faktorami.

It is shown that the action of ultraviolet rays (UVR) or magnetic field (MF) on the erythrocytes of intact Wistar rats by weight 140-170 g induces the property to stimulate the immune response on sheep's T-dependent antigen-erythrocytes and on bull serum albumin in allogenic transference. The most expressed immune stimulated effect is induced by the heavy erythrocytes, which are effected by magnetic field. The warming and vibration don't induce the immunomodulating characteristics in erythrocytes. By spleen cells sticking to glass, the modified UVR and MF, the heavy erythrocytes induce factors (differentiated in mass), which stimulate the development of immune response to T-dependent antigens of sheep's erythrocytes and of bull serum albumin. These factors depress the function of antigen-specific T-suppressors and induce the immune-suppressive characteristics in light erythrocytes.

[Comparative studies on immunomodulation via hyperfractionated whole-body irradiation]. / Vergleichende Untersuchungen über die Immunomodulation durch hyperfraktionierte Ganzkörperbestrahlung.

The effect of the humoral immune response on Sprague-Dawley rats by hyperfractionated total body irradiation was investigated. These experiments included single doses of total body irradiation (1 Gy, 2 Gy, 3 Gy) and the administration of 1 Gy and 2 Gy in two or three fractions 2 x 0.5 Gy, 3 x 0.33 Gy and 2 x 1.0 Gy, 3 x 0.66 Gy. The time between the fractions was six hours. The primary production of antibodies against erythrocytes of sheep were determined as immunity parameter. The immune stimulation could be achieved, when single doses of 1 or 2 Gy were delivered. The single dose of 3 Gy total body irradiation showed a small, not significant immunosuppression. A significant immunosuppression was observed in all our hyperfractionated experiments. The degree of immunosuppression was nearly equivalent in the hyperfractionated group (2 x 0.5, 3 x 0.33 Gy and 2 x 1.0, 3 x 0.66 Gy).

Photoreversible antigen-antibody reactions.

A monoclonal antibody (Z1H01) for an oligopeptide carrying an azobenzene group, was prepared under conditions where the azobenzene group is in the trans form. The antibody bound the hapten peptide effectively when the hapten peptide is in the trans form (K = 5 x 10(7) M-1), but the antibody released the hapten under irradiation with UV light where the hapten is in the cis form. The antibody bound the hapten again, when the hapten reverted to the trans form after irradiation with visible light.

Enhancement of monoclonal antibody uptake in human colon tumor xenografts following irradiation.

Indium-111-labeled AUA1 tumor-associated monoclonal antibody raised against an antigen of colon adenocarcinoma was used to evaluate the effect of ionizing radiation on antibody uptake by the LoVo adenocarcinoma cell line grown as a xenograft in nude mice. Tumors were exposed to single doses of external X-irradiation of between 400 and 1600 cGy followed, 24 h later, by administration of specific or nonspecific antibody. Animals were sacrificed 3 days after antibody administration. At doses higher than 400 cGy, tumor uptake with both specific and nonspecific antibody was significantly increased. No difference in changes in tumor volume was observed between the groups receiving irradiation and the controls. Specific antibody uptake by tumors was always significantly higher than nonspecific having an approximate 4-fold binding advantage. Vascular permeability and the vascular volume of irradiated and control tumors was measured 24 and 72 h after irradiation, using iodine-125-labeled nonspecific antibody and labelling of the red blood cells in vivo with 99mTcO4. At doses higher than 400 cGy, vascular permeability in the tumor 24 h after irradiation was significantly increased (P less than 0.05), while the vascular volume decreased (P less than 0.001) compared to control values. However at 72 h after irradiation there was no difference between treated and control groups. The results obtained in this study suggest a potential value of external irradiation to increase monoclonal antibody uptake by tumors governed mainly by the increased vascular permeability of the tumor vasculature soon after the irradiation exposure.

Inactivation of Coxiella burnetii by gamma irradiation.

The gamma radiation inactivation kinetics for Coxiella burnetii at -79 degrees C were exponential. The radiation dose needed to reduce the number of infective C. burnetii by 90% varied from 0.64 to 1.2 kGy depending on the phase of the micro-organism, purity of the culture and composition of suspending menstruum. The viability of preparations containing 10(11) C. burnetii ml-1 was completely abolished by 10 kGy without diminishing antigenicity or ability to elicit a protective immune response in vaccinated mice. Immunocytochemical examinations using monoclonal antibodies and electron microscopy demonstrated that radiation doses of 20 kGy did not alter cell-wall morphology or cell-surface antigenic epitopes.

