Homologous Rabbit Antisera Research Articles

Characterization of the mitogenic activity elicited by Neisseria gonorrhoeae ribosomal fractions

Ribosomal preparations from Neisseria gonorrhoeae types 1 and 4 were examined for their in vitro stimulation of mouse splenocytes to determine the ribosomal moiety or contaminant responsible for the immunoproliferative activity. In immunodiffusion tests with homologous rabbit antiserum, crude 70S ribosomes formed four precipitin bands while the purified 30S and 50S subunits showed one major line. The same antiserum reacted with lysed N. gonorrhoeae and Neisseria meningitidis A cells but no precipitation occurred with Escherichia coli cells purified N. gonorrhoeae lipopolysaccharide (LPS). No membrane or LPS contaminant was detected in the purified 30S and 50S preparations. All the ribosomal preparations from virulent and non-virulent N. gonorrhoeae consistently stimulated the murine splenocytes. The mitogenic activity of the 30S and 50S ribosomal preparation was destroyed by treatment with trypsin but only slightly decreased by ribonuclease. It is suggested that the lymphoproliferative response elicited by gonococcal ribosomes is triggered by the protein moiety of the 30S or 50S subunits.

Interaction of Mycoplasma arthritidis and Other Mycoplasmas with Murine Peritoneal Macrophages

Neither mouse nor rat peritoneal macrophages were able to kill Mycoplasma arthritidis to any observable degree in the absence of specific hyperimmune rabbit antiserum. Although convalescent mouse and rat serum were somewhat inhibitory to M. arthritidis in the absence of macrophages, these sera did not promote active phagocytosis by peritoneal macrophages. In fact, the macrophages appeared to protect the mycoplasmas against the inhibitory effects of the antisera by stimulating their growth. Hyperimmune rabbit antiserum against M. arthritidis initiated phagocytic action and resulted in a 50-fold decrease in numbers of viable mycoplasmas by 6 h. In contrast with M. arthritidis, M. pulmonis rapidly adsorbed to the surface of peritoneal macrophages. Upon addition of specific rabbit antiserum, a rapid decrease in viable organisms occurred, and a more complete destruction of organisms ensued in comparison with M. arthritidis. M. gallinarum, as with M. arthritidis, did not adsorb to the macrophages to any great extent. Phagocytic action was observed only in the presence of homologous rabbit antiserum and was not marked until after 6 h of incubation.

Immunochemical studies on the lipopolysaccharides of Bordetella pertussis.

Lipopolysaccharide (LPS) was extracted from three Phase I strains (18334, 1494, and 134) and one Phase IV strain (11615) of B. pertussis. LPS was also obtained from ATCC 190 and NCTC 8632 which are listed as B. pertussis Phase IV but proved to be Lophomonas sp. and Brucella melitensis type 1. Chromatographic studies and chemical analysis revealed that all four B. pertussis LPS contained hexosamine, heptose, and hexose.The antigenic structure of the extracted LPS was studied by the techniques of passive immune lysis, lysis inhibition, and Ouchterlony immunodiffusion using homologous and heterologous rabbit antisera. The four B. pertussis LPS possessed at least six antigenic determinants. Four of these, designated A, C, E, and F, were found only in LPS of strains 18334 and 1494 (Phase I). Determinant D was found only in strain 134 (Phase I) and strain 11615 (Phase IV). All four strains of B. pertussis shared a sixth determinant, B. Antigen C was shared with the LPS of Lophomonas sp. (ATCC 190) and antigen F with the LPS of Brucella melitensis (NCTC 8632).There was no close correlation between the pattern of antigenic determinants on the LPS and other properties of B. pertussis, such as growth characteristics and possession of the protective antigen and the histamine-sensitizing factor.

Response of mice to injection of ribosomal fraction from group B Neisseria meningitidis.

Ribosomes of strain NOR-7 of group B Neisseria meningitidis were isolated by a procedure that included treatment of the cells with sodium dodecyl sulfate, disruption in a French pressure cell, and differential centrifugation. These preparations consisted of 66% ribonucleic acid and 24% protein and sedimented as a single component with a constant of approximately 66S. When used in immunodiffusion tests with homologous rabbit antiserum, untreated ribosomes formed two precipitin lines, when treated with ribonuclease three lines, and when Pronase-digested only one distinct line. Qualitatively indistinguishable reactions were obtained with the same antiserum and ribosomes from group A meningococci, but no precipitation occurred with those of Escherichia coli. When injected into mice, group B ribosomes elicited an increase in the number of antibody-producing spleen cells demonstrable by the hemolytic plaque technique using unsensitized sheep erythrocytes. Sensitization of the erythrocytes with increasing amounts of supernatant fluid of meningococcal cultures progressively reduced the number of demonstrable plaque-forming cells. Neuraminidase treatment of the erythrocytes increased immune hemolysis, whereas Pronase digestion reduced it. Injected mice were protected against homologous and heterologous meningococcal challenge. Both hemolysis and protection-inducing activities of the ribosomes were unimpaired by ribonuclease, but were reduced by Pronase. It is concluded that the immunological response elicited by the meningococcal ribosomes does not involve the group-specific carbohydrate antigen. The immunological mechanism by which the mice are protected against meningococcal challenge remains unknown.

