Fluorescein-labeled Antiserum Research Articles

Diagnosis of rickettsial diseases: A perspective

Rickettsioses have nonspecific clinical manifestations, making them difficult to diagnose in a clinical setting. Laboratory testing is usually needed to confirm the diagnosis. Rickettsial isolation is a sensitive and specific diagnostic technique, but the hazards associated with handling pathogenic rickettsiae usually preclude isolation attempts in most laboratories. Rickettsiae can also be detected in infected tissues by fluorescein-labeled antisera or by immunoperoxidase staining, but these techniques lack sensitivity, except when applied to postmortem tissue specimens. However, rickettsial DNA can be detected in acute phase blood specimens by polymerase chain reaction (PCR) technology, and this technique offers the prospect of prompt diagnosis and treatment. Serologic testing remains the most frequently used approach to diagnosis, although antibody tests usually fail to identify rickettsioses early enough to affect the management of individual patients. Available serologic techniques vary considerably in their sensitivity and specificity. Enzyme-linked immunosorbent assays (ELISA) are extremely sensitive, but the general unavailability of specific diagnostic antigens reduces the specificity of this and other serologic techniques. Molecular characterization of rickettsial antigens may soon allow the production of peptide antigens that are specific for each species and could maximize the specificity of test results. No diagnostic technique has any value unless it is applied successfully to the appropriate patient population. Improved surveillance of rickettsial diseases is urgently needed to identify specific areas in which rickettsioses are endemic. Such surveillance data would promote awareness of rickettsioses among local physicians and increase the probability that individual patients with rickettsioses would be identified promptly and receive appropriate therapy early in the course of their illness.

Unusual characteristics of a parvovirus isolated from a clinically ill steer

A parvovirus was isolated from the feces of an 8- to 9-month-old steer that died acutely with hemorrhagic diarrhea and microscopic evidence of a coccidial infection. The concurrent intestinal parasitism in this steer appeared to play a role in the development of clinical disease. The viral isolate was identified as a bovine parvovirus (BPV) on the basis of its size (22 nm) and icosahedral morphology, the neutralization of viral cytopathology by antiserum to BPV, a strong immunofluorescent reaction with fluorescein-labeled antiserum to BPV, and the inhibition of viral hemagglutination of guinea-pig erythrocytes by antiserum to BPV. Cell cultures infected with this isolate showed a slight nuclear fluorescent reaction with fluorescein-labeled antiserum to canine parvovirus, suggesting an antigenic relationship to canine parvovirus. Patterns of hemagglutination for this isolate with human erythrocytes from 20 donors of various blood types differed from those obtained with the reference Abinanti strain of BPV. These results indicate that blood from multiple donors of a species may be necessary to confirm the presence or absence of viral hemagglutinating activity with clinical isolates of BPV.

