Presence Of Specific Antiserum Research Articles

Real-time monitoring of flavivirus induced cytopathogenesis using cell electric impedance technology

A real-time cell analysis (RTCA) system based on cell-substrate electric impedance technology was used to monitor cytopathic effects (CPE) in Vero cell cultures infected with West Nile virus (WNV) and St. Louis encephalitis virus (SLEV) at infectious doses ranging from 101 to 106 plaque forming units (PFU) of virus. A kinetic parameter characterizing virus-induced CPE, CIT50 or the time to 50% decrease in cell impedance, was inversely proportional to virus infectious dose. In WNV-infected cells, the onset and rate of CPE was earlier and faster than in SLEV-infected cells, which was consistent with viral cytolytic activity. A mathematical model simulating impedance-based CPE kinetic curves indicated that the replication rate of WNV was about 3 times faster than SLEV. The RTCA system also was used for quantifying the level of cell protection by specific neutralizing antibodies against WNV and SLEV. The onset of WNV or SLEV-induced CPE was delayed in the presence of specific anti-sera, and this delay in the CIT50 was well correlated with the titer of the neutralizing antibody as measured independently by plaque reduction neutralization tests (PRNT). The RTCA system provided a high throughput and quantitative method for real-time monitoring viral growth in cell culture and its inhibition by neutralizing antibodies.

Cell-to-cell interactions in cultures of swine bone marrow cells and CPK-66b cells infected with african swine fever virus

In the presence of specific antiserum, cultured bone marrow cells from pigs infected with African swine fever virus form cell-to-cell junctions which are morphologically similar to those observed in antibody-dependent cellular cytotoxicity. This type of cytotoxicity does not determine the reaction of delayed hemadsorption, since this reaction is realized in a long-term culture of CPK-66b cells in the absence of the effectors of antibody-dependent cellular cytotoxicity. The sensitivity of delayed hemadsorption depends on the variant of infecting virus. A negative correlation is established between the area of the contact between erythrocytes and cells infected with different variants of the virus, on the one hand, and titers of antibodies in the delayed hemadsorption reaction, on the other.

Microparticle-enhanced nephelometric immunoassay with microsphere-antigen conjugates

gamma-Irradiation of acrolein and other acrylic monomers allowed the synthesis of spherical polyfunctional hydrophilic microparticles in the size range of 50 to 300 nm, on which antigens (immunoglobulins G, chorionic gonadotropin hormone, prealbumin) could be covalently bound. Microsphere-antigen conjugates clustered together in the presence of specific antiserum or monoclonal antibodies and their agglutination was quantified by light-scattering measurement performed with a specially designed nephelometer. Essential factors concerning the conjugate agglutination and its quantitation (size of microsphere, amount of antigen bound on microsphere, concentration of conjugate, concentration of agglutinating reagent, angle of light-scattering observation) were successively studied. A microparticle-enhanced nephelometric immunoassay for prealbumin was finally developed as an example of application. It was based on the inhibition of the immunoagglutination of microspheres-prealbumin conjugate by free prealbumin. This prealbumin immunoassay was easy to perform (one-step assay without washing or phase separation), fast (30 min), reliable (variation coefficients ranged from 3.6% to 7.5% for within- and between-assay determination), and sensitive (1 microgram/L detected). It was correlated with conventional immunonephelometry and radial immunodiffusion (correlation coefficients, 0.98). Microparticle-enhanced nephelometric immunoassay offered many advantages over the last two methods. Its better sensitivity allowed a lower reagent consumption and a larger sample dilution (contrary to the conventional immunonephelometry, sample pretreatment and sample blank measurement were unnecessary). Its inhibition mode induced a total accuracy for sample with high analyte concentration (a risk of underevaluation in antigen excess conditions existed in all method based on a noncompetitive antigen-antibody reaction) and provided the possibility to quantify haptens.(ABSTRACT TRUNCATED AT 250 WORDS)

Helminthotoxic responses of intestinal eosinophils to Trichinella spiralis newborn larvae

Because the gastrointestinal lamina propria is the first line of defense against invasion with Trichinella spiralis newborn larvae, we investigated the helminthotoxic characteristics of isolated lamina propria eosinophils. Eosinophils were isolated from the intestinal lamina propria of rats and purified to nearly 90% purity by a combination of velocity sedimentation through Percoll and unit gravity sedimentation through a continuous gradient of bovine serum albumin. Isolated eosinophils were of high viability and responded to surface receptor stimulation. Freshly isolated intestinal eosinophils lacked cytotoxic capacity when incubated with newborn larvae in the presence of specific antiserum. Peritoneal eosinophils from the same rats exhibited 100% helminthotoxicity after 24 h. Cytotoxicity could be stimulated in the intestinal eosinophils by the addition of recombinant murine interleukin-5.