Serologic changes associated with the encystment of axenically grown Hartmannella culbertsoni.

SYNOPSIS. Serologic reactions elicited by sonically ruptured trophic and cystic forms of Hartmannella culbertsoni were studied. The antigens of trophic amoebae reacted with their homologous rabbit antiserum showing multiple precipitin lines which could not be seen when the reacting antigens were treated with trypsin prior to application on the Ouchterlony plates. Antigens of trophic amoeba did not react with antiserum against cysts. Cyst antigens reacted with their homologous antiserum only after trypsin treatment. Antigens prepared from trophozoites excysting from cysts reacted positively with the antiserum against antigens of trophic amoebae.Antigens of trophic as well as cystic forms fixed guinea pig complement in presence of their homologous antisera. With the trophic form, this property was abolished after trypsin treatment. Non‐specific complement fixation mediated by cyst antigens was abolished by treatment with cellulase. Antiserum against trophic amoebae immobilized trophozoites and, in the presence of guinea pig complement, led to their lysis.

Rapid detection of viral antibody by cellulose acetate electrophoresis.

An immunoelectrophoretic procedure utilizing microprecipitation and cellulose acetate electrophoresis was developed for detection of antibody to specific virus. A model system of tobacco mosaic virus and homologous rabbit antiserum is described.

The effect of homologous rabbit antiserum on the growth of Leishmania tarentolae--a fine structure study.

SYNOPSIS. The fine structure of Leishmania tarentolae growing in the presence of homologous rabbit antiserum was investigated. Immediately after agglutination of the promastigotes, a dense band between pellicles of adjacent cells could be seen, and a surface coat was rendered visible. The cells did not fuse. At dilutions of antiserum from 1:100–1:1200 the promastigotes continued to grow, forming large masses. By 5 days these consisted of individual cells adhering to one another. In cross‐section, any 2 cells were separated by 2 membranes with associated subpellicular fibrils and a dense band between the 2 membranes.The term syncytium generally refers to a multinucleate cell originating by fusion of several cells and has been used to mean a multinucleate cell arising from nuclear division unaccompanied by cytokinesis. Therefore, it is unlikely that the masses formed in the presence of homologous antiserum are syncytia.

Rapid Detection of Viral Antibody by Cellulose Acetate Electrophoresis

An immunoelectrophoretic procedure utilizing microprecipitation and cellulose acetate electrophoresis was developed for detection of antibody to specific virus. A model system of tobacco mosaic virus and homologous rabbit antiserum is described.

Labeling of antibodies with radioactive isotopes in plant virus serology

Antibodies isolated from antiserum against plant viruses were labeled with the isotope35S as follows: the mixture of antibodies with radioactive cysteine hydrochloride was allowed to stand for half an hour, run on a Sephadex G-25 column and individual fractions were collected. Sephadex G-50 bed was equilibrated and washed with saline (0,85 % NaCl) phosphate buffer (0,01 m) pH 7,2. Fractions showing the highest radioactivity and at the same time the most evident serological reaction were combined and used as35S labeled antibodies. The labeled antibodies were used for rubbing leaves; the leaves were afterwards incubated, washed, killed, dried and then subjected to autoradiography. The method of indirect serological reaction also proved to be very good. Using this method, pig gamma globulin against rabbit gamma globulin was labeled with35 S; this labeled gamma globulin was then used to detect serological reaction on leaves between the virus and homologous rabbit antiserum and/or antibodies. The results of those reactions were also determined by autoradiography.

The chemistry of allergens XX. New antigens generated by pepsin hydrolysis of bovine milk proteins

Bovine serum albumin, α-lactalbumin, β-lactoglobulin, and casein were hydrolyzed at pH 2 with pepsin for 8 minutes. Hydrolyzates were separated into 2 fractions by dialysis—the dialysate and the endo fraction. Antigencity was determined by the Schultz-Dale technique with the use of uterine strips from guinea pigs sensitized with Freund's complete adjuvant. A new antigen was demonstrated in the dialysate of each of bovine serum albumin, α-lactalbumim, β-lactoglobulin, and casein. Casein was significantly less effective than the other proteins in the production of a new antigen. None of the antigens in the dialysates gave a precipitate with homologous rabbit antiserum. The endo fractions of β-lactoglobulin and casein contained no new antigens, and only one of 10 tests with the endo fraction of α-lactalbumin showed a new antigen. The endo fraction of bovine serum albumin contained a new antigen which gave a precipitate with homologous rabbit antiserum. Guinea pig antibodies for the dialysate of the pepsin digest of α-lactalbumin were stable when heated 4 hours at 56° C. These results may explain why milk proteins and possibly other foods in some cases do not give positive skin reactions on persons who give an immediate allergic response on ingestion of the food. Such persons may be sensitive to this type of new antigen formed during digestion.

