Type-specific Rabbit Antisera Research Articles

Characterisation of a South African human astrovirus as type 8 by antigenic and genetic analyses.

Human astroviruses (HAstV) can, on the basis of immunoassays using type-specific rabbit antisera, be classified into eight serotypes that correlate with genotypes. Very few isolates of HAstV type 8 have been described and there is a paucity of data available with regard to the antigenic and genetic relationships between HAstV type 8 (HAstV-8) and HAstV types 1 (HAstV-1) to 7 (HAstV-7). A wild-type HAstV from a South African paediatric patient with diarrhoea was analysed antigenically, by immune electron microscopy and enzyme immunoassay, and genetically in selected regions of the ORF1a, ORF1b and ORF2 and characterised as a HAstV-8. This HAstV-8 strain exhibited greatest homology with HAstV-4 in the 5' end of the capsid gene and ORF1a and 1b, and greatest homology with HAstV-5 in the 3' end of the capsid region. This study confirms, by both antigenic and genetic analyses, that HAstV-8 represents a distinct antigenic and genotype and is the first report of a HAstV-8 from a hospitalised paediatric patient with diarrhoea in southern Africa.

Polysaccharide capsule-mediated resistance to opsonophagocytosis in Klebsiella pneumoniae.

The polysaccharide capsule of Klebsiella pneumoniae is an important virulence factor that confers resistance to phagocytosis. The treatment of encapsulated bacteria with salicylate to inhibit capsule expression was found to enhance the phagocytosis of encapsulated bacteria by human neutrophils only in the presence of cell surface-specific antibodies. Both type-specific rabbit antisera and anticapsular human hyperimmune globulin were employed as opsonins. Salicylate significantly enhanced phagocytosis with homologous, but not heterologous, whole-cell antisera. Antisera, diluted 1:40, no longer opsonized fully encapsulated bacteria but promoted the uptake of multiple salicylate-treated bacteria in > 90% of neutrophils. Salicylate (0.25 to 1.0 mM) also enhanced opsonization with globulin against homologous bacteria. Higher salicylate levels (1 to 2.5 mM) enhanced the opsonization of heterologous serotypes with human globulin. The nature of antibody attachment to encapsulated bacteria was determined by immunofluorescence. Even after the addition of purified capsular polysaccharide to prevent phagocytosis, K-specific antibodies attached in large amounts to bacteria. K-specific antibodies reacted with antigens throughout the capsule and showed a predilection for a denser inner layer of the capsule, indicating that many of the K-specific antibodies may be masked underneath the capsule surface. K-specific antibodies can also be rendered nonfunctional by soluble, cell-free capsular antigen. In culture, large quantities of soluble capsular polysaccharide extrude from bacteria after overnight growth. The reduction in capsule expression caused by salicylate largely affected the soluble, cell-free fraction. Purified capsular polysaccharide was shown to retard the opsonophagocytosis of salicylate-treated bacteria in a concentration-dependent manner. However, extensive washing of encapsulated bacteria to remove loosely attached capsular material did not significantly enhance opsonophagocytosis. In conclusion, cell-free capsule and cell-associated capsule are antiphagocytic; both act to neutralize K-specific antibodies by binding or concealment. Salicylate-mediated inhibition of capsule expression, particularly of the cell-free fraction, improved K-specific opsonization dramatically.

Antigenic Characterization of Cell-Cultivated Astrovirus Serotypes and Development of Astrovirus-Specific Monoclonal Antibodies

Cultivation of human astroviruses in human embryonic kidney or LLCMK2 cell cultures was corroborated for four of the five serotypes originally reported (types 1, 2, 4, and 5). By using type-specific rabbit antisera and immunofluorescence of virus-infected cells, we readily distinguished between serotypes of astrovirus; however, these serotypes showed a high degree of cross-reactivity by enzyme-linked immunoassay, a result indicating the presence of a group antigen. We prepared monoclonal antibodies to astrovirus type 2 antigen and selected them on the basis of group antigen reactivity. The antibodies were reactive with the four astrovirus serotypes that we could cultivate, as well as with the Marin County strain of astrovirus. A previously reported cell-cultivated astrovirus type 3 also reacted with the monoclonal antibodies. These monoclonal antibodies, and the finding of group reactivity among the human astroviruses, should facilitate studies on the importance of these viruses as agents of viral gastroenteritis.