[The immunomodulating action of microwaves in the induction of an immune response to Vi antigen]. / Immunomoduliruiushchee deistvie mikrovoln pri induktsii immunnogo otveta na Vi-antigen.

In this work the possibility of using microwaves for immunomodulation in the immunization of animals with thymus-independent antigen was studied. The projection zones of the thymus and adrenal glands of the test animals were subjected to the action of decimeter, or ultrahigh frequency (UHF), waves, while the corresponding zones of the control animals were subjected to imitation UHF irradiation. Vi-antigen was shown to be a thymus-independent antigen for rabbits (according to the results of the evaluation of the functional state of thymocytes, as manifested by the adsorption of acridine orange by the chromatin of thymocyte nuclei and by the content of RNA and DNA in the thymus). The action of UHF waves on the projection zone of the thymus was accompanied by a decrease in the glucocorticoid activity of the adrenal cortex, observed simultaneously with a pronounced immunostimulating effect. The UHF irradiation of the zone of the adrenal glands was accompanied by immunosuppression in combination with enhanced glucocorticoid activity of the adrenal cortex.

Internalization and re-expression of antigens of human melanoma cells following exposure to monoclonal antibody.

Modulation of the surface membrane of human Sk-Mel-28 melanoma cells by monoclonal antibody (MoAb) 96.5 recognizing p97 determinants was examined using direct radioimmunoassay and indirect fluorescent antibody-staining techniques. It was determined that the majority of 111In-labeled antibody that remained associated with cells after a 24-hr incubation at 37 degrees C had been internalized because MoAb 96.5 was no longer visible on the cell surface. A second treatment of these cells with the same antibody 24 hr later not only increased the cell-associated radioactivity, reflecting an increase of total antibody bound, but also rendered these cells membrane immunofluorescent again, indicating the re-expression of surface antigens. Autoradiographs of the electrophoretically analyzed membrane components of Sk-Mel-28 cells further demonstrated the appearance of newly synthesized 97-kDa proteins that were immunoprecipitable with MoAb 96.5. Taken together, the present findings suggest that p97 antigens undergo endocytosis in Sk-Mel-28 cells following exposure to MoAb 96.5. However, the same antigens were regenerated and expressed on the cell surface within a period of 24 hr. The re-expression of tumor cell surface antigen following initial internalization of the MoAb-antigen complex may have implications for diagnosis and therapy.

[New data on the biological action of normal immunoglobulins]. / Novye dannye o biologicheskom deistvii normal'nykh immunoglobulinov.

Experiments on 2,520 CBA mice (CBA X X C57BL) F1 nice have shown that the injection of homologous serum immunoglobulins (obtained from intact and blood-stimulated animals), made 2 hours after gamma irradiation from a 60Co source, prevents the development of intestinal dysbacteriosis and endogenous infection. The injection of mouse and human immunoglobulins to nonirradiated mice improved their resistance to experimental infection with Escherichia coli live culture, increased the expression of receptors to the Fc-fragments of IgG in peritoneal macrophages and enhanced the physical working capacity of the animals. The preparations containing normal antitissular antibodies have proved to be particularly effective. In mice, rabbits and dogs the preparations under test have produced no changes in the general state of the animals, no local reactions and no disturbances in the cardiovascular activity.

[Immunological reactivity in a radiation microclimate]. / Imunologichna reaktivnost v usloviiata na radiatsionen mikroklimat.

Bearing in mind the complexity of the radiation factor in industrial conditions the aim of work was to investigate the immunological reactivity in the conditions of glass production. The following methods were used: bactericide properties of skin, quantity of deep microflora of the skin, lysosome activity of the saliva, phagocytic activity of neutrophils in blood, bactericide properties of serum and serum complement. A decrease in the immunological reactivity of the organism was established which was most strongly expressed in relation to the bactericide properties of the skin, lysosome activity of the saliva and the phagocyte activity of the neutrophils in blood.

[Changes in immunobiological reactivity under the combined action of microwave, infrasonic and gamma irradiation]. / Izmenenie immunobiologicheskoi reaktivnosti pri kombinirovannom deistvii mikrovolnovogo, infrazvukovogo i gamma-izlucheniia.

