Indirect Fluorescent Antibody Staining Research Articles

Antisera to human cytomegalovirus produced in hamsters: reactivity in radioimmunoassay and other antibody assay systems

Hamsters immunized with human cytomegalovirus (CMV) concentrated and purified by polyethylene glycol precipitation and density gradient centrifugation produced antisera with high titers of specific viral antibody, and which showed no significant reactivity with human host cell components. The antisera had high titers of CMV antibody in complement fixation, indirect fluorescent-antibody (FA), and neutralization tests, but titers obtained by indirect radioimmunoassay (RIA) were markedly higher. The antisera were used to follow the development of CMV antigen in infected host cells by indirect RIA and indirect FA staining. Virus-specific antigen was first detectable by RIA at 8 h after infection, and by FA staining at 16 h; cells contained optimal amounts of antigen for RIA and FA assays at 72 to 100 h postinfection. Immune globulins from the antisera were labeled with 125I for use in direct RIA. The labeled globulins gave highly specific reactions with CMV-infected cells, including those infected with low-passage isolates, and showed no reactivity with cells infected with other human herpesviruses or certain other human viruses.

Detection of Thiobacillus ferrooxidans in acid mine environments by indirect fluorescent antibody staining.

An indirect fluorescent antibody (FA) staining technique was developed for the rapid detection of Thiobacillus ferrooxidans. The specificity of the FA stain for T. ferrooxidans was demonstrated with both laboratory and environmental samples. Coal refuse examined by scanning electron microscopy exhibited a rough, porous surface, which was characteristically covered by water-soluble crystals. Significant numbers of T. ferrooxidans were detected in the refuse pores. A positive correlation between numbers of T. ferrooxidans and acid production in coal refuse in the laboratory was demonstrated with the FA technique.

The infection of cucumber mesophyll protoplasts with tobacco mosaic virus.

Protoplasts from the first leaf mesophyll of cucumber plants were isolated by an 18 hours combined petinase/cellulase treatment. Conditions favouring the infection of these protoplasts with tobacco mosaic virus (TMV), and the accumulation of infective virus up to 96 hours after inoculation have been studied. Infection of approximately 5--10 per cent of the protoplats, revealed by indirect fluorescent antibody staining, was achieved by pre-treatment of the cells in 0.01 M citrate-buffered mannitol (CBM), pH 5.2 with 2 mug/ml poly-L-ornithine followed by centrifugation and direct resuspension of the cells in the same mixture together with 2 to 4 mug/ml TMV. Higher concentrations of the polycation and buffer were toxic to the protoplasts. Under the best conditions, virus yields of approximately 10-20 mug TMV/10(6) protoplasts were attained, while after 72 hours incubation, significant amounts of virus could often be recovered from the incubation medium. Addition of actinomycin D to cultures of protoplasts 2 hours post-inoculation partially inhibited development of infectivity.

Differentiation in cultures derived from embryonic chicken muscle: II. Phosphorylase histochemistry and fluorescent antibody staining for creatine kinase and aldolase

The presence or absence of five proteins (glycogen phosphorylase, aldolase A, aldolase C, creatine kinase M, creatine kinase B) in the various classes of cells found in primary cultures derived from embryonic chick breast muscle was investigated using cytological staining methods. Histochemical staining for phosphorylase and indirect fluorescent antibody staining for aldolase A and C as well as for creatine kinases M and B showed the following: All five proteins were found in the many myotubes present in standard medium cultures and in the very few myotubes found in cultures containing 5-bromodeoxyuridine (10 −5 M). The elongated bipolar cells prevented from fusing in medium containing EGTA also contain all five proteins. The flattened myogenic cells that predominate in the 5-bromodeoxyuridine-treated cultures contain no phosphorylase or creatine kinase M, though many of them contain creatine kinase B and aldolases A and C. These results are interpreted as indicating that: (1) phosphorylase and creatine kinase M, but not aldolase A, are suitable all-or-none markers for terminal muscle differentiation; (2) the small amounts of creatine kinase M detected in electrophoreses of 5-bromodeoxyruridine-treated cultures can be accounted for by the few myotubes present and are not due to “protodifferentiation” of large numbers of cells; (3) proteins typical of differentiated muscle are produced only in cells that have passed through the last step in myogenesis that is susceptible to 5-bromodeoxyuridine inhibition, and (4) if fusion is blocked by reducing the concentration of calcium ions, accumulation of characteristic muscle proteins can continue in those cells that have initiated terminal differentiation.