Immunoblotting to demonstrate antigenic and immunogenic differences among nine standard strains of Clostridium difficile

The epidemiology of Clostridium difficile-associated disease is being elucidated with the development of typing schemes for the organism. We recently described a new typing scheme based on the incorporation of [35S]methionine into bacterial proteins followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Nine standard strains were identified. We report here some observations on the antigenic differences among these nine strains when studied by immunoblotting. Type-specific rabbit antiserum was raised against each of the nine standard strains. Immunoblotting of the strains with these antisera demonstrated, in addition to the presence of shared, common proteins, a type-specific response with homologous antisera. When [35S]methionine-labeled C. difficile proteins were immunoblotted with homologous and heterologous antisera, both the immunoblots and the autoradiographs demonstrated the same strain-specific response. These strain-specific proteins, which have been so useful for epidemiological and typing purposes, were also immunogenic.

Factors influencing adherence of group B streptococci to human vaginal epithelial cells.

Factors affecting the adherence of group B streptococci to human vaginal epithelial cells in vitro were examined. Maximal adherence was achieved within 15 min of incubation of bacteria with epithelial cells. Adherence was temperature and pH dependent; maximal adherence occurred at 37 degrees C and pH 5.5. Killing of streptococci with ultraviolet light or penicillin did not affect adherence. Similarly, adherence was not altered by preincubating epithelial cells at 65 degrees C for 30 min. Thus neither bacterial nor epithelial cell viability appears to be a prerequisite for adherence. Preincubation of streptococci at 65 degrees C for 30 min resulted in a marked decrease in adherence, whereas preincubation of group B streptococci with neuraminidase was associated with a significant increase in adherence. The adherence of strains belonging to five different group B streptococcal serotypes was not altered by group-specific or type-specific rabbit antisera. These findings suggest that the site for adherence on the bacterial cell wall is heat sensitive and is marked by sialic acid, but is not related to either group-specific or type-specific antigens.

Effects of surface-active agents on neutrophil receptors.

An easily performed assay to identify the C3b and Fc receptors on human neutrophils was developed. Salmonella typhimurium were treated with fluorescein and then incubated in nonimmune fresh human serum, which led to C3b fixation via activation of the alternative pathway. Similarly, type II pneumococci were treated with fluorescein and opsonized with type-specific rabbit antiserum. Neutrophils bearing C3b and Fc receptors formed rosettes with the respective bacteria, which were easily readable because of their bright fluorescence. Incubation of neutrophils at 37 degrees C with C3-coated bacteria generated 54 +/ 4% C3b rosettes, whereas neutrophils incubated with immunoglobulin G-coated bacteria yielded 75 +/ 7% rosettes. Incubation at 4 degrees C inhibited the formation of C3b rosettes but not Fc rosettes. Heat inactivation of the fresh human serum at 56 degrees C for 30 min completely inhibited the formation of the C3b rosettes, and addition of heat-aggregated immunoglobulin G to the polymorphonuclear leukocyte blocked the ability of the polymorphonuclear leukocyte to bind immunoglobulin G-coated bacteria. Addition of 1.0 mM N-ethylmaleimide, 0.1 mg of trypsin per ml, 10 mM H2O2, O2- generated by xanthine-xanthine oxidase, and 8 times 10(-4) M hydrocortisone inhibited the C3b receptor, but did not inhibit the Fc receptor. In neutrophils, the selective effect of the various inhibitors suggests that the Fc and C3b receptors are distinct entities.

EFFECTS OF SURFACE ACTIVE AGENTS ON NEUTROPHIL RECEPTORS

An easily performed assay to identify the C3b and Fc receptor on human polymorphonuclear leukocytes (PMN) was developed. Salmonella typhi were directly fluoresceinated and then incubated in non-immune fresh human serum which led to C3b fixation via activation of the alternative pathway. Similarly, Type II pneumococci were fluoresceinated and opsonized with type specific rabbit anti-serum. PMN bearing C3b and Fc receptors formed rosettes with the respective bacteria which were easily readable because of their bright fluorescence. Studies employing various surface agents were employed to provide information on the physical properties of PMN receptors. Incubation of PMN at 37° C with C3-coated bacteria generated 54±5% C3b rosettes whereas PMN incubated with IgG coated bacteria yielded 78±8% rosettes. Heat inactivation of the fresh human serum at 56° C for 30 minutes completely inhibited the formation of the C3b and addition of goat anti-rabbit IgG inhibited the formation of the Fc rosettes. Pre-incubation of PMN with 1 mg/ml Trypsin, 1 mM N-ethyl maleimide, xanthine-xanthine oxidase to generate 20 nm superoxide anion and hydrogen peroxide (H2O2), 10 mM H2O2, and 8×10−4 M hydrocortisone reduced the number of C3b rosettes to values of 25±6%, 7±6%, 22±4%, 36±2%, and 24±5%, respectively, but had no significant effect on the number of Fc rosettes. Thus, this study provides evidence for selective receptors for C3b and Fc on PMN membranes.

