Direct Fluorescent Antibody Method Research Articles

Development and Evaluation of a Direct Fluorescent Antibody Method for the Diagnosis of Pneumocystis carinii Infections in Experimental Animals

Because no fully satisfactory diagnostic method has been available for use in pneumocystis infection, an attempt was made to apply the fluorescent antibody technique in the identification of Pneumocystis carinii . Hyperimmune sera were prepared in rabbits against P. carinii from human and rat sources. After proper adsorption, these antisera were conjugated with fluorescein isothiocyanate and used as reagents in a direct fluorescent antibody procedure. Each of the two reagents was found to stain trypsin-treated P. carinii organisms from either human or rat sources, indicating the presence of common antigens. Stained organisms were demonstrated in the hypopharyngeal material from rats in which pneumocystis infection had been activated by the administration of corticosteroid. From the results reported here, the procedure outlined is considered sufficiently sensitive and specific to justify tests on pneumocystis infections in man. The findings in a series of specimens from human subjects will be reported separately. The method also provides an extended approach to related research problems. The need for controls of the procedure at all points is emphasized.

Cloning of dengue virus type 2 by the direct fluorescent antibody technique.

SummaryDengue virus type 2 has been cloned in hamster kidney cells, a noncyto-pathic, noncytocidal virus-cell system, utilizing a direct fluorescent antibody method. A clone is considered to have arisen from the infection of a susceptible cell by a single infectious particle of DEN-2.This work was performed under the sponsorship of the Commission on Viral Infections, Armed Forces Epidemiological Board, and was supported by the U. S. Army Medical Research and Development Command under Contract No. DADA17-69-C-9048 and by Public Health Service Research Grant No. AI-02686 from the National Institute of Allergy and Infectious Diseases. The application of direct fluorescent antibody methods to dengue viruses was achieved by Drs. Robert W. Atchison and J. Jayavasu, who also prepared the conjugated immune ascitic fluids. Most able technical assistance was provided by Alma Tillman, Annie McCoy, Carlie White, and Frances Brisset.

Development and Evaluation of a Direct Fluorescent Antibody Method for the Diagnosis of Pneumocystis carinii Infections in Experimental Animals

Because no fully satisfactory diagnostic method has been available for use in pneumocystis infection, an attempt was made to apply the fluorescent antibody technique in the identification of Pneumocystis carinii. Hyperimmune sera were prepared in rabbits against P. carinii from human and rat sources. After proper adsorption, these antisera were conjugated with fluorescein isothiocyanate and used as reagents in a direct fluorescent antibody procedure. Each of the two reagents was found to stain trypsin-treated P. carinii organisms from either human or rat sources, indicating the presence of common antigens. Stained organisms were demonstrated in the hypopharyngeal material from rats in which pneumocystis infection had been activated by the administration of corticosteroid. From the results reported here, the procedure outlined is considered sufficiently sensitive and specific to justify tests on pneumocystis infections in man. The findings in a series of specimens from human subjects will be reported separately. The method also provides an extended approach to related research problems. The need for controls of the procedure at all points is emphasized.

Rapid, direct fluorescent-antibody method for the detection of salmonellae in food and feeds.

An improved immunofluorescent-antibody (FA) method for the detection of salmonellae in foods and feeds was developed. This FA method combines a rapid cultural phase and a serological phase that allow for propagation of salmonellae in a minimum time, employing the industrial 8-hr work day as a guide. Two hundred fifty naturally contaminated human food and animal feed samples, representing 647 trials, were tested by the FA method. A total of 18 different food and feed samples was used. The method used by the Association of Official Analytical Chemists (AOAC) for the detection of salmonellae was the control method. The percent agreement when comparing the FA slide method to the AOAC method ranged from 87.1 to 95.3%, depending upon the conjugated antisera used in comparative studies.

Rapid, Direct Fluorescent-Antibody Method for the Detection of Salmonellae in Food and Feeds

An improved immunofluorescent-antibody (FA) method for the detection of salmonellae in foods and feeds was developed. This FA method combines a rapid cultural phase and a serological phase that allow for propagation of salmonellae in a minimum time, employing the industrial 8-hr work day as a guide. Two hundred fifty naturally contaminated human food and animal feed samples, representing 647 trials, were tested by the FA method. A total of 18 different food and feed samples was used. The method used by the Association of Official Analytical Chemists (AOAC) for the detection of salmonellae was the control method. The percent agreement when comparing the FA slide method to the AOAC method ranged from 87.1 to 95.3%, depending upon the conjugated antisera used in comparative studies.