Purification of El Tor cholera enterotoxins and comparisons with classical toxin

In 55 clinical isolates of Vibrio cholerae biotype El Tor, cholera toxin (CT) production was higher after growth in liquid medium first under relatively anaerobic conditions followed by excessive aeration (AKI conditions) as compared with growth under the optimal conditions for CT production from V. cholerae of classical biotype (median toxin level being 400 ng ml-1 and 1 ng ml-1 respectively, for the two different growth conditions). Large growth volumes further enhanced El Tor toxin production to levels at or above 3-5 micrograms ml-1 from several strains, which allowed for easy purification of toxin by salt precipitation, aluminium hydroxide adsorption and/or GM1 ganglioside affinity chromatography. However, such purified El Tor CT completely lacked the A subunit when examined by SDS-PAGE or by monoclonal anti-A subunit antibody GM1-ELISA. In contrast, when El Tor CT was prepared from bacteria grown in the presence of specific antiserum against soluble haemagglutinin/protease it contained the A subunit (unnicked) in the same proportion to the B subunit (1A:5B) as classical CT. Immunodiffusion-in-gel tests revealed that the B subunits of El Tor and classical CTs share major epitopes but also have one or more weaker biotype-specific epitopes. The two types of toxin were practically indistinguishable in various GM1-ELISA tests, and antisera raised against El Tor and classical CT, respectively, could also completely neutralize the heterologous as well as the homologous toxin activity in vivo. The results indicate that CTs from El Tor and classical V. cholerae, despite demonstrable epitope differences, are predominantly cross-reactive and give rise to antisera with strong cross-neutralizing activity.

Bactericidal effect of normal swine sera on Treponema hyodysenteriae.

Treponema hyodysenteriae was incubated in 20% normal swine sera (NSS) at 37 degrees C for 4 h, and viability was determined by a plate dilution method. NSS was bactericidal for nonpathogenic T. innocens and avirulent T. hyodysenteriae, but not for virulent T. hyodysenteriae isolates. Heat inactivation at 56 degrees C for 30 min, treatment with EDTA or EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid], or removal of immunoglobulin M eliminated the bactericidal activity of NSS. However, removal of the alternate complement pathway with 10 mg of bentonite per ml did not remove bactericidal activity of NSS. Incubation of virulent isolates of T. hyodysenteriae in the presence of specific antisera plus NSS resulted in bactericidal activity. These data suggest that complement and natural antibody may be involved in protecting the host from T. innocens or avirulent T. hyodysenteriae and that T. hyodysenteriae antibody plus complement are involved in protecting convalescent pigs from re-exposure to swine dysentery.

Adhesion of leptospires to mouse fibroblasts (L929) and its enhancement by specific antibody.

The adhesion of leptospires (Leptospira interrogans serovar. copenhageni L45) to mouse L-cells was studied by microscopic observations. Within 3 h of infection of monolayers many leptospires adhered to 95-100% of the cells, and intracellular leptospires were demonstrated by electron microscopy. No specific site of attachment on the cells or the leptospires was observed. Avirulent or dead leptospires adhered poorly but attachment of the saprophytic leptospire L. biflexa serovar. patoc occurred on cell and glass surfaces. After adhesion, microvilli on the cell surfaces disappeared within 6 h of infection and cell damage was observed after 12 h. The adhesion was greatly enhanced by the presence of specific antiserum at a subagglutinating concentration. No direct penetration by leptospires of the host cells was observed with transmission and scanning electron microscopy. It appears that (1) adhesion of leptospires to L-cells precedes cell damage, and (2) leptospires may enter cells either through damaged membranes, or by a phagocytosis-like mechanism.

Antibody-resistant spread of vesicular stomatitis virus infection in cell lines of epithelial origin.

In MDCK cells, vesicular stomatitis virus (VSV) buds exclusively from the basolateral plasma membranes beneath tight junctions, whereas influenza virus forms only at the free apical surface. Anti-VSV antiserum did not prevent the formation of plaques on MDCK cell monolayers infected with VSV, whereas plaque formation in BHK-21 cells was completely inhibited by such antiserum. Under similar conditions, homologous antiserum completely prevented plaque formation by influenza virus on MDCK cells. In several other epithelioid cell lines, VSV also formed plaques in the presence of specific antiserum. These results suggest that VSV receptors are present on basolateral membranes in the cells studied and that junctional complexes present between cells may exclude antibody from intercellular spaces and thus permit the lateral spread of virus infection in the presence of neutralizing antibody.