Haemophilus vaginalis--further investigations into its identity.

SUMMARY Haemophilis vaginalis strains isolated at Lewisham Hospital were shown to be anti-genically similar if not identical with those isolated by Gardner and Dukes (1955). No similarity was shown to exist between H. vaginalis and the strains of Haemophilus, Coryne-bacterium and Lactobacillus that were examined. Indirect fluorescent-antibody staining specifically demonstrated the presence of H. vaginalis in direct films of vaginal exudate. This agreed with the isolation of this organism from the same sources and could be used as a diagnostic procedure.

Transformation of hamster embryo and human embryo cells by temperature sensitive mutants of herpes simplex virus type 2

Transformation of hamster embryo (HaE) cells and human embryo (HuE) cells was attempted with temperature sensitive mutants of herpes simplex virus type 2 (HSV-2) at the nonpermissive temperature of 38.5°. Among 12 ts mutants tested, transformation of HaE cells has been observed with three mutants ( ts 4, 9, 35), and of HuE cells with one mutant ( ts 9). Inoculation of transformed HaE cells into newborn hamsters resulted in tumor formation. The continued presence of the HSV-2 genome in transformed HaE and HuE cells was suggested by indirect fluorescent antibody staining using anti-HSV rabbit serum and antirabbit goat serum.

The anemia-induced reversible switch from hemoglobin A to hemoglobin C in caprine ruminants: Immunochemical evidence that both hemoglobins are found in the same cell

During anemic episodes, goats and certain sheep replace hemoglobin A (HbA = α 2 β 2 A) with hemoglobin C (HbC = α 2 β 2 C). Rabbit serum directed against either purified sheep HbA or purified sheep HbC was prepared. Both types were used to test whether the two hemoglobins are found in the same cell during switching by an indirect fluorescent antibody assay. Unabsorbed antisheep HbA cross-reacted extensively with goat HbA but to a lesser extent with goat or sheep HbC. Similarly, unabsorbed antisheep HbC reacted with these antigens in the order: Sheep HbC > goat HbC > sheep HbA > goat HbA. Cross-absorption resulted in sera specific either for sheep and goat HbA or for sheep and goat HbC. The specificities were confirmed by indirect fluorescent antibody staining of sheep and goat erythrocytes containing either at least 99% HbA or at least 99% HbC. Smears of erythrocytes from sheep and goats in the process of switching were reacted with one of the absorbed sera then with fluorescein conjugated antirabbit immunoglobulin G. The sum of the fractions stained both by anti-HbA and by anti-HbC exceeded 100% during the switch. Most strikingly when HbA was replacing HbC, nearly all cells stained for HbC while more than half stained for HbA. Thus, the two hemoglobins are found in the same cell during switching.

Immunocytochemical localization of the extracellular polysaccharide alginic acid in the brown seaweed, Fucus distichus.

Fluorescent and peroxidase-labeled antibody techniques were employed to localize alginate, which is one component in the complex extracellular acidic polysaccharide situation of the brown seaweed, Fucus distichus. The alginate antigen was extracted and characterized by a variety of methods. It had a low degree of polymerization and a mannuronic acid to guluronic acid ratio of 2.2. No contaminants were detected. The specificity of rabbit antisera was tested by immunodiffusion. Antisera reacted with alginate and fractions of alginate but not with other brown algal polysaccharides. When cryostat sections of Fucus proved unsuitable, 1-µ sections embedded in glycol methacrylate were used for indirect fluorescent or peroxidase antibody staining. Highly sulfated extracellular polysaccharides and intracellular phenolic compounds were implicated in nonspecific staining. Texture artifacts were caused by solubility of alginates and other polysaccharides from embedded sections. Alginate was localized in variable patterns in all cell walls and some matrix regions. The applicability of the methods is discussed.

Immunofluorescence staining of group B coxsackieviruses.