Toxoplasma Cystozoite Antigenic Change in Tissue Culture

Antigenic differences between the endozoite and cystozoite of Toxoplasma gondii were revealed by fluorescent antibody staining (Lunde and Jacobs, 1983, Journal of Parasitology 69: 806-808). Antisera against the cystozoite reacted only against the cystozoite while anti-endozoite sera reacted partially against the cystozoite as well as against the endozoite. Absorption of sera with endozoites removed only positive reactions with endozoites. It was of interest to determine whether or not cystozoite antigens persisted on the surface of organisms derived from tissue cultures infected with cystozoites. Bovine embryo skeletal muscle cell cultures were infected with cystozoites derived from Toxoplasma strain ME 49 cysts separated from triturated mouse brain by centrifugation at 5,000 g for 30 min in 17% Ficoll-PBS (pH 7.2). The cystozoites were obtained by digestion with pepsin-HCl (Jacobs et al., 1960, Journal of Parasitology 46: 11-21) for 10 min and were removed from the digestion medium by 2 washings and centrifugations and resuspended in Eagle's complete medium with glutamine and 10% fetal calf serum. Approximately 100 x 103 cystozoites were added to each of 2 tissue cultures in 35 x 100 mm disposable culture dishes and incubated at 37 C with 5% CO2. The tissue cultures were confluent when inoculated. Gentamicin, 50 ,g/ml, was added to the culture medium to inhibit possible bacteria contaminants. On day 3 the cultures showed cytopathic effect, cell lysis and rounding, and were examined. Viable zoites were observed. Cells were mechanically removed from the culture dishes, centrifuged at 1,000 g and washed once with PBS pH 7.2. Portions of the sedimented zoites were then placed within circumscribed areas on microslides. Toxoplasma endozoites obtained from bovine embryo skeletal muscle cells infected with endozoites, RH strain, were also added to similar slides. The zoites were air dried and fixed briefly in absolute methanol. Fluorescein-labeled rabbit antiserum against either endozoites or cystozoites (Lunde and Jacobs, 1983, loc. cit.) was then added to the slides. After 60 min at 37 C the slides were washed and examined for immunofluorescence. Toxoplasma derived from tissue cultures infected with cystozoites or with endozoites reacted identically with the fluorescein-tagged anti-sera. The organisms all reacted positively with anti-endozoite antibody and showed no reaction with anti-cystozoite antibody. It appears, therefore, that only a few generations [we estimate about seven (Kaufman et al., 1958, American Journal of Ophthalmology 46: 255-260)] of the parasites within cultured cells results in the loss of the specific surface determinants of the cystozoites. The fact that this transformation occurs suggests that it probably takes place at the first division of the cystozoites within the cultured cells, because new membranes are formed in the division process. It was difficult to obtain enough cystozoites to infect the cultures, and recover and identify adequate numbers of individual parasites to determine their surface antigens. Because of this, a series of experiments to determine precisely when this change occurred was not done.

Periodontal disease and resistance factor. I. Immunopathological study for detection of antibiotic factors in the pocket fluid (author's transl)

This study was made to clarify the existense of antibiotic factors to oral bacteria in the gingival pocket fluid of individuals with periodontal disease by using fluorescent antibody techniques. Fluorescein-labeled antisera were obtained from human oral bacteria, Clq, C3, properdin, IgG, IgA, IgM, IgD, IgE, Fab, fibrinogen, and serious frozen sections were stained by them.The results were as follows:1) Each substance was detcted in the bacterial mass as showing Table 1 and Fig. 1.2) IgG, IgA, Fab, and fibrinogen were demonstrated most frequently as Fig. 3.From above results, we arrived at following conclusions:1) Main defensive system to the oral bacteria in the gingival pocket were immunoglobulins and activated complement system.2) The complement was the alternative pathway in addition to classical pathway.3) We were suggested that both complement systems raised to activity of phagocytosis, and finally caused to bacterial lysis in the gingival pocket.

Immunofluorescence findings in human renal amyloidosis.

Positive direct immunofluorescence findings have been occasionally reported for human renal amyloidosis. However, no substantial information describing the frequency of immunomicroscopic pattern has been reported. Twenty-seven renal biopsy specimens with proven amyloidosis were studied by direct immunofluorescence using monospecific antisera against IgG, IgA, IgM, IgD, IgE, kappa and lambda light chains, C3, C4 properdin, alpha-2 macroglobulin, albumin, transferrin, and fibrinogen. Ten biopsy specimens were negative. The remaining specimens showed principally a diffuse immunofluorescent staining of the mesangial areas with various of the above fluorescein-labeled antisera except IgE, albumin, and transferrin antisera, which gave a consistently negative reaction. IgD, properdin, or fibrinogen were weakly identified in only one case. Immunoglobulins and C3 were observed in 14 cases. Kappa and/or lambda light chains were found in nine of ten biopsy specimens so tested. Positive immunofluorescent staining of other elements of the nephron was rarely observed. These results suggest that various immunoreactants, in addition to light chains, can be frequently detected in renal amyloidosis and that passive nonselective absorption of plasma proteins does not explain these immunomicroscopic findings.