Quantitative detection of type A staphylococcal enterotoxin by Laurell electroimmunodiffusion.

The detection of staphylococcal enterotoxin A by the quantitative technique of electroimmunodiffusion is described. High dilutions of type-specific rabbit antiserum were used in 1% agarose gels, 1 mm thick, and prepared in 0.05-mug barbital buffer, pH 8.6. Volumes of 10 muliters containing 1.5 to 10 ng of toxin were electrophoresed out of 4-mm diameter wells at 5 mA/cm width of gel. The precipitin cones formed were made visible by first immersing the agarose gels in 0.2 M NaCl and then overlaying the surface with the purified globulin fraction of sheep serum against rabbit globulin, followed by soaking of the gels in 1% aqueous cadmium acetate and staining with 0.1% thiazine red in 1% glacial acetic acid. Fully extended cones, 4 to 23 mm in length depending on toxin concentration and antiserum dilution, were developed in 2 to 5 h of electrophoresis, and visualization was achieved within 2 to 3 h. Because the method is qualitative, quantitative, simple, rapid, and sensitive, it offers a practical tool for the detection of small amounts of bacterial toxins in contaminated foods. The method should also qualify as a sensitive detection device in biochemical procedures which attempt to trace, detect, and identify biological substances in nanogram quantities, provided these substances are antigenic and capable of forming a precipitate with their specific antibodies.

Quantitative Detection of Type A Staphylococcal Enterotoxin by Laurell Electroimmunodiffusion

The detection of staphylococcal enterotoxin A by the quantitative technique of electroimmunodiffusion is described. High dilutions of type-specific rabbit antiserum were used in 1% agarose gels, 1 mm thick, and prepared in 0.05-mug barbital buffer, pH 8.6. Volumes of 10 muliters containing 1.5 to 10 ng of toxin were electrophoresed out of 4-mm diameter wells at 5 mA/cm width of gel. The precipitin cones formed were made visible by first immersing the agarose gels in 0.2 M NaCl and then overlaying the surface with the purified globulin fraction of sheep serum against rabbit globulin, followed by soaking of the gels in 1% aqueous cadmium acetate and staining with 0.1% thiazine red in 1% glacial acetic acid. Fully extended cones, 4 to 23 mm in length depending on toxin concentration and antiserum dilution, were developed in 2 to 5 h of electrophoresis, and visualization was achieved within 2 to 3 h. Because the method is qualitative, quantitative, simple, rapid, and sensitive, it offers a practical tool for the detection of small amounts of bacterial toxins in contaminated foods. The method should also qualify as a sensitive detection device in biochemical procedures which attempt to trace, detect, and identify biological substances in nanogram quantities, provided these substances are antigenic and capable of forming a precipitate with their specific antibodies.

A Type-Specific Immunodiffusion Technique for the California Encephalitis Virus Group

Abstract The development and evaluation of a California encephalitis virus (CEV) typespecific immunodiffusion (ID) technique is described. Agreement between this typing method and the more conventional complement-fixation test ranged from 96.2% to 100%. To date, type-specific rabbit antisera used in the system have been prepared against Keystone, trivittatus, BFS 283, snowshoe hare, LaCrosse and Jamestown Canyon antigens. A type-specific reaction was obtained with rabbit anti-San Angelo serum following absorption with BFS 283 antigens. However similar absorption of rabbit anti-Tahyna, anti-Lumbo and anti-Jerry Slough sera failed to produce a type-specific response. By screening isolates initially in the ID test using a CEV grouping serum, reacting isolates can quickly be identified by the typespecific antisera in the ID test. The technique is relatively inexpensive and can be performed readily by a technically competent technician in a modicum of time.

Relationship between Metabolism-inhibiting Antibodies and Colonial Growth of Mycoplasma hyorhinis and other Porcine Mycoplasma in the Presence of Rabbit and Pig Sera

Metabolism-inhibition tests with the porcine mycoplasma strains Mycoplasma hyorhinis, M. granularum and mycoplasma B3 showed that two of these strains, M. hyorhinis and M. granularum, produced colonies in the presence of type-specific rabbit antisera. This type of reaction is referred to as colonial growth of mycoplasma in fluid medium. The colonies were never found in the presence of heterotypic or negative rabbit sera. M. hyorhinis strains showed quantitative and quantitative differences in colonial growth. They produced colonies in the presence of pig sera obtained from herds with a known incidence of M. hyorhinis whereas M. granularum failed to do so in the presence of these sera. Neither of the strains produced colonies in the presence of pig sera obtained from herds from which M. hyorhinis could not be recovered.