Measurement of mutagenesis in mammalian cells.

A method using mammalian cells in vitro for detection and quantitation of mutagenic actions that appears to be useful for screening for carcinogenesis and genetic damage by environmental agents is presented. The method involves use of stable human--Chinese hamster ovary hybrid cells that have retained a single human chromosome not necessary for cell reproduction. Forward mutations are detected in genes necessary for production of specific human cell surface antigens. Such mutants form colonies in the presence of specific antisera and complement that destroy the unmutagenized cells. Use of the method is illustrated for the action of x-irradiation, N-methyl-N'-nitro-N-nitrosoguanidine, and caffeine. The method appears to be unique in that it permits assessment of lesions that cause loss of all or most of the chromosome as well as various localized gene mutations. The former action is particularly important because of the major involvement of chromosomal lesions in an extremely important class of human genetic disease.

Ultrastructural and immunological aspects of the phagocytosis of Trypanosoma brucei by mouse peritoneal macrophages.

Trypanosome-activated mouse peritoneal macrophages phagocytized and digested Trypanosoma brucei in vitro and in vivo, but in the absence of specific antiserum and complement the degree of phagocytosis was minimal. Ultrastructurally, the parasites attached to the macrophage by their flagella, and ingestion proceeded flagellum first. Once ingested, T. brucei was degraded, presumably due to fusion of the parasite-containing phagosome with lysosomes. Contrariwise, normal mouse peritoneal macrophages displayed negligible ability to ingest T. brucei, even in the presence of specific antiserum and complement. During trypanosomiasis in deer mice (Peromyscus maniculatus), the development of hypergammaglobulinemia correlated with enhanced phagocytosis of T. brucei by macrophages, but only at early post-inoculation days (PID 5 to 15). Complement lysis of trypanosomes was not identified in these experiments. Between PID 20 to 30, antiserum and complement either had no phagocytosis-promoting ability or depressed the phagocytosis of T. brucei by macrophages. These results indicate that both specific antibody and complement contribute to the ingestion of T. brucei by activated macrophages, but that parasite antigenic variation effectively abrogates the phagocytic defense mechanism.

Simple immunochemical method for measuring DNA repair rate in u.v.-irradiated bacteria

A straightforward immunochemical method is reported to determine the repair rate of DNA in u.v. (ultraviolet-irradiated bacteria based on use of antibodies specifically interacting with u.v.-irradiated DNA. The method involves quantitative precipitation of labeled 3H-DNA on nitrocellulose filters in the presence of specific antiserum. The method is justified that the dimers are almost completely eliminated in cells of wild strain during postradiation incubation after u.v.-irradiation with 400 ergs/mm 2. In the cells of u.v.-sensitive uvr A6 mutant the dimers are practically not affected, whereas in those of uvr 502 strain excision of the dimers does take place, though more slowly than in wild strain bacteria. The reported approach gives results in good accord with those obtained by other chromatographically.

Immunity to the group B streptococci: interaction of serum and macrophages with types Ia, Ib, and Ic.

The opsonization and phagocytosis of group B streptococci of types Ia, Ib, and Ic were studied in vitro by measuring the uptake of radioactivity by coverslip cultures of rabbit alevolar macrophages during incubation with radiolabeled, nonviable bacteria which had been exposed to rabbit serum. The uptake of counts per minute was quantitative, reproducible, and reversibly inhibited by cold, indicating that it was largely a measurement of phagocytic ingestion rather than of attachment of bacteria-immunoglobulin complexes to macrophage membranes. Moreover, suspended macrophages killed approximately 90% of viable streptococci in the presence of specific antiserum. The opsonic activity of immune serum was heat stable, and phagocytosis of streptococci was insignificant after incubation with normal serum and antiserum to some heterologous group B streptococci. By absorption studies, it was possible to identify the effect of antibodies to specific bacterial antigens. Phagocytosis of streptococci containing the corresponding antigens was maximal after opsonization with homologous or heterologous sera containing antibody to IaCHO, IbCHO, or Ibc protein. Phagocytosis of all three serotypes was intermediate when opsonization could be attributed to anti-IabcCHO. The opsonization of a specific group B streptococcus is complex and may involve two or more antigen-antibody systems.