Studies were conducted on the sensitivity and specificity of indirect fluorescent-antibody (FA) staining for identification of group B coxsackieviruses. Antisera produced in four different species (monkeys, rabbits, horses, hamsters) and immune ascitic fluids prepared in mice were compared for suitability in FA staining. The horse antisera showed high titers of nonspecific staining, and the rabbit antisera showed relatively low homologous FA titers. Immune reagents from monkeys, hamsters, and mice were used for homologous and heterologous testing against cell cultures infected with the various group B coxsackieviruses. Antisera or immune ascitic fluids produced in these three species showed some heterotypic and nonspecific staining at low dilutions, with the monkey antisera showing the highest heterotypic titers. However, the immune reagents could be diluted to a point where they gave no heterotypic reactivity, but still showed characteristic homotypic staining. Heterotypic staining appeared as diffuse, low-level staining of the cells, whereas homotypic staining revealed characteristic, brightly staining aggregates of viral antigen in the cytoplasm of the infected cells. By using hamster immune sera, appropriately diluted to eliminate heterotypic staining and yet give strong homotypic staining, it was possible to identify correctly 79 (93%) of 85 field strains of group B coxsackieviruses at the first passage level in BS-C-1 cells; the remainder of the strains were identified after two passages in BS-CS-1 cells. No incorrect identifications were made. A limited number of field strains of group B coxsackieviruses were passed into rhesus monkey kidney and human fetal diploid kidney cells, and these were all correctly identified by FA staining, even the strains which failed to produce a cytopathic effect in the human fetal diploid kidney cells. Two human heart and brain tissues from which coxsackievirus type B4 had been isolated failed to show homotypic FA staining in excess of nonspecific or heterotypic staining.

Immunofluorescence Staining of Group B Coxsackieviruses

Studies were conducted on the sensitivity and specificity of indirect fluorescent-antibody (FA) staining for identification of group B coxsackieviruses. Antisera produced in four different species (monkeys, rabbits, horses, hamsters) and immune ascitic fluids prepared in mice were compared for suitability in FA staining. The horse antisera showed high titers of nonspecific staining, and the rabbit antisera showed relatively low homologous FA titers. Immune reagents from monkeys, hamsters, and mice were used for homologous and heterologous testing against cell cultures infected with the various group B coxsackieviruses. Antisera or immune ascitic fluids produced in these three species showed some heterotypic and nonspecific staining at low dilutions, with the monkey antisera showing the highest heterotypic titers. However, the immune reagents could be diluted to a point where they gave no heterotypic reactivity, but still showed characteristic homotypic staining. Heterotypic staining appeared as diffuse, low-level staining of the cells, whereas homotypic staining revealed characteristic, brightly staining aggregates of viral antigen in the cytoplasm of the infected cells. By using hamster immune sera, appropriately diluted to eliminate heterotypic staining and yet give strong homotypic staining, it was possible to identify correctly 79 (93%) of 85 field strains of group B coxsackieviruses at the first passage level in BS-C-1 cells; the remainder of the strains were identified after two passages in BS-CS-1 cells. No incorrect identifications were made. A limited number of field strains of group B coxsackieviruses were passed into rhesus monkey kidney and human fetal diploid kidney cells, and these were all correctly identified by FA staining, even the strains which failed to produce a cytopathic effect in the human fetal diploid kidney cells. Two human heart and brain tissues from which coxsackievirus type B4 had been isolated failed to show homotypic FA staining in excess of nonspecific or heterotypic staining.

The adaptation of a fluorescent antibody staining technique for the quantitative study of potentially pathogenic Escherichia coli in calf faeces.

SUMMARY An indirect fluorescent antibody staining technique was adapted to study the population of potentially pathogenic E. coli serotypes relative to the total coliform population in faeces from calves. Preliminary experiments were carried out with pig faeces in which the haemolytic property of a pathogenic serotype was utilised to facilitate comparison of the results with those obtained from plate counts. In a natural outbreak of colibacillosis in calves, diarrhoea in all except one case was associated with an increase in the proportion of pathogenic serotypes to at least 50 per cent. of the total coliform population. In some other cases, a similar increase occurred in the absence of clinical disease. The significance of these results is discussed in relation to the practical value of the method as a diagnostic procedure and as a means of studying the influence of environmental predisposing factors on the E. coli population of the gastro-intestinal tract.

Direct and indirect fluorescent antibody staining of Ophiobolus graminis Sacc. in culture and in the rhizosphere of cereal plants.