Determination of the Specificity of Plasma Cells for Bacterial Antigens In Situ

We developed a method for determining the specificity of plasma cells for bacterial antigens in situ. The method consists of applying rhodamine-labeled whole bacteria to frozen sections of tissue in which inflammation had been induced by injection of the same bacterial strain. After appropriate washings, the tissue sections were briefly treated with a fluorescein-labeled antiserum to endogenous immunoglobulins to detect plasma cells. Plasma cells which could recognize bacterial somatic antigens appeared as clumps of orange-fluorescing bacteria overlying a green-fluorescing cell. The fidelity of the procedure was determined by performing several competition experiments, which showed that pretreatment of the inflamed tissue sections with homologous unlabeled bacteria (i.e., the same bacteria used to induce the tissue inflammation) decreased binding of the labeled bacteria to a significant extent. Pretreatment of the inflamed tissue sections with heterologous unlabeled bacteria, however, did not significantly decrease binding of the labeled bacterium. Additionally, binding of rhodamine-labeled heterologous bacteria to the tissue sections was insignificant. In further experiments, we demonstrated that there was no natural affinity between the bacterial strains employed and lymphocytes taken from the strain of rabbit used and that pretreatment of the sections with anti-immunoglobulin abrogated the binding of the bacteria to which the animal had been specifically sensitized.

Detection by Indirect Immunofluorescence of Fc Receptors in Cells Acutely Infected with Herpes simplex Virus

In attempting to use the indirect fluorescent antibody test (IFA) to measure antibodies to herpes simplex virus (HSV), we found that all human sera gave a positive reaction with Chang liver cells infected with type 1 (HSV). All sera gave equivalent titers of 320-640 for acetone-fixed cells and about 40 for live cells (membrane fluorescence) in the presence of fluorescein-labeled antisera to human Ig; none of the sera reacted with uninfected cells. The fluorescence seen in fixed cells was primarily cytoplasmic; some cells showed a diffuse fluorescence, obscuring the demarcation between the nucleus and cytoplasm. Purified IgG from antibody-negative human sera and a purified Fc fragment of IgG were positive both for cytoplasmic and membrane fluorescence, whereas F(ab')2, IgM and IgA were unreactive. The reaction was also seen when an antiserum conjugate specific for the Fc fragment of IgG was used. The reactive IgG was present in freshly prepared plasma and serum; it could not be removed from serum either by ultracentrifugation or by serial absorption with HSV-infected cells. These findings suggest that the nonspecificity of the IFA results from the formation of low-avidity bonds between the large mass of native serum IgG and an Fc receptor on the plasma membrane and in the cytoplasm of cells infected with HSV. The results also suggest that extreme caution be exercised in attempting to use the IFA in the serodiagnosis of infections with HSV and perhaps the other human herpes-viruses.

Cross-immunity to Schistosoma mansoni and S. haematobium in the hamster

Hamsters (WO strain) with a primary infection of Schistosoma mansoni or S. haematobium rapidly developed immunity to homologous challenge judged by the lung recovery assay. Immunity was detected at 4-5 weeks and reached a plateau 6 weeks after infection. Using this information, hamsters with an 8-week primary infection with S. mansoni or S. haematobium were tested for resistance to homologous reinfection and resistance to a challenge with the other species of schistosome. Primary infection with S. mansoni or S. haematobium conferred a high level of immunity to reinfection with either species of schistosome judged by the perfusion assay, involving recovery of adult worms 6-10 weeks following challenge. Estimation of the level of immunity with the lung recovery assay, 5 days after challenge, indicated that immunity due to a primary infection with S. mansoni acted at or before migration of the challenge through the lungs but immunity stimulated by a primary S. haematobium infection was only partially effective at the lung stage and substantial destruction of challenging organisms occurred at a later stage of development. Antibodies in immune sera of hamsters with a primary S. mansoni or or S. haematobium infection were shown to bind to common antigens on the surface of young schistosomula of either species by u.v. microscopy using as detecting agent a fluorescein-labelled rabbit antiserum directed against hamster globulins.

The identification and ultrastructure of macrophages from the mammary gland of the ewe.