Desensitization of delayed-type hypersensitivity by antigen and specific antibody

The role of antibody in the desensitization of delayed-type hypersnsitivity (DTH) to dinitrophenylated bovine gammaglobulin (DNP-BGG) was studied in rats. Rats sensitized by a subcutaneous injection of DNP 32-BGG in Freund's complete adjuvant (FCA) were desensitized 14 days later with various doses of DNP 32-BGG injected intravenously. It was found that only certain doses (100–500 μg) of DNP-BGG effectively desensitized, antigen doses outside this optimum range being ineffective in suppressing DTH. In adoptive cell transfer experiments, it was shown that sensitized peritoneal cells incubated with optimum doses of the antigen in the presence of specific antiserum in vitro failed to transfer the delayed response to normal recipients, whereas the treatment of the sensitized cells with the antigen or with the antiserum separately did not impair the ability of these cells to transfer DTH. The effect of desensitization is specific and is not permanent. The DTH reappears 3–4 wk after desensitizing injection.

Vitamin A in liposomes. Inhibition of complement binding and alteration of membrane structure

Incorporation of vitamin A aldehyde (retinal) into liposomes had an inhibitory effect on the amount of human complement protein bound in the presence of specific antiserum. The total membrane-bound protein was directly measured on liposomes which were washed after incubation in antiserum and fresh human serum (complement). At every concentration of complement, decreased protein binding was found with liposomes which contained retinal. Binding of the third component of complement (C3) was also measured directly on washed liposomes and was found to be decreased in the presence of retinal. The diminution in protein binding due to retinal was not caused by differences in the amount of antibody bound and this was shown by two experiments. First, specific antibody protein binding to liposomes was directly measured and was essentially unaffected by retinal. Second, liposomes were prepared from lipid extracts of sheep erythrocytes. These liposomes were used as as immunoadsorbants to remove antisheep erythrocyte antibodies. The immunoadsorbant capacity was the same in both the presence and the absence of retinal. A further conclusion from these experiments was that retinal did not change the number of liposomal glycolipid antigen molecules available for antibody binding and thus presumably did not change the total number of lipid molecules present on the outer surface of the liposomes. Retinal did have an effect on the geometric structure of the liposomes. Size distribution measurements were performed in the diameter range of 1–6.35 μm by using an electronic particle size analyzer (Coulter Counter). Liposomes containing retinal were shifted toward smaller sizes and had less total surface are and volume. It was suggested that retinal-containing liposomes may have had a tighter packing of the molecules in the phospholipid bilayer. This effect of retinal on liposomal structure may have been responsible for the observed decreased binding of C3 and total complement protein.

Specific Susceptibility of Sensitized (Memory) B Cells to Suppression and Antigenic Alteration by Murine Leukemia Virus

Mice infected with murine oncogenic RNA viruses, either Friend virus (FV) or Moloney leukemia virus (MuLV-M) reacted well to a primary immunization with antigen(s) of Vibrio cholerae; both the kinetics and magnitude of the direct (IgM) primary vibriolytic antibody plaque forming cell (PFC) response in the spleen was the same as in non-infected control mice. In contrast, the anamnestic PFC (IgM) response of mice preimmunized with cholera and later (2 to 9 months following priming) infected with either FV or MuLV-M, before the secondary challenge, was inhibited 90% or more when compared to the anamnestic response of controls. Hemolytic PFC response to a single (primary) immunization with sheep red blood cells (sRBC, an antigen to which laboratory mice have a natural immune background) was suppressed by infection with both viruses in a manner similar to the suppression of the secondary vibiolytic response. The suppressive effect of FV, and the lack of it, on these different immune responses was correlated with the appearance of FV-induced cell membrane antigen, FVMA, on respective individual PFC from the spleen. FVMA was detected by inhibition of PFC in the presence of specific antiserum, anti-FVMA, and complement (C). In particular, about 80% of the FV-infected mice with an anamnestic response to a secondary challenge with cholera antigen had a significant decline in the proportion of PFC (33% or more) following incubation with anti-FVMA plus C. A similar degree of inhibition was found with hemolytic (anti-sRBC) PFC from FV-infected mice. In contrast, primary vibriolytic PFC from FV-infected mice were not inhibited by anti-FVMA, i.e., they did not carry the virus-induced antigen. These results indicate absolute correlation between viral suppression of a given clone of immunocompetent cells, and antigenic alteration of individual antibody-forming cells due to the virus-infection. Furthermore, the absence of both functional inhibition and virus-induced antigen(s) in the primary immunocompetent cells and the presence of both these virus-related functions in the memory cells (either experimentally sensitized cholera memory cells or naturally sensitized sRBC memory cells) strongly suggested that the latter are selectively susceptible to oncornavirus.

Biochemical comparison of some classical Tetrahymena pyriformis and T. vorax strains.