Low molecular weight L-chain components related to Bence-Jones proteins.

Abstract During preparation of Bence-Jones proteins from urines of a group of 15 patients with multiple myeloma, a patient was found to excrete 2 molecular forms of L-chain-related materials of 1.8S and 3.0S. The smaller L-chain component had a molecular weight of 11,600 and was antigenically deficient with respect to the larger component (Mol. wt. 33,900) by Ouchterlony analysis and quantitative precipitin curves. Relative proportions of the lower molecular weight L-chain component excreted in urines examined over a 12 month interval declined with coincident slight improvement in clinical renal function. Both the low molecular weight and higher molecular weight L-chain components reacted with kappa type-specific rabbit antisera.

Studies on Streptococcus pyogenes. IV. Effect of whole cells, M and C antigens, and other components of the cell on adrenalectomized rats.

Heat-killed cells of Group A streptococci caused death of the adrenalectomized rat. While the adrenalectomized rat readily succumbed to intraperitoneal infection with living cells, death was due primarily to toxicity. The normal rat was highly resistant under either condition. For studies on the toxic materials, the cells of numerous serological types of group A streptococci, and of a Group B and a Group D streptococcus, were extracted with 0.1 N HCl at 100 degrees C. or by sonic oscillation. The extracts, containing macromolecular components, were subjected to chemical fractionation and purification. C substance and M protein of Group A streptococci released from the cell by sonic oscillation were toxic to the adrenalectomized rat in quantities of 1 mg./100 gm. rat. Death usually occurred within 2 hours. On the other hand, C substance and M protein released from the cell with HCl at 100 degrees C. were relatively non-toxic to the adrenalectomized rat. The sonic-extracted C substance of streptococcal Groups B, C, and D was also toxic. The toxic property of the C and M preparations was neutralized in vitro in each case by group and type-specific rabbit antiserum. Heterologous antiserum was without effect. Adrenalectomized rats which received homologous antiserum 18 hours before challenge were also resistant to the toxicity of the C and M preparations. Trypsin destroyed the toxic effect of the M protein preparations and was without effect on the toxicity of the C substance. The R antigen and a nucleoprotein component of Group A streptococci, preparations of protein from Groups B and D streptococci, and coagulase from Staphylococcus aureus were all found to be essentially non-toxicic for the adrenalectomized rat. Large quantities of peptone, crystalline albumin, and rabbit serum were also without effect.

INFECTIOUS CROUP: III. HEMOPHILUS INFLUENZAE TYPE B CROUP

Croup due to H. influenzae type B occurred in 8% of a group of 347 cases of infectious croup. Bacteriologically and pathologically the disease was distinct from diphtheritic and so-called "virus" croup. Clinically this form of croup is characterized by the extreme rapidity of onset and the severity of symptoms. The outstanding clinical signs and symptoms encountered were: severe sore throat, epiglottitis with supraglottic edema, extreme early prostration, marked leukocytosis with a high polynudear percentage, tender anterior cervical adenitis, and necessity for very early tracheotomy in selected cases. Treatment consists of two important parts: relief of respiratory obstruction and treatment of the accompanying bacteremia and toxemia. The former calls for early tracheotomy to avoid the frequent adverse results of thoracic extra-alveolar air; intubation is contraindicated. Treatment of the second problem calls for transfusions of whole blood plus early administration of type-specific rabbit antiserum and sulfadiazine and streptomycin in high dosage. Death from H. influenzae type B croup is attributed to bacteremia and toxemia, aided by progressive respiratory tract obstruction.

Treatment of influenzal meningitis with sulfadiazine: Further report

Summary Twenty-three children with influenzal meningitis have been treated at the Charity Hospital in the past two years with sulfadiazine. Sixteen patients have been discharged as cured and have remained well except one patient who developed and still has a residual generalized spasticity. Six fatalities occurred, all in young infants below 8 months of age. In this group only one recovery occurred in an infant 4 months old. In our series no fatalities occurred above the age of 8 months. Since sulfadiazine alone does not seem sufficient in these young infants, it is imperative that type-specific rabbit antiserum be given together with large doses of sulfadiazine. In older infants and children the recovery rate is very high with sulfadiazine alone. Very few spinal punctures and drainages were performed. Leucopenia in one patient, hematuria in one patient, and drug fever in one patient were the only toxic conditions encountered that can be attributed to the drug.