In experiments on rats and rabbits a study was made of the combined effect of 9.3 gHz and 0.1 gHz (200 and 1530 muW/cm2, respectively), infrasound (8 Hz, 115 db), and gamma-radiation (cumulative dose of 5.5 Gy) on cell and humoral immunity, and on autoimmune processes. It was shown that preirradiation with microwaves increased the resistance of the organism to gamma-radiation, and microwaves combined with infrasound enhanced the biological effect of gamma-radiation.

[Immunobiological effects of a decimeter-range electromagnetic field in its exposure over the area of the thyroid gland]. / Immunobiologicheskie éffekty élektromagnitnogo polia detsimetrovogo diapazona pri ego vozdeistvii na oblasti shchitovidnoi zhelezy.

The immunological effect caused by the influence of the local action of the ultrahigh frequency (UHF) electromagnetic field on the projection zone of the thyroid gland was studied. The action of UHF waves shown to be capable of producing both immunostimulating and immunosuppressing effects; their manifestation depended on the period of immunogenesis, affected by the action of UHF waves. The synthesis of nonspecific immunoglobulins showed greater resistance to the suppressive action of UHF waves than the synthesis of specific immunoglobulins, i.e. antibodies. The activation of the thyroid gland under the influence of UHF waves occurred before the immunostimulating effect was observed.

Effects of microwave exposure on the hamster immune system. II. Peritoneal macrophage function.

Acute exposure to hamsters to microwave energy (2.45 GHz; 25 mW/cm2 for 60 min) resulted in activation of peritoneal macrophages that were significantly more viricidal to vaccinia virus as compared to sham-exposed or normal (minimum-handling) controls. Macrophages from microwave-exposed hamsters became activated as early as 6 h after exposure and remained activated for up to 12 days. The activation of macrophages by microwave exposure paralleled the macrophage activation after vaccinia virus immunization. Activated macrophages from vaccinia-immunized hamsters did not differ in their viricidal activity when the hamsters were microwave- or sham-exposed. Exposure for 60 min at 15 mW/cm2 did not activate the macrophages while 40 mW/cm2 exposure was harmful to some hamsters. Average maximum core temperatures in the exposed (25 mW/cm2) and sham groups were 40.5 degrees C (+/- 0.35 SD) and 38.4 degrees C (+/- 0.5 SD), respectively. In vitro heating of macrophages to 40.5 degrees C was not as effective as in vivo microwave exposure in activating macrophages to the viricidal state. Macrophages from normal, sham-exposed, and microwave-exposed hamsters were not morphologically different, and they all phagocytosed India ink particles. Moreover, immune macrophage cytotoxicity for virus-infected or noninfected target cells was not suppressed in the microwave-irradiated group (25 mW/cm2, 1 h) as compared to sham-exposed controls, indicating that peritoneal macrophages were not functionally suppressed or injured by microwave hyperthermia.

Effects of microwave exposure on the hamster immune system. I. Natural killer cell activity.

Hamsters were exposed to repeated or single doses of microwave energy and monitored for changes in core body temperature, circulating leukocyte profiles, serum corticosteroid levels, and natural killer (NK) cell activity in various tissues. NK cytotoxicity was measured in a 51Cr-release assay employing baby hamster kidney (BHK) targets or BHK infected with herpes simplex virus. Repeated exposure of hamsters at 15 mW/cm2 for 60 min/day had no significant effect on natural levels of spleen-cell NK activity against BHK targets. Similarly, repeated exposure at 15 mW/cm2 over a 5-day period had no demonstrable effect on the induction of spleen NK activity by vaccinia virus immunization, that is, comparable levels of NK were induced in untreated and microwave-treated animals. In contrast, treatment of hamsters with a single 60-min microwave exposure at 25 mW/cm2 caused a significant suppression in induced spleen NK activity. A similar but less marked decrease in spleen NK activity was observed in sham-exposed animals. Moreover, the sham effects on NK activity were not predictable and appeared to represent large individual animal variations in the response to stress factors. Depressed spleen NK activity was evident as early as 4 h postmicrowave treatment and returned to normal levels by 8 h. Hamsters exposed at 25 mW/cm2 showed an elevated temperature of 3.0-3.5 degrees C that returned to normal within 60 min after termination of microwave exposure. These animals also showed a marked lymphopenia and neutrophilia by 1 h posttreatment that returned to normal by 8-10 h. Serum glucocorticosteroids were elevated between 1 aNd 8 h after microwave treatment. Sham-exposed animals did not demonstrate significant changes in core body temperature, peripheral blood leukocyte (PBL) profile, or glucocorticosteroid levels as compared to minimum-handling controls.