Comparison of direct and indirect fluorescent antibody methods for staining Treponema pallidum.

Comparison of direct and indirect fluorescent antibody methods for staining Treponema pallidum.

The olfactory bulb lesion in the hibernating squirrel (Citellus lateralis) infected with coxsackievirus B-3. II. Immunological study.

The immunological response was studied in the adult hibernating squirrel (Citellus lateralis) infected with coxsackievirus B-3. The complement activity was considerably reduced between the 4th and 9th day of infection, but was restored to the normal level by the 11th day. The neutralizing antibody was first detected in the blood on the fourth day, reached an average ND50 of 10−2.62 on the seventh day after inoculation, and remained at this level.Gamma globulin was demonstrated in the infected olfactory bulb by the direct fluorescent antibody method on the seventh day after inoculation. The specific fluorescence appeared as a surface staining of the infected granule cells, cellule nervose piccole, and within numerous inflammatory cells. On staining with methyl green – pyronin these cells resembled the antibody-producing cells in the spleen.A sequential correlation existed between the development and appearance of the gamma-globulin-producing cells in the spleen and the olfactory bulb which indicated that the lymphoid cells infiltrated into and contributed to the tissue destruction in the olfactory bulb of the infected squirrel.

The olfactory bulb lesion in the hibernating squirrel (Citellus lateralis) infected with coxsackievirus B-3. I. Virus assay, light microscopic and immunofluorescent study.

The olfactory bulb was studied in the normal and infected adult hibernating squirrel (Citellus lateralis). Severe damage developed in the olfactory bulbs of all the squirrels which were inoculated into the axillary adipose pad with coxsackievirus B-3. The earliest changes were observed in the small blood vessels and the large neurons. The blood vessels showed endothelial swelling but no virus antigen. Most of the large neurons showed the presence of virus antigen by the direct fluorescent antibody method. Concurrently, the virus was recovered from the olfactory bulb at a relatively high titer (LD50 10−5.25), but only traces of virus had remained in the blood. In the small neurons (the cellule nervose piccole and the granule cells) the viral antigen appeared later, by which time the large neurons had undergone complete lysis. The granule cells retained the normal morphology throughout the infection. The cellule nervose piccole showed reversible changes, and the glia cells were unaffected.

IMMUNOFLUORESCENT STUDIES OF PLASMODIUM BERGHEI: A "NATURAL" ANTIBODY IN WHITE MICE.

Summary Pooled or individual sera from a proportion of normal mice from a standard laboratory colony were shown to contain an antibody which reacted specifically with Plasmodium berghei antigen, as demonstrated by the indirect and direct fluorescent antibody methods. This antibody could be demonstrated in vitro when reacted with P. berghei antigen, then treated with FITC conjugated antiserum, or as in vivo bound antibody on antigen derived from P. berghei-infected mice. The source of antigen giving rise to this “natural” antibody has not been identified.

Serologic Differentiation of Trachoma Strains and Other Agents of the Psittacosis - Lymphogranuloma Venereum-Trachoma Group with the Aid of the Direct Fluorescent Antibody Method

Summary The serologic relationship of various strains of the psittacosis-lymphogranuloma venereum-trachoma group of agents was examined with the aid of rabbit immune sera and antigens partially purified with fluorocarbon or diethylaminoethyl cellulose. The direct fluorescent antibody technique was employed. Major sharing of antigens between trachoma and inclusion conjuctivitis (TRIC) agents and two strains of lymphogranuloma venereum was demonstrated. No cross-reactions were observed between these strains and a strain of psittacosis. By the use of cross-absorbed immune sera, a subdivision of TRIC agents was obtained according to which strains Dari and Camal, isolated in Israel, and strains Bour and T'ang fell in Group I and strains Asgh, SA-1 and G-17 in Group II. The strain of inclusion conjunctivitis IC Cal-3 showed cross-reactions with both groups of trachoma and, in addition, some specificity of its own. With the aid of cross-absorbed rabbit immune sera it was possible to group trachoma agents directly from infected yolk sac smears.