Fluorescein isothiocyanate (FITC) conjugates were prepared to whole-cell and cell-wall fractions of four cultures ofOphiobolus graminis W1, W2, O1 and O2. Homologous and heterologous direct and indirect FA staining of the four cultures ofO. graminis gave positive staining in all reactions. This indicated that the four cultures could not be differentiated by fluorescent antibody (FA) staining. Species specificity of the conjugates was shown by the staining ofO. graminis hyphae in the rhizosphere of wheat and oat roots. Plant tissues were not stained. Furthermore out of 52 rhizosphere isolates stained with whole-cell and cell-wall conjugates of the four cultures ofO. graminis, only 7 cross-reacted with the whole-cell conjugates whereas none cross-reacted with the cell-wall conjugates.

Survey for antibodies to arboviruses in the serum of various animals in Israel during 1965-1966.

Abstract Serum from 2,294 wild birds, 128 mammals, 22 reptiles, and 96 amphibians caught from May 1965 to June 1966 in Israel were examined for antibodies against Eastern equine encephalomyelitis. Semliki Forest, Sindbis, Langat, Israel turkey meningoencephalitis, and West Nile (WN) viruses by hemagglutination-inhibition (HI) tests. The incidence of positive serum was 14.4% for wild birds, 5% for mammals, and 9% for reptiles. No antibodies were detected in the amphibian serum; 73% of the positive avian serum had antibodies against group B viruses. The rest of the positive serum was divided among that positive for group A and that positive for both group A and group B viruses. Serologic conversions in sentinel chickens confirmed the activity of WN virus in the Hadera region during the period studied. Use of acetone extraction followed by kaolin treatment of serum minimized the number of falsely positive results obtained in the HI tests. Good correlation was found between results obtained in HI tests and indirect fluorescent-antibody staining of WN antigen among serum samples from 116 Columbidae examined by these two methods.

Transitional forms of Corynebacterium acnes in disease.

A clear-cut triad of sequential Corynebacterium acnes transitional forms from disease has been discovered. This entity includes three major forms which are capable of stabilization in culture, the spherical, the intermediate, and the definitive C. acnes. During conversion or reversion among the three forms, a variety of forms with mixed characteristics was observed. The spherical form was gram-negative and osmotically fragile, but it possessed a vestigial cell wall and mesosomes which excluded it from the L forms. In lieu of the L-form designation, the term "transitional" was adopted for all forms leading up to the definitive C. acnes. Culture of the spherical form was successful only on Mycoplasma-type media. The intermediate form was gram-negative, had mixed spherical and filamentous morphology, and bore a striking resemblance to Streptobacillus moniliformis. Like the spherical form, it was nutritionally exacting. The definitive form of C. acnes was preceded by gram-positive transitional forms of C. acnes morphology. It lacked, however, the carbohydrases and proteinases of C. acnes and susceptibility to C. acnes bacteriophages. Reversion was often blocked at this stage. A series of blood cultures from a patient with endocarditis was studied. Postmortem stain sections of the heart-valve lesion included intracellular masses of gram-negative spherical organisms. Indirect fluorescent antibody staining of these masses was strongly positive with antiserum to the spherical form and weakly positive with antiserum to the intermediate form.

Localization of avian tumor virus group-specific antigen in cell and virus

Nonproducing Rous sarcoma cells of the chicken contain an antigen which is group specific for the avian leukosis and sarcoma complex and can be demonstrated throughout the cytoplasm of such cells by indirect fluorescent antibody staining with immune sera from Rous sarcoma-bearing Syrian hamsters. In chick fibroblasts producing infectious avian tumor virus this group-specific ( gs) antigen is localized at the cell periphery, where it is distributed in a fashion that is typical of released, cell-associated virus. This indicates that the gs antigen is closely associated with mature virus. Such gs antigen affiliated with extracellular, cell-associated virus is accessible to the immunochemical stain only after histologic fixation and is therefore considered to be an internal component of the virion. The type-specific viral envelope antigen, on the other hand, can be stained in extracellular virus prior to fixation. Gs antibody is readily demonstrable in the sera of tumor-bearing Syrian hamsters. In chicken sera prepared against avian tumor viruses, only type-specific antibodies have so far been detected, but the occurrence of a minor gs component has not been excluded with certainty.