SYNOPSIS. The classical Tetrahymena pyriformis and T. vorax strains have been classified in antigenic subgroups based on immobilization and agar double diffusion in the presence of specific antisera. The present study utilized disc electrophoresis of proteins and lactate dehydrogenase isozymes for comparison of 11 strains. In agreement with similarities reported for agar diffusion data, close electrophoretic relationships were observed between strains GL and H, and V2 and W with aniline black staining. Altho LDH isozyme production was more variable and related to cultural growth phase, similarities were recognized between GL and H, and F and PR.

Adsorption of 3H-fatty acid esters of streptococcal groups A and E cell wall polysaccharide antigens by red blood cells and their effect on hemagglutination.

The streptococcal group A and E cell wall polysaccharide (PS) antigens were esterified under identical conditions with four fatty acid chlorides (lauroyl, myristoyl, palmitoyl, and stearoyl), varying from 12 to 18 carbon atoms. With group A PS, it was shown that the four resulting esters varied in their ability to sensitize red blood cells (RBC) to agglutination in the presence of specific antiserum. The most active was palmitoyl (16C) followed by myristoyl (14C). The least active was the lauroyl ester (12C). One-tenth as much palmitoyl ester was required as stearoyl group A PS ester. Such variation in the ability to sensitize RBC was not demonstrated with the group E esters, with the exception of the lauroyl ester which was the least active. Removal of N-acetylglucosamine from the esterified and the nonesterified group A PS by enzyme action resulted in a significant loss of serological activity of both antigens. No appreciable difference in the rate or total loss of activity was found in either case. It was demonstrated that both tritium-labeled stearic and palmitic acids and their respective PS esters were adsorbed in significant amounts to RBC. The results indicate that the esterified antigens were adsorbed to the RBC because of the presence of the fatty acid in the PS ester. Attempts to block the receptor sites on the red cell by presensitizing the cells with fatty acid were negative. Likewise, the adsorbed ester did not prevent the uptake of fatty acid at the levels tested. Tritium-labeled esterified group A PS and group E PS were used to show that the amount of antigen required to produce maximal agglutination was the same when cells from the same individual were used, whereas this was not the case when cells from different individuals were used. The amount of antigen required to produce maximal agglutination varied from one batch of sheep RBC to another. Once the optimal concentration of antigen was reached, any additional adsorption did not increase the titer of agglutination.

Simian virus 40: replication in the presence of specific antiserum and adenovirus 4.

Twelve consecuttive passages of simian virus 40, made in the presence of adenovirus 4 and antiserum to simian virus 40, indicated that simian virus 40 would multiply almost indefinitely under these conditions. The adenovirus and the simian virus had previously replicated together in the absence of antiserum. These data support the conclusion that the SV-40 genome is incorporated within the protein coats of adenovirus 4.

ANTIGENIC SIMILARITY AMONG CULTURE FORMS OF THE 'BRUCEI' GROUP OF TRYPANOSOMES.

Extensive cross-reactions have been found between cultural forms of trypanosomes of the ‘brucei’ group by the agglutination reaction. The antigen (or antigens) responsible for agglutination are apparently specific to the ‘brucei’ group of trypanosomes. These antigens are also quite stable to the presence of specific antiserum in contrast to the serotype of the blood trypanosomes which are known to be altered in the presence of specific antiserum.

An Immunologic Study of the “Lipovirus”

Summary Sheep erythrocytes treated with lipovirus were so altered that they became agglutinated in the presence of specific antiserum. The serum agglutinating factor was stable to heat at 56°C for 30 min and was absorbed by lipovirus-treated red cells. This agglutination was specifically inhibited by pretreatment of antiserum with lipovirus. Rabbits injected with lipovirus responded with the formation of antibody in high titers as demonstrated by this reaction. Serums from 28 rabbits injected with a variety of viral, rickettsial, and bacterial agents and cell components did not agglutinate lipovirus-sensitized erythrocytes. Complement-fixing antigen specific for the lipovirus was prepared by concentrating the specific involved shrunken cells. Antibody responses were demonstrated in all rabbits injected with the lipovirus but not in those injected with uninfected cells. Some animals immunized with lipovirus grown in human cells showed rises in antibody against uninfected chick cell antigen. Similarly, some injected with lipovirus in chick cells showed rises in antibody against human cells. A variety of antisera or paired acute and convalescent sera were tested for “lipovirus” antibody. There is no evidence that the lipovirus is immunologically related to any of the following viruses: vaccinia, herpes simplex, varicella, cytomegalo-, adeno-, SV40, polyoma, influenza types A and B, para-influenza types 1, 2, 3 and 4, measles, mumps, respiratory syncytial and Newcastle disease viruses.