Cells in the secretion of involuting and non-lactating mammary glands of the ewe were studied. Light microscopy studies revealed that the majority of the cells were mononucleated, but it was impossible to distinguish macrophages from sloughed epithelial cells. Electron microscopy studies showed that the majority of the cells were macrophages and that they phagocytosed polystyrene latex spheres. Very few epithelial cells were found and they were distinguished from macrophages by numerous microfilaments and blunt microvilli. Epithelial cells could be further distinguished from macrophages by the presence of tight junctions. Tissue cultures of cells in secretion of mammary glands revealed the predominant cell type to be the macrophage. After 3 days in culture these macrophages formed multinucleated giant cells which contained acid-phosphatase granules. Fluorescein-labelled antiserum prepared against the cell cultures, stained cells in the lumens of alveoli and ducts, but not epithelial cells, in frozen sections of mammary tissue. The study confirms previous findings that the large majority of cells from mammary glands in late involution are macrophages. It also points to the usefulness of non-lactating mammary glands as a source of sheep and cattle macrophages.

The Localization And Distribution Of Gram-Positive Cocci In Normal Skin And In Lesions Of Acne Vulgaris

The localization of gram-positive cocci in the normal skin and in the lesions of acne vulgaris was investigated using fluorescein-labeled antiserum raised to gram-positive, coagulase-negative cocci. The cocci were found in 10 of 19 specimens from normal facial skin and in 3 of 11 specimens from the normal skin of the rest of the body. The bacteria were found mostly in the openings of follicles, but in 6 of 10 facial skin specimens, they were also present deeply in the lumina of the dilated sebaceous follicles near the sebaceous glands. Cocci were found in 5 of 6 noninflammatory acne comedones. In inflammatory acne they were demonstrated not only in the follicular canals but also sparsely in the infiltrate surrounding the follicles.

Fluorescent-antibody study of natural finger-like zoogloeae.

Fluorescent-antibody techniques using Zoogloea ramigera 106 antiserum were used to study fresh activated sludge flocs and finger-like zoogloeae in the microbial film that developed over stored samples of activated sludge. Few cells in fresh activated sludge reacted positively with the fluorescein-labeled antiserum. Finger-like zoogloeae containing reactive cells were readily observed in the microbial film layer over stored activated sludge. Certain of the naturel finger-like projections were entirely composed of cells that reacted positively to the labeled Z. ramigera 106 antiserum, whereas other projections were devoid of reactive cells.

Fluorescent-Antibody Study of Natural Finger-Like Zooloeae

Fluorescent-antibody techniques using Zoogloea ramigera 106 antiserum were used to study fresh activated sludge flocs and finger-like zoogloeae in the microbial film that developed over stored samples of activated sludge. Few cells in fresh activated sludge reacted positively with the fluorescein-labeled antiserum. Finger-like zoogloeae containing reactive cells were readily observed in the microbial film layer over stored activated sludge. Certain of the naturel finger-like projections were entirely composed of cells that reacted positively to the labeled Z. ramigera 106 antiserum, whereas other projections were devoid of reactive cells.

Tissue Immunoglobulins in Nodular Lymphomas as Compared With Reactive Follicular Hyperplasias

Abstract Frozen tissue sections of lymph nodes and spleens from twelve patients with nodular (follicular) lymphomas were studied with fluorescein-labeled antisera against human immunoglobulins. Immunoglobulins detected by this method in the neoplastic nodules were minimal or absent. Furthermore, in no instance could the characteristic network pattern of immunoglobulin distribution which is readily demonstrable in active germinal centers be observed within these nodules. It is suggested that the preservation of the follicular network of immunoglobulins as observed by fluorescent antihuman IgG, IgM, or polyvalent antisera indicates functionally intact germinal centers. The detection, by this rather simple method, of large amounts of immunoglobulin distributed in such a distinctive pattern may thus be very useful for the differentiation between severe benign follicular hyperplasia and malignant lymphoma with nodular pattern, which may be difficult to distinguish by routine histological methods.