The fluorescent antibody technique in the estimation of immunity in patients infected with Plasmodium malariae

Investigations were performed in 15 psychiatric patients therapeutically infected with Plasmodium malariae (strain VS) in whom antibody titres in the blood serum were determined by the indirect fluorescent antibody staining technique. The results were compared with clinical findings, immunobiological data and gamma-globulin levels as determined by paper electrophoresis. These investigations indicate that the negative phase of the primary response to the antigenic stimulus lasts 4–6 days after the onset of parasitaemia. After primary stimulation the immunological response varies according to the persistence and intensity of the antigenic stimulus. In the case of prolonged antigenic stimulation followed by a state of parasitic tolerance (symptomless parasitaemia), antibody titres ranged from 2000 to 3000, at 596 and 827 days after the onset of parasitaemia. In the re-inoculated patients, the secondary immunological response was characterized by increased antibody titres, attaining figures of 3000 to 5000. In one case (V.C.) antibodies were demonstrable at a titre of 1000, 665 days after the subsidence of parasitaemia. The fluorescent antibody technique is a highly valuable test for demonstrating and assaying immunobiological response in malaria. The value of this technique is increased by its specificity, as demonstrated by the following findings: 1. 1. The test was negative in non-immune controls and in patients tested before inoculation of the parasite. 2. 2. It was likewise negative in patients re-inoculated with strain VS, who displayed no clinical symptoms of malarial infection, because the secondary infection had been preceded by efficient chemoprophylaxis which suppressed the antigenic stimulus.

Immunofluorescence studies on Coxsackie group a viruses

The indirect fluorescent antibody staining technique was found suitable for the detection and specific identification of viral antigens in primary human amnion cells infected with tissue culture adapted Coxsackie group A viruses.

EXPERIMENTAL INFECTION WITH MYCOPLASMA PNEUMONIAE (EATON'S AGENT).

The pathogenesis of Mycoplasma pneumoniae infection was studied in the Syrian hamster with qualitative and quantitative culture methods and special histopathologic techniques. The animals were readily infected with the mycoplasma, which multiplied throughout the respiratory tract. Sensitivity of this experimental host to infection was indicated by the 50 per cent infective dose, which was 10 colony-forming units of the organism. Inoculation consistently resulted in the production of peribronchial pneumonitis which was induced by the mycoplasma. The organisms were visualized in a superficial location in the mucosa of involved bronchi, by means of indirect fluorescent antibody staining and by a modification of the Brown and Brenn technique. The data indicate applicability of the hamster to the study of problems concerned with M. pneumoniae disease which are impractical or impossible to resolve in the human host.

Antigenic characteristics of Leptotrichia buccalis (Fusobacterium fusiforme).

Gustafson, Robert L. (University of Illinois College of Medicine, Chicago) and Arthur V. Kroeger. Antigenic characteristics of Leptotrichia buccalis (Fusobacterium fusiforme). J. Bacteriol. 84:1313-1320. 1962.-Thirty-nine isolates of an oral filamentous organism, usually termed Leptotrichia buccalis Trevisan (Fusobacterium fusiforme), were isolated from materia alba or saliva. The cultural, morphological, and biochemical characteristics were similar to those reported by other investigators. Agglutination reactions were not reproducible with all strains, since spontaneous agglutination frequently occurred. When sonic extracts of nine strains were tested with antisera by precipitin ring tests, some strains cross-reacted extensively. Indirect fluorescent antibody staining reactions revealed significant antigenic differences among fresh isolates. However, strains which had received 12 or more passages on artificial media cross-reacted with one or several heterologous antisera. Currently, the results do not allow classification of L. buccalis into a given number of antigenic types. From the data obtained by the various serological techniques, a tentative picture of the antigenic complex was formulated. The outermost layer of freshly isolated strains appears to be type-specific, and may be lost or altered upon passage on artificial media. Beneath this surface component lie one or more antigens, which may prove to be group antigens shared by several strains of L. buccalis.

Studies on primary atypical pneumonia. III. A factor in normal serum which enhances the reaction between PAP virus and convalescent serum.

A factor present in fresh normal human or guinea pig serum enhances the primary atypical pneumonia (PAP) antibody titers in serological reaction with its homologous virus as tested by indirect fluorescent antibody staining. This factor is not properdin. It has many properties similar to complement but may not be identical with complement. The addition of this enhancement factor to human sera convalescent from primary atypical pneumonia has made the serological test more sensitive without affecting its specificity.