Radiation treatment of foods. II. Public health significance of irradiation-recycled Salmonella.

Salmonellae resistant to gamma irradiation were developed by repeated irradiation and subculturing in a nutrient broth-yeast extract medium. Few differences were noted in the biochemical characteristics of parent and resistant cultures; however, microculture studies revealed variations in morphology and in cell division patterns. A considerable decrease in pathogenicity for day-old chicks was apparent with resistant cultures, but their phenol-water extracts were as toxic as parent material for 10-day chick embryos. Five serial chick passages did not reverse the reduced pathogenicity or aberrant morphology of a resistant Salmonella typhimurium culture. Results of phage typing of both parent and serially irradiated S. typhimurium were inconclusive, whereas the O-1 genus-specific phage lysed all parent serotypes tested but only one of the serially irradiated cultures. Agglutination of parent S. typhimurium cells with their homologous rabbit antiserum was unaffected by prior absorption with resistant strains. The results indicate that radiation recycling altered Salmonella into strains of lesser public health significance.

Red cell binding of gamma heavy chains isolated from polyalanylated anti-Rho IgG.

To extend biochemical studies of isolated anti-Rh IgG subunits (unpublished results of S. J. G.), experiments have been designed in which human anti-Rh0 heavy chains rendered soluble in neutral aqueous solution show binding specificity for the Rh determinant. Antigenicity of the solubilized heavy chains remains partially intact when tested with homologous rabbit antisera.

Previously unrecognized virus from submaxillary glands of gnotobiotic and conventional rats.

Ashe, Warren K. (National Institute of Dental Research, Bethesda, Md.), Henry W. Scherp, and Robert J. Fitzgerald. Previously unrecognized virus from submaxillary glands of gnotobiotic and conventional rats. J. Bacteriol. 90:1719-1729. 1965.-A serially transmissible cytopathic agent was isolated from histologically normal submaxillary glands, but not from various other tissues or specimens, from 74 of 97 gnotobiotic and conventional rats. Triturates of the glands or subsequent culture supernatant fluids induced specific cytopathic effects (CPE) in monolayer cultures of primary rabbit kidney cells (14 passages), a line of human skin cells (8 passages), and HeLa cells (17 passages). Transfer of supernatant fluids containing infected cells enhanced transmissibility. Neutralization of the CPE was demonstrated with sera of gnotobiotic and conventional rats and with homologous rabbit antiserum. A cold hemagglutinin specific for rabbit erythrocytes is associated with, but separable from, the infectious particle. Cultures of the agent induced no discernible effect on inoculation by various routes into suckling, weanling, or adult conventional mice, rats, and hamsters. Weanling and adult rabbits were also unaffected. Cultures for bacteria in gland extracts and infected cell supernatant fluids were uniformly negative. Negative cultures on PPLO media and negative arginine deiminase tests indicated that this agent is not a Mycoplasma. The data indicate that it is a virus whose biological and physical properties distinguish it from the known cytomegaloviruses. Because it has been found so far only in rat submaxillary glands, this agent is designated provisionally as RSMG virus.

STUDIES ON THE LIVETINS OF HEN'S EGG YOLK. II. IMMUNOELECTROPHORETIC IDENTIFICATION OF LIVETINS WITH SERUM PROTEINS.

Ten antigens have been distinguished in the sera of cocks and non-laying hens by immunoelectrophoretic analysis (IEA) against homologous rabbit antisera. An additional antigen has been found in the sera of laying hens by IEA against anti-laying-hen serum.Four of the serum antigens obtained by IEA have been correlated electrophoretically with the filter paper electrophoretic fractions serum albumin, serum α2-globulin, serum β-globulin, and serum γ-globulin.Serum albumin, serum α1-globulin, serum β-globulin, and serum γ-globulin have been identified immunologically with alpha-livetin, a livetin antigen (livetin 2), gamma1-livetin and gamma2-livetin respectively. Another antigen of cock serum and hen serum has been identified by IEA with a livetin antigen detected by IEA of livetin against anti-cock, anti-hen, and anti-yolk sera.

INACTIVATION OF DUPONOL-TREATED TOBACCO MOSAIC VIRUS WITH RABBIT ANTISERUM.

Abstract The common strain of tobacco mosaic virus, Ul, was partly degraded with Duponol (sodium lauryl sulfate). Infectious units surviving the detergent treatment were RNase sensitive as a consequence of having a segment of their RNA exposed. These infectious units displayed no marked change in their sensitivity to ultraviolet light or homologous rabbit antiserum, compared with intact virus, suggesting that the remaining protein coat still functioned during infection in a manner comparable to that of the intact virus.