Reovirus myocarditis in mice: An electron microscopic, immunofluorescent, and virus assay study

Newborn white mice (Webster Swiss) uniformly died with extensive myocarditis 5–7 days after intraperitoneal inoculation of reovirus type 1. Myocardial damage was often so severe that by the 5th postinoculation day it was apparent on gross inspection of the heart. Virus was recovered in relatively high titers from the heart (10 6 PFU per gram) during the period when myocardial injury occurred; viremia was also present, but titers in the bloodstream remained much below those in the heart. Cytoplasmic inclusion bodies, distinctive of reovirus, were seen on histologic examination of myocardial lesions. Viral antigen, demonstrated by the direct fluorescent antibody method, was observed in and around foci of myocardial necrosis. Reovirus particles were numerous and readily visualized by electron microscopy in a distribution similar to that of viral antigen. Both complete and coreless particles were seen measuring 65–75 mμ in diameter; crystalline arrays were occasionally noted. Virus particles were embedded in granular matrices—the apparent sites of their replication. These virus nests were located in the cytoplasm of myocytes between myofibrils. Fine strands formed a filamentous network throughout such matrices. The earliest pathologic changes of fine structures in myocytes were nonspecific ones consisting of swelling and vesiculation of mitochondria and dilatation of the sarcoplasmic reticulum. Cellular necrosis was seen with release of virus into the surrounding tissue. Examples of a less severe cellular injury, focal cytoplasmic degradation, were also encountered.

Direct FA Technique Using Flazo Orange Counterstain in Identification of Neisseria gonorrhoeae

IN THE immunofluoreseent diagnosis of gonorrhea previously described (1-3), a direct and a delayed procedure for identifying gonococci in exudates have been used. When sufficient number of gonococci are present in the exudate, direct fluorescent antibody (FA) staining of smears permits detection and identification of the gonococci within less than an hour even when many contaminating bacteria are present. When the direct FA method is used, the presence of only a few gonococci in an exudate results in a 50 percent loss of sensitivity compared with results by the delayed FA method (1). By the delayed FA method, exudate collected with a swab is incubated 16 to 20 hours on chocolate agar slants, and gonococci too few to detect by the direct FA technique groiw to easily observable numbers. Rapid diagnosis of gonorrhea is often necessary, and the longer time required with the delayed FA method is undesirable. When the simpler, faster, direct FA procedure is used, differentiation of specific Neisseria gonorrhoeae fluorescence is difficult even when the organisms are present in sufficient numbers to permit identification. Various counterstains have been reported to increase the effectiveness of the FA method (4-6). These counterstains allow faster and more specific detection of microorganisms in smears by increasing the background contrast. Careful attention to the collection of specimens for direct smears is necessary for the successful detection of gonococci. It has been suggested (7) that the use of a bacteriological loop is preferable to a swab for the collection of specimens from female gonorrheal patients, particularly when a small amount of exudate is present. In this study the results of examination of smears by the direct FA (DFA) method and by the direct FA method using Flazo orange counterstain (FFA) were compared with the cultural results when specimens were obtained with a bacteriological loop.

Fluorescent-antibody staining of exo-erythrocytic stages of Plasmodium gallinaceum

The exo-erythrocytic stages of P. gallinaceum were stained in tissues by the direct fluorescent antibody method following a freeze-substitution procedure.

IDENTIFICATION OFMYCOPLASMATACEAEBY THE FLUORESCENT ANTIBODY METHOD

Clark, Harold W. (The George Washington University, Washington, D.C.), Jack S. Bailey, Richard C. Fowler, and Thomas McP. Brown. Identification of Mycoplasmataceae by the fluorescent antibody method. J. Bacteriol. 85:111-118. 1963.-The conditions of the fluorescent antibody reactions were studied in relation to their application to Mycoplasmataceae or pleuropneumonia-like organisms (PPLO). Mycoplasma hominis type 1 and 2 antigens and their homologous antisera were used to determine the activity and specificity of these and other strains. Fluorescein isothiocyanate conjugated antiserum globulin preparations were used in both the direct and indirect fluorescent antibody methods. A direct tube technique was used for the detection and measurement of growth in broth cultures by the addition of conjugated antiserum. The specific fluorescent staining and recognition of hot water fixed M. hominis colonies was presented as a suitable identification standard. The antigenic activity was found to remain in the insoluble residue after exposure of M. hominis strains to sonic vibration (9 kc) for 30 min and centrifugation. Brief 2-min exposures of tissue cells to vibration (9 kc) caused the disruption of tissues, with the release of viable and "bound" nonwashable strains that reacted specifically with fluorescent antibody. It is proposed to apply both the sonic vibration and the fluorescent antibody techniques for the identification of Mycoplasmataceae in human tissues.