North Carolina Turkey Enteric Virus: Further Characterization

SUMMARY The North Carolina turkey enteric virus was further characterized on the basis of certain physical and chemical properties. Intracytoplasmic staining of infected turkey kidney cells was demonstrated by fluorescein-labeled antiserum. May-GrunwaldGiemsa staining revealed very large syncytia. An intense greenish color was observed in syncytia stained with acridine orange. The virus was relatively heat-stable and pH-stable, and plaques of slightly increased size were produced in the presence of magnesium chloride. The virus did not hemagglutinate when a number of avian and mammalian red blood cells were employed. The growth curve was typical of reoviruses. Pathogenicity for 13-day-old embryonated turkey eggs was demonstrated.

Properties of microtubule proteins in different organelles in Tetrahymena pyriformis

Attempts were made to elucidate whether or not microtubules within cilia, oral apparatus and macronuclei in Tetrahymena pyriformis include common proteins, by making use of antiserum to microtubule proteins of cilia. The microtubule fraction containing two protein components was used as antigen and the antiserum to the microtubule proteins was proved to be specific by analysing electrophoretic patterns in the antigen absorption experiments. The antiserum reacted with the dissolved proteins of isolated oral apparatus or macronuclei, forming precipitin lines common to those of cilia. Furthermore, the two organelles were positively stained with the fluorescein-labelled antiserum. These results offered important clues to understand multifariousness in function and behavior of morphologically identical microtubules; that is, various microtubules in the cell appear to include a common protein(s) one another.

Developmental pattern of enzyme secretion in the embryonic venom glands of Vipera palaestinae (Ophidia, Reptilia)

The relationship between morphogenesis and the appearance of secretory components was studied in the venom glands of Vipera palaestinae embryos. Twenty days after oviposition (hatching after 40 days at 30°C), membrane-bound ribosomes, a well developed Golgi apparatus, and some vesicles are evident in the secretory cells. In 25-day-old embryos venom components were detected with fluorescein-labeled antisera against whole venom or against neurotoxin. At this stage the cells have the characteristic columnar shape and tubular lumina are formed. The activity of three enzymes, l-amino acid oxidase, phosphodiesterase, and benzoyl arginine ethyl esterase, which are found in the venom, was first demonstrated in the gland epithelium at about the 30th day of incubation, when well differentiated cells with a high content of succinate dehydrogenase appear among the secretory epithelium. A steepincrease in specific activity of the three exportable enzymes becomes evident at that stage, with the highest level attained at 39 days after oviposition. Shortly after hatching the venom glands have a pattern similar to that of the adults, with both central and tubular lumina filled with secretion material. The specific activity of the enzymes in the venom at hatching, especially that of benzoyl arginine ethyl esterase, is lower than the average specific activity reported for adult snakes.

A pathological study on dental caries with labeled antibody techniques

The hypothesis that immunologic mechanism may play an important role in modifying the carious process was suggested by Blechman and Mori (1965). Presently an attempt was made to find the localization of salivary components, immunoglobulins, serum albumin and oral microorganisms in the human sound and carious dentin with a fluorescent antibody technique (Coons and Kaplan, 1950), an enzyme (peroxidase-labeled) antibody technique (Nakane and Pierce, 1967) and other staining techniques, as well as by microradiography.Seventy freshly extracted sound and carious human permanent teeth (Table 1) were immediately put in cold 95% ethanol for fixation. These were divided into 2 segments. A segment from each tooth was hand polished to a thickness of about 70μ, cooled with cold saline and used for microradiographic studies.Then it was demineralized in 0.5M pH7.4 EDTA (2Na-salt) at 4°C for 2-3 days. Another segment was demineralized in the same solution at 4°C for 7-14 days and embedded with paraffin. Microtome sections were made from each segment to a thickness of 7μ(Table 2).Rabbit antiserums to human saliva, human immunoglobulins*, human serum albumin* and oral microorganisms were prepared. Fluorescein isothiocyanate**, tetramethylrhodamine isothiocyanate** and horseradish peroxidase*** were conjugated with the globulin fraction of rabbit antiserum. Commercially available fluoresceinlabeled anti human immunoglobulins serum** was also used. Control studies of specificity were made with labeled antiserum from which specific antibody had been absorbed by the addition of antigen.When stained with labeled anti human saliva serum, labeled anti human immunoglobulins serum and labeled anti human immunoglobulins serum and labeled anti human serum albumin serum, specific reaction was usually observed in dentinal tubules (Figs. 6 and 7), dentinal fibers, predentin and the odontoblast layer of dentin (Fig. 8). These labeled antiserums also resulted in the most intense reaction in dentinal tubules beneath a translucent zone in the ground section, a radiopaque zone in microradiogram, of carious dentin (Figs. 10, 11, 14, 16, 17 and 18). Azure A eosin B stain, PAS stain and bromphenol blue stain produced a staining similar to that given by these labeled antiserums (Figs. 14, 15 and 19). Moreover, labeled anti human saliva serum resulted in diffuse reaction in soft carious dentin (Figs. 9 and 19). Although microorganisms were localized in dentinal tubules and the matrix of carious dentin (Figs. 20 and 21), no specific relation was observed between the localization of salivary components and that of oral microorganisms.*Nutritional Biochemicals Co., Cleveland, Ohio, U. S. A.**Baltimore Biological Laboratory, Baltimore, Maryland, U. S. A.***Sigma Chemical Co., St. Louis, Missouri, U. S. A.