A Study of Bentonite Flocculation Test for Measurement of Type-Specific Streptococcal Antibodies.

SummaryA flocculation test for detection and measurement of type-specific antibody to Group A streptococci has been evaluated. A single strain of each of 3 serological types of streptococcus was employed. When a suspension of bentonite particles between 1.0 and 1.5 μ in size was exposed to an unad-sorbed homologous rabbit antiserum after being coated with partially purified M protein antigen, flocculation was obtained. The highest homologous flocculation titer encountered was 1-32. A weak non-reciprocal cross-reaction between types 36 and 14 M antigens was found: while M antigen of strain S-94 (type 36) did not react with anti-S-23 (type 14) antisera, M protein of strain S-23 exhibited some cross-reactivity with anti-S-94 antisera. The test antigen could be stored at 4°C for at least 6 weeks without loss of potency. No group-specific or type-specific flocculation occurred when the test antigen was prepared by exposing the bentonite particles to a crude antigen mixture, obtained by hot HCl extraction of st...

Dissociation of Infective Poliomyelitis Virus from Neutralizing Antibody by Fluorocarbon

Summary Varying doses of poliomyelitis type I virus were mixed with appropriately diluted homologous rabbit antiserum and incubated at 37°C for 12–30 hr. The resulting noninfectious mixtures were homogenized with fluorocarbon and the amount of dissociated virus was determined by plaque assay. Similarly treated mixtures of virus and normal rabbit serum served as controls for the small losses of infectivity during incubation and homogenization. The use of optimal procedures permitted total recovery of as little as 90 to 270 plaque forming units from mixtures neutralized by an excess of antibody. Homogenization of these mixtures in absence of fluorocarbon did not yield infectious virus, indicating that the fluorocarbon is responsible for dissociation of virus from antibody.

Studies on the bacteriophages of hemolytic streptococci. II. Antigens released from the streptococcal cell wall by a phage-associated lysin.

The lysis of cell walls of hemolytic streptococci by a phage-associated lysin has been described. A method is presented for preparing the lysin from Group C streptococcal phage lysates. Following lysis almost all of the cell wall carbohydrate is recovered in solution. This material has the serological reactivity, physical-chemical properties, and values for nitrogen, rhamnose, and glucosamine similar to those of the carbohydrate isolated from the cell walls by the Streptomyces albus enzyme. Group C carbohydrate isolated by either enzyme inactivates Group C bacteriophage. The protein liberated by the lysin from Group A Type 6 cell walls gives a type-specific precipitin reaction with homologous rabbit antiserum. Preliminary data are presented on the ammonium sulfate fractionation and the electrophoretic separation of a protein fraction with the serological reactivity of M protein.

The Specific Hapten of Group C (Group II α) Meningococcus

Summary Soluble specific substances were prepared from 3 representative strains of Group C (formerly Group II α) meningococci. Dilutions as high as 1:4,000,000 of all preparations gave positive precipitin reactions with homologous rabbit antiserum. However, all evidenced some degradation of immunologic integrity, e.g., the best preparation absorbed only ¾ of the homologous mouse-protective activity from a rabbit Group C antiserum and it failed to engender significant active immunity in mice. On the other hand, intraabdominal injection of 30 μg of this preparation/animal sharply diminished the immune response of mice to subsequent injections of an effective Group C vaccine. The precipitin reactions of these substances with an equine polyvalent antimeningococcal serum prepared in 1918 indicated that Group C meningococci had been isolated in the United States at that time, although they were not recognized as a distinct group until 1940.

The Reaction of Chicken Ovalbumin with Homologous Antibody in the Region of Antibody Excess

Summary A pronounced decrease in the amount of precipitation in the region of antibody excess has been observed in the reaction of chicken ovalbumin with the concentrated globulin fraction of its homologous rabbit antiserum. A significant decrease was observed at a molar ratio of antibody to antigen of about 50:1, with almost complete inhibition at a ratio of 300:1. The results were confirmed with radioiodinated antigen; a much larger proportion of the antigen appeared in the supernatant in the prozone. The amount of precipitate formed is dependent on the method used for mixing the two reactants. For maximum inhibition by excess antibody, it is necessary to add the antigen to antibody and to stir during the addition, so as to maintain constantly a high antibody to antigen ratio. A large effect of order of addition was observed; the inhibiting effect of excess antibody was greatly decreased when antibody was added slowly to antigen so that the proportions in the mixture passed through the equivalence zone. Attempts to dissolve precipitates by extraction with excess antibody at 0° or 56°C have been unsuccessful. A high free energy of activation in the transformation of precipitate to soluble complexes is indicated.