Immunofluorescence Studies on the Anatomic Distribution of the Soluble Antigen Responsible for the Production of Nephrotoxic Serum Nephritis

Abstract Normal human kidney, lung, aorta, heart, muscle and liver were stained with fluorescein-labeled antiserum to intact human ST-GBM (the tryptic-digested ultrasupernatant substance of intact human glomerular basement membrane) for the purpose of elucidating the anatomic distribution of the specific antigen that is responsible for the production of nephrotoxic serum nephritis. These particular organs were investigated because we have previously demonstrated that antiserum against the ST of these organs (rat) could produce typical glomerulonephritis in rats. The characteristic distribution of fluorescence in these organs was as follows: kidney, glomerular basement membrane and tubular basement membrane; lung, alveolar capillary basement membrane; aorta, probably the sarcolemma of muscle cells in the tunica media; skeletal and cardiac muscle, sarcolemma of muscle cells; and liver, sinusoidal walls. Besides this specific distribution, fluorescence was also found in blood vessels and epithelial basement membrane. Further investigation is necessary to clarify the relationship between the antigenic substance in epithelial basement membrane and the production of nephrotoxic antiserum.

Immunofluorescent localization of Tamm-Horsfall mucoprotein in human kidney.

A fluorescein-labelled antiserum to human Tamm-Horsfall mucoprotein applied to frozen human kidney sections gave strong specific labelling, mainly of cells of tubules in the outer medulla. By comparison with adjacent serial sections stained for alkaline phosphatase and succinic dehydrogenase, it is suggested that material reacting immunologically as Tamm-Horsfall muco-protein is found particularly in the cells of the ascending limb of the loop of Henle and the macula densa segment of the distal tubule.

Comparison of Fluorescent Antibody with Cultural Technique for Isolation of Enteropathogenic Escherichia coli from Swine1

In an investigation of hogs as possible reservoirs of human strains of enteropathogenic Escherichia coli (EEC), 92 six-month-old grain- and garbage-fed hogs were examined on the farm and again at the packing plant. Of the 331 specimens obtained by swabbing the rectum, cecum, and edible meat carcass of these hogs, 125 were presumptively positive for EEC when screened by the fluorescent-antibody (FA) technique. These "presumptive positive" specimens then underwent extensive bacteriological examination and complete serological typing. The FA technique proved to be an easier, simpler, and more economical procedure than culture when a large number of specimens were examined for possible EEC serogroups. It was found especially valuable for identification of multiple serogroups of EEC within a single specimen. It also appeared to be more sensitive than cultural examination, since results were not dependent on the presence of large numbers of organisms in the specimen, or even on their viability. However, the FA technique was found to be less specific than culture because of cross-reactivity with antigenically related Enterobacteriaceae when fluorescein-labeled antisera were used. Therefore, any specimen found positive on FA examination should be considered as presumptive positive until confirmed by bacteriological examination and